Stachydrine, a Major Constituent of the Chinese Herb Leonurus Heterophyllus Sweet, Ameliorates Human...

January 21, 2010 10:21 WSPC WS-AJCM SPI-J000 00773

The American Journal of Chinese Medicine, Vol. 38, No. 1, 157–171© 2010 World Scientific Publishing Company

Institute for Advanced Research in Asian Science and Medicine

Stachydrine, a Major Constituent of theChinese Herb Leonurus Heterophyllus Sweet,

Ameliorates Human Umbilical VeinEndothelial Cells Injury Induced by

Anoxia-Reoxygenation

Jun Yin, Ze-Wen Zhang, Wen-Jun Yu, Jing-Yuan Liao, Xin-Guo Luo and You-Jin Shen

Division of Hematology, The Second Hospital Affiliated to Medical College

of Shantou University, Shantou, Guangdong Province, China

Abstract: Stachydrine is a major constituent of Chinese herb leonurus heterophyllus sweet,which is used in clinics to promote blood circulation and dispel blood stasis. Our studyaimed to investigate the role of stachydrine in human umbilical vein endothelial cells(HUVECs) injury induced by anoxia-reoxygenation. Cultured HUVECs were divided ran-domly into control group, anoxia-reoxygenation (A/R) group and 4 A/R+stachydrine groups.HUVECs in the control group were exposed to normoxia for 5hours, while in all A/R groups,HUVECs underwent 3hours anoxia followed by 2hours reoxygenation, and HUVECs in the4 A/R + stachydrine groups were treated with 10−8 M, 10−7 M, 10−6 M and 10−5 M (finalconcentration) of stachydrine respectively. After anoxia-reoxygenation, tissue factor (TF) wasover-expressed, cell viability and the concentrations of SOD, GSH-PX and NO were declined,while LDH, MDA and ET-1 were over-produced (p< 0.05 to 0.001 vs. the control group).However, in stachydrine treated groups, TF expression was inhibited at both mRNA and pro-tein levels, while the declined cell viability and SOD, GSH-PX, NO as well as the enhancedLDH, MDA and ET-1 levels occurred during anoxia-reoxygenation were ameliorated andreversed effectively (p< 0.05 to 0.01 versus A/R group). Consequently, our findings indi-cate that TF plays an important role in the development of anoxia-reoxygenation injury ofHUVECs, stachydrine ameliorates HUVECs injury induced by anoxia-reoxygenation and itsputative mechanisms are related to inhibition of TF expression.

Keywords: Stachydrine; Human Umbilical Vein Endothelial Cells; Anoxia-Reoxygenation;Tissue Factor.

Correspondence to: Dr. Jun Yin, Division of Hematology, The Second Hospital Affiliated to Medical College ofShantou University, Dongxia Road North, Shantou, Guangdong Province, 515041, China. Tel: (+86) 754-8891-5950 (office) or (+86) 138-2960-8816 (cell phone), Fax: (+86) 754-8852-0097, E-mail: [email protected]

157

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


158 J. YINet al.

Introduction

Vascular endothelium provides a barrier in the body and inhibits the formation of thrombosisand leukocyte transmigration during normal vascular homeostasis. Endothelial cells, how-ever, can undergo cellular activation or death following ischemia and reperfusion, whichinvolves oxidative stress and is likely to play an important role in the pathogenesis of awide variety of clinical conditions such as peripheral vascular disease, stroke and myocar-dial infarction (Zhaoet al., 2003; Huanget al., 2007). There is much evidence that vas-cular endothelium is a crucial site of ischemia and reperfusion injury. Data have shownthat endothelial cells, under ischemic or hypoxic conditions, present dysfunction of energymetabolism, over-production of oxygen free radicals and malondialdehyde (MDA), etc.which activate endothelial cells themselves and lead to the destruction of the integrity of thevascular endothelium (Eltzschig and Collard, 2004). The integrity of the vascular endothe-lium is necessary for maintaining the normal structure and function of the vessel. Therefore,it is an important and imminent work to study and explore novel endothelium-protectingdrugs. According to this strategy, our group has been searching for some effective endothe-lium protecting drugs from Chinese traditional medicinal herbs.

Traditional Chinese medicine (TCM) has over 5000-years of history. Many edible andmedicinal plants described in classical Chinese Materia Medica provide a potential resourcefor research and development of ameliorating endothelial cells injury with relatively lowtoxicity. Leonurus heterophyllus sweet (LHS), also called a Chinese motherwort herb, func-tions by promoting blood circulation, clearing clots, treating breast pain and swelling, as wellas detoxification. LHS might have been used for promoting blood circulation and dispellingblood stasis (Zouet al., 1989; Kuanget al., 1988). Our previous studies have shown thatLHS could effectively protect the heartagainst myocardium injury inan ischemia-reperfusioninduced rat myocardium injury modelin vivo. However, stachydrine is the major constituentof LHS, the role of which in endothelial cells injury induced by anoxia-reoxygenationremains unclear. In the present study, we attempted to investigate the role of stachydrinein cultured human umbilical vein endothelialcells injury induced by anoxia-reoxygenationand its putative mechanisms.

