| Background and ObjectiveCoronary artery disease and stroke are the two greatest lethal factors in the world,and their "root cause" is atherosclerosis(AS)lesions.When the lipid metabolism disorder,lipid plaque blocking the arterial wall,the blood flow difficulties lead to coronary heart disease or stroke attack.AS etiology is complex,it involves the theory of inflammation and lipid metabolic disorder,which are still the focus at this stage.Further study of these two theories is helpful to fundamentally improve the pathological basis of AS formation and prevent the occurrence of acute cardiovascular and cerebrovascular events.In this study,we used the combination of animal experiments and cell experiments with Lipid metabolism disorder as the starting point to observe the two lipid metabolism pathways:PPARy-LXR-ABCA1 and autophagy-lysosomal pathway,and explore the Rho/Rock signal pathway with their relevance.Through the experiment,we observed the therapeutic effect of TiaoZhiTongMai Granules on lipid plaque,and from the perspective of lipid metabolism disorder to explain the pharmacological mechanism of AS.We hope to provide a new idea for the prevention and treatment of AS,and also provide experimental basis for the late promotion of the compound.MethodsExperiment 1:The rabbit model of AS was established by high fat diet plus balloon injury of common carotid artery.The rabbits were divided into 5 groups:norrmal group,sham operation group,model group,TiaoZhiTongMai group and Atorvastatin group.Two treatment groups were given intragastric administration.The remaining three groups were fed with the same volume of distilled water.6 weeks and 12 weeks were selected as observation points.At that two time points we do evaluation:ultrasound evaluation of plaque formation;blood lipid;H&E staining was used to observe the carotid artery and liver pathology;enzyme linked immunosorbent assay(ELISA)detection of serum endothelial cell markers of endothelial nitric oxide synthase(eNOS),endothelin 1(ET-1),prostacyclin(PGI2)and thromboxane(TXA2)level.RT-PCR method for detection of cholesterol transporter related proteins:the mRNA expression of neutral cholesteryl ester hydrolase(nCEH)and Adipophilin.Through the mentioned above indicators to evaluate the lipid regulating effects of TiaoZhiTongmai Granules on AS.Experiment 2:Using ox-LDL-induced RAW264.7 macrophage foam cell model.The experiment was divided into 5 groups:normal group(Con),model group(Mod),model+ROCK inhibitor group(Mod+Y-27632),TiaoZhiTongMai granules containing-serum group(TZTM)and atorvastatin containing-serum group(Atorva).CCK8 method was used to determine the optimum dosage,administration time,and detact survival rate of each groups.Oil red O staining was used to identify the formation of foam cells;AnnexinV-FITC double staining method were used to detect apoptosis cells;Spectrophotometric Determination of cholesterol(TC),free cholesterol(FC)content,calculation of cholesterol ester(CE)/total cholesterol(TC)ratio;WesternBlot method to detect the PPARy,LXR and ABCA1 protein expression.Observe the effects of lipid-lowering Tongmai Granules on reverse cholesterol transport pathway.Experiment 3:Using ox-LDL-induced RAW264.7 macrophage foam cell model.The experiment was divided into 6 groups:normal group(Con),model group(Mod),model+inducer of autophagy group(Mod+Rapa),model+inducer of autophagy+Y-27632 group(Mod+Rapa+Y-27632),TiaoZhiTongMai granules containing-serum group(TZTM+Rapa)and atorvastatin containing-serum group(Atorva+Rapa).Immunofluorescence was used to detect the expression level of autophagosome formation;WesternBlot was used to detect the protein expression of Beclinl,p62,LC3?,Rab7;the protein binding of Beclinl and Bcl-2 were measured by Co-immunoprecipitation.This experiment was not only focus on the relationship between Rho/Rock signaling pathway and autophagy,but also on the molecular mechanism of TiaoZhiTongMai granules regulating autophagy.Results Experiment 1(1)Compared with the model group,the TC level of the traditional Chinese medicine group decreased(P<0.05).The calculation of atherosclerosis index(AI)showed that Tiaozhi Tongmai granules showed the best reduction of hyperlipidemia at 12 weeks(P<0.05).(2)At 6 weeks,H&E staining showed that the common carotid artery plaque in the traditional Chinese medicine group and the western medicine group got a certain degree of control,the disease was mild,prompting us the treatment was effective.At 12 weeks,carotid plaques in the model group were almost covered with the entire lumen,forming a typical lipid plaque.Compared with the model group,only a small amount of lipid was found in the traditional Chinese medicine group and the western medicine group,the lesion was light.The results showed that,compared with the model group,the liver cells of the traditional Chinese medicine group had a certain degree of damage,but there was no evidence of liver inflammation and fibrosis.(3)Serum ELISA results:At the two time points,compared with the sham operation group,the serum ET-1 and TXA2 levels were significantly increased in the model group,and the levels of eNOS and PGI2 were significantly decreased(P<0.01).Compared with the model group,the levels of ET-1 and TXA2 were decreased,eNOS and PGI2 levels were significantly increased in the control group and atorvastatin group(P<0.01).