TBHQ Alleviated Endoplasmic Reticulum Stress-Apoptosis...

Research ArticleTBHQ Alleviated Endoplasmic ReticulumStress-Apoptosis and Oxidative Stress by PERK-Nrf2Crosstalk in Methamphetamine-Induced ChronicPulmonary Toxicity

Yun Wang1 Yu-Han Gu1 Ming Liu2 Yang Bai1 Li-Ye Liang1 and Huai-Liang Wang13

1Department of Clinical Pharmacology School of Pharmacy China Medical University Shenyang 110122 China2Department of Drug Control China Criminal Police University Shenyang 110035 China3National Key Subject Institute of Respiratory Diseases China Medical University Shenyang 110001 China

Correspondence should be addressed to Yun Wang ywang28cmueducn

Received 3 November 2016 Revised 6 January 2017 Accepted 17 January 2017 Published 20 February 2017

Academic Editor Silvana Hrelia

Copyright copy 2017 Yun Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Methamphetamine (MA) leads to cardiac and pulmonary toxicity expressed as increases in inflammatory responses and oxidativestress However some interactions may exist between oxidative stress and endoplasmic reticulum stress (ERS)The current study isdesigned to investigate if both oxidative stress and ERS are involved in MA-induced chronic pulmonary toxicity and if antioxidanttertiary butylhydroquinone (TBHQ) alleviated ERS-apoptosis and oxidative stress by PERK-Nrf2 crosstalk In this study the ratswere randomly divided into control group MA-treated group (MA) and MA plus TBHQ-treated group (MA + TBHQ) Chronicexposure to MA resulted in slower growth of weight and pulmonary toxicity of the rats by increasing the pulmonary arterialpressure promoting the hypertrophy of right ventricle and the remodeling of pulmonary arteries MA inhibited the Nrf2-mediatedantioxidative stress by downregulation of Nrf2 GCS and HO-1 and upregulation of SOD2 MA increased GRP78 to induce ERSOverexpression and phosphorylation of PERK rapidly phosphorylated eIF2120572 increased ATF4 CHOP bax caspase 3 and caspase12 and decreased bcl-2 These changes can be reversed by antioxidant TBHQ through upregulating expression of Nrf2 The aboveresults indicated that TBHQ can alleviate MA-induced oxidative stress which can accelerate ERS to initiate PERK-dependentapoptosis and that PERKNrf2 is likely to be the key crosstalk between oxidative stress and ERS inMA-induced chronic pulmonarytoxicity

1 Introduction

Methamphetamine (MA) abuse is a major public health con-cern [1] For methamphetamine abusers one of the impor-tant death causes is cardiovascular complications includinghypertension aortic dissection acute coronary syndromesmethamphetamine-associated cardiomyopathy and pulmo-nary arterial hypertension (PAH) [2] Chronic metham-phetamine abuse is an increasingly common cause of PAH[3] Increasing evidences suggested that methamphetamineadministration leads to cardiac and pulmonary toxicityexpressed as increases in inflammatory responses and oxida-tive stress [3ndash5]

Oxidative stress is a susceptible factor for endoplasmicreticulum stress (ERS) [6] ERS has recently been paid moreand more attention ERS may initiate the unfolded proteinresponse (UPR) to restore proteostasis or to lead to apop-tosis [7] Recent reports have suggested that the UPR sig-naling switches from prosurvival to proapoptosis throughthree ER transmembrane sensors protein kinase-like ERkinase (PERK) activating transcription factor 6 (ATF6)and inositol-requiring enzyme-1120572 (IRE1) [8] Remarkableevidence has indicated that ERS is accelerated by accu-mulation of unfoldedmisfolded proteins after endoplasmicreticulum environment disturbance triggered by a varietyof physiological and pathological factors such as nutrient

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 4310475 12 pageshttpsdoiorg10115520174310475

2 Oxidative Medicine and Cellular Longevity

deprivation altered glycosylation calcium depletion DNAdamage energy disturbance and oxidative stress [9] Inthe condition of ERS the overexpression of PERK willphosphorylate eIF2120572 and then the p-eIF2120572 will transcribesome transcription factors such as activating transcriptionfactor 4 (ATF-4) CEBP homologous protein (CHOP) andcaspase 12 [10] which affect the apoptosis of cells or tissuesthrough PERK-dependent pathway

Activation of PERK signal can induce conformationalchange of nuclear factor E2-related factor 2 (Nrf2) whichtriggers the dissociation of Kelch-like ECH-associated pro-tein 1- (Keap1-) Nrf2 complex to adjust the oxidation-redoxcondition of cells [11] Nrf2 can enhance the transcription ofcytoprotective genes during oxidative stress [12] In additionit has been reported that Nrf2 is involved in increasing thelevels of endogenous antioxidants attenuating apoptosis andincreasing mitochondrial biogenesis [13ndash15] According tothe above it is prompted that there are some interactionsbetween oxidative stress and ERS and that oxidative stresscan cause ERS meanwhile ERS can also cause or aggravateoxidative stress [16 17] However the mechanism of theinteractions between oxidative stress and ERS is still not fullyunderstood Taken together we will put forward a hypothesisthat PERK-Nrf2 is likely to be the key crosstalk linked tooxidative stress and ERS

ERS-initiated apoptotic signaling has been implicatedin various chronic diseases including diabetes cancer andinflammation [18ndash20] However it has been not clear if ERS-initiated apoptosis is associated with the chronic lung injurycaused by methamphetamine Tertiary butylhydroquinone(TBHQ) an Nrf2 signaling pathway inducer is demonstratedto induce remarkable antioxidant activity in a variety ofcells and tissues [21] On the basis of our hypothesis thecurrent study is designed to investigate if oxidative stress andERS are involved in pulmonary toxicity and if PERKNrf2 isassociated with the interaction between oxidative stress andERS-initiated apoptosis in MA-induced pulmonary toxicity

2 Materials and Methods

21 Chronic Methamphetamine Exposure Forty-five maleWistar rats (200 plusmn 10 g) were purchased from Animal Re-source Center China Medical University (Certificate num-ber Liaoning SCSK 2012-0005) All of the 45 rats wererandomly divided into control group methamphetamine-treated group (MA) and methamphetamine plus TBHQ-treated group (MA + TBHQ) At the first week intraperi-toneal injection of methamphetamine with the dosage of10mgkg for one week was administrated to the rats in MAgroup and MA + TBHQ group and then increasing dailydosage of 1mgkg was administrated per week until the sixthweek a daily dosage was increased to 15mgkg respectively(twice per day for 6 weeks) [5 22] Meanwhile the rats incontrol group were injected with an equal volume of 09physiological saline solution After administration of MArats in the MA + TBHQ groups then received intragastricadministration of 125mgkg TBHQ [23] Rats in the controland MA groups were intragastrically administered with anequal volume of 05 gum tragacanth All animals were

housed in a roomwith controlled temperature (18ndash22∘C) andhumidity (50ndash70) and were fed solid food and water adlibitum in an alternating 12 h light and 12 h dark cycle Allprocedures followed the Guide for the Care and Use of Lab-oratory Animals of the National Institutes of Health (NIH)and all protocols were approved by the Institutional AnimalCare and Use Committee of China Medical University