Materials and Methods

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were acquired as follows (McCormicket al., 2001): human umbilical veins were flushed with phosphate buffered saline (PBS), andfilled with PBS containing collagenase (200mg/ml) and incubated for 30minat roomtemper-ature. Then the veins were washed with PBS, the collagenase solution and the wash were col-lected and centrifuged for 10min at 200×g. Cells were re-suspended in RPMI 1640 completemedium (GIBCO) supplemented with 20% fetal bovine serum (FBS) (HyClone), penicillin(100U/ml) and streptomycin (100µg/ml), and were seeded on glass slides (approximately1.8× 106 cells per slide) coated with gelatin and incubated at 37◦C with 95% humidity and5% CO2. Upon reaching 75% confluence, cells were subcultured by the ratio of 1:3. When

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


STACHYDRINE AMELIORATES HUVECS INJURY INDUCED 159

grown into monolayer like cobble, cells wereidentified as HUVECs by morphological crite-ria (uniform polygonal shape) and by immunofluorescent demonstration of von Willebrandfactor (vWF) antigen.

MTT Assay

In order to determine whether stachydrinewas toxic to HUVECs, cell viability was observed.Cytotoxicity was determined by colorimetric MTT cleavage assay (Tansuwanwonget al.,2006). Briefly, HUVECs (1× 104 per well) were plated in triplicate in 96-well cultureplates, and treated with different final concentrations (10−8 M, 10−7 M, 10−6 M, 10−5 Mand 10−4 M) of stachydrine (Nanjing TCM Instituteof Chinese Materia Medica, Nanjing,China) respectively for 24hours. After incubation, culture media were discarded and newculture media containing 0.5mg/ml of MTT were added. The plates were further incu-bated at 37◦C for 4hours. After the incubation, culture media were discarded and 0.1ml ofdimethyl sulfoxide (DMSO) was added to each well to solubilize the formazine crystals.The absorbance (OD) was measured at 540 nm using a microplate reader. The viability ofHUVECs was expressed as: relative viability= (Ae×100)/Ac, where Ae is the absorbanceof treated cells and Ac is that of the control.

Anoxia-Reoxygenation of HUVECs

HUVECs were grown on gelatinized 35-mm dishes to confluence in endothelial growthmedium (25mmM HEPES, 2mM L-glutamine, 100U/ml penicillin and 100µg/ml strepto-mycin, and 20% heat-inactivated FBS in RPMI 1640 medium) and were randomly dividedinto 6 groups, the control group, anoxia-reoxygenation(A/R) group and 4 A/R+stachydrinegroups with 4 different final concentrationsof stachydrine, with each group containing 6dishes (approximately 6× 105 viable cells /dish) of HUVECs. Confluent cell monolayerswere washed three times with PBS. In 4 A/R+ stachydrine groups, stachydrine with finalconcentrations of 10−8 M, 10−7 M, 10−6 M and 10−5 M were added respectively into theculture medium. The same volume of culture medium was added in the control group andA/R group. HUVECs in all groups were incubated for 6hours at 37◦C in the 95% humidityand 5% CO2.

Cells in 5 A/R groups were maintained under anoxic stress at 37◦C in a 0% O2/5%CO2/∼12% H/balance N2 atmosphere in the anoxic chamber (Forma Scientific). Thechamber environment was rendered anoxic by a palladium catalyst, and O2 concentra-tion was maintained at 0 PPM (Coy Laboratories products). The cells were removed fromthe anoxic chamber after 3hours, and placed in a 95% air/5% CO2 incubator at 37◦C toundergo a period of reoxygenation for 2hours (Zhaoet al., 2003). The cells in the con-trol group were incubated in a 95% air/5% CO2 incubator (normoxia) at 37◦C for totally5hours. After anoxia-reoxygenation, both supernatant and HUVECs were harvested to beanalyzed.

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


160 J. YINet al.

Isolation of Nuclei

HUVECs were washed in PBS, the cell pellets were harvested and lyzed by lysis buffer(10mM NaCl, 3mM MgCl2, 10mM Tris-HCl, pH 7.4, 0.5% Nonidet P-40). The pelletswere dispersed with repeated pipetting and spun for 3sec in cold-room microcentrifuge topellet the nuclei. The nuclear pellets were estimated in volume and re-suspended in an equalvolume of nuclei storage buffer (40% glycerol, 50mM Tris-HCl, pH 8.5, 5mM MgCl2, and0.1mM EDTA) and stored at−80◦C.

cDNA Slot Blot Preparation

Tenµg linear fragments of human tissue factor (TF) cDNA and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) cDNA were denatured respectively by chilling on ice and neutral-ized with one volume of 2M ammonium acetate after adding 30µl of 10N NaOH to eachsample, 1µg (150µl) of the cDNA solution was filtered on to a nitrocellulose membrane onthe top of one piece of 3MM Whatman paper by using slot blot apparatus. The nitrocellulosemembrane was cut into strips, with each containing one set of cDNA samples to be tested,and baked in a vacuum oven for 2hours at 80◦C and stored at room temperature.

Transcription Reaction

The nuclei was thawed on ice, 50µl of nuclei (using wide-bore tip) was added to 50µl offreshly prepared 2× reaction cocktail [50µl 32P-UTP (500µCi), 250µl 4 × reaction mix(100mM HEPES, pH 7.5, 10mM MgCl2, 10mM DTT, 300mM KCl, 20% glycerol), 125µl8× tri-phosphate mix (14µl of each 25mM ATP, 25mM GTP and 25mM CTP, 0.4µl 1mMUTP and 83µl ddH2O, with the final concentrations of 2.8mM ATP, GTP, CTP and 3.2mMUTP), 75µl ddH2O] and incubated at room temperature for 20min. The reaction was stoppedby adding 2µl of DNase I and incubating at 37◦C for 10min. Three hundredµl stop buffer(2% SDS, 7M urea, 0.35M NaCl, 1mM EDTA, 10mM Tris-HCl, pH 8.0), proteinase Kwith a final concentration of 1mg/ml and 100µg tRNA were added to each reaction, whichwas then mixed by pipetting and incubated at 45◦C for 2hours. The reaction was stoppedby adding ice cold trichloroacetic acid (TCA)to each reaction to a finalconcentration of10%, incubation on ice for 20min and spin for 15min, the nucleic acid pellet was washedwith cold absolute ethanol to remove any trace of TCA and incubated at 65◦C for 25min todissolve the RNA after air-drying and re-suspending in 50µl of TE containing 0.5% sodiumdodecylsulphate (SDS).

