Study On The Effect Of Toniy Qi Of The Kidney Compound Ruanmaijian On Autophagy In Rabbit Model Of Atherosclerosis Based On PI3K/Akt/mTOR Signaling Pathway

Objective Through the establishment of atherosclerosis(AS)rabbit model,the changes of serum lipids and vascular morphology of the AS rabbit model were observed by the kidneytonifying and qi-invigorating Compound Ruanmaijian decoction,and the effects of Ruanmai decoction on autophagy of AS were observed based on the PI3K/Akt/mTOR signaling pathway,so AS to explore new targets for the treatment of AS by ruanmai decoction.Methods 1.Rabbits were divided into normal group,model group and Ruanmaijian group.The AS model was established by air drying and feeding with high-fat feed.2.Blood samples were taken from the abdominal aorta of rabbits,the serum lipid index was detected by spectrophotometer: triglyceride(TG),total cholesterol(TC),high-density lipoprotein(HDL-C),and low-density lipoprotein(LDL-C)content;3.Common carotid artery was taken for paraffin section and HE staining,and plaque morphology was observed under the microscope.4.Autophagy marker LC3 protein: Western Blot,immunofluorescence,immunohistochemical and laser confocal methods.5.Atg5,the key protein of autophagosome formation,and PI3K/Akt/mTOR signaling pathway proteins were detected by Western Blot and immunohistochemistryResults 1.Comparison of blood lipid indexes: compared with the normal group,TC,TG,and ldl-c levels were all increased in the model group and the Ruanmaijian group(P < 0.05),while hdl-c levels were decreased in the model group(P > 0.05).Compared with the model group,the levels of TC,TG,and ldl-c in the Ruanmaijian group were all decreased(P < 0.05),while the levels of hdl-c were increased(P < 0.05).2.HE staining observation: the area of common carotid artery plaque in rabbits was significantly smaller in the normal group than in the model group and the Ruanmaijian group.The plaque area of the Ruanmaijian group was significantly smaller than that of the normal group.3.Results of autophagy marker LC3II:(1)Results of Western Blot.At the tissue of the plaque,compared with the normal group,the LC3II/LC3 I ratio in the model group increased,but there was no significant change(P>0.05),and compared with the model group,the LC3II/LC3 I ratio in the Ruanmaijian group decreased,and the difference was statistically significant(P<0.05).At the lesion tissue beside the plaque,compared with the normal group,the LC3II/LC3 I ratio in the model group decreased,but there was no significant change(P>0.05),and compared with the model group,the LC3II/LC3 I ratio in the Ruanmaijian group increased significantly(P<0.01).(2)Results of immunohistochemical detection.At the tissue of the plaque,compared with the normal group,the expression of LC3 II protein in the model group increased,but there was no significant change(P>0.05),and compared with the model group,the expression of LC3 II protein in the Ruanmaijian group decreased significantly(P<0.001).At the lesion tissue beside the plaque,LC3 II protein expression was significantly decreased in the model group compared with the normal group(P<0.001),and significantly increased in the Ruanmaijian group compared with the model group(P<0.01).(3)Results of immunofluorescence test.At the tissue of the plaque,compared with the normal group,LC3 II protein expression increased in the model group,and compared with the model group,LC3 II protein expression decreased in the Ruanmaijian group.At the lesion tissue beside the plaque,compared with the normal group,LC3 II protein expression decreased in the model group,and compared with the model group,LC3 II protein expression increased in the Ruanmaijian group.