induced by thyroxine through PI3K/AKT signaling Hydrogen ...

©The Japan Endocrine Society

2018, 65 (7), 769-781

Original

Hydrogen sulfide ameliorates rat myocardial fibrosisinduced by thyroxine through PI3K/AKT signalingpathwayMaojun Liu1) *, Zining Li1) *, Biao Liang1), Ling Li1), Shengquan Liu1), Wenting Tan1), Junrong Long1),Fen Tang1), Chun Chu2) and Jun Yang1)

1) Department of Cardiology, the First Affiliated Hospital of University of South China, Hunan 421001, China2) Department of Pharmacy, the Second Affiliated Hospital of University of South China, Hunan 421001, China

Abstract. This study aims to investigate the role and regulatory mechanism of the Hydrogen sulfide (H2S) in amelioration of ratmyocardial fibrosis induced by thyroxine through interfering the autophagy via regulating the activity of PI3K/AKT1 signalingpathway and the expression of relative miRNA. 40 adult male SD rats were randomly divided into 4 groups (n = 10): the controlgroup, the thyroxine model group (TH group), the model group with H2S intervention (TH + H2S group) and the normal group withH2S intervention (H2S group). Pathological changes were observed via H&E staining and Masson staining, Expressions of MMPs/TIMPs, PI3K/AKT, autophagy-related proteins in myocardial tissues were detected via Western blotting, and the expressions ofmiR-21, miR-34a, miR-214 and miR-221 were detected via RT-qPCR. Compared with the control group, in the TH group, myocardialfibrosis was more significant, the expressions of proteins in PI3K/AKT and autophagy-related proteins were significantly decreased,as well as the expression of miR-221; while the expressions of miR-21, miR-34a and miR-214 were significantly elevated. Bycontrast, all above-mentioned changes were obviously reversed with H2S treatment, which demonstrated the positive function of H2Sin amelioration of rat myocardial fibrosis induced by thyroxine. The mechanism of such amelioration may be correlated withautophagy activated by the upregulation of expression of PI3K/AKT signaling pathway and downregulation of expressions of miR-21,miR-34a and miR-214.

Key words: Hydrogen sulfide, Myocardial fibrosis, Autophagy, Thyroxine, PI3K/AKT

HYPERTHYROIDISM refers to the thyrotoxicosiscaused by excessive generation of thyroxine from thyroid.It always brings damages to organs, tissues and cells at

Submitted Oct. 20, 2017; Accepted Apr. 9, 2018 as EJ17-0445Released online in J-STAGE as advance publication May 9, 2018Correspondence to: Jun Yang, Department of Cardiology, The FirstAffiliated Hospital of University of South China, No.69, Chuan‐shanRoad, ShiGuDistrict, Hengyang, Hunan 421001, China.E-mail: [email protected] to: Chun Chu, Department of Pharmacy, The Sec‐ond Affiliated Hospital of University of South China, No.30, Jie‐fangRoad, ShiGuDistrict, Hengyang, Hunan 421001, China.E-mail: [email protected]*These authors contributed to the work equally and should beregarded as co-first authors.Abbreviations: H2S, hydrogen-sulfide; NaHS, sodiumhy-drosulfide;RAAS, Renin-angiotensin-aldosterone System; MMPs, Matrixmetalloproteinases; TIMPs, Tissue inhibitor of metalloproteinases;TEM, Transmission electron microscope; BCA, bicinchoninic acid;PVDF, Polyvinylidene fluoride membrane; PMSF, phenylmethyl‐sulfonyl fluoride

varying degrees. As a key target organ sensitive to thy‐roxine, heart under direct or indirect action of excessivethyroxine in the circulation suffers hyperthyroid heartdisease with apparent clinical symptoms and pathologi‐cal changes, such as myocardial hypertrophy, myocardialfibrosis, arrhythmia or cardiac insufficiency [1, 2].Among them, myocardial fibrosis is a key marker indi‐cating myocardial remodeling in hyperthyroid heartdisease, and also is the major cause leading to insuffi‐ciency of left heart [3, 4]. Although the pathogenesis ofmyocardial fibrosis has not been fully understood due toits complexity, it has been suggested by existing eviden‐ces that such pathogenesis is associated with manyfactors, including the metabolic disorder in myocardialcells, activation of Renin-angiotensin-aldosterone Sys‐tem (RAAS), imbalance between the sympathetic toneand vagal tone, and excessive inflammatory responsesand stress responses in myocardium [5-7].