Hybridization Reaction

Filter-bound TF cDNA and human GAPDH cDNA were prehybridized in 2.5ml hybridiza-tion buffer (50% formamide, 6× SSC, 10× Denhardt’s solution, 0.2% SDS) at 42◦C for8 hours and hybridized at 42◦C for 72hours after adding 50µl 32P-labeled RNA to thehybridization buffer. Then the cDNA blots were washed once with 6× SSC and 0.2% SDS

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.



at room temperature for 10min, twice with 2×SSC and 0.2% SDS, followed by two washesin 0.2 × SSC and 0.2% SDS at 65◦C for 20min. The cDNA blot strips were placed on anX-ray film as backing, covered with plasticwrap, and analyzed by autoradiography.

Tissue Factor Assay

TF procoagulant activity (TF:C) in HUVECs lysate was determined with one stage clottingassay (Saliolaet al., 1998).Briefly, 100µl of HUVECs lysate were added to 100µl of normalpooled citrated plasma; after 150sec incubation at 37◦C, 100µl of 0.025mM CaCl2 wasadded and clotting time was recorded. All samples were tested in duplicate. Consequently,clotting time was converted to arbitrary TF units (U)/2×105 HUVECs by using logarithmicplots with clotting time vs. dilution of a standard TF solution, which was obtained by meansof commercial thromboplastin (Dade). Undiluted thromboplastin was assigned a value of1000TF units, corresponding to a clotting time of 14sec. This procoagulant activity was notseen with plasma deficient in factors VII, X, or V.

The enzyme linked immunosorbent assay (ELISA) was performed to measure antigenof TF (TF:Ag) in HUVECs lysate by using a commercial kit (Imubind Tissue Factor ELISAKit, American Diagnostica Inc.). The assay recognized TF-apo, TF, and TF-VII complexesand is designed such that there is no interference from other coagulation factors or inhibitorsof procoagulant activity (Saliolaet al., 1998).

Assay of Cell Viability and Lactate Dehydrogenase Activity

Cell viability was determined by trypan blue exclusion. HUVECs were seeded in 96-wellplates with 1× 105 cells/well. After treatment withanoxia-reoxygenation, subconfluentHUVECs were washed and harvested in PBS to prepare a single-cell suspension. 50µl ofthe suspension were added to 0.95 ml 0.4% trypan blue solution. The total number of livingand dead cells was counted with a haemacytometer (Huanget al., 2007).

The activity of lactate dehydrogenase (LDH) in cultured supernatant was assayed by aLDH assay kit (Jiancheng Biology Engineering Institute, Nanjing, China) according to themanufacturer’s instructions.

Assay of Malondialdehyde, Superoxide Dismutase and Glutathione Peroxidase

Lipid peroxidationwas quantified by assaying the concentrationof malondialdehyde(MDA)in HUVECs, the activities of superoxide dismutase (SOD) and glutathione peroxidase(GSH-PX) in HUVECs were determined by using nitroblue tetrazolium method and 5,5’-dithiobis-2- nitrobenzoic acid method respectively. At the end of anoxia-reoxygenation, theHUVECs were washed with ice-cold PBS for three times. Then they were lysed in cold lysisbuffer, collected with a rubberpoliceman, and sonicated in cold 20mM HEPES buffer (1mMEDTA, 210mM mannitol, 70mM sucrose, pH 7.2) followed by centrifugation at 1500× gfor 5min at 4◦C. The supernatant was evaluated for MDA, SOD and GSH-PX with an assaykit (Jiancheng Biology Engineering Institute, Nanjing, China) respectively according to the

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


162 J. YINet al.

manufacturer’s protocols, the absorbance of samples was read at 733nm, 550nm and 412nmrespectively. Total cell protein was determined by the Lowry method.

Assay of Nitric Oxide and Endothelin-1

After anoxia-reoxygenation, the medium was collected, and the amount of nitric oxide (NO)released by cells was determined by using a NO assay kit (Jiancheng Biology EngineeringInstitute, Nanjing, China) according to the manufacturer’s protocol. The method involvedmeasuring the amount of NO metabolites (nitriteand nitrate), which were more stable thanNO. The absorbance of samples was read at 550nm.

The concentration of endothelin-1 (ET-1) was measured with a sandwich-enzymeimmunoassay kit (R&D Systems, Inc.) according to the manufacturer’s instructions. Theabsorbance of samples was read at 650nm.

Statistics

Results were expressed as mean±SD. Inter-group comparisons were performed with 1-wayANOVA to test for difference. Statistical Package for the Social Sciences software (SPSSInc.) was used for all analyses. Differenceswere considered to be significant at a value ofp < 0.05.

Results

MTT Assay

Cells were incubated for 24hours with increasing concentrations of stachydrine. Twelvehours of exposure to stachydrine (the final concentrations of 10−8 M, 10−7 M, 10−6 Mand 10−5 M respectively) did not reduce the viability of HUVECs in the MTT assay,therefore we chose 11hours exposure of stachydrine with series final concentrations of10−8 M, 10−7 M, 10−6 M and 10−5 M as the incubation time in the present study (Fig. 1).