22 Hemodynamic Measurements and Tissue CollectionAfter 6 weeks rats in all groups were anaesthetized with 3pentobarbital sodium (45mgkg ip) prior to hemodynamicmeasurements as described previously Briefly the rightjugular vein was dissected and catheterized with a PV-1catheter The catheter was advanced via the right ventricleinto the pulmonary artery for direct measurement of pul-monary arterial pressure (PAP) The right carotid artery wasdissected and intubated with a PE-50 catheter to measuresystemic blood pressure (SBP) Prior to catheterization thePV-1 and PE-50 catheters were filled with saline containing1 heparin PAP and SBP were recorded using a polygraphsystem (RM6000 Nihon Kohden Tokyo Japan)

Of the 45 rats 15 rats (five per group) were perfusedwith paraformaldehyde The right lower lung tissues weredissected and then they were paraformaldehyde-fixed andparaffin-embedded The lung sections (5 120583m thick) wereprepared for hematoxylin-eosin (HE) staining Eosin VanGieson (EVG) staining immunohistochemical (IHC) stain-ing immunofluorescence (IF) and TUNEL assay

The other rats were then killed by overdose pentobarbitalsodium The rat lungs and pulmonary arteries were quicklydissected and removed on ice and stored at minus80∘C untilfurther use for real-time PCR and western blotting

23 Assessment of Remodeling of the Heart and PulmonaryArteries The hearts were dissected and taken out Rightventricle (RV) and left ventricle plus interventricular septum(LV + S) were dissected and weighed separately to evaluatethe extent of the right ventricular hypertrophy expressed asright ventricular index (RVI) which is calculated by weightratio of the RV and LV + S

Lung sections (5 120583m thick) were stained with hema-toxylin and eosin (HampE) watched under light microscopyand analyzed by MetamorphyBX41 (UICOLYMPUSUSAJAP) Vessels gt 50120583m were identified and measured atthe 2 ends of the shortest external diameter of the distal PAsand the percentage of medial wall thickness (medial wallthickness) is calculated by ([2timeswall thicknessexternal dia-meter] times 100)

Lung sections (5 120583m thick) were stained with EVG tolocalize collagen (staining red) and elastin (staining blackbrown) in lungs watched under light microscopy and ana-lyzed by MetamorphyBX41 (UICOLYMPUS USAJAP)The degree of muscularization was calculated as nonmuscu-larized (no evidence of any vessel wall muscularization) par-tially muscularized (smooth muscle cells (SMC) identifiablein less than three-quarters of the vessel circumference) orfully muscularized (SMC in more than three-quarters of thevessel circumference) to assess the remodeling of pulmonaryarteries

Oxidative Medicine and Cellular Longevity 3

Table 1

Accession number Species Primer Primer sequences (51015840 to 31015840)

RA016146 Rat (Nrf2)-Forward AGAGATGGAACTGACTTGGCAAGAG

RA016146 Rat (Nrf2)-Reversed TGCATCTGGATGAATTGAACAGG

RA015375 Rat (120573-actin)-Forward GGAGATTACTGCCCTGGCTCCTA

RA015375 Rat (120573-actin)-Reversed GACTCATCGTACTCCTGCTTGCTG

24 Immunohistochemical Staining After processing the tis-sue and embedding in paraffin 5 120583m thick sections werestained by immunohistochemical (IHC) procedures withUltrasensitive TM S-P Kit and DAB Staining Kit (Maxin-Bio Co China) IHC staining followed a basic indirectprotocol using a citrate antigen retrieval method Primaryantibodies of mouse were anti-rat CHOP (1 100 BioworldUSA) and goat anti-mouse 120573-actin (1 2000 Santa CruzCalifornia USA) in TBS-T with 5 BSA overnight at4∘C For the negative control the primary antibody wasreplaced with 001M phosphate-buffered saline (PBS) in theincubation step The positive expression of these primaryantibodies was examined and analyzed by BX51Metamorphy(OLYMPUSUIC JAPUSA) At least 6 medium and smallpulmonary arteries were examined for each slide For theconvenience of understanding and statistical process thecontent of protein in the pulmonary arteries was calculatedas optical density average

25 Immunofluorescence Staining Tissue paraffin sections(5 120583m) were mounted on slides and placed in a 55∘C ovenfor 10min deparaffinized in xylene (3x 5min) hydratedusing an alcohol series 100 95 and 70 alcohol (each3x 5min) and rinsed in water The sections were processedfor antigen retrieval by boiling the slides in 10mM CitrateBuffer (pH 60) The slides were cooled at room temperaturefor 20min washed in PBS and blocked in 10 normalserum overnight at 4∘C Immunofluorescence was performedon serial sections from each group using rabbit anti-baxand anti-bcl-2 antibodies (Proteintech USA) The sectionswere incubated with primary antibodies for 1 h at roomtemperature Subsequently sections were stained with AlexaFluor 488 goat anti-rabbit secondary antibodies (Molec-ular Probes Inc) The fluorescent-stained sections werewatched by Laser Confocal Scanning Microscope (FV1000S-SIMIX81 OLYMPUS JAP) and analyzed by Software ImageJ to calculate as the average intensity of the fluorescent signalSix visual fields from each slide were imaged and quantifiedto assess the final mean fluorescent signal for each sample

26 Western Blotting Lung samples were homogenized inlysis buffer Total protein from each sample was separatedby sodiumdodecyl sulfate polyacrylamide gel electrophoresisand transferred to PVDF membrane (Bio-Rad) The mem-branes were blocked by TBS-005 Tween-20 (TBS-T) with5 nonfat dry milk for 60min and were then incubated with

mouse anti-ratNRF2 (1 500 ProteintechUSA)GCS (1 200Beijing Biosynthesis Biotechnology Co Ltd) HO-1 (1 200Beijing Biosynthesis Biotechnology Co Ltd) SOD 2 (SantaCruz Biotechnology Inc) GRP 78 (1 2000 ProteintechUSA) PERK (1 1000 BioworldUSA) p-PERK (1 200 SantaCruz Biotechnology Inc) eIF2120572 (1 1000 Abcam USA) p-eIF2120572 (1 1000 Cell Signaling USA) ATF4 (1 1000 Proteintech USA) CHOP (1 500 Biolworld USA) bax (1 1000Proteintech USA) caspase 12 (1 1000 Proteintech USA)and goat anti-mouse 120573-actin (1 2000 Santa Cruz CaliforniaUSA) antibodies in TBS-T with 5 BSA overnight at 4∘Crespectively A corresponding secondary antibody treatmentis followed by enhanced chemiluminescence (ECL) Therelative protein expression was quantified by densitometryusing image quant software (MolecularDynamics)The resultof NRF2 GCS HO-2 SOD 2 GRP78 p-PERKPERK p-eIF2120572eIF2120572 ATF4 CHOP bax caspase 3 and caspase 12wasrepresented by the relative yield to the 120573-actin respectively