As a specific metabolic process, autophagy in eukary‐

ote can degrade the damaged, degenerated and agedproteins, and organelles in cells with the involvement oflysosome. Therefore, it is critically significant to main‐tain the regular intracellular homeostasis and self-renewal in cells [8]. Moderate autophagy is a kind ofself-protective mechanism surviving cells in adverseenvironment [9, 10]. Current studies have shown that cellautophagy involves in the pathogenesis of myocardialfibrosis and myocardial remodeling. However, fewstudies focused on the relation between autophagy andthe occurrence and progression of myocardial fibrosiscaused by hyperthyroid heart disease.

PI3K/AKT signaling pathway, one of the signalingpathways widely existing in animals, not only acts asclassic signaling pathway that regulates the cell au‐tophagy, but also involves the regulation of a series ofpathophysiological processes, such as cell proliferation,apoptosis and stress response [11].

In addition, the PI3K/AKT signaling pathway is alsoimportant for insulin signaling and glucose metabolism,lymphocyte migration, proliferation and differentiation,and TGF-β signaling regulation. Thus it is an crucialtherapeutic target for the treatment of various disease,including cancer, diabetes and others [12]. Actually,many studies indicated the presence of activation ofPI3K/AKT signaling pathway in many cancers. Forinstances, Gamal Badr et al. reported that apoptosis ofhuman breast carcinoma cells are induced by samsum antvenom through PI3K/AKT signaling pathway [13]. PeiLi et al. suggested that resveratrol apoptosis and senes‐cence of attenuates high glucose-induced nucleus pulpo‐sus cell through activating the ROS-mediated PI3K/Aktpathway [14].

It has been confirmed that H2S, a novel gas signalmodule, has a wide variety of biological effects, includ‐ing protecting the myocardium in various cardiovasculardiseases, such as hypertension and myocardial ischemia-reperfusion injuries through expanding the vessels, andregulating the mechanisms of autophagy, anti-oxidation,anti-inflammation and anti-apoptosis [15]. However,when it comes to the question of whether H2S can exertthe protective effect on hyperthyroid heart disease andmyocardial fibrosis induced by high-concentration thy‐roxine, and its signaling regulation mechanism, moreefforts are required. Therefore, a rat myocardial fibrosismodel induced by thyroxine at a high concentration wasestablished in this study to investigate the effect of H2Son rat myocardial fibrosis induced by high-concentrationthyroxine, and to determine the role of H2S on the regu‐

lation of cell autophagy and expressions of myocardialfibrosis-related miRNAs (miR-21, miR-34a, miR-214and miR-221). In conclusion, the research aimed to pro‐vide experimental evidence for further understanding ofpathogenesis of hyperthyroid heart disease and discoveryof new therapeutic targets.

Materials and Methods

Animals and reagentsThe experimental protocol, which had been approved

by the Animal Ethics Committee of University of SouthChina (Hengyang, China), complied with the provisionson experimental animal management in the People ’ sRepublic of China. 40 adult male Sprague Dawley (SD)rats, weighing 140–160 g, were purchased from the SJAExperimental Animal Center of Changsha (Changsha,Chinese), and then kept in separate cages under control‐led temperature and allowed to access to food and waterfreely. Artificial lighting was alternated every 12 hours.Sodium hydrogen sulfide (NaHS) was supplied bySigma-Aldrich (St. Louis, MO, USA). L-Thyroxine wasfrom Merck Drugs & Biotechnology (Darmstadt, Ger‐many). Cell lysis buffer was bought for western blotting,and phenylmethylsulfonyl fluoride (PMSF), bicincho‐ninic acid (BCA) protein assay kit, tris buffered saline,SDS-PAGE gel preparation kit and chloral hydrate werepurchased from Beyotime Biotechnology Co., Ltd.(Shanghai, Chinese). Polyvinylidene fluoride (PVDF)membrane and pre-stained protein molecular markerwere supplied from Merck Millipore (Billerica, MA,USA); Rabbit polyclonal anti matrix metalloproteinase11 (MMP11), rabbit polyclonal anti-MMP12, rabbitpolyclonal anti-MMP14, rabbit polyclonal anti-TIMP-1,TIMP-4 and rabbit, rabbit polyclonal anti-MMP17 andpolyclonal anti-glyceraldehyde 3-phosphate dehydroge‐nase (GAPDH) were procured from Wuhan Boster Bio‐logical Technology, Ltd. (Wuhan, China). The dilutionrate of above these antibodies was 1:400. Rabbit anti-autophagy related gene 5 (ATG5), rabbit monoclonalanti-ATG7, rabbit monoclonal anti-A16L1, rabbit mono‐clonal anti-Beclin1, rabbit monoclonal anti-LC3A, rabbitpolyclonal anti-PI3K, rabbit polyclonal anti-AKT1 wereprovided by Cell Signaling Technology, Inc. (Danvers,MA, USA), and the dilution rate was 1:1,000. Further‐more, anti-rabbit secondary antibody and anti-rat secon‐dary antibody were also purchased from ProteintechGroup, and the dilution rate for which was 1:2,000.