Nuclear Transcription

After anoxia-reoxygenation,HUVECs in 5 A/R groups displayed more increased TF mRNAthan those in the control group, reflecting enhanced TF gene transcription. However, TFmRNA was decreased in stachydrine-treated HUVECs than those incubated without stachy-drine in A/R group (Fig. 2).

TF : C and TF : Ag in HUVECs

HUVECs expressed stronger TF procoagulantactivity after anoxia-reoxygenation. Beforeanoxia-reoxygenation, TF : C was 2.59± 0.53U/2× 105 cells, 2.47± 0.46 U/2× 105 cells,2.62± 0.59 U/2× 105 cells, 2.56± 0.52 U/2× 105 cells, 2.61± 0.63 U/2× 105 cells and

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.



Figure 1. Effects of stachydrine on the viability of HUVECs. HUVECs were exposed to various concentrationsof stachydrine for 24hours. The percentage of cell viability was determined by the MTT assay. Values are themeans± SD of triplicate analyses (n = 3, ✩ p < 0.05 and�p < 0.01 compared to the cell viability withoutstachydrine for different time respectively).

Figure 2. Nuclear transcription assay for HUVECs. Contr: HUVECs from control group; A/R: HUVECs fromA/R group; Std-a: HUVECs from A/R+ stachydrine (10−8 M) group; Std-b: HUVECs from A/R+ stachydrine(10−7 M) group; Std-c: HUVECs from A/R+stachydrine (10−6 M) group; Std-d: HUVECs from A/R+stachydrine(10−5 M) group. The thickness of each line represents relative TF mRNA strength. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was served as inner control in each slot blotting.

2.63± 0.57 U/2× 105 cells respectively in HUVECs of the control group, A/R group and 4A/R + stachydrine groups with increased final concentrations of stachydrine from 10−8 Mto 10−5 M. While after anoxia-reoxygenation, TF : C was 2.52 ± 0.49 U/2× 105 cells,63.28± 5.76 U/2× 105 cells, 59.28± 5.38 U/2× 105 cells, 46.72± 4.25 U/2× 105 cells,34.85± 3.78 U/2× 105 cells and 23.15± 2.64 U/2× 105 cells respectively. TF : Ag was11.56± 1.92 pg/2× 105 cells, 10.56± 1.72 pg/2× 105 cells, 11.82± 1.97 pg/2× 105 cells,10.95±1.88pg/2×105cells, 11.85±1.84pg/2×105cells and 12.06±2.03pg/2×105cellsrespectively in HUVECs of the control group, A/R group and 4 A/R+ stachydrine groupswith increased final concentrations of stachydrine from 10−8 M to 10−5M before anoxia-reoxygenation, however, it was 10.95±1.85 pg/2×105cells, 95.38±5.69 pg/2×105cells,90.32±5.17pg/2×105cells, 76.35±4.53pg/2×105cells, 59.76±3.65pg/2×105cells and46.66±3.25 pg/2×105cells after anoxia-reoxygenation. TF : C and TF : Ag were much lower

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


164 J. YINet al.

(A)

(B)

Figure 3. TF : C and TF : Ag in HUVECs. (A) TF : C in HUVECs; (B) TF : Ag in HUVECs. Contr: TF fromHUVECs in control group; A/R: TF from HUVECs in A/R group; Std-a: TF from HUVECs in A/R+ stachydrine(10−8 M) group; Std-b: TF from HUVECs in A/R+ stachydrine (10−7 M) group; Std-c: TF from HUVECs inA/R + stachydrine (10−6 M) group; Std-d: TF from HUVECs in A/R+ stachydrine (10−5 M) group.Be: beforeanoxia-reoxygenation;Af: after anoxia-reoxygenation,�p < 0.001 compared to the control group;�p < 0.05compared to A/R group;♦p < 0.01 compared to A/R group.

in stachydrine treated HUVECs than those from A/R group respectively (p< 0.05 ∼ 0.01,Fig. 3).

Cell Viability and LDH Activity in HUVECs

After anoxia-reoxygenation,cell viability declined from 96.2±6.3% to 71.5±4.8% in A/Rgroup, 95.3±6.3% to 74.6±4.1% in 10−8 M stachydrine treated HUVECs, 96.5±6.8% to

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.



79.4±4.3% in 10−7M stachydrine treated HUVECs, 95.5±6.5% to 85.3±5.8% in 10−6 Mstachydrine treated HUVECs and 95.9±7.1% to 89.2±6.2% in 10−5 M stachydrine treatedHUVECs. There was a significant difference between HUVECs from the control group95.8± 6.5% to 94.1± 6.3% and A/R groups except 10−5M stachydrine treated group afteranoxia-reoxygenation (p< 0.05 ∼ 0.01 versus the control group), however, there was nostatistical difference between those from the control group and 10−5 M stachydrine treatedgroup.

The activity of LDH in the supernatant was increased from 26.9 ± 1.2U/L to 263.5 ±12.6U/L in A/R group, 27.1 ± 1.5U/L to 250.8 ± 12.3U/L in 10−8 M stachydrine treatedHUVECs, 25.9 ± 1.1U/L to 205.9 ± 10.4U/L in 10−7 M stachydrine treated HUVECs,26.3±1.4U/L to 158.8±9.7U/L in 10−6 M stachydrine treated HUVECs and 26.6±1.3U/Lto 103.5±8.5U/L in 10−5M stachydrine treated HUVECs after anoxia-reoxygenation. Therewas a significant difference between HUVECs from the control group (26.5 ± 1.3U/L to27.2± 1.4U/L) and other groups after anoxia-reoxygenation (p< 0.001 versus the controlgroup).

Stachydrine (10−7 M, 10−6 M and 10−5M) attenuated the decline of HUVECs viabilityand the increase of LDH activity induced by anoxia-reoxygenation (p< 0.05 to 0.01 vs.A/R group) (Fig. 4).