27 RNA Extraction and Quantitative Real-Time PCR TotalRNA was extracted with TRIzol Reagent (Invitrogen CoUSA) as described in its directions The concentration andpurification of total RNA were estimated with ultraspec-trophotometer The extracted RNA was reverse-transcribedwith oligo dT primers and PrimeScript RT reagent KitPerfect Real Time (Takara Co Japan) PCR experiments weredone using theChroma4RT-PCRDetection System (Bio-RadCFX96 CA USA) and SYBR Green Supermix (Takara CoJapan) according to the manufacturerrsquos protocol Sequencesfor gene specific primers corresponding to PCR targets weredesigned and synthesized by Lian-xing Bio-Tech Inc Thesequences for the Nrf2 and 120573-actin were described as Table 1

28 TUNEL Assay Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)TdT-mediated dUTP Nick-End Labeling was performedon lung sections according to the manufacturerrsquos speci-fications (Beyotime Institute of Biotechnology ShanghaiChina) Addition of the chromogen diaminobenzidine tetrahydrochloride (DAB) resulted in a brown reaction productthat was evaluated by light microscopy The proportion ofapoptosis was calculated as the number of apoptotic cellsdivided by the total number of cells Six visual fields fromeach slidewere imaged and quantified to assess the finalmeanpercentage of apoptotic cells in the lungs


Table 2 Comparison of the rat weights in different groups

Preweight (g) Postweight (g) Percentage of weight change ()Control (119899 = 15) 1987 plusmn 98 2994 plusmn 184 508 plusmn 88MA (119899 = 10) 2036 plusmn 58 2326 plusmn 121lowastlowast 143 plusmn 60lowastlowast

MA + TBHQ (119899 = 12) 1994 plusmn 77 2500 plusmn 119lowastlowast 254 plusmn 59lowastlowast

Data are means plusmn SD lowastlowast119875 lt 001 versus control group 119875 lt 005 119875 lt 001 versusMA group MAmethamphetamine TBHQ tertiary butylhydroquinone

Table 3 Comparison of HR mSAP mPAP and RVI in different groups

HR (bmp) mSAP (mmHg) mPAP (mmHg) RVIControl (119899 = 15) 375 plusmn 31 1389 plusmn 85 157 plusmn 19 023 plusmn 002MA (119899 = 10) 376 plusmn 39 1381 plusmn 167 205 plusmn 20lowast 037 plusmn 003lowastlowast

MA + TBHQ (119899 = 12) 378 plusmn 37 1394 plusmn 173 174 plusmn 24 027 plusmn 004

Data are means plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control group 119875 lt 005 119875 lt 001 versus MA group HR heart rate mSAP mean systemic arterialpressure mPAP mean pulmonary arterial pressure RVI right ventricular index MA methamphetamine TBHQ tertiary butylhydroquinone

29 Statistical Analysis All data are expressed as the mean plusmnSD Statistical comparisons weremade by one-way analysis ofvariance and statistical differences between two groups wereestablished using the least significant difference test Valuesof 119875 lt 005 were considered statistically significant

3 Results

31 Comparison of Weight and Hemodynamic Indexes Theweight of rats was increased in all groups but the percentageof weight changes was different between the groups after6 weeks The percentage of weight change was significantlylower in MA group than in control group (119875 lt 001 versuscontrol) (Table 2) MA has no significant effect on HR andSAP In MA group the PAP and RVI were significantlyincreased but they can be reversed by TBHQ (Table 3)

32 Effect of TBHQ on MA-Induced Pulmonary VascularRemodeling Hypertrophy of pulmonary vessel wall was eval-uated as the percentage of medial wall thickness (medial wallthickness) by HampE staining (Figure 1(a)) The percentageof medial wall thickness was increased from 341 plusmn 21in control group to 645 plusmn 64 in MA group and wasmarkedly inhibited in MA + TBHQ at 447 plusmn 23 (Fig-ure 1(c)) The muscularization of pulmonary arteries wasdetected by EVG staining and was investigated under lightmicroscopy (Figure 1(b)) Elastin was stained in black andcollagen was stained in redThe rates of nonmuscularizationpartial muscularization and full muscularization in the con-trol group were 649 255 and 116 respectively in the MAgroup these values were 495 155 and 35 respectively andin the MA + TBHQ group these values were 609 236 and155 respectively (Figure 1(d)) These results indicate thatMA significantly promotes pulmonary vascular remodelingwhereas TBHQ attenuates the effects of MA

33 Effect of MA and TBHQ on Nrf2-Mediated AntioxidativeStress in Lungs Protein extracts were subjected to west-ern blot Compared with control group MA significantlydownregulated the Nrf2 expression which was markedly

upregulated by TBHQ (Figure 2(a)) Additionally the resultsof Nrf2 by real-time PCR were coincided with the westernblot (Figure 2(b)) And as shown in Figure 2(c) the antiox-idative enzymes GCS andHO-1 were decreased but oxidativeenzyme SOD2 was increased by MA which was reversedby TBHQ These results indicated that MA impeded Nrf2-medicated antioxidative stress and impaired the ability ofantioxidative stress which aggravated the oxidative stress

34 Effect of TBHQ on the Lasting-ERS Chronically Exposedto MA GRP78 is the key signal of the ERS The western blotassay demonstrated that the expression of the GRP78 ishigher in MA group than in the control group (Figure 3(a))Additionally immunohistochemistry with anti-GRP78 anti-body revealed strong GRP78 expression in rat lungs com-pared with the control group (Figure 3(b)) However TBHQcan obviously downregulate the expression of GRP78 inrat lungs These suggested that the prolonged stimulationexcessively activated ERS which can be alleviated by TBHQ

35 Effect of MA and TBHQ on the PERK Signaling PathwayResults from western blot analysis (Figure 4(a)) demon-strated that the expressions of PERK p-PERK and ratio ofp-PERKPERK were all significantly upregulated in lungs inMA group compared with control group and that they weredecreased in MA + TBHQ group compared with MA group(Figure 4(b)) PERK signaling was activated to induce rapidphosphorylation of eIF2120572 Western blot was carried out tofurther demonstrate that in MA group signal eIF2120572 wasphosphorylated compared with control group And p-eIF2120572expression was decreased after administration of TBHQ(Figure 4(c)) Downstream signal ATF4 expression is alsoreversed by TBHQ from MA (Figure 4(d)) These resultsindicated that chronic exposure to MA induced lasting ERSby excessively phosphorylating PERKeIF2120572 signaling

36 Effect of MA and TBHQ on ERS-Initiated ApoptosisResults from western blot analysis demonstrate that com-pared with control group CHOP bax caspase 3 and caspase12 protein expressions were significantly increased in MA


Control MA MA + TBHQ

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NonmuscularizationPartial muscularizationFull muscularization