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Establishment of modelThese experimental animals were randomly divided

into four groups (n = 10 for each group): normal group(control group), L-thyroxine (TH)-treated group (THgroup), and hyperthyroidism treated with H2S group (TH+ H2S group), normal rats treated with H2S group (H2Sgroup). TH group and TH + H2S group were injectedintraperitoneally with L-thyroxine (2.5 mg/kg/day). Ratsin the control group and H2S group were injected intra‐peritoneally with normal saline. While TH + H2S groupand H2S group was treated with intraperitoneally injectedsodium hydrogen sulfide (100 μmol/kg). The rats in thecontrol group and TH group were daily treated with PBS.After 4 weeks, all rats were weighed, then they wereanesthetized with chloral hydrate (350 mg/kg) to lavagetheir hearts with ice-cold normal saline; afterward thehearts were removed and weighed, and then reserved at–80°C for experiment.

Pathological analysis of myocardial fibersMyocardial tissue was firstly rinsed with sterile saline,

and then was fixed to 4% paraformaldehyde (BeyotimeInstitute of Biotechnology, Shanghai, China), alcoholdehydration, Paraffin embedded. After that, they weremade into 4 μm thick slices and stained with Hematoxylinand Eosin staining kits and Masson kits. Finally, theseslices were observed under optical microscope.

Transmission electron microscope (TEM)observation

The myocardial tissue was firstly fixed with 2.5%glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd.).Then it was sequentially fixed with 1% osmium tetroxide(Absin Bioscience Inc., Shanghai, China), rinsed withPhosphoric acid rinse solution (Beyotime Institute ofBiotechnology, Shanghai, China), dehydrated at differentconcentrations of acetone (Beyotime Institute of Biotech‐nology, Shanghai, China), and solidified into 50–100 nmthick slices. After being stained with 3% uranyl acetate(Johnson Biotechnology Co., Ltd., Shanghai, China) andlead nitrate (Tianyuan Industrial Fine Chemical Co.,Ltd., Yingkou, China), the ultrathin slices were observedunder TEM.

Western blot analysisProteins were extracted by cell lysis buffer containing

protease inhibitors, and then quantified by BCA proteintest kit. Denatured proteins were separated by SDS-PAGE electrophoresis, and then transferred to the PVDF

membrane by wet transfer method. The Tris membranewas blocked in Tris buffered saline containing Twain 20and 5% skim milk at 37°C, and then it was incubatedwith blocking solution containing primary antibody for 1hour, and stayed overnight at 4°C. After washing withTBST for three times, the membrane was sequentiallyincubated with horseradish peroxidase-conjugated sec‐ondary antibody for 1 hour. After that, the membranewas again rinsed for three times with TBST buffer.Finally, the membranes was subjected to chemilumine‐scence detection assay. The bands were analyzed with aMolecular Imager VersaDoc MP 5000 system (Bio-RadLaboratories, Inc., Hercules, CA, USA) and AlphaImager (San Leandro, USA) analysis strip, with GAPDHas a reference.

