The Study On The Mechanism Of Berberine Anti-atherosclerosis With Damp-heat Syndrome Based On Autophagy Theory

ObjectiveTo explore the effects of damp-heat syndrome on atherosclerosis and autophagy,and the molecular mechanism of berberine in preventing and treating atherosclerosis.MethodsIn this study,a model of atherosclerosis in rats with damp-heat syndrome was established to observe the changes in serum lipids,aortic histopathology and autophagy level during the formation of damp-heat syndrome,and to explore whether damp-heat syndrome could promote atherosclerosis and its possible mechanism.Then,we treated atherosclerosis model of rats with damp-heat syndrome by berberine to observe the changes of serum lipids,aortic histopathology and autophagy level to explore whether berberine can prevent and treat atherosclerosis,and to explore whether its effect is related to its regulation of autophagy level.The specific method is as f'ollows:Experiment one:Atherosclerosis in rats was induced by high-fat and high-sugar diet,and damp-heat syndrome was induced by intraperitoneal injection of vitamin D3+tail vein injection of IPS endotoxin?SPF grade SD rats were randomly divided into control group(CON),atherosclerosis group(AS),atherosclerosis damp-heat syndrome group(AS+DH),thirty rats in each group,Obtain specimens at two,four,and eight weeks,take ten pieces each time.The expressions of serum TC,TG,LDL-C and HDL-C were measured by automatic biochemical analyzer.The pathological changes of aortic tissue sections were evaluated by HE staining,oil red 0 staining and Masson staining.Immunofluorescence staining CD68 detection tissue the change of macrophages content,and to detected the expression of autophagy marker proteins LC3-? and P62 by immunohistochemistry and Western blot.Experiment two:Based on the experimental one results,a model of atherosclerosis in rats with damp heat syndrome was established.It was randomly divided into atherosclerosis group(CON),atherosclerosis+damp-heat syndrome group(AS+DH),atherosclerosis+damp-heat syndrome+berberine group(AS+DH+BBR),atherosclerosis+damp-heat syndrome+berberine+3-methyladenine group(AS+DH+BBR+3-MA),after four weeks of administration,the expressions of serum TC,TG,LDL-C and HDL-C were measured by automatic biochemical analyzer.To evaluate the pathological changes of aortic tissue sections by HE staining and Masson staining.Immunofluorescence staining CD68 detection tissue the change of macrophages content,and the expression of autophagy marker proteins LC3-? and P62 were detected by immunohistochemistry and Western blot.ResultsExperiment one:Compared with CON group,At the 8th week of modeling,the serum levels of TC,TG and LDL-C in the AS group and the AS+DH group were significantly increased(P<0.01),and the HDL-C was significantly decreased(P<0.01).The levels of TC,TG and LDL-C in AS+DH group were significantly higher than those in AS group(P<0.01),and the decrease in HDL-C was more significant(P<0.01).HE staining,oil red 0 staining and Masson staining showed that at the 8th week,the aortic tissue sections of AS group and AS+DH group gradually showed atherosclerosis typical such as intimal thickening,disordered cell arrangement,and different nuclear size.A large number of foam cells were observed,and the content of collagen fibers decreased,and this change was more obvious in AS+DH group.Immunofluorescence staining showed that the immunofluorescence signal was enhanced in AS group and AS+DH group,and the latter was more significant.Immunohistochemistry and Western blot showed that the expression of LC3-? was significantly decreased in the AS group and the AS+DH group(P<0.01),and the expression of P62 was significantly increased(P<0.01).The expression of LC3-? was more lower?P62 expression was higher in the AS+DH group than in the AS group,and there was a significant difference(P<0.01).Experiment two:After four weeks of berberine intervention,compared with AS+DH group,the serum levels of TC,TG and LDL-C were significantly decreased in AS+DH+BBR group(P<0.01),and HDL-C was significantly increased(P<0.01).The results of HE staining showed that,compared with the AS+DH group,the aortic tissue sections of the AS+DH+BBR group were arranged more neatly,the nucleus size became uniform,the intima became thinner,and the number of foam cells decreased.Masson staining showed an increase in collagen content in aortic tissue slices of rats after berberine treatment.Immunofluorescence staining showed a decrease in macrophage content in the AS+DH+BBR group.Iumunohistochemistry and Western blot showed that the expression of autophagy marker protein LC3-? was significantly increased in the AS+DH+BBR group compared with the AS+DH group(P<0.01),and the expression of P62 was significantly decreased(P<0.01).Compared with AS+DH+BBR group,the expression of LC3-? protein in AS+DH+BBR+3-MA group supplemented with autophagy inhibitor 3-methyladenine was significantly decreased and the expression of p62 was significantly increased.The difference was statistically significant(P<0.01).ConclusionOne.Damp-heat syndrome promotes the formation of atherosclerosis and aggravates plaque instability.Two.Damp-heat syndrome can lead to a decrease in the level of autophagy in atherosclerotic rats.Three.Berberine has the effect of preventing and treating atherosclerosis and stabilizing plaque,and its mechanism may be related to the increase of autophagy activity.