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Figure 1 Effect of TBHQ on MA-induced pulmonary vascular remodeling (a) Hypertrophy of pulmonary vessel wall by HampE staining(Olympus BX 51 Japan times400) (b) the muscularization of pulmonary arteries by EVG staining (c) the percentage of medial wall thicknessin different groups (Olympus BX 51 Japan times400) (d) muscularization degree of pulmonary arteries in different groups The changes ofpulmonary arteries were marked by black arrows Compared with the control group in MA group the lumen was significantly narrowedand the wall of pulmonary arteries was markedly thickened which were attenuated by TBHQ Values are expressed as mean plusmn SD (119899 = 5)lowast119875 lt 005 lowastlowast119875 lt 001 compared with the control group 119875 lt 005 compared with the MA group MA methamphetamine THBQ tertiarybutylhydroquinone

groups which were markedly decreased after administrationof TBHQ (Figure 5(a)) The immunofluorescence stainingresults (Figure 5(b)) showed that there is higher positiveexpression (green) of apoptotic cytokine bax in MA groupthan in control group and it is ameliorated by TBHQHowever it is contrary to the expression of anti-apoptoticcytokine bcl-2 which implied that TBHQ can ameliorate theERS-initiated apoptosis by MA TUNEL-positive staining isindicated by dark brown TUNEL-positive cells are infre-quently observed in the control group The proportion ofapoptosis significantly increased in the MA group (119875 lt 005versus control) Yet it decreased in the MA + TBHQ group(119875 lt 005 versus MCT) (Figure 5(c))

4 Discussion

Results from the present study showed that chronic expo-sure to methamphetamine reduced weight growth of ratsand induced pulmonary toxicity of rats by increasing thepulmonary arterial pressure promoting the hypertrophy ofright ventricle and the remodeling of pulmonary arteriesMA inhibited the Nrf2-mediated antioxidative stress bydownregulation of Nrf2 GCS and HO-1 and upregulationof SOD2 MA damaged the ability of anti-oxidative stress toaggravate oxidative stress Overexpression and phosphory-lation of PERK rapidly phosphorylated eIF2120572 and activatedthe PERKeIF2120572ATF4 signaling This chronic stimulation


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Figure 2 Effect of MA and TBHQ on Nrf2-mediated antioxidative stress in lungs (a) Nrf2 protein expression in lungs (b) Nrf2 level indifferent groups by real-time PCR (c) oxidative genes expression in different groups Values are expressed as mean plusmn SD (119899 = 5) lowast119875 lt 005lowastlowast119875 lt 001 compared with the control group 119875 lt 005 119875 lt 001 compared with the MA group MA methamphetamine THBQ tertiary

butylhydroquinone


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Figure 3 Effect of TBHQ on the lasting ERS chronically exposed to MA (a) GRP78 expression in rat lungs by western blot (b) GRP78protein expression illustrated by immunohistochemical analysis (times400) Cross sections were stained with rabbit anti-GRP78 (brown) andcounterstained with hematoxylin (blue) in different groups Values are expressed as mean plusmn SD (119899 = 5) lowastlowast119875 lt 001 compared with thecontrol group 119875 lt 005 compared with the MA group MA methamphetamine THBQ tertiary butylhydroquinone

caused lasting ERS and further induced apoptosis by theincrease in CHOP bax caspase 3 caspase 12 and decrease inbcl-2 These changes can be reversed by antioxidant TBHQthrough upregulating expression of Nrf2 The above resultsindicated that TBHQ can alleviate MA-induced oxidativestress which can accelerate ERS to initiate PERK-dependentapoptosis and that PERKNrf2 is likely to be the key crosstalkbetween oxidative stress and ERS in MA-induced chronicpulmonary toxicity

As a powerful addictive drug MA leads to multipleorgans damage such as brain heart and lung [24ndash26] Theredox imbalances and generation of free radicals such asROS can lead to oxidative stress [27] Oxidative stress is oneof the reasons of MA-induced neurotoxicity [28] In thisstudy it is found that in rat lungs the antioxidative enzymesGCS and HO-1 were decreased but oxidative enzyme SOD2was increased by MA which was reversed by TBHQ Theseresults are in accord with the previous reports that the


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Figure 4 Effect of MA and TBHQ on the PERK signaling pathway (a) PERK signaling expression by western blot (b) p-PERKPERK inlungs in different groups (c) p-eIF2120572eIF2120572 in different groups (d) ATF4 protein expression in different groups Values are expressed asmean plusmn SD (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 compared with the control group 119875 lt 005 119875 lt 001 compared with the MA group MAmethamphetamine THBQ tertiary butylhydroquinone

damage in nervous system caused by MA can be attenuatedby antioxidants [29 30] Therefore TBHQ attenuated MA-induced pulmonary toxicity by increasing and activatingNrf2to strengthen the ability of antioxidative stress

Oxidative stress can disrupt the function of endoplasmicreticulum (ER) and lead to endoplasmic reticulum stress(ERS) [31] To survive the ERS the three mechanisms un-folded protein response (UPR) the ER-overload response(EOR) and the ER-associated degradation (ERAD) will beactivated [28] UPR is vital to ensure the ER function and cellsurvival [32] GRP78 is one of the most highly expressed ERresident chaperones in the condition of UPR which belongsto heat-shock protein (Hsp70) family [32 33] When theunfoldedmisfolded proteins accumulate in the ER the threeER transmembrane receptors will release GRP78 and activateand initiate signaling cascades designed to protect the cells orlead to apoptosis [34 35] GRP78 is the sign of UPR and ERS[36] In our studyMA induced the high expression ofGRP78

which indicated that ERSwas involved in chronic lung injuryTheGRP78 inMA+ TBHQ group was less thanMA group Itdemonstrated that TBHQ alleviated the lasting ERS to someextent

ER transmembrane sensors include PERK ATF6 andIRE1 [8] PERK is the one of three transmembrane proteinsof the UPR signaling and activated by the ERS [37 38] Thephosphorylation of PERK and its downstream factor eIF2120572attenuate the synthesis of protein and restore ER homeostasis[39 40] The Nrf2 is known as a substrate of PERK [41]Phosphorylation of PERK can cause a conformational changeof Nrf2 protein by triggering the dissociation of Keap1-Nrf2 complex and the dissociation of Nrf2 into the nucleusupregulated expression of antioxidant genes [42] Thereforeoxidative stress and endoplasmic reticulum stress has somecertain relevance through PERKNrf2 pathway which is whywe choose PERK-dependent pathway of ERS In our study itis found that MA inhibited the expression of Nrf2 in lungs


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Figure 5 Effect of MA and TBHQ on ERS-initiated apoptosis (a) Apoptotic cytokines CHOP bax caspase 12 and caspase 3 expressed indifferent groups by western blot (b) immunofluorescence assay for bax and bcl-2 in different groups (times400) Higher positive expression(green) of bax in MA group than in control group but it is contrary to the expression of bcl-2 which can be reversed by TBHQ (c) TUNELassay in different groups (times400) TUNEL-positive staining is indicated by dark brown particles in cell nucleus The proportion of apoptosissignificantly increased in the MA group compared with control group Yet it decreased in the MA + TBHQ group Values are expressed asmean plusmn SD (119899 = 5) lowast119875 lt 005 lowastlowast119875 lt 001 compared with the control group 119875 lt 005 119875 lt 001 compared with the MA group MAmethamphetamine THBQ tertiary butylhydroquinone

to phosphorylate PERKeIF2120572 and activate ATF4CHOP-dependent apoptosis Hence interfering with Nrf2 responsecaused accumulation of damaged proteins within the ERleading to PERK-dependent apoptosis [43] It is a hint thatoxidative stress induced endoplasmic reticulum stress andexcessive endoplasmic reticulum stress can also cause or agg-ravate oxidative stress due to PERKNrf2 crosstalk [41 44]

