Study On The Protective Mechanism Of Liuwei Dihuang Pill On Apoptosis Of Endothelial Cells Induced By

Purpose：To investigate and observe the protective effect of rat serumcontaining Liuweidihuang pill, one of the classic prescriptions, on impairedhuman umbilical vascular endothelial cells by high concentrations of palmiticacid and reveal its mechanism at the cellular and molecular level. The followingtechnologic methods such as cell culture, biochemical detection kit, RT-PCR andwestern blot and so on were used in order to find a new target for the preventionof diabetes and vascular lesions. This study is also aimed at providing a newangle of theory with our experiment and building a new research platform in thetreatment of diabetes and its complications by using Liuweidihuang pill, whichworks well with the insulin receptor substrate-2/phosphatidy linositide-3kinase/protein kinases B signal transduction pathway and nicotinamide adeninedinucleotide phosphate oxidase-reactive oxygen species-asymmetricdimethylarginine system of the endothelial cells.Materials and methods：Part1:1. Preparation of rat serum containing liuweidihuang pill.52SPFSprague-Dawley rats were randomly divided into normal controlled group and theliuweidihuang pill (LWDHP) group according to numeral. Traditional Chinesemedicine group was fed with liuweidihuang pill powder water solution(pharmacognostic mass concentration of15g/kg·d) by gavage administration for6days, while the normal controlled group with equal volume of saline. And thenall the rats were given anesthesia by10%chloral hydrate in abdominal cavityand blood was collected from abdominal artery. Blood coagulated naturally atroom temperature. Serum was separated by centrifugation, inactivated at56℃water bath and then filtrated, sterilized, packed, sealed and reserved at-80℃.2. The culture of the EA. hy926human umbilical vascular endothelial cells (HUVEC)and the establishment of the cell model. EA. hy926HUVEC were regularly cultured in incubation box with37℃and5%CO2, by changing culture medium once every2days and passaging once every3or4days. EA. hy926HUVEC of the logarithmicphase were adjusted with its density of0.5×105/mL and were inoculated in96-wellculture plate, which the normal control group, and five different concentrationgradient of palmitic acid (PA) groups, each of which with six holes were set.After the cells growing into fusion by90%, they were processed with PA in finalconcentration of200μmol/L,300μmol/L,400μmol/L,500μmol/L, and600μmol/L respectively and incubated for1hour. Then the cells were processedwith insulin in final concentration of50nmol/L. Twenty four hours later afterPA affecting, the endothelial cells (EC) were induced to injured, and then20μL MTT was added in concentration of5mg/mL for4hours into incubation boxand dimethyl sulfoxide (DMSO) with150μL for15minutes at the same timeshocking cells. The optical density (OD) values were detected in microplatereader with490nm wavelength and the cell survival rate of the PA groups wascalculated about50%～60%compared with normal controlled group, which wassignificantly different. And then the best concentration of PA to realize injuryto the model cells could be found out for following study.3. Effect of rat serumcontaining liuweidihuang pill on proliferation of impaired EA.hy926HUVEC byPA. The density of EA. hy926HUVEC of the logarithmic phase were adjusted in0.5×105/mL and inoculated in96-well culture plate, which the normal controlledgroup, PA group with400μmol/L, liuweidihuang pill Chinese medicine treatmentgroup with serum containing drug of2.5%,5%,10%,20%and metformin (MET) groupwere set. After the cells growing into fusion by90%, they were processed withDMEM culture mediums containing liuweidihuang pill rat serum with differentconcentration gradient of2.5%,5%,10%and20%, MET with final concentrationof2mmol/L, and to be continued to culture and intervene to protect for24hours.The cells were processed with insulin in final concentration of50nmol/L afterPA with400μmol/L for1hour. Twenty four hours later after PA affecting, themorphological characteristics of EC growth in every group was observed undermicroscope, and then20μL MTT was added in concentration of5mg/mL for4hours in incubation box and DMSO with150μL for15minutes at the same time shockingcells. The OD values were detected in microplate reader with490nm wavelengthand the cell survival rate was calculated about70%～80%compared with normalcontrolled group, which was significantly different in liuweidihuang pill group.The rat serum containing liuweidihuang pill of suboptimal concentration couldbe discarded, and the remaining concentration gradients could be been for thefollowing experiments.Part2:1. Detection of nitric oxide (NO) content in cell culture medium.The density of EA. hy926HUVEC of the logarithmic phase were adjusted in0.5×105/mL and were inoculated in culture plate, which the normal controlled group,PA group with400μmol/L, liuweidihuang pill Chinese medicine treatment groupwith serum containing drug of2.5%,5%,10%and MET group were set. After thecells growing into fusion by90%, they were processed with DMEM culture