(4)Results of laser confocal detection.At the tissue of the plaque,compared with the normal group,LC3 II expression increased in the model group,and compared with the model group,LC3 II expression decreased in the Ruanmaijian group.At the lesion tissue beside the plaque,compared with the normal group,LC3 II expression decreased in the model group,and compared with the model group,LC3 II expression increased in the Ruanmaijian group.4.Detection results of Atg5,a key protein in autophagosome formation,were as follows:(1)Western Blot detection.At the tissue of the plaque,compared with the normal group,the expression of Atg5 protein was significantly increased in the model group(P<0.05),and compared with the model group,the expression of Atg5 protein in the Ruanmaijian group decreased significantly(P<0.05).At the lesion tissue beside the plaque,the expression of Atg5 protein was significantly decreased in the model group compared with the normal group(P<0.001),and compared with the model group,the expression of Atg5 protein in the Ruanmaijian group was significantly increased(P<0.05).(2)immunohistochemical detection.At the tissue of the plaque,compared with the normal group,the expression of Atg5 protein in the model group increased,but there was no significant change(P>0.05),and compared with the model group,the expression of Atg5 protein in the Ruanmaijian group was significantly decreased(P<0.01).At the lesion tissue beside the plaque,the expression of Atg5 protein in the model group decreased compared with the normal group,but there was no significant difference(P>0.05),and compared with the model group,the Atg5 protein expression was significantly increased in the Ruanmaijian group(P<0.01).5.Detection results of PI3K/Akt/mTOR signaling pathway:(1)Western Blot detection.At the tissue of the plaque,compared with the normal group,the expression of PI3 K and p-Akt decreased in the model group,but there was no significant change(P>0.05),and the expression of Akt,mTOR and p-mTOR decreased in the model group,and the difference was statistically significant(P<0.05),and compared with the model group,the expressions of PI3 K and mTOR were significantly increased(P<0.001),and Akt and p-Akt were significantly increased(P<0.01),and p-mTOR war significantly increased in the Ruanmaijian group(P<0.05).At the lesion tissue beside the plaque,compared with the model group,the expressions of PI3 K,Akt,p-Akt,mTOR and p-mTOR decreased in the Ruanmaijian group,and the differences were statistically significant(p<0.05).(2)immunohistochemical detection.At the tissue of the plaque,compared with the normal group,the expression of PI3 K,p-Akt,mTOR in the plaque tissue of the model group decreased,and the difference was statistically significant(p < 0.05),and the expression of Akt,p-mTOR decreased,but there was no significant change(p>0.05),and compared with the model group,the expressions of PI3 K,Akt,p-Akt,mTOR and p-mTOR in the Ruanmaijian group increased,and the differences were statistically significant(p<0.05).At the lesion tissue beside the plaque,compared with the normal group,the expression of PI3 K,p-Akt and mTOR in the model group increased,and the difference was statistically significant(p < 0.05).The expression of Akt and pmTOR increased,but there was no significant change(p>0.05),and compared with the model group,the expressions of PI3 K,p-Akt,mTOR and p-mTOR in the Ruanmaijian group decreased,and the differences were statistically significant(p<0.05),and the expression of Akt decreased,but there was no significant change(p>0.05).Conclusions 1.Ruanmaijian can reduce the area of carotid artery plaque in AS rabbit model,reduce the levels of TC,TG and LDL-C in blood lipids,and increase the levels of HDL-C.2.In the plaque organization,Ruanmaijian can cut autophagy related gene Atg5,LC3 ? proteins,increase Akt,p-Akt,PI3 K,mTOR,p-mTOR protein level,promote the PI3K/Akt/mTOR signaling pathways,inhibit excessive autophagy.3.Beside the plaques in the diseased tissue,Ruanmaijian can increase autophagy related gene Atg5,LC3 ? proteins,by Akt,p-Akt,PI3 K,mTOR,p-mTOR protein levels,inhibition of PI3K/Akt/mTOR signaling pathways,to promote a certain level of cell autophagy,effectively prevent and control of the AS.4.Toniy Qi of the kidney Chinese medicine compound is effective in the treatment of AS and regulates different degrees of autophagy.In the early stage of AS plaque formation,AS was prevented and treated by promoting autophagy.In the late stage of AS plaque formation,AS plaque was stabilized by inhibiting excessive autophagy.The PI3K/Akt/mTOR signaling pathway may be an effective mechanism and target for the prevention and treatment of AS by Ruanmaijian.