A rich body of evidence has demonstrated that TBHQcanactivate antioxidant responsive element (ARE) and upreg-ulate Nrf2 expression to attenuate the damage of nervoussystem [45 46] The antioxidant TBHQ is effective in pro-tecting against cellular dysfunction induced by oxidativestress inducers in various cell types [47] In our studyit is found that in rat lungs TBHQ not only upregu-lated the level of Nrf2 and its related antioxidative genesbut also inhibited the expression of GRP78 and abrogatedthe PERKeIF2120572ATF4CHOP-dependent apoptosis Takentogether with the above results it is indicated that TBHQattenuated ER stress to abrogate ERS-initiated apoptosisby activating antioxidative stress in MA-induced chronicpulmonary toxicity It hinted that the lasting endoplasmicreticulum stress-apoptosis can be alleviated by the adjustingthe balance between oxidation and reduction [42 48]

In summary both oxidative stress and ERS may beinvolved in MA-induced pulmonary toxicity and oxidativestress can accelerate ERS to further initiate PERK-dependentapoptosis which can be alleviated by the antioxidant TBHQ

These results from our study implied that PERKNrf2 is likelyto be the key crosstalk between oxidative stress and ERS inMA-induced chronic pulmonary toxicity

Competing Interests

The authors declare that there are no competing interests

Acknowledgments

This research was funded by National Natural Science Foun-dation of China (no 81503058) and Natural Science foun-dation of Liaoning Province (no 2014021065) The authorswould like to acknowledge Zai-Xing Chen Yan Wang andWei-Fan Yao for providing technical assistance

References

[1] K A Stout A R Dunn K M Lohr et al ldquoSelective enhan-cement of dopamine release in the ventral pallidum ofmethamphetamine-sensitized micerdquo ACS Chemical Neuro-science vol 7 no 10 pp 1364ndash1373 2016

[2] E D Paratz N J Cunningham and A I MacIsaac ldquoThecardiac complications of methamphetaminesrdquo Heart Lung andCirculation vol 25 no 4 pp 325ndash332 2016


[3] C A Thompson ldquoPulmonary arterial hypertension seen inmethamphetamine abusersrdquoAmerican Journal of Health-SystemPharmacy vol 65 no 12 pp 1109ndash1110 2008

[4] Z V Varga P Ferdinandy L Liaudet and P Pacher ldquoDrug-induced mitochondrial dysfunction and cardiotoxicityrdquo Amer-ican Journal of PhysiologymdashHeart and Circulatory Physiologyvol 309 no 9 pp H1453ndashH1467 2015

[5] Y Wang M Liu H M Wang et al ldquoInvolvement of serotoninmechanism in methamphetamine-induced chronic pulmonarytoxicity in ratsrdquo Human and Experimental Toxicology vol 32no 7 pp 736ndash746 2013

[6] S Muratsu-Ikeda M Nangaku Y Ikeda T Tanaka T Wadaand R Inagi ldquoDownregulation of miR-205 modulates cellsusceptibility to oxidative and endoplasmic reticulum stressesin renal tubular cellsrdquo PLOSONE vol 7 no 7 Article ID e414622012

[7] Y-P Vandewynckel D Laukens A Geerts et al ldquoThe para-dox of the unfolded protein response in cancerrdquo AnticancerResearch vol 33 no 11 pp 4683ndash4694 2013

[8] R Dai J Li J Fu et al ldquoDisturbance of Ca2+ homeostasis con-verts pro-Met into non-canonical tyrosine kinase p190MetNCinresponse to endoplasmic reticulum stress in MHCC97 cellsrdquoJournal of Biological Chemistry vol 287 no 18 pp 14586ndash145972012

[9] A H Schonthal ldquoEndoplasmic reticulum stress its role indisease and novel prospects for therapyrdquo Scientifica vol 2012Article ID 857516 26 pages 2012

[10] C Koumenis C Naczki M Koritzinsky et al ldquoRegulation ofprotein synthesis by hypoxia via activation of the endoplasmicreticulum kinase PERK and phosphorylation of the translationinitiation factor eIF2120572rdquoMolecular and Cellular Biology vol 22no 21 pp 7405ndash7416 2002

[11] Y F Zhu X H Li Z P Yuan et al ldquoAllicin improves endoplas-mic reticulum stress-related cognitive deficits via PERKNrf2antioxidative signaling pathwayrdquo European Journal of Pharma-cology vol 762 pp 239ndash246 2015

[12] L Baird and A T Dinkova-Kostova ldquoThe cytoprotective role ofthe Keap1-Nrf2 pathwayrdquo Archives of Toxicology vol 85 no 4pp 241ndash272 2011

[13] J L Lim M M M Wilhelmus H E de Vries B Drukarch JJ M Hoozemans and J van Horssen ldquoAntioxidative defensemechanisms controlled by Nrf2 state-of-the-art and clinicalperspectives in neurodegenerative diseasesrdquoArchives of Toxicol-ogy vol 88 no 10 pp 1773ndash1786 2014

[14] A Glory andD A Averill-Bates ldquoThe antioxidant transcriptionfactor Nrf2 contributes to the protective effect of mild ther-motolerance (40∘C) against heat shock-induced apoptosisrdquo FreeRadical Biology and Medicine vol 99 pp 485ndash497 2016

[15] X Pan D Yan D Wang et al ldquoMitochondrion-mediated apo-ptosis induced by acrylamide is regulated by a balance betweenNrf2 antioxidant and MAPK signaling pathways in PC12 cellsrdquoMolecular Neurobiology pp 1ndash14 2016

[16] S Thummayot C Tocharus A Suksamrarn and J TocharusldquoNeuroprotective effects of cyanidin against A120573-induced oxida-tive and ER stress in SK-N-SH cellsrdquo Neurochemistry Interna-tional vol 101 pp 15ndash21 2016

[17] B Yang Y Xu Y Hu et al ldquoMadecassic Acid protects agai-nst hypoxia-induced oxidative stress in retinal microvascularendothelial cells via ROS-mediated endoplasmic reticulum

stressrdquo Biomedicine amp Pharmacotherapy vol 84 pp 845ndash8522016

[18] Z Wang Y Huang Y Cheng et al ldquoEndoplasmic reticulumstress-induced neuronal inflammatory response and apopto-sis likely plays a key role in the development of diabeticencephalopathyrdquo Oncotarget 2016

[19] K Miyahara H Kazama H Kokuba et al ldquoTargeting borte-zomib-induced aggresome formation using vinorelbine enhan-ces the cytotoxic effect along with ER stress loading in breastcancer cell linesrdquo International Journal of Oncology vol 49 no5 pp 1848ndash1858 2016

[20] P Xue B Li Y An et al ldquoDecreased MORF leads to pro-longed endoplasmic reticulum stress in periodontitis-associa-ted chronic inflammationrdquo Cell Death and Differentiation vol23 pp 1862ndash1872 2016