RT-qPCR analysisTotal RNA was extracted from myocardial tissue of

mice from each group using Trizol reagent (Invitrogen,California, USA). The concentration of the extractedRNA was measured by ultraviolet Spectrophotometer(Agilent Technologies, CA, USA). and the integrity ofRNA was analyzed by gel imaging system (Bio-RadLaboratories, Inc., Hercules, CA, USA). Reverse tran‐scription reaction was manipulated with mi-RNA spe‐cific RT primer (GenScript USA Inc., Nanjing, China)using reverse transcription polymerase chain reaction kit(MBI Fermentas, the Republic of Lithuania). The expres‐sion levels of miR-21-TAGCTTATCAGACTGATGTTGA,miR-34a-TGGCAGTGTCTTAGCTGGTTGT, miR-214-AGAGTTGTCATGTGTCT and miR-221-ACCTGGCATACAATGTAGATTTC were detected by Real-time PCRusing Taqman mi-RNA assay probe (Applied Biosys‐tems, Shanghai, China), and normalized by endogenousU6 small RNA. Reverse transcription was manipulatedunder following circumstances: 37°C for 15 minutes,42°C for 50 minutes and 85°C for 5 minutes. The cDNAobtained was subjected to real-time PCR under the fol‐lowing circumstances: 50°C for 2 minutes, 95°C for 10minutes, 95°C for 5 seconds, and 60°C for 30 seconds.

Statistical analysisData were expressed as mean ± standard deviation

(SD). Group differences were assessed by one-way analy‐sis of variance using SPSS software 18.0 (Chicago,USA). Differences between two groups were analyzedusing the Student-Newman-Keuls test. p < 0.05 was con‐sidered as statistically significant.

H2S ameliorates myocardial fibrosis 771

Results

Effect of hydrogen sulfide on body weight, heartweight and heart weight/body weight ratio

Eight weeks later, 10, 9, 8, 10 mice were survived incontrol group, TH group, TH + H2S group, H2S grouprespectively. As shown in Table 1, the differences inbody weight, heart weight and heart weight/body weightratio between groups were not significant (Table 1).

Effect of hydrogen sulfide on myocardial fibrosisinduced by thyroxine

To observe the morphological changes in myocardiumof heart tissues, HE-staining (Fig. 1a), Masson-staining(Fig. 1b) and collagen Volume Fraction (CVF) of theMasson staining (Fig. 1c) were respectively conducted toidentify the pathological changes in myocardial tissues,and the morphology of myocardial fiber and collagendeposition in myocardial tissues. As shown in the Fig.1a, purplish blue area stands for the composition of cellnucleus, while red is for the compositions in cytoplasmand extracellular matrix. In the control group, the mor‐phology of myocardium with a clear structure was nor‐mal; in TH group, the diameter of myocardial cells wereincreased, and the myocardial tissues were irregularlyarranged; compared with TH group, the diameter ofmyocardial cells was shortened and the structure ofmyocardial tissues was improved in TH + H2S group.However, changes in myocardial tissue structure anddiameter of myocardial cells in H2S group were similarto those in the control group. The figure shows that theregions in blue between myocardial cells are collagenfibers. It can be seen from the Fig. 1b that in comparisonto the control group, myocardial fibers of rats in THgroup were arranged irregularly, and the quantity ofblue-stained fibers was increased with obvious myocar‐dial fibrosis. In comparison with TH group, disorderedarrangement of myocardial cells was significantly

improved, the quantity of blue-stained collagen fiberswas decreased, and the myocardial fibrosis was im‐proved in TH + H2S group, while there was no signifi‐cant difference in changes of myocardial tissues betweenthe control group and H2S group was observed.

Effects of H2S on collagen III expressions ofthyroxine rats

The expression of collagen III in myocardium tissues,which can be used to reflect fibrosis in some degree, wasmeasured by immunohistochemically analysis. As aresult, it is found that when compared with the controlgroup, the positive expression of collagen III was signifi‐cantly decreased in TH group, and the myocardialexpression of collagen III was remarkably reduced in TH+ H2S group. While as for TH group and TH + H2Sgroup, there is no significant difference was observed.(Fig. 2).