MDA Concentration and the Activity of SOD and GSH-PX

After anoxia-reoxygenation, the expression of MDA was increased, while SOD and GSH-PX were less produced in HUVECs subjected to anoxia-reoxygenation (p< 0.05 to 0.01 vs.the control group). Stachydrine inhibited the increased expression of MDA and reduction ofSOD and GSH-PX induced by anoxia-reoxygenation (p< 0.05 to 0.01 versus A/R group,Table 1).

Concentrations of NO and ET-1

To investigate the release of NO and ET-1from HUVECs subjected toanoxia-reoxygenation,NO and ET-1 release were measured simultaneously. Background NO release was decreasedand ET-1 production was increased significantly after anoxia-reoxygenation (p< 0.01 to0.001 vs. the controlgroup).However, theywere inhibitedbystachydrine inadose-dependentmanner (p< 0.05 to 0.01 versus A/R group, Table 2).

Discussion

Ischemia-reperfusion injury is a serious clinical problem, which involves oxidative stressand leads to tissue injury. Endothelial cells are the tissue’s first line of defense; oxidativestress activates endothelial cells and leads to the injury or even death of the cells (Scarabelliet al., 2002).

In this study, we found that TF expression at both mRNA and protein levels was increasedin HUVECs subjected to anoxia-reoxygenation.TF, often called thromboplastin or factor III,

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


166 J. YINet al.

(A)

(B)

Figure 4. Cell viability and LDH activity in HUVECs. (A)cell viability; (B) LDH activity. Contr: HUVECs fromcontrol group; A/R: HUVECs from A/R group; Std-a: HUVECs from A/R+ stachydrine (10−8 M) group; Std-b:HUVECs from A/R+ stachydrine (10−7 M) group; Std-c: HUVECs from A/R+ stachydrine (10−6 M) group;Std-d: HUVECs from A/R+ stachydrine (10−5 M) group. Be: before anoxia-reoxygenation;Af: after anoxia-reoxygenation,∗p<0.05 compared with control group;�p<0.01 compared with control group;�p < 0.001compared to the control group;�p < 0.05 compared to A/R group;♦p < 0.01 compared to A/R group.

plays an indispensable role in the activation of factor VII (FVII) through the formation ofcomplex and activation factors X and IX by the production of FVIIa. Therefore, TF isregarded as an initiation factor of blood coagulation (Butenaset al., 2007). Data have shownthat in addition to thrombosis, TF takes part in a set of cellular processes including inflam-mation, cancer, atherosclerosis, and embryogenesis, etc. (Pawlinskiet al., 2004; Bluffet al.,2008; Keyet al., 2007). In fact, not only anoxia-reoxygenation induced the over-expression

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.



Table 1. Concentration of MDA, Activities of SOD and GSH-PX

MDA Concentration SOD Activity GSH-PX Activity(nmol/mg Pro) (mIU/min./mg Pro) (µmol/min./mg Pro)

Groups Before A/R After A/R Before A/R After A/R Before A/R After A/R

Control 15.35± 3.56 15.65± 3.78 20.95± 4.86 19.53± 4.32 56.35± 9.18 55.89± 8.76A/R 14.85± 3.42 29.66± 6.55� 21.15± 5.12 8.83± 2.16� 55.91± 8.89 25.86± 5.27�A/R+Std (10−8 M) 15.16± 3.51 27.87± 5.95� 20.67± 4.77 9.03± 2.56� 55.85± 8.28 26.83± 5.76�A/R+Std (10−7 M) 14.87± 3.46 24.02± 5.44�� 21.05± 4.96 11.15± 2.86�� 56.95± 9.35 31.26± 6.05��A/R+Std (10−6 M) 14.65± 3.07 20.25± 4.87∗♦ 19.96± 4.15 13.16± 3.41�� 54.87± 7.86 39.53± 6.94��A/R+Std (10−5 M) 15.06± 3.33 18.18± 4.12∗♦ 19.96± 4.15 14.86± 3.46∗� 53.85± 7.81 46.38± 7.92∗♦

(mean± SD) ∗p < 0.05 compared to the control group;�p < 0.01 compared to the control group;�p < 0.05compared to A/R group;♦p < 0.01 compared to A/R group.

Table 2. Concentrations of NO and ET-1

NO (nmol/ml) ET-1 (pg/ml)

Groups Before A/R After A/R Before A/R After A/R

Control 69.18± 7.84 68.26± 7.72 26.53± 2.55 26.81± 3.01A/R 68.86± 8.25 24.29± 4.53� 26.91± 2.46 94.75± 5.86�A/R+Std (10−8 M) 68.58± 7.69 26.42± 4.15� 25.62± 2.73 91.88± 5.21�A/R+Std (10−7 M) 66.84± 6.29 36.25± 5.64�� 26.93± 2.59 75.56± 5.09��A/R+Std (10−6 M) 68.56± 7.15 45.78± 6.23�♦ 27.32± 2.92 62.84± 6.42�♦A/R+Std (10−5 M) 67.98± 8.02 53.75± 6.23�♦ 27.32± 2.92 43.69± 4.68�♦

(mean± SD)�p < 0.01 compared with control group;�p < 0.001 compared to the control group;�p < 0.05compared to A/R group;♦p < 0.01 compared to A/R group.

of TF, studiesin vivo andin vitro showed that ischemia and hypoxia also produced solublefactors that either directly or indirectly stimulated the expression of TF (Sevastoset al.,2007; Compeauet al., 1994). TF plays a pivotal role in anoxia-reoxygenationand ischemia-reperfusion injury.