[21] X Shi Y Li J Hu and B Yu ldquoTert-butylhydroquinoneattenuates the ethanol-induced apoptosis of and activates theNrf2 antioxidant defense pathway in H9c2 cardiomyocytesrdquoInternational Journal of Molecular Medicine vol 38 no 1 pp123ndash130 2016

[22] J Lan X Deng and H Wang ldquoResearch of nitration modifi-cation in chronic methamphetamine treated rats and humanserum and the neurotoxicityrdquo Chinese Journal of Drug Depen-dence vol 20 no 3 pp 177ndash181 2011

[23] Z Wang C Ji L Wu et al ldquoTert-butylhydroquinone alleviatesearly brain injury and cognitive dysfunction after experimentalsubarachnoid hemorrhage role of Keap1Nrf2ARE pathwayrdquoPLoS ONE vol 9 no 5 Article ID e97685 2014

[24] S M Wells M C Buford S N Braseth J D Hutchison andA Holian ldquoAcute inhalation exposure to vaporized metham-phetamine causes lung injury in micerdquo Inhalation Toxicologyvol 20 no 9 pp 829ndash838 2008

[25] S J Kish I Boileau R C Callaghan J Tong and P BolamldquoBrain dopamine neurone lsquodamagersquo methamphetamine usersvs Parkinsonrsquos diseasemdasha critical assessment of the evidencerdquoEuropean Journal of Neuroscience vol 45 no 1 pp 58ndash66 2017

[26] S Sliman J Waalen and D Shaw ldquoMethamphetamine-associa-ted congestive heart failure increasing prevalence and rela-tionship of clinical outcomes to continued use or abstinencerdquoCardiovascular Toxicology vol 16 no 4 pp 381ndash389 2016

[27] M Birch-Machin and A Bowman ldquoOxidative stress and age-ingrdquo British Journal of Dermatology vol 175 supplement 2 pp26ndash29 2016

[28] D Wen M An H Gou et al ldquoCholecystokinin-8 inhibitsmethamphetamine-induced neurotoxicity via an anti-oxidativestress pathwayrdquo NeuroToxicology vol 57 pp 31ndash38 2016

[29] E A Kiyatkin and H S Sharma ldquoAcute methamphetamine in-toxication brain hyperthermia blood-brain barrier brainedema and morphological cell abnormalitiesrdquo InternationalReview of Neurobiology vol 88 pp 65ndash100 2009

[30] M Shen L Wang B Wang et al ldquoActivation of volume-sen-sitive outwardly rectifying chloride channel by ROS contributesto ER stress and cardiac contractile dysfunction involvementof CHOP through Wntrdquo Cell Death and Disease vol 5 no 11Article ID e1528 2014

[31] R V Rao H M Ellerby and D E Bredesen ldquoCoupling endo-plasmic reticulum stress to the cell death programrdquo Cell Deathand Differentiation vol 11 no 4 pp 372ndash380 2004


[32] J Wu and R J Kaufman ldquoFrom acute ER stress to physiologicalroles of the unfolded protein responserdquo Cell Death and Differ-entiation vol 13 no 3 pp 374ndash384 2006

[33] H Tanjore T S Blackwell and W E Lawson ldquoEmergingevidence for endoplasmic reticulum stress in the pathogen-esis of idiopathic pulmonary fibrosisrdquo American Journal ofPhysiologymdashLung Cellular and Molecular Physiology vol 302no 8 pp L721ndashL729 2012

[34] A Bertolotti Y Zhang LMHendershot H P Harding andDRon ldquoDynamic interaction of BiP and ER stress transducers inthe unfolded-protein responserdquo Nature Cell Biology vol 2 no6 pp 326ndash332 2000

[35] M Schroder and R J Kaufman ldquoThe mammalian unfoldedprotein responserdquo Annual Review of Biochemistry vol 74 pp739ndash789 2005

[36] G Zhu and A S Lee ldquoRole of the unfolded protein responseGRP78 and GRP94 in organ homeostasisrdquo Journal of CellularPhysiology vol 230 no 7 pp 1413ndash1420 2015

[37] K Kohno ldquoHow transmembrane proteins sense endoplasmicreticulum stressrdquo Antioxidants and Redox Signaling vol 9 no12 pp 2295ndash2303 2007

[38] H P Harding Y Zhang and D Ron ldquoProtein translationand folding are coupled by an endoplasmic-reticulum-residentkinaserdquo Nature vol 397 pp 271ndash274 1999

[39] H Yoshida K Haze H Yanagi T Yura and K Mori ldquoIdenti-fication of the cis-acting endoplasmic reticulum stress responseelement responsible for transcriptional induction of mam-malian glucose-regulated proteins involvement of basic leucinezipper transcription factorsrdquo Journal of Biological Chemistryvol 273 no 50 pp 33741ndash33749 1998

[40] X Liu D Kwak Z Lu et al ldquoEndoplasmic reticulum stress sen-sor protein kinase R-like endoplasmic reticulum kinase (PERK)protects against pressure overload-induced heart failure andlung remodelingrdquo Hypertension vol 64 no 4 pp 738ndash7442014

[41] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[42] Y-F Zhu X-H Li Z-P Yuan et al ldquoNeuropharmacologyand analgesia Allicin improves endoplasmic reticulum stress-related cognitive deficits via PERKNrf2 antioxidative signalingpathwayrdquo European Journal of Pharmacology vol 762 ArticleID 70031 pp 239ndash246 2015

[43] A Zanotto-Filho V P Masamsetti E Loranc et al ldquoAlkylatingagent-induced NRF2 blocks endoplasmic reticulum stress-mediated apoptosis via control of glutathione pools and proteinthiol homeostasisrdquo Molecular Cancer Therapeutics vol 15 no12 pp 3000ndash3014 2016

[44] T Hayashi A Saito S Okuno M Ferrand-Drake R LDodd and P H Chan ldquoDamage to the endoplasmic reticulumand activation of apoptotic machinery by oxidative stressin ischemic neuronsrdquo Journal of Cerebral Blood Flow andMetabolism vol 25 no 1 pp 41ndash53 2005

[45] A D Kraft D A Johnson and J A Johnson ldquoNuclear factorE2-related factor 2-dependent antioxidant response elementactivation by tert-butylhydroquinone and sulforaphane occur-ring preferentially in astrocytes conditions neurons againstoxidative insultrdquo Journal of Neuroscience vol 24 no 5 pp 1101ndash1112 2004

[46] J-M Lee M J Calkins K Chan Y W Kan and J A JohnsonldquoIdentification of the NF-E2-related factor-2-dependent genesconferring protection against oxidative stress in primary cor-tical astrocytes using oligonucleotide microarray analysisrdquo TheJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003

[47] S Li J Li C Shen et al ldquoTert-Butylhydroquinone (tBHQ) pro-tects hepatocytes against lipotoxicity via inducing autophagyindependently of Nrf2 activationrdquo Biochimica et BiophysicaActamdashMolecular and Cell Biology of Lipids vol 1841 no 1 pp22ndash33 2014

[48] P Palsamy K R Bidasee and T Shinohara ldquoValproic acid sup-presses Nrf2Keap1 dependent antioxidant protection throughinduction of endoplasmic reticulum stress and Keap1 promoterDNA demethylation in human lens epithelial cellsrdquo Experimen-tal Eye Research vol 121 pp 26ndash34 2014