H2S content in each groupTo discover the correlation of the myocardial fibrosis

induced by high-concentration thyroxine with thechanges in endogenous H2S, the level of endogenousH2S in myocardial tissues of all groups by ELISA wasanalyzed in the very study. Results indicate that in com‐parison to the control group, the expression level of H2Sin myocardial tissues of rats in TH group was dramati‐cally down-regulated; however, compared with THgroup, H2S expression in myocardial tissues of rats inTH + H2S group was significantly upregulated. Overall,the expression level of H2S in myocardial tissues is thehighest in TH group, and there is no statistical differencebetween TH group and TH + H2S group (Fig. 3).

Effects of H2S on myocardial ultrastructural inthyroxine rats

To observe the changes in myocardial ultrastructural,and effect of H2S on autophagy of myocardial cells and

Table 1 Body weight (BW), heart weight (HW) and heart weight/body weight(HW/BW) ratio in each group

Group Number BW (g) HW (mg) HW/BW (mg/g)

Control 10 191.60 + 6.17 766.20 + 47.00 4.00 + 0.32

TH 9 187.33 + 8.56 765.78 + 68.77 4.10 + 0.48

TH + H2S 8 196.50 + 10.20 766.62 + 59.12 3.90 + 0.21

H2S 10 188.00 + 11.55 756.10 + 38.15 4.04 + 0.35

Values are expressed as means ± SD.

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myocardial fibers in rats with hyperthyroidism inducedby H2S, autophagosomes and myocardial fibers in allgroups were examined under TEM. As a result, auto‐phagosomes were found in control group, TH + H2Sgroup and H2S group, while absent in TH group.Myocardial fibers were clearly observed in normallyarranged without swelling in the control group. In theTH group, the myocardial fibers from some myocardialtissues were arranged disorderly and dropsically; andswelling of the myocardial fibers were observed as well.However, all these changes observed in TH group wereobviously augmented in TH + H2S group. Similarly,

there was no significant changes were found between thecontrol group and H2S group (Fig. 4).

Effects of H2S on MMP11, MMP12, MMP14,MMP17, TIMP1 and TIMP4 expressions ofthyroxine rats

Since dynamic balance between MMPs and TIMPs isclosely correlated with the formation and degradation ofcollagen in myocardial tissues, Western blotting assaywas performed to detect the changes in expressions ofMMPs/TIMPs-related proteins in myocardial tissues.This then helps reveal the correlation between the colla‐

Fig. 1 H2S improves myocardial fibrosis in thyroxine rats. Morphological changes in myocardium assessed by eosin (HE) staining (a),Masson staining (b) and Collagen Volume Fraction (CVF) of the Masson staining (c). Images were acquired at ×400magnification.

Fig. 2 Effects of H2S on collagen III expressions of thyroxine rats measured using immunohistochemical analysis.


gen deposition in myocardial tissues and interstitialfibrosis. Western blotting assay results indicated proteinexpressions of MMP11, MMP12, MMP17, TIMP1 andTIMP4 were significantly upregulated, and howeverthe protein expression of MMP14 was significantlydecreased in TH group when compared with the controlgroup. Compared with TH group, all the protein expres‐sions of MMP11, MMP12, MMP17, TIMP1 and TIMP4were significantly decreased, except the MMP14, whichwas upregulated in TH + H2S group; However, in com‐parison between H2S group and control group, therewere no statistically significant differences were ob‐served (Fig. 5).

Effect of H2S on autophagy protein markersexpression in thyroxine rats

Similarly, Western blotting assay covering detection ofthe protein expressions of ATG5, ATG7, ATG16L1,

Fig. 3 H2S content in each group detected by ELISA. Data areexpressed as mean ± SD (n = 3). *p < 0.05 vs. Controlgroup; #p < 0.05 vs. TH group.

Beclin1 and LC3A in all groups, was also conducted toobserve the level of autophagy in myocardial tissues andeffect of H2S on expression level of autophagy. Theresults showed that compared with the control group,protein expressions of ATG5, ATG7, ATG16L1, Beclin1and LC3A in myocardial tissues of rat in TH group weresignificantly decreased; and on the contrary expressionsof all these proteins in myocardial tissues of rat weresignificantly increased in TH + H2S group when com‐pared to TH group. While the comparison between H2Sgroup and the control group suggested that these twogroups possess statistically comparable protein expres‐sions of ATG5, ATG7, ATG16L1, Beclin1 and LC3A(Fig. 6).