(4)RT-PCR results:At the two time points,compared with the sham group,the nCEH content in the model group decreased by 20.81%and 40.35%(P<0.05).Compared with the model group,the content of nCEH in Chinese medicine group increased by 2.08%and 5.69%(P>0.05).The content of nCEH in atorvastatin group increased by 20.61%and 29.48%(P<0.05).Compared with the sham group,the expression of Adipophilin in the model group was increased by 26.94%and 37.15%(P<0.05).Compared with the model group,the content of Adipophilin decreased by 6.93%and 10.28%(P>0.05),the atorvastatin group reduced the expression of Adipophilin(P<0.05).Experiment 2(1)CCK8 results:Compared with the control hole,the OD value of the model group was significantly decreased(P<0.01).The cell survival rate is the highest(70%-80%)when TiaoZhiTongMai granules containing-serum concentration is 5%.5%concentration of TiaoZhiTongMai granules containing-serum,the survival rate of the cells was the most significant when the administration time was 24h.So the final concentration was 5%and the incubation time was 24h.TiaoZhiTongMai granules had no effect on ox-LDL-stimulated macrophages,suggesting that TiaoZhiTongMai granules do not have cytotoxic effects.After 24h,compared with the model group,the survival rate of the model+inhibitor group,TiaoZhiTongMai granules group and atorvastatin group were increased(P<0.05).(2)Oil red O staining results:80p,g/ml ox-LDL-induced macrophages visible lipid droplets can be seen everywhere,more than 50%of the cells were stained with red blood cells,which are consistent with the characteristics of macrophage foam.The model+Rock inhibitor group was seen across the dispersed lipid droplets.Compared with the model group,the lipid droplets of TiaoZhiTongMai granules group and atorvastatin group were few.The lipid droplets in the inner layer of the cell membrane were lighter in red color and dispersed in the small lipid droplets.(3)Annexin V-FITC double labeling method results:Compared with the model group,TiaoZhiTongMai granules group and model+inhibitor group apoptotic cells decreased significantly(13.2±1.68%and 18.2±0.73%)(P<0.05),atorvastatin group the apoptosis rate also reduced(11.9±0.73%)(P<0.05).(4)TC,FC,CE/TC ratio:In TiaoZhiTongMai Granules group,the TC content was decreased,FC content increased,CE/TC ratio was 38.19%(P<0.05).The ratio of TC,FC and CE/TC in the atorvastatin group was significantly different from that in the model group(P<0.01).(5)WesternBlot results:In the control group,the expression of Rho and Rock protein was few.After ox-LDL induction,the levels of Rho and Rock protein increased significantly(P<0.05).Compared with the model group,the TiaoZhiTongMai granules group and atorvastatin group can improve the lipid metabolism disorder by increasing the expression of PPARy-LXR-ABCA1(P<0.05).Experiment 3(1)Immunofluorescence results:Compared with the model group,the cell gap of the foam cell + rapamycin group was increased,and the fluorescence intensity increased.The fluorescence intensity of the foam cell+rapamycin+Y-27632 was enhanced and the green granule was increased.Compared with the model control group,the green fluorescence intensity of TiaoZhiTongMai granules group and atorvastatin group was significantly enhanced.(2)WesternBlot results:Compared with model group,the expression of Beclinl protein in foam cells+rapamycin group and foam cell+rapamycin+Y-27632 group increased by 20.4%and 29.1%(P<0.05).The expression of LC3? protein increased by 30.2%and 43.9%(P<0.05).The expression of p62 protein was decreased by 31.7%and 47.3%(P<0.05).Rab7 levels were increased by 10.0%and 15.7%(P<0.05).Compared with the foam cell+rapamycin group,the content of Beclinl protein in the TiaoZhiTongMai granules group and atorvastatin group increased by 19.8%and 23.4%(P<0.05),and the expression of LC3?protein increased by 29.8%and 35.5%(P<0.05);p62 content decreased by 41.9%and 36.8%(P<0.05);Rab7 protein level increased by 34.9%(P>0.05)and 16,4%(F<0.05).(3)Co-Immunoprecipitation results:Compared with the normal group,the protein binding degree of beclinl and bcl-2 in the model group was significantly increased(P<0.05).Compared with the model group,the protein binding degree of foam cells+rapamycin group,foam cell+rapamycin+Y-27632 group was significantly decreased(P<0.05).Compared with the foam cell+rapamycin group,the degree of protein binding was also decreased(P>0.05).The degree of protein binding of the beclinl and bcl-2 was decreased in atorvastatin group(P<0.05).Conclusions(1)TiaoZhiTongMai granules could regulating endothelial cell marker ET-1,eNOS,PGI2,TXA2 levels,Repair of atherosclerotic rabbit models of endothelial cell injury,regulate the cholesterol transport pathway associated with the protein,and improve the AS plaque.(2)Y-27632 can inhibit Rho/Rock signaling pathway and reduce the protein binding of Bcl-2 and Beclinl at the beginning of autophagy,increase the activity of intracellular autophagy,then reduce the formation of foam cells.(3)TiaoZhiTongMai granules can be used to reduce the formation of foam cells and promote cholesterol efflux in foam cells,which may be achieved by regulating the PPAR?-LXR-ABCA1 signaling pathway and increase the expression of Beclinl,LC3? and Rab7 protein in the autophagy-lysosome system,and decrease the expression of p62. |
PDF Full Text Request