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


deprivation altered glycosylation calcium depletion DNAdamage energy disturbance and oxidative stress [9] Inthe condition of ERS the overexpression of PERK willphosphorylate eIF2120572 and then the p-eIF2120572 will transcribesome transcription factors such as activating transcriptionfactor 4 (ATF-4) CEBP homologous protein (CHOP) andcaspase 12 [10] which affect the apoptosis of cells or tissuesthrough PERK-dependent pathway

Activation of PERK signal can induce conformationalchange of nuclear factor E2-related factor 2 (Nrf2) whichtriggers the dissociation of Kelch-like ECH-associated pro-tein 1- (Keap1-) Nrf2 complex to adjust the oxidation-redoxcondition of cells [11] Nrf2 can enhance the transcription ofcytoprotective genes during oxidative stress [12] In additionit has been reported that Nrf2 is involved in increasing thelevels of endogenous antioxidants attenuating apoptosis andincreasing mitochondrial biogenesis [13ndash15] According tothe above it is prompted that there are some interactionsbetween oxidative stress and ERS and that oxidative stresscan cause ERS meanwhile ERS can also cause or aggravateoxidative stress [16 17] However the mechanism of theinteractions between oxidative stress and ERS is still not fullyunderstood Taken together we will put forward a hypothesisthat PERK-Nrf2 is likely to be the key crosstalk linked tooxidative stress and ERS

ERS-initiated apoptotic signaling has been implicatedin various chronic diseases including diabetes cancer andinflammation [18ndash20] However it has been not clear if ERS-initiated apoptosis is associated with the chronic lung injurycaused by methamphetamine Tertiary butylhydroquinone(TBHQ) an Nrf2 signaling pathway inducer is demonstratedto induce remarkable antioxidant activity in a variety ofcells and tissues [21] On the basis of our hypothesis thecurrent study is designed to investigate if oxidative stress andERS are involved in pulmonary toxicity and if PERKNrf2 isassociated with the interaction between oxidative stress andERS-initiated apoptosis in MA-induced pulmonary toxicity

2 Materials and Methods

21 Chronic Methamphetamine Exposure Forty-five maleWistar rats (200 plusmn 10 g) were purchased from Animal Re-source Center China Medical University (Certificate num-ber Liaoning SCSK 2012-0005) All of the 45 rats wererandomly divided into control group methamphetamine-treated group (MA) and methamphetamine plus TBHQ-treated group (MA + TBHQ) At the first week intraperi-toneal injection of methamphetamine with the dosage of10mgkg for one week was administrated to the rats in MAgroup and MA + TBHQ group and then increasing dailydosage of 1mgkg was administrated per week until the sixthweek a daily dosage was increased to 15mgkg respectively(twice per day for 6 weeks) [5 22] Meanwhile the rats incontrol group were injected with an equal volume of 09physiological saline solution After administration of MArats in the MA + TBHQ groups then received intragastricadministration of 125mgkg TBHQ [23] Rats in the controland MA groups were intragastrically administered with anequal volume of 05 gum tragacanth All animals were

housed in a roomwith controlled temperature (18ndash22∘C) andhumidity (50ndash70) and were fed solid food and water adlibitum in an alternating 12 h light and 12 h dark cycle Allprocedures followed the Guide for the Care and Use of Lab-oratory Animals of the National Institutes of Health (NIH)and all protocols were approved by the Institutional AnimalCare and Use Committee of China Medical University

22 Hemodynamic Measurements and Tissue CollectionAfter 6 weeks rats in all groups were anaesthetized with 3pentobarbital sodium (45mgkg ip) prior to hemodynamicmeasurements as described previously Briefly the rightjugular vein was dissected and catheterized with a PV-1catheter The catheter was advanced via the right ventricleinto the pulmonary artery for direct measurement of pul-monary arterial pressure (PAP) The right carotid artery wasdissected and intubated with a PE-50 catheter to measuresystemic blood pressure (SBP) Prior to catheterization thePV-1 and PE-50 catheters were filled with saline containing1 heparin PAP and SBP were recorded using a polygraphsystem (RM6000 Nihon Kohden Tokyo Japan)

Of the 45 rats 15 rats (five per group) were perfusedwith paraformaldehyde The right lower lung tissues weredissected and then they were paraformaldehyde-fixed andparaffin-embedded The lung sections (5 120583m thick) wereprepared for hematoxylin-eosin (HE) staining Eosin VanGieson (EVG) staining immunohistochemical (IHC) stain-ing immunofluorescence (IF) and TUNEL assay

The other rats were then killed by overdose pentobarbitalsodium The rat lungs and pulmonary arteries were quicklydissected and removed on ice and stored at minus80∘C untilfurther use for real-time PCR and western blotting

23 Assessment of Remodeling of the Heart and PulmonaryArteries The hearts were dissected and taken out Rightventricle (RV) and left ventricle plus interventricular septum(LV + S) were dissected and weighed separately to evaluatethe extent of the right ventricular hypertrophy expressed asright ventricular index (RVI) which is calculated by weightratio of the RV and LV + S

Lung sections (5 120583m thick) were stained with hema-toxylin and eosin (HampE) watched under light microscopyand analyzed by MetamorphyBX41 (UICOLYMPUSUSAJAP) Vessels gt 50120583m were identified and measured atthe 2 ends of the shortest external diameter of the distal PAsand the percentage of medial wall thickness (medial wallthickness) is calculated by ([2timeswall thicknessexternal dia-meter] times 100)

Lung sections (5 120583m thick) were stained with EVG tolocalize collagen (staining red) and elastin (staining blackbrown) in lungs watched under light microscopy and ana-lyzed by MetamorphyBX41 (UICOLYMPUS USAJAP)The degree of muscularization was calculated as nonmuscu-larized (no evidence of any vessel wall muscularization) par-tially muscularized (smooth muscle cells (SMC) identifiablein less than three-quarters of the vessel circumference) orfully muscularized (SMC in more than three-quarters of thevessel circumference) to assess the remodeling of pulmonaryarteries


Table 1






















3 Results










(a)


(b)


0102030405060

Perc

enta

ge o

f med

ial t

hick

ness

of

pulm

onar

y ar

terie

s (

) 7080

lowastlowast

(c)



01020304050607080

Pulm

onar

y ar

teria

l rem

odel

ing

()

lowast

lowastlowast

(d)



4 Discussion



Nrf2

120573-Actin



0010203040506070809

1

Nrf2

pro

tein

expr

esse

d in

lung

s

lowast

68kDa

43kDa

(a)


002040608

11214

Relat

ive n

orm

aliz

ed ex

pres

sion

of N

rff2

by re

al-ti

me P

CR

16

lowast

(b)



GCS

HO-1

SOD2

0

01

02

03

04

05

06

07

08

09

1

Oxi

dativ

e stre

ss g

enes

expr

esse

d in

lung

s

GCSHO-1SOD2

120573-Actin

73kDa

32kDa

25kDa

43kDa

lowastlowast

lowastlowast

(c)


butylhydroquinone


GRP78



0

02

04

06

08

1

12G

RP78

pro

tein

expr

esse

d in

lung

slowastlowast

120573-Actin

78kDa

43kDa

(a)