Effects of H2S on the PI3K/AKT1 signalling pathwayin thyroxine rats

To determine the potential role of PI3K/AKT1 signal‐ing pathway in the regulatory mechanism of myocardialfibrosis caused by high-concentration thyroxine, andautophagy, Western blotting assay was performed tomeasure the expressions of PI3K and AKT1 proteins,and the key proteins in PI3K/AKT1 signaling pathway.The results indicated that the protein expressions of PI3Kand AKT1 in the myocardial tissues of rat in controlgroup, TH group and TH + H2S group were firstly signif‐icantly increased and then significantly decreased. How‐ever, the expression between H2S group and the controlgroup were almost same without statistical difference(Fig. 7).

Effects of H2S on miRNA21, miRNA34a,miRNA214, miRNA221 expression in thyroxine rats

To figure out the potential role of miRNA21,miRNA34a, miRNA214 and miRNA221 in the occur‐rence and progression of hyperthyroid heart disease, andthe effect of H2S on expressions, RT-qPCR was

Fig. 4 Effects of H2S on myocardial ultrastructural in thyroxine rats. Transmission electron micrographs at bars of 2 μm.

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employed to detect the expressions of these miRNAs inmyocardial tissues of rats in all groups. The original dataof miRNA was provided in the supplementary file. The

results revealed that when compared with the controlgroup, the expressions of miRNA21, miRNA34a andmiRNA214 in the myocardial tissues of rat in TH group

Fig. 5 Effects of H2S on MMP11, MMP12, MMP14, MMP17, TIMP1 and TIMP4 expressions of thyroxine rats. Data are expressed asmean ± SD (n = 3). *p < 0.05 vs. Control group; #p < 0.05 vs. TH group.


were significantly increased, while the expression ofmiRNA221 was downregulated. Similar tendency forthese all miRNAs was observed from TH group to TH +H2S group. Specifically, miRNA21, miRNA34a andmiRNA214 were statically significantly increased andthe expression of miR-221 was insignificantly increased.Similarly, the changes in expressions of all miRNAs inH2S group and the control group were statistically equiv‐alent (Fig. 8).

Discussion

Hyperthyroidism may augment metabolism, and affectalmost all of the physiological systems, especially thecardiovascular system, resulting in symptoms of hyper‐

tension, arrhythmia and cardiac hypertrophy. In somesevere conditions, patients even suffer from hyperthyroidheart disease [16-18]. As for hyperthyroid heart disease,major pathological changes are associated with the directtoxicity and indirect effect of thyroxine on myocardium,including cardiac hypertrophy, myocardial fibrosis anddysfunction of left heart. Hyperthyroidism may activatethe sympathetic nervous-catecholamine system and theRAAS system to induce the proliferation of cardiacfibroblast and mass deposition of collagen, thereby lead‐ing to the interstitial fibrosis in myocardium. Myocardialfibrosis, the major pathological change in myocardialremodeling, is closely associated with the microvascularinjuries and damages of the myocardial structures, andalso plays a critical role in the occurrence and progres‐

Fig. 6 Effect of H2S on autophagy protein markers expression in thyroxine rats. Data are expressed as mean ± SD (n = 3). *p < 0.05 vs.Control group; #p < 0.05 vs. TH group.

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sion of hyperthyroid heart disease [19, 20]. In this study,a rat model of myocardial fibrosis was prepared underthe induction of mass thyroxine through intraperitonealinjection of L-Thy. Based on this, it was then found thatthe collagen deposition was significantly increased in themyocardial tissues of rat with the induction of thyroxinethrough Masson-staining; meanwhile, imbalance in

MMPs/TIMPs was also identified in the myocardialtissues of rat. Furthermore, immunohistochemistry resultindicated that the expression of collagen III in the myo‐cardial tissues was significantly increased after inductionof thyroxine. As a whole, significant interstitial fibrosisoccurred in the myocardial tissues after induction ofthyroxine, which resulted in remodeling in myocardial

Fig. 7 Effects of H2S on the PI3K/AKT1 signalling pathway in thyroxine rats. Data are expressed as mean ± SD (n = 3). *p < 0.05 vs.Control group; #p < 0.05 vs. TH group.

