| Objective: To study the anti-atherosclerosis effect and mechanism of Mongolian medicine Zhurihen Dropping Pill.Methods: 1.Study on the anti-atherosclerosis effect of Mongolian medicine Zhurihen Dropping Pills.Sixty Japanese big-eared white rabbits were fed for one week.They were randomly divided into six groups: solvent control group(Control),model group(Model),high dose group(HZRH,1160 mg/kg),medium dose group(MZRH,367 mg/kg),low dose group(LZRH,116 mg/kg)and probucol group(26mg/kg).High fat feed combined with bovine serum albumin injection(250mg/kg)was used to intervene Japanese big ear white rabbits and establish AS animal model.On the basis of feeding in the model group,Zhurihen dropping pills group was given every dose of Zhurihen dropping pills once a day for 4 weeks,while the probucol group was given probucol solution once a day for 4 weeks,and was executed at the 12 th week.The following indicators were detected:(1)The changes of feed intake and body mass in rabbits were measured at the first,second and seventh and eighth weeks of the modeling period;(2)The contents of total cholesterol(CHOL),low density lipoprotein(LDL),triglyceride(TG)and high density lipoprotein(HDL)in serum were measured at the 0th,4th,8th and 12 th weeks from the beginning of the modeling to the administration of drugs;(3)The aortic plaque Coverage rate was observed by oil red O staining;(4)The proliferation of vascular smooth muscle and pathological changes of intima were observed by hematoxylin-eosin(HE)staining.2 Study on antioxidant stress of Mongolian medicine Zhurihen Dropping Pills.(1)The activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)and oxidized low density lipoprotein(ox-LDL)in the serum of each group at the 12 th week of "1" were determined by Elisa method;(2)The atherosclerotic vascular endothelial injury model was established by co-incubation of oxidized low density lipoprotein(ox-LDL)with human umbilical vein endothelial cells(HUVEC),and Zhurihen Dropping Pills containing serum was given at the same time.The experiment was divided into seven groups: control group,model group,VSC group,HZRHS group,MZRHS group,LZRHS group and Vit C control group.(1)MTS method was used to determine the cell survival rate and the concentration and time of ox-LDL injured HUVEC;(2)MTS method was used to determine the cell survival rate,and the dosage and time of each dose group when ZRH containing serum exerted protective effect on HUVEC;(3)MTS method was used to determine the cell survival rate,and the protective effect of each dose of ZRH containing serum on HUVEC injured by ox-LDL was investigated;(4)Flow cytometry was used to determine of ZRH containing serum on apoptosis of HUVEC by ox-LDL injured.(5)The effect of ZRH containing serum on cell cycle of HUVEC injured by ox-LDL was analyzed by flow cytometry.(6)The expression of HO-1 and the activity of SOD in HUVEC injured by ox-LDL were detected by Elisa kit.3.Study on the relationship between antioxidant stress and Nrf2 signaling pathway of Mongolian medicine Zhurihen Dropping Pills.To detect the expression of Nrf2 protein in each group of HUVEC by "2-(2)" as follows:(1)Quantitative real-time fluorescence PCR(q PCR)was used to detect the level of m RNA of Nrf2 gene in each group of HUVEC;(2)Western blot was used to detect the expression of Nrf2 protein in each group of HUVEC;(3)Western blot was used to detect the expression of Nrf2 protein in cytoplasm and nucleus of each group of HUVEC;(4)The nuclear translocation of Nrf2 in HUVEC was detected by immunofluorescence assay.(5)The interference RNA(si RNA)of Nrf2 was added.The proliferation rate of HUVEC was detected by MTS method.The correlation between antioxidant stress and Nrf2 signaling pathway of Zhurihen Dropping Pills was further explored.Results:1.Study on the anti-atherosclerosis effect of Mongolian medicine Zhurihen Dropping Pills.(1)Changes in feed intake and body mass during the modeling period: Compared with the Control group,the feed intake of Model group,ZRH dose group and Probucol group decreased significantly(P < 0.05),but there was no significant difference in body weight(P > 0.05).(2)The serum levels of TG,CHOL,LDL and HDL were measured by automatic biochemical analyzer at 0,4,8 and 12 weeks.Compared with the control group,the levels of TG,CHOL and LDL in model group were significantly increased at 8 weeks(P < 0.01),indicating that the atherosclerosis model of rabbits was successfully established.At the end of the 12 th week,the serum levels of TG,LDL and CHOL in ZRH groups were lower than those in Model group,and HDL levels were higher than those in Model group,but there was no significant difference(P > 0.05).