MA MA + TBHQControl


0

01

02

03

04

05

06

Opt

ical

den

sity

aver

age o

f GRP

78 lowastlowast

(b)





PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1






















3 Results










(a)


(b)


0102030405060

Perc

enta

ge o

f med

ial t

hick

ness

of

pulm

onar

y ar

terie

s (

) 7080

lowastlowast

(c)



01020304050607080

Pulm

onar

y ar

teria

l rem

odel

ing

()

lowast

lowastlowast

(d)



4 Discussion



Nrf2

120573-Actin



0010203040506070809

1

Nrf2

pro

tein

expr

esse

d in

lung

s

lowast

68kDa

43kDa

(a)


002040608

11214

Relat

ive n

orm

aliz

ed ex

pres

sion

of N

rff2

by re

al-ti

me P

CR

16

lowast

(b)



GCS

HO-1

SOD2

0

01

02

03

04

05

06

07

08

09

1

Oxi

dativ

e stre

ss g

enes

expr

esse

d in

lung

s

GCSHO-1SOD2

120573-Actin

73kDa

32kDa

25kDa

43kDa

lowastlowast

lowastlowast

(c)


butylhydroquinone


GRP78



0

02

04

06

08

1

12G

RP78

pro

tein

expr

esse

d in

lung

slowastlowast

120573-Actin

78kDa

43kDa

(a)

MA MA + TBHQControl


0

01

02

03

04

05

06

Opt

ical

den

sity

aver

age o

f GRP

78 lowastlowast

(b)





PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results










(a)


(b)


0102030405060

Perc

enta

ge o

f med

ial t

hick

ness

of

pulm

onar

y ar

terie

s (

) 7080

lowastlowast

(c)



01020304050607080

Pulm

onar

y ar

teria

l rem

odel

ing

()

lowast

lowastlowast

(d)



4 Discussion



Nrf2

120573-Actin



0010203040506070809

1

Nrf2

pro

tein

expr

esse

d in

lung

s

lowast

68kDa

43kDa

(a)


002040608

11214

Relat

ive n

orm

aliz

ed ex

pres

sion

of N

rff2

by re

al-ti

me P

CR

16

lowast

(b)



GCS

HO-1

SOD2

0

01

02

03

04

05

06

07

08

09

1

Oxi

dativ

e stre

ss g

enes

expr

esse

d in

lung

s

GCSHO-1SOD2

120573-Actin

73kDa

32kDa

25kDa

43kDa

lowastlowast

lowastlowast

(c)


butylhydroquinone


GRP78



0

02

04

06

08

1

12G

RP78

pro

tein

expr

esse

d in

lung

slowastlowast

120573-Actin

78kDa

43kDa

(a)

MA MA + TBHQControl


0

01

02

03

04

05

06

Opt

ical

den

sity

aver

age o

f GRP

78 lowastlowast

(b)





PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)


0102030405060

Perc

enta

ge o

f med

ial t

hick

ness

of

pulm

onar

y ar

terie

s (

) 7080

lowastlowast

(c)



01020304050607080

Pulm

onar

y ar

teria

l rem

odel

ing

()

lowast

lowastlowast

(d)



4 Discussion



Nrf2

120573-Actin



0010203040506070809

1

Nrf2

pro

tein

expr

esse

d in

lung

s

lowast

68kDa

43kDa

(a)


002040608

11214

Relat

ive n

orm

aliz

ed ex

pres

sion

of N

rff2

by re

al-ti

me P

CR

16

lowast

(b)



GCS

HO-1

SOD2

0

01

02

03

04

05

06

07

08

09

1

Oxi

dativ

e stre

ss g

enes

expr

esse

d in

lung

s

GCSHO-1SOD2

120573-Actin

73kDa

32kDa

25kDa

43kDa

lowastlowast

lowastlowast

(c)


butylhydroquinone


GRP78



0

02

04

06

08

1

12G

RP78

pro

tein

expr

esse

d in

lung

slowastlowast

120573-Actin

78kDa

43kDa

(a)

MA MA + TBHQControl


0

01

02

03

04

05

06

Opt

ical

den

sity

aver

age o

f GRP

78 lowastlowast

(b)





PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nrf2

120573-Actin



0010203040506070809

1

Nrf2

pro

tein

expr

esse

d in

lung

s

lowast

68kDa

43kDa

(a)


002040608

11214

Relat

ive n

orm

aliz

ed ex

pres

sion

of N

rff2

by re

al-ti

me P

CR

16

lowast

(b)



GCS

HO-1

SOD2

0

01

02

03

04

05

06

07

08

09

1

Oxi

dativ

e stre

ss g

enes

expr

esse

d in

lung

s

GCSHO-1SOD2

120573-Actin

73kDa

32kDa

25kDa

43kDa

lowastlowast

lowastlowast

(c)


butylhydroquinone


GRP78



0

02

04

06

08

1

12G

RP78

pro

tein

expr

esse

d in

lung

slowastlowast

120573-Actin

78kDa

43kDa

(a)

MA MA + TBHQControl


0

01

02

03

04

05

06

Opt

ical

den

sity

aver

age o

f GRP

78 lowastlowast

(b)





PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GRP78



0

02

04

06

08

1

12G

RP78

pro

tein

expr

esse

d in

lung

slowastlowast

120573-Actin

78kDa

43kDa

(a)

MA MA + TBHQControl


0

01

02

03

04

05

06

Opt

ical

den

sity

aver

age o

f GRP

78 lowastlowast

(b)





PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PERK

eIF2120572

p-eIF2120572

ATF4

p-PERK


120573-Actin

125 kDa

125 kDa

65kDa

38kDa

39kDa

43kDa

(a)


0005

01015

02025

03035

04045

05

p-PE

RKP

ERK

in ra

t lun

gs lowastlowast

(b)


lowast

0

01

02

03

04

05

06

07

08

p-eI

F2120572

eIF

2120572 in

rat l

ungs

(c)

0010203040506070809

1


Expr

essio

n of

ATF

4 in

lung

s

lowast

(d)







CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CHOP

Caspase 12

Caspase 3

bax


120573-Actin

39kDa

35kDa

43kDa

21kDa

27kDa


0

02

04

06

08

Rela

tive e

xpre

ssio

n of

apop

totic

prot

ein

in lu

ngs

1

CHOPbax

Caspase 12Caspase 3


lowastlowast

(a)


bax

bcl-2


Aver

age i

nten

sity

of b

cl-2

in lu

ngs

lowast

0

50

100

150

200

250

300

Aver

age i

nten

sity

of b

ax in

lung

s

lowastlowast

050

100150200250300350400450


(b)

Figure 5 Continued


Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control MA

MA + TBHQ


01020304050607080

Perc

enta

ge o

f apo

ptot

icce

lls in

rat l

ungs

()

lowast

(c)






Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















TBHQ Alleviated Endoplasmic Reticulum Stress-Apoptosis...

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Transcript of TBHQ Alleviated Endoplasmic Reticulum Stress-Apoptosis...