Fig. 8 Effects of H2S on miRNA21, miRNA34a, miRNA214, miRNA221 expression in thyroxine rats. Data are expressed as mean ± SD(n = 3). *p < 0.05 vs. Control group; #p < 0.05 vs. TH group.


tissues and collagen deposition. Myocardial fibrosis hasbeen considered as the key link in identifying the occur‐rence and progression of hyperthyroid heart disease, butthe pathogenesis of myocardial fibrosis was induced byhigh concentration of thyroxine remains unclear.

Autophagy is a process of self-elimination of damagedproteins and organelles in cells, through which the con‐tent in cytoplasm could be degraded and reused byeukaryotes. Recent evidences show that in basal condi‐tions, autophagy serves as a key homeostasis mechanismto maintain normal cardiovascular functions and mor‐phology, as well as a major way of cell protection[21-23]. Dysfunction in autophagy is correlated with theoccurrence of some myocardium-related diseases. Thedecreased autophagy usually results in the aggregation ofabnormal proteins, thereby inducing dysfunction ofcytoskeleton proteins and decrease in cellular functions,which finally contributes to cell death [24]. Xiao Y et al.reported that cucurbitacin-b can ameliorate the hyper‐trophy and myocardial fibrosis in pressure-overloadmouse cells [25]. Liu S et al. also suggested that acti‐vated autophagy can improve the myocardial fibrosisinduced by angiotensin II [26]. Therefore, it is believedthat autophagy is involved in cellular homeostasis andmaintenance of cellular function, and plays a criticalrole in mechanisms of occurrence and progression ofmyocardial remodeling. ATG5 and ATG7 are the neces‐sary genes that participate in autophagy in mammals[27]. In fact, due to insufficient energy [28], ATG5 andATG7 gene-knockout mice are very likely to die.Although some mice survived, they may still sufferedsevere dilated cardiomyopathy. As key components inautophagy, LC3 in microtubule-associated protein, whichmakes up the isolating membrane of autophagosome,and Beclin1 exert critical roles in regulation of cellautophagy and serve as the markers of cell autophagy[29-31]. In this experiment, the rat models of myocardialfibrosis were prepared through the induction of thyroxineat high concentration, and upon which, a series of assayswere performed to detect the levels of autophagy-relatedproteins of ATG5, ATG7, ATG16L1, Beclin1 and LC3A.The results showed that compared with the controlgroup, the expressions of all these proteins were signifi‐cantly decreased in myocardial tissues of rats, suggestingthat the decrease of autophagy may trigger the occur‐rence and progression of myocardial fibrosis in rat withhyperthyroidism heart disease.

PI3K/AKT signaling pathway is a kind of classic path‐ways regulating cell autophagy, and is the key signaling

transduction pathway in cells with extremely significantbiological functions in cell apoptosis and autophagy [32,33]. It can directly regulate the level of downstreammTOR to inhibit autophagy, thus participating in theregulation of cell proliferation, migration and differentia‐tion [34, 35]. Existing studies showed that up-regulatedexpression of PI3K/AKT signaling pathway can suppressautophagy [36]. In the very study, obvious myocardialfibrosis and suppression in autophagy were found inmyocardium of Hyperthyroidism rats, and the proteinexpressions of PI3K and AKT in PI3K/AKT signalingpathway were obviously upregulated. Therefore, it couldbe concluded that the mechanism of high-concentrationthyroxine leading to myocardial fibrosis may be associ‐ated with the decreased autophagy caused by upregulatedexpression of PI3K/AKT signaling pathway.

Current studies suggest that miRNA involves in thedevelopment of heart and the differentiation of stemcells through translational repression [37]. While stud‐ies also indicated that miR-34a and miR-21 participate inthe regulatory mechanism of cell autophagy. Somestudies revealed that miR-21 can ameliorate hypoxia/reoxygenation-induced myocardial injuries by inhibitingcell autophagy via AKT/mTOR signaling pathway [38].Similarly, there are studies confirming that the mecha‐nism of miR-21 inhibiting autophagy is correlated withthe downregulation of PI3K-AKT-mTOR signaling path‐way [39]. Moreover, miR-34a inhibits cell autophagy byregulating the activity of TNF-α [40]. In addition, asdemonstrated by a large number of studies, all miRNAsof miR-21, miR-34a and miR-214 are all involved in theregulatory mechanism of myocardial fibrosis [41-43].The results of this study showed that under the stimula‐tion of high-concentration thyroxine significant fibrosiswas found in myocardium of rat, significant upregula‐tions of the expression levels of miR-21, miR-34a andmiR-214 in myocardial tissues were observed, as well asevident decrease of autophagy in myocardial tissues.Consequently, it is evident that myocardial fibrosis in ratinduced by high-concentration thyroxine may be correla‐ted with the inhibition of cell autophagy and myocardialfibrosis through the up-regulation of miR-21 andmiR-34a expressions.