mediumscontaining liuweidihuang pill rat serum with different concentration gradientof2.5%,5%and10%, MET with final concentration of2mmol/L, and to be continuedto culture and intervene to protect cells for24hours. The cells were processedwith insulin in final concentration of50nmol/L after PA with400μmol/L for1hour. Twenty four hours later after PA affecting, the culture medium of allgroups were collected respectively and the experiments were operated accordingto detection kit instructions. The OD values were detected in microplate readerwith540nm wavelength and the content of NO in cell culture medium was detected.Thus assist in selecting the best concentration of serum containingliuweidihuang pill for the following experiments.2. The mRNA expression ofinsulin receptor substrate-2(IRS-2), phosphatidy linositide-3kinase (PI3K),protein kinases B (PKB/Akt), and endothelial nitric oxide synthase (eNOS) incells were evaluated by RT-PCR. The density of EA. hy926HUVEC of the logarithmicphase were adjusted in4×105/mL and inoculated in25cm2culture bottles with4mL culture medium per bottle, which the normal controlled group, PA group with400μmol/L, liuweidihuang pill Chinese medicine treatment group with serumcontaining drug of5%and MET group were set. After the cells growing into fusion by90%, they were processed with DMEM culture mediums containing liuweidihuangpill rat serum with concentration of5%and MET with final concentration of2mmol/L, and to be continued to culture and intervene to protect for24hours.The cells were processed with insulin in final concentration of50nmol/L afterPA with400μmol/L for1hour. Twenty four hours later after PA affecting, theculture medium was discarded, cells were collected and the cellular total RNAwas extracted, then reverse transcription and polymerase chain reaction shouldbe carried on by RT-PCR Kit. Electrophoresis images were analyzed by gel imagingsystem, with which the ratio of band optical density values between target geneand the corresponding β-actin in each sample indicated the mRNA expression levelof each target gene.3. The protein expression of IRS-2, PI3K, P-Akt, and eNOSin cells was evaluated by western blot. The density of EA. hy926HUVEC of thelogarithmic phase were adjusted in5×105/mL and inoculated in25cm2culturebottles with5mL culture medium per bottle, which the normal controlled group,PA group with400μmol/L, liuweidihuang pill Chinese medicine treatment groupwith serum containing drug of5%and MET group were set. After the cells growinginto fusion by90%, they were processed with DMEM culture mediums containingliuweidihuang pill rat serum with concentration of5%and MET with finalconcentration of2mmol/L, and to be continued to culture and intervene to protectfor24hours. The cells were processed with insulin in final concentration of50nmol/L after PA with400μmol/L for1hour. Twenty four hours later afterPA affecting, the culture medium was discarded, cells were collected and thecellular total proteins were extracted, protein concentration in each samplewas determined by BCA protein concentration detection kit. To adjust proteinsample volume about55μg of each group and carry on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transfer the protein onpolyvinylidene fluoride (PVDF) membrane, tightly seal the PVDF membrane with5%skimmed milk powder, then apply the primary and secondary antibodies and colordevelopment by diaminobenzidine (DAB). Electrophoresis images were scanned andanalyzed by software, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was took as internal reference, with which the ratio of protein expressionintensity between target protein and the corresponding GAPDH in each sampleindicated the protein expression level of each target protein.Part3:1. Detection of malonyldialdehyde (MDA) content and total superoxidedismutase (SOD) activity in cell lysate supernatant fluid and asymmetricdimethylarginine (ADMA) concentration in cell culture medium. The density ofEA. hy926HUVEC of the logarithmic phase were adjusted in0.5×105/mL and wereinoculated in culture plate, which the normal controlled group, PA group with400μmol/L, liuweidihuang pill Chinese medicine treatment group with serumcontaining drug of2.5%,5%,10%and MET group were set. After the cells growinginto fusion by90%, they were processed with DMEM culture mediums containingliuweidihuang pill rat serum with different concentration gradient of2.5%,5%and10%, MET with final concentration of2mmol/L, and to be continued to cultureand intervene to protect cells for24hours. The cells were processed with insulinin final concentration of50nmol/L after PA with400μmol/L for1hour. Twentyfour hours later after PA affecting, the culture medium and the cells of allgroups were collected and the experiments were operated according to detectionkit instructions respectively. The OD values were detected in microplate readerwith532nm,560nm and450nm wavelength and MDA content and total SOD activityin cell culture medium and ADMA concentration in cell culture medium weredetected. Thus in the end the best concentration of serum containingliuweidihuang pill was selected for the following