Anoxia-reoxygenation induces an upregulation of TF mRNA in HUVECs by stimulat-ing the TF promoter via activating transcription factors such as activator protein-1 (AP-1),nuclear factor-κB (NF-κB) and early growth response protein-1 (EGR-1), etc. (Bavendieket al., 2002; Mackman, 1997). The transcription factor NF-κB plays an essential role in TFgene expression, it translocates from the cytoplasm to the nucleus, and binds to the putativeκB site in the promoter of the TF gene (Bavendieket al., 2002), ultimately resulting in TFover-expression.

TF may contribute to endothelial cells injury during anoxia-reoxygenation via two path-ways, reactive oxygen species (ROS) production and inflammation. TF induces phosphory-lation of xanthine oxidase (XO) in hypoxic vascular endothelial cells via p38 MAPK andcasein kinase II signalling (Kayyaliet al., 2001; Steffelet al., 2005). XO normally occursas an NAD+-dependent dehydrogenase [xanthine dehydrogenase (XD)] incapable of ROSproduction. XD activity converts by sulfhydryloxidation or limited proteolysis (conditionsthat exist during reoxygenation) to XO. It was confirmed that phosphorylation of XO wasan essential step for producing ROS including O2·-, H2O2 and·OH during reoxygenation

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


168 J. YINet al.

(Li and Jackson, 2002). Under physiological conditions, the amount of ROS, e.g. O2·-,H2O2 and ·OH stay at a relatively stable level by regulating the balance between ROS-forming enzymes, such as XO and ROS-scavenging enzymes, such as SOD and GSH-PX.Under pathological conditions such as anoxia and reoxygenation, however, over-expressionof TF induces phosphorylation of XO and subsequently generates much more ROS, andcauses ROS accumulation and lipid peroxidation, which result in over-production of MDA,a biomarker of oxidative stress level, and consumptive decrease of SOD and GSH-PX activ-ities. SOD and GSH-PX play key roles in detoxification of reactive oxygen metabolites(peroxides) and reactive electrophilic compounds and are commonly considered as anti-peroxidative biomarkers (Kalpakcioglu and Senel, 2008; Nakamura, 2003). Therefore, wedetected the concentrations of SOD, GSH-PX and MDA to study HUVECs’ ability in scav-enging ROS and lipid peroxidation injury in anoxia-reoxygenation.

On the other hand, the over-expressed TF maycontribute to anoxia-reoxygenation injuryby subsequent G-protein-coupled protease-activated receptors (PARs) signaling (Mandalet al., 2007). The PAR family contains four members, PAR1, PAR2, PAR3 and PAR4. TFirritates vascular endothelial cells to produce many mediators such as tissue necrosis fac-tor (TNF), interleukins, adhesion molecules [monocyte chemoattractant protein (MCP-1),intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), selectins, etc.] and growth factors [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), etc.] by activation ofPAR-1 and PAR-2 expressed on endothelialcells during anoxia-reoxygenation, and subse-quently elicits inflammation which leads to the injury of the cells (Chu, 2005; Hagi-Pavliet al., 2004). In addition, TF also contributes to ROS generation by activation of PAR-2, themechanism of which still remains unclear (Limet al., 2006).

Both lipid peroxidation and inflammation subsequently result in endothelial cells injuryeven death, which lead to ET-1 release. ET-1, a 21-amino acid peptide produced primarilyin endothelial cells, is the most powerful endogenous vasoconstrictor agent and has beenidentified as a key player in endothelial dysfunction resulting from endothelial cell activa-tion and injury (Abraham and Dashwood, 2008). NO is an unstable substance produced inendothelial cells and a sensitive biomarker of endothelial cell injury, which is involved inthe regulation of blood pressure, inhibition of the adhesion of leukocytes to endothelium,decreased platelet and blood vessel wall interaction, inhibition of apoptosis induced by vari-ous apoptotic stimuli and proliferationand migration of vascular smooth muscle cells. Due tothe very unstable chemical nature of NO, endothelium-derivedNO can be rapidly inactivatedby oxygen-derived free radicals due to the formation of the complex between NO and O2·-,and thus, an enhanced production of ROS appears to be involved in the accelerated break-down of NO. Excess vascular oxidative stress leads to the impairment of NO production,resulting in reduced NO in endothelial cells (Wanget al., 2005). Therefore, we detected theconcentrations of ET-1 and NO to assess HUVECs’ function in regulating vasoconstrictionand vasodilation and injury of HUVECs.

Impaired functions of endothelial cells in scavenging ROS and regulating vasodilationand vasoconstriction lead to a decline of cell viability and an increase of LDH activity,which indicate directly the degree of cells injury. Therefore, we measured the cell viability

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.



and LDH activity to evaluate the HUVECs injury induced by anoxia-reoxygenation in thepresent study.

The results showed that after anoxia-reoxygenation, HUVECs displayed lower activi-ties of SOD and GSH-PX as well as higher concentration of MDA, which revealed thatHUVECs’ ability in scavenging ROS was damaged and HUVECs were injured by lipid per-oxidation. While the HUVECs in the A/R group presented a higher concentration of ET-1and a lower concentration of NO after anoxia-reoxygenation, which demonstrated dysfunc-tion of HUVECs in regulating vasoconstriction and vasodilation and injury of HUVECs. Inaddition, HUVECs viability declined and LDH activity increased to a certain degree in A/Rgroup, which indicated HUVECs were injured by anoxia-reoxygenation.This suggested thatthe experimental model was reliable and successful (Huanget al., 2007).