(3)Observing aortic plaque coverage by oil red O staining: Compared with model group,the aortic plaque coverage of ZRH groups decreased significantly(P < 0.01).(4)The pathological changes of vascular intima were observed by HE staining.Compared with group Model,the cells in HZRH group were arranged orderly,the proliferation of smooth muscle cells was not obvious,the intimal layer foam cells were less,and the lesions were lighter.The corresponding lesions in MZRH and LZRH groups were alleviated.2.Study on antioxidant stress of Mongolian medicine Zhurihen Dropping Pills.(1)The content of MDA,ox-LDL and SOD activity in serum of AS model rabbits were detected by Elisa method.Compared with model group,the content of MDA in serum of ZRH groups decreased significantly(HZRH: P < 0.01,MZRH: P < 0.01,LZRH: P < 0.05),and the content of ox-LDL decreased significantly(HZRH: P < 0.01,MZRH: P < 0.01,LZRH: P < 0.05).The activity of SOD increased significantly(P < 0.01).(2)(1)The activity of HUVEC was determined by MTS method,and the best damage condition was that ox-LDL of 200 ug/m L acted on HUVEC for 12 hours.(2)The activity of HUVEC was determined by MTS method.Finally,20% of each dose of ZRHS for 24 hours was selected as the best condition to investigate the protective effect of ZRHS on HUVEC.(3)The activity of HUVEC was measured by MTS.Compared with model group,the cell survival rate of ZRH serum group was significantly increased(P < 0.01),and there was a dose-dependent relationship among the groups.(4)Flow cytometry was used to detect the effect of ZRH-containing serum on apoptosis of injured HUVEC.Compared with model group,ZRH-containing serum at different doses could significantly reduce the apoptotic rate(HZRHS: P< 0.01,MZRHS: P < 0.05,LZRHS: P < 0.05),and there was a dose-dependent relationship between each group.(5)Flow cytometry was used to detect the effect of ZRH-containing serum on cell cycle of damaged HUVEC.Compared with model group,HZRHS group could significantly increase the ratio of S+G2/M cells and promote cell proliferation(P < 0.01),while MZRHS and LZRHS did not change significantly(P > 0.05).(6)The content of HO-1 and SOD activity in HUVEC of each group were detected by Elisa method.Compared with model group,the content of HO-1 and SOD activity in cells were significantly increased in ZRH serum group at different doses(P < 0.01),and there was a dose-dependent relationship among the groups.3.Study on the relationship between antioxidant stress and Nrf2 signaling pathway of Mongolian medicine Zhurihen Dropping Pills.(1)The level of Nrf2's m RNA in HUVEC was detected by real-time fluorescence quantitative PCR(q PCR).Compared with model group,the level of Nrf2's m RNA in serum containing ZRH increased significantly(P < 0.01)in a dose-dependent manner.(2)Western blot was used to detect the expression of Nrf2 protein in HUVEC of each group.Compared with model group,the content of Nrf2 protein in ZRH serum group increased significantly(P < 0.01),and showed dose-dependent manner.(3)Western blot was used to detect the expression of Nrf2 protein in cytoplasm and nucleus of HUVEC.Compared with model group,the content of Nrf2 protein in cytoplasm of serum containing ZRH increased significantly(HZRHS: P < 0.01,MZRHS: P < 0.01,LZRHS: P < 0.05),in a dose-dependent manner;the content of Nrf2 protein in nucleus of HUVEC cells increased significantly(HZRHS: P < 0.01,LZRHS: P < 0.05).The difference was statistically significant(P < 0.01),which was dose-dependent.(4)The nuclear transfer of Nrf2 in HUVEC of each group was detected by immunofluorescence assay.Compared with model group,the amount of Nrf2 in cell nucleus of ZRH serum group increased(P < 0.05)in a dose-dependent manner.(5)The protective effect of ZRH serum on HUVEC was detected by MTS method after adding interfering RNA(si RNA)of Nrf2.Compared with model group,si RNA + HZRHS,si RNA + MZRHS,si RNA + LZRHS did not significantly improve the cell survival rate(P > 0.05),indicating that ZRH dropping pills mainly play a protective role on HUVEC by regulating the antioxidant signal pathway of Nrf2.Conclusion:The Mongolian medicine Zhurihen Dropping Pills may play an anti-oxidative stress role mainly based on Nrf2 signaling pathway,thus showing an anti-atherosclerosis effect. |
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