experiments.2. The mRNAexpression of nicotinamide adenine dinucleotide phosphate oxidase-4(NOX4),protein arginine methyltransferase-1(PRMT1) and dimethylargininedimethylaminohydrolase-2(DDAH2) in cells were evaluated by RT-PCR. The densityof EA. hy926HUVEC of the logarithmic phase were adjusted in4×105/mL andinoculated in25cm2culture bottles with4mL culture medium per bottle, whichthe normal controlled group, PA group with400μmol/L, liuweidihuang pillChinese medicine treatment group with serum containing drug of5%and MET groupwere set. After the cells growing into fusion by90%, they were processed with DMEM culture mediums containing liuweidihuang pill rat serum with concentrationof5%and MET with final concentration of2mmol/L, and to be continued to cultureand intervene to protect for24hours. The cells were processed with insulinin final concentration of50nmol/L after PA with400μmol/L for1hour. Twentyfour hours later after PA affecting, the culture medium was discarded, cellswere collected and the cellular total RNA was extracted, then reversetranscription and polymerase chain reaction should be carried on by RT-PCR Kit.Electrophoresis images were analyzed by gel imaging system, with which the ratioof band optical density values between target gene and the corresponding β-actinin each sample indicated the mRNA expression level of each target gene.3. Theprotein expression of NOX4, PRMT1and DDAH2in cells was evaluated by westernblot. The density of EA. hy926HUVEC of the logarithmic phase were adjusted in5×105/mL and inoculated in25cm2culture bottles with5mL culture medium perbottle, which the normal controlled group, PA group with400μmol/L,liuweidihuang pill Chinese medicine treatment group with serum containing drugof5%and MET group were set. After the cells growing into fusion by90%, theywere processed with DMEM culture mediums containing liuweidihuang pill rat serumwith concentration of5%and MET with final concentration of2mmol/L, and tobe continued to culture and intervene to protect for24hours. The cells wereprocessed with insulin in final concentration of50nmol/L after PA with400μmol/L for1hour. Twenty four hours later after PA affecting, the culture mediumwas discarded, cells were collected and the cellular total proteins wereextracted, protein concentration in each sample was determined by BCA proteinconcentration detection kit. To adjust protein sample volume about55μg ofeach group and carry on sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), transfer the protein on polyvinylidene fluoride(PVDF) membrane, tightly seal the PVDF membrane with5%skimmed milk powder,then apply the primary and secondary antibodies and color development bydiaminobenzidine (DAB). Electrophoresis images were scanned and analyzed bysoftware, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was took as internal reference, with which the ratio of protein expression intensity betweentarget protein and the corresponding GAPDH in each sample indicated the proteinexpression level of each target protein.All data were showed by mean±standard (x s) and tested by One-WayAnalysis of Variance (ANOVA) and LSD test. P＜0.05meaned significant differenceand P＜0.01meaned highly significant difference.Results：1. Selecting the best concentration of PA of model cells. After being processedof PA with different concentrations gradient (200μmol/L～600μmol/L) onEA.hy926cells, the OD values and cell survival rates were gradually decreased,which were significantly different (P＜0.01) compared with the normal controlledgroup. The optimal concentration of cells modeling of PA concentration was400μmol/L when the cell survival rate was50.48%.2. The observation of cell morphology in each group. Cells of normal controlledgroup were flat polygonal or spindle-shaped and clear boundary, and they werearranged in a single layer paving stone like mosaic, while cells of PA groupwere shrinked and deformed to more stellate shape or oval form. The connectionsof cells became fracture and separation, with the outline of cells was unclearand dark granules came into being within the cytoplasm. The number of cells in2.5%serum containing liuweidihuang pill group were smallest, with the lowestdensity; and the number and morphology of cells in5%serum containingliuweidihuang pill group got close to that of the normal controlled group. Thecell number in10%serum containing liuweidihuang pill group increased and wereand density of which were higher than those of the normal controlled group. Thecell number and density of cells in20%serum containing liuweidihuang pill groupalmost the same as that of5%serum containing liuweidihuang pill group. Cellsin MET group closed to the normal controlled group.3. Effect of serum containing liuweidihuang pill on survival rate of EA.hy926HUVEC. The cells intervened by different concentrations of serum containing liuweidihuang pill got different degree of protection. The cell survival ratein2.5%serum containing liuweidihuang pill group was31.67%; while the cellsurvival rates in5%,10%and20%serum containing liuweidihuang pill were77.46%,130.87%and85.86%respectively, which were significantly higher than those ofthe