Study On The Mechanism Of Action Of Total Flavonoids Of Ziziphora Clinopodioides Lam Regulating PI3K/Akt/mTOR Pathway To Improve AS

Object:To observe the effects of total flavonoids of Ziziphora clinopodioides Lam（TFZC）on autophagy and phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）/mammalian target of rapamycin（m TOR）signaling pathway in apolipoprotein E gene knockout(Apo E^-/-)mice fed with high-fat diet,and to explore the mechanism of TFZC against atherosclerosis（AS）.Methods:Sixty healthy male Apo E^-/-mice aged 6-8 weeks and weighing 18-22 g were fed with high-fat diet for 8 weeks to establish an AS model in vivo.They were randomly divided into 5 groups:model group（Model）,atorvastatin group（Atorvastatin）,low-dose group of total flavonoids of Ziziphora clinopodioides Lam（TFZC-L）,medium-dose group of total flavonoids of Ziziphora clinopodioides Lam（TFZC-M）,and high-dose group of total flavonoids of Ziziphora clinopodioides Lam（TFZC-H）.Twelve healthy C57BL/6 J mice of the same age and sex were fed with normal diet for 8 weeks to establish a blank group（Blank）.The Blank group and the Model group were given distilled water by gavage.After12 weeks of administration,the mice were dissected,and the serum,liver tissue and aorta were taken out for subsequent experiments.（1）The effect of TFZC on the general condition and aortic plaque of Apo E^-/-mice.1 Observe and record the changes of mental state,activity,body weight,diet and water intake of mice before and after administration.2 HE staining was used to observe the plaque in the aortic root lumen;3 Oil red O staining was used to observe the distribution and size of aortic root plaques.4 Masson staining was used to observe the collagen level in aortic root plaque.5 Oil red O staining The aortic wall plaque area was observed.（2）Effects of TFZC on lipid metabolism disorder and inflammatory factor levels in Apo E^-/-mice.1 The contents of TC,TG,LDL-C and HDL-C in serum of mice were detected by automatic biochemical analyzer.2 The changes of liver coefficient in each group were calculated.3 Oil red O staining and HE staining were used to observe liver fat vacuoles and lipid deposition in mice.4 Masson staining of liver tissue to observe the level of collagen in the liver of mice;5 the expression levels of TC,TG,NEFA and FC in liver homogenate were determined by kit.6 The expression levels of MMP-2,MMP-9,ICAM-1,VCAM-1,IL-18,IL-1βand MCP-1 in serum were detected by enzyme-linked immunosorbent assay（ELISA）.（3）Effects of TFZC on autophagy and PI3K/Akt/m TOR signaling pathway in Apo E^-/-mice.1 Transmission electron microscopy（TEM）was used to observe autophagosomes in mouse aortic root.2 The expression of P62 and LC3 in the aortic root of mice was detected by immunofluorescence.3The m RNA expression of PI3K,Akt,m TOR,LC3,Beclin-1 and P62 in aorta was detected by q RT-PCR.4Western-Blot was used to detect p-PI3K,p-Akt,p-m TOR,LC3 II/I,Beclin-1 and P in aortic tissue.Results:（1）The changes of activity,body weight and diet and water intake of mice before and after administration were observed and recorded.It was found that there were slight changes in mice within 1week of administration,but gradually returned to a stable state with the increase of administration time.Aortic pathological observation showed that compared with the Blank group,the plaque area in the aorta of the Model group increased significantly（P<0.01）,the collagen level in the plaque decreased significantly,and the inflammatory cell infiltration was obvious.Compared with the Model group,with the administration of TFZC in each dose group,the area of aortic plaque was significantly reduced（P<0.01）,the content of foam cells in the vascular wall and the degree of vascular stenosis were down-regulated,and the level of collagen in the plaque was up-regulated.（2）Compared with the Blank group,the levels of TC,TG and LDL-C in the serum of the Model group were significantly increased（P<0.05）,and the level of HDL-C was significantly decreased（P<0.05）.Liver staining showed that lipid deposition and collagen levels were significantly increased（P<0.01）.The levels of TC,TG,NEFA and FC in liver homogenate were significantly increased（P<0.01）.The levels of ICAM-1,VCAM-1,MMP-2,MMP-9,IL-18,IL-1βand MCP-1 in serum were significantly increased（P<0.01 or P<0.05）.Compared with the Model group,TFZC-M and TFZC-H groups showed that liver lipid deposition and collagen levels were significantly down-regulated（P<0.01）,blood lipid TC,TG,LDL-C and inflammatory factors ICAM-1,VCAM-1,MMP-2,MMP-9,IL-18,IL-1βand MCP-1 levels were significantly decreased（P<0.01 or P<0.05）,HDL-C expression levels were significantly increased（P<0.01 or P<0.05）,and TC,TG,NEFA and FC levels in liver homogenate were significantly increased（P<0.01）.（3）Compared with the Blank group,TEM observation showed that autophagosomes decreased in the Model group.The expression of LC3 in the Model group was significantly down-regulated and the expression of P62 was significantly increased（P<0.01 or P<0.05）.The results of q RT-PCR and Western-Blot showed that the protein and m RNA expression of LC3 II/I and Beclin-1 in the Model group were significantly down-regulated,and the protein and m RNA expression of p-PI3K,p-Akt,p-m TOR and P62 were significantly up-regulated（P<0.01）.Compared with the Model group,the number of autophagosomes in each dose group of TFZC increased significantly.The protein and m RNA expression levels of LC3 and LC3 II/I,Beclin-1 in the aorta and lysate were significantly increased,and the protein and m RNA expression levels of p-PI3K,p-Akt,p-m TOR and P62 in the aorta and lysate were significantly decreased（P<0.01 or P<0.05）.Conclusion:TFZC can reduce the plaque area and collagen level of Apo E^-/-mice,inhibit liver lipid deposition and collagen expression,regulate autophagy,inhibit inflammatory response,and slow AS.The mechanism may be related to the regulation of PI3K/Akt/m TOR signaling pathway.