From the aforementioned, TF appears to be responsible for anoxia-reoxygenation inducedinjury in HUVECs. Therefore, inhibition of TF expression would protect against anoxia-reoxygenation injury.

In the present study, we found that stachydrine blocked the expression of TF in bothmRNA and protein levels. This TF inhibition was not caused by cell death elicited bystachydrine, since MTT assay showed that HUVECs exposed to a series of the final concen-trations of stachydrine (10−8 M, 10−7 M, 10−6 M, 10−5 M and 10−4 M) for 24hours, only10−4 M stachydrine showed a certain degree of toxicity to HUVECs, the cell viability wasslightly decreased from 18thhour for 10−5 M and 6thhour for 10−4 M. Therefore, there wasno cytotoxicity of stachydrine with the concentrations and incubation time used here.

Stachydrine is a major constituent in the Chinese herb leonurus heterophyllus sweet,which has a low toxicity, multiple and long-term oral doses have producedno toxic reactions,and an intramuscular injection solution of the herb does not cause any side effects exceptdry mouth and shortened sleep. The LD50 value of the injection solution of the herb is30∼60g/kg in mice by intravenous administration. Male rats that consumed feed containing50% of the powderedherb for 80 days did not exhibit toxic reactions nor was fertility affected(Zhanget al., 1982; Zhu, 1998).

The results showed that stachydrine inhibited the transcription and expression of TFgene in a dose-dependent manner. While TF contributes to anoxia-reoxygenation injuryboth by producing ROS through XO phosphorylation via p38 MAPK and casein kinase IIsignal pathway, and by initiating inflammation through inducing endothelial cells to expressvarious inflammatory mediators such as adhesion molecules, cytokines and chemokines viaactivation of PAR-1 and PAR-2 (Chu, 2005; Penn and Topol, 2001). This TF-dependentROS generation and inflammation subsequently lead to endothelial cell damage. Therefore,stachydrine blocked ROS production and inflammationby interrupting the two TF-dependentsignal pathways, and subsequently reversed the changes of SOD, GSH-PX, NO, MDA andET-1, ameliorated the decline of HUVECs viability and the enhancement of LDH activity thatoccurred during anoxia-reoxygenation. This indicated that stachydrine obviously reducedthe degree of anoxia-reoxygenation injury toHUVECs and ultimately improved endothelialcell survival.

Consequently, our findings indicate that TF plays an important role in the developmentofanoxia-reoxygenation injury of HUVECs. Stachydrine, a major constituent of the Chinese

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.


170 J. YINet al.

herb leonurus heterophyllus sweet, protects HUVECs against the injury induced by anoxia-reoxygenation, the putative mechanisms of which are related to inhibition of TF expression.Whereas, anoxia-reoxygenation is a pathological condition anda complex process, whichis the consequence of many diseases, it is also a delicate subject to understand becauseseveral signal pathways are involved. The further role of stachydrine in endothelial injurystill remains unclear, e.g. by what signalingdoes stachydrine use toinhibit TF expression?Whether stachydrine directly reduces lipid peroxidation and ameliorates endothelial injuryin a TF-independent manner? How is the influence of stachydrine on endothelial metabolismand morphology? Besides reducing lipid peroxidation and inflammation, is there any othersignal pathway which stachydrine takes part in? Therefore, further study of the underly-ing mechanisms of stachydrine protecting HUVECs against anoxia-reoxygenation inducedinjury needs to be investigated.

Acknowledgments

This research was supported by a grant from Guangdong Provincial Administration of Tra-ditional Chinese Medicine (No. 403016). The authors wish to thank Dr. Yong Xiao and Dr.Eric Hui for editing the article.

References

Abraham, D. and M. Dashwood. Endothelin — role in vascular disease.Rheumatology (Oxford).47(Suppl. 5): v23–24, 2008.

Bavendiek, U., P. Libby, M. Kilbride, R. Reynolds, N. Mackman and U. Schönbeck. Induction of tissuefactor expression in human endothelial cells by CD40 ligand is mediated via activator protein1, nuclear factor kappa B, and Egr-1.J. Biol. Chem. 277: 25032–25039, 2002.

Bluff, J.E., N.J. Brown, M.W. Reed and C.A. Staton. Tissue factor, angiogenesis and tumour progres-sion.Breast Cancer Res. 10: 204, 2008.

Butenas, S., T. Orfeo, K.E. Brummel-Ziedins and K.G. Mann. Tissue factor in thrombosis and hem-orrhage.Surgery. 142: S2–14, 2007.

Chu, A.J. Tissue factor mediates inflammation.Arch. Biochem. Biophys. 440: 123–132, 2005.Compeau, C.G., J. Ma, K.N. DeCampos, T.K. Waddell, G.F. Brisseau, A.S. Slutsky and O.D. Rotstein.

In situ ischemia and hypoxia enhance alveolar macrophage tissue factor expression.Am. J.Respir. Cell Mol. Biol. 11: 446–455, 1994.

Eltzschig, H.K. and C.D. Collard. Vascular ischaemia and reperfusion injury.Br. Med. Bull. 70: 71–86,2004.

Hagi-Pavli, E., P.M. Farthing and S. Kapas. Stimulation of adhesion molecule expression in humanendothelial cells (HUVEC) by adrenomedullin and corticotrophin.Am. J. Physiol. Cell Physiol.286: C239–246, 2004.

Huang, Q., M. He, H. Chen, L. Shao, D. Liu, Y. Luo and Y. Dai. Protective effects of sasanquasaponin oninjury of endothelial cells induced by anoxia and reoxygenationin vitro. Basic Clin. Pharmacol.Toxicol. 101: 301–308, 2007.