model group (P＜0.01). Compared with those of5%group there was nosignificant difference (P＞0.05) in cell survival rate in20%serum containingliuweidihuang pill group, Therefore,5%serum containing liuweidihuang pillshould be the best concentration in treatment.4. NO content of the culture medium in the PA-induced impairment of EA.hy926cells decreased, there was significant difference (P＜0.01) compared with thenormal controlled group. NO content in2.5%serum containing liuweidihuang pillgroup further reduced; NO content in the cell culture medium significantlyincreased in5%and10%serum containing liuweidihuang pill groups and MET group,there was significant difference (P＜0.01) compared with the PA group, but therewas no significant difference (P＞0.05) within the three groups comparison witheach other.5. The expression level of intracellular IRS-2, PI3K, Akt and eNOS mRNA andprotein in model group decreased significantly (P＜0.01) compared with thenormal controlled group. The expression level of intracellular IRS-2, PI3K, Aktand eNOS mRNA and protein in5%serum containing liuweidihuang pill group andMET group increased significantly (P＜0.01) compared with the PA group, whichmeaned liuweidihuang pill could promote the expression of intracellular IRS-2,PI3K, Akt and eNOS mRNA and protein in the PA-induced impairment of EA.hy926HUVEC after intervention.6. MDA content increased and total SOD activity of the cell lysate supernatantfluid in impaired cells by PA significantly decreased. ADMA concentration incell culture medium significantly increased, there were significant difference(P＜0.01) compared with the normal controlled group. MDA content in2.5%serumcontaining liuweidihuang pill group slightly decreased, there was difference(P＜0.05) compared with the PA group; ADMA concentration decreased indistinctly, there was no difference (P＞0.05) compared with the PA group; total SOD activityalmost had no change there was no difference (P＞0.05) compared with the PA group.MDA content and total SOD activity in cell lysate supernatant fluid in5%and10%serum containing liuweidihuang pill groups and MET group significantlydecreased and increased and ADMA concentration in cell culture mediumsignificantly decreased, there were significant difference (P＜0.01) comparedwith the PA group.7. The expression level of intracellular NOX4and PRMT1mRNA and protein in modelgroup significantly increased, but those of DDAH2significantly decreased, bothwith significant difference (P＜0.01), compared with the normal controlled group.The expression level of intracellular NOX4and PRMT1mRNA and protein in5%and10%serum containing liuweidihuang pill groups and MET group significantlydecreased and those of DDAH2significantly increased, there were significantdifference (P＜0.01) compared with the PA group.Conclusions：1. PA has a very strong toxicity on EA.hy926HUVEC, and the insulin resistance(IR) cell model simulated by PA in our experiment is successful.2. Cell damage in serum containing liuweidihuang pill group significantly couldbe reduced, which indicated that it has a significantly protective effect onendothelial cells.3. Certain concentration of serum containing liuweidihuang pill is capable ofpromoting proliferation of impaired EA.hy926HUVEC by PA and increase cellsurvival rate.4. The serum containing liuweidihuang pill is capable of increasing NO contentof the cell culture medium in impaired EA.hy926cells by PA, and at the sametime promote the expression level of intracellular eNOS mRNA and protein, whichindicates liuweidihuang pill is capable of effectively promote synthesis of NOby changing the expression of eNOS mRNA and protein.5. The serum containing liuweidihuang pill could promote IRS-2, PI3K, and Akt mRNA and protein expression levels of endothelial cells, indicating that it isable to further increase the expression of eNOS mRNA and protein and the synthesisof NO through PI3K/Akt ways, and thus protect the damaged cells, by improvingand correcting the IR.6. The serum containing liuweidihuang pill is able to significantly decreaseMDA content and increase total SOD activity in impaired EA.hy926cells by PA,and at the same time it could lower the expression of NOX4mRNA and protein inthe damaged cells, which proves that liuweidihuang pill is able to reduce thedegree of oxidative damage of PA to the HUVEC, by enhancing the activity ofthe antioxidant enzyme, and improving the impaired EC state, which indicatingliuweidihuang pill plays its antioxidant role through inhibiting the expressionof NOX4.7. The serum containing liuweidihuang pill is able to decrease the expressionof PRMT1significantly and increase the expression of DDAH2significantly, andsignificantly reduced ADMA levels, which proved that liuweidihuang pill mightalso regulate the expression of PRMT1-DDAH2-ADMA in a certain extent with itsantioxidant protection, by inhibiting oxidative stress effect and oxidativestress impairment.8. The data further indicate that there is a close relationship betweenliuweidihuang pill and the increased NO level in protecting endothelial cellsthrough PI3K and Akt pathway and regulating NOX4-ROS (MDA) and PRMT1-DDAH2-ADMApathway.