H2S, a colorless gas with the characteristic odor ofrotten eggs, is the third endogenous gas signal moleculewith extensive physiological functions secondary to NOand CO. Existing studies have revealed that endogenousH2S is involved in regulation of a series of physiologicaland pathological processes, and has extensive effects in

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cardiovascular system to protect the heart, such as theanti-inflammation, anti-fibrosis and pro-angiogenesis[44]. Meanwhile, our previous studies also demonstratedthat H2S can improve the myocardial fibrosis in rat withdiabetes mellitus, which is associated with the activationof cell autophagy [45]. However, there is no studyfocused on the role and relevant regulatory mechanismof myocardial fibrosis induced by high-concentrationthyroxine. The results of this study showed that after H2Sintervention, collagen deposition in myocardial intersti‐tium was significantly decreased under the stimulation ofthyroxine at high concentration, and at the same time, theimbalance of MMPs/TIMPs was obviously ameliorated.Thus, it can be concluded that H2S can improve themyocardial fibrosis of rat under the induction of high-concentration thyroxine. Additionally, researches alsoshowed that under the induction of high-concentrationthyroxine, the autophagy level in the myocardial tissuesof rat was decreased, but after the intervention of H2S,the expression of autophagy-related proteins in myocar‐dial tissues of rat were significantly elevated. Moreover,some studies reported that in H2S group, the proteinexpressions of PI3K and AKT, the key proteins inPI3K/AKT signaling pathway, were significantlydecreased in rats compared with the model group. Inother words, the mechanism of H2S upregulating theautophagy is possibly associated with the downregula‐tion of PI3K/AKT signaling pathway, and H2S mayactivate cell autophagy by suppressing the PI3K/AKTsignaling pathway. Furthermore, the study also foundthat the intervention of H2S can significantly downregu‐lated the expression levels of miR-21, miR-34a andmiR-1 in myocardial tissues. Namely, H2S may inhibitcell autophagy and myocardial fibrosis by downregulat‐ing the expressions of miR-21, miR-34a and miR-1.

In this study, it is found that by up-regulating theautophagy level, H2S can ameliorate the myocardialfibrosis induced by thyroxine at high concentration. The

mechanism of this may be associated with the downregu‐lation of PI3K/AKT signaling pathway and expressionsof miR-21, miR-34a and miR-214. All these resultsprovide new evidence for better understanding of thepathogenesis of hyperthyroid heart disease, and suggestthat H2S, an endogenous gas signal, may be a new inter‐vention target for prophylaxis and treatment of hyper‐thyroid heart disease and myocardial fibrosis. On theone hand, with these new knowledge a new drug thatcan release the endogenous gas signal of H2S in the myo‐cardial tissue may be developed as a novel approach toimprove the heart function and to alleviate the myocar‐dial fibrosis of hyperthyroid heart disease in some de‐gree. On the other hand, PI3K/AKT and relevant miRNAsmay be a potential a novel target for the treatment ofhyperthyroid heart disease. For example, an inhibitor oractivator of the PI3K/AKT signaling pathway which canregulate PI3K/AKT and relevant miRNAs may be devel‐oped, along with the gene knockout technology, tojointly intervene the signaling pathway related to ourfinding. However, more studies are further required todiscover the specific molecular mechanism by whichH2S regulates the activity of PI3K/AKT signaling path‐way and the expressions of relevant miRNAs and inter‐feres with cell autophagy.

Acknowledgements

This study was supported by the National Natural Sci‐ence Foundation of China (Grant No. 81202830) and theNatural Science Foundation of Hunan Province, China(Grant No. 2017JJ3271).

Competing Financial Interests Statement

The authors declare no conflict of competing financiainterest.

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