Kalpakcioglu, B. and K. Senel. The interrelation of glutathione reductase, catalase, glutathione per-oxidase, superoxide dismutase, and glucose-6-phosphate in the pathogenesis of rheumatoidarthritis.Clin. Rheumatol. 27: 141–145, 2008.

Kayyali, U.S., C. Donaldson, H. Huang, R. Abdelnour and P.M. Hassoun. Phosphorylation of xanthinedehydrogenase/oxidase in hypoxia.J. Biol. Chem. 276: 14359–14365, 2001.

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.



Key, N.S., J.G. Geng and R.R. Bach. Tissue factor; from Morawitz to microparticles.Trans. Am. Clin.Climatol. Assoc. 118: 165–173, 2007.

Kuang, P.G., X.F. Zhou, F.Y. Zhang and S.Y. Lang. Motherwort and cerebral ischemia.J. Tradit. Chin.Med. 8: 37–40, 1988.

Li, C. and R.M. Jackson. Reactive species mechanisms of cellular hypoxia-reoxygenation injury.Am.J. Physiol. Cell Physiol. 282: C227–241, 2002.

Lim, S.Y., G.M. Tennant, S. Kennedy, C.L. Wainwright and K.A. Kane. Activation of mouse protease-activated receptor-2 induces lymphocyte adhesion and generation of reactive oxygen species.Br. J. Pharmacol. 149: 591–599, 2006.

Mackman, N. Regulation of the tissue factor gene.Thromb. Haemost. 78: 747–754, 1997.Mandal, S.K., U.R. Pendurthi and L.V. Rao. Tissue factor trafficking in fibroblasts: involvement of

protease-activated receptor-mediated cell signaling.Blood. 110: 161–170, 2007.McCormick, S.M., S.G. Eskin, L.V. McIntire, C.L. Teng, C.M. Lu, C.G. Russell and K.K. Chittur.

DNA microarray reveals changes in gene expression of shear stressed human umbilical veinendothelial cells.Proc. Natl. Acad. Sci. USA. 98: 8955–8960, 2001.

Nakamura, H. Experimental and clinical aspects of oxidative stress and redox regulation.Rinsho Byori51: 109–114, 2003 (in Japanese).

Pawlinski, R., B. Pedersen, J. Erlich and N. Mackman. Role of tissue factor in haemostasis, thrombosis,angiogenesis and inflammation: lessons from low tissue factor mice.Thromb. Haemost. 92: 444–450, 2004.

Penn, M.S. and E.J. Topol. Tissue factor, the emerging link between inflammation, thrombosis, andvascular remodeling.Circ. Res. 89: 1–2, 2001.

Saliola, M., R. Lorenzet, D. Ferro, S. Basili, C. Caroselli, A.D. Santo, M. Sallese and F. Violi. Enhancedexpression of monocyte tissue factor in patients with liver cirrhosis.Gut. 43: 428–432, 1998.

Scarabelli, T.M., A. Stephanou, E. Pasini, L. Comini, R. Raddino, R.A. Knight and D.S. Latchman.Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes duringischemia/reperfusion injury.Circ. Res. 90: 745–748, 2002.

Sevastos, J., S.E. Kennedy, D.R. Davis, M. Sam, P.W. Peake, J.A. Charlesworth, N. Mackman andJ.H. Erlich. Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemiareperfusion injury.Blood. 109: 577–583, 2007.

Steffel, J., M. Hermann, H. Greutert, S. Gay, T.F. Lüscher, F. Ruschitzka and F.C. Tanner. Celecoxibdecreases endothelial tissue factor expression throughinhibition of c-Jun terminal NH2 kinasephosphorylation.Circulation. 111: 1685–1689, 2005.

Tansuwanwong, S., Y. Hiroyuki, I. Kohzoh and U. Vinitketkumnuen. Induction of apoptosis in RKOcolon cancer cell line by an aqueous extract ofMillingtonia hortensis. Asian Pac. J. CancerPrev. 7: 641–644, 2006.

Wang, Y.K., Y.J. Hong and Z.Q. Huang. Protective effects of silybin on human umbilical vein endothe-lial cell injury induced by H2O2 in vitro. Vascul. Pharmacol. 43: 198–206, 2005.

Zhang, C.F., Y.S. Jia, H.C. Wei, X.M. Zhu, Y.M. Hui, C.Y. Zhang, Q.Z. Mo and B. Gong. Studies onactions of extract of motherwort.J. Tradit. Chin. Med. 2: 267–270, 1982.

Zhao, H., M. Miller, K. Pfeiffer, J.A. Burasand G.L. Stahl. Anoxia and reoxygenation of humanendothelial cells decrease ceramide glucosyltransferase expression and activates caspases.FASEB J. 17: 723–724, 2003.

Zhu, Y.P.Chinese Materia Medica: Chemistry, Pharmacology and Applications. Harwood AcademicPublishers, Singapore, 1998.

Zou, Q.Z., R.G. Bi, J.M. Li, J.B. Feng, A.M. Yu, H.P. Chan and M.X. Zhen. Effect of motherwort onblood hyperviscosity.Am. J. Chin. Med. 17: 65–70, 1989.

Am

. J. C

hin.

Med

. 201

0.38

:157

-171

. Dow

nloa

ded

from

ww

w.w

orld

scie

ntif

ic.c

omby

OH

IO U

NIV

ER

SIT

Y o

n 10

/05/

13. F

or p

erso

nal u

se o

nly.

Stachydrine, a Major Constituent of the Chinese Herb Leonurus Heterophyllus Sweet, Ameliorates Human...

Documents

Transcript of Stachydrine, a Major Constituent of the Chinese Herb Leonurus Heterophyllus Sweet, Ameliorates Human...