Notoginsenoside R1 Mitigates Atherosclerosis By Activating SIRT1/AMPK Mediated VSMC Autophagy In Mouse Atherosclerosis Model

Objective:Atherosclerosis is a chronic atheromatous disease caused by a combination of lipid deposition,intimal damage and inflammatory response,and is the most common factor leading to cardiovascular events and even death,posing a serious threat to human life and health.Notoginsenoside R1（NGR1）has been shown to have a protective effect against atherosclerosis,and autophagy of vascular smooth muscle cells（VSMC）was found to be involved in regulating the development of atherosclerosis.Silent Information Regulator1（SIRT1）is an NAD⁺dependent histone deacetylase,and its high expression can protect against oxidative stress and inflammatory response in cardiovascular disease,however,whether SIRT1 can regulate VSMC autophagy to participate in NGR1 protection against atherosclerosis and its mechanism are not clear.In this study,we intend to establish an atherosclerosis model by high-fat feeding Apo E^-/-male mice,and use immunoblotting,transmission electron microscopy and gene-specific silencing techniques to elucidate the mechanism of NGR1 promoting VSMC cell autophagy to prevent and treat atherosclerosis from the perspective of cellular autophagy,and take SIRT1 as an entry point to understand the mechanism of NGR1 promoting VSMC autophagy in depth.This will provide an experimental basis for NGR1to prevent atherosclerosis by regulating VSMC autophagy and to promote precision medicine for the prevention and treatment of atherosclerosis in Chinese medicine.Methods: Chapter 1: theoretical part.Review the literature to review and analyze the etiology causing VSMC autophagy and atherosclerosis,the role of VSMC autophagy in atherosclerosis,the preventive and curative effects of Panax ginseng saponin R1 on cardiovascular diseases,and the research progress of SIRT1 in atherosclerosis.Taking SIRT1 as the entry point,we will deeply understand the mechanism of NGR1 promoting VSMC autophagy,and provide the basis for NGR1 to prevent and treat atherosclerosis by promoting VSMC autophagy and promoting Chinese medicine to accurately prevent and treat atherosclerosis.Chapter 2: animal experiments.Firstly,Apo E-/-mice were fed with highfat feeding method for 8 weeks to construct atherosclerosis model,and 20 modeled Apo E-/-mice were randomly and equally divided into model group（Model group）and NGR1 group.To verify whether the model was successfully constructed,10 C57BL/6 mice（before knockdown of Apo E gene）were used as normal control group（Control group）.25 mg/kg gavage was administered daily to the NGR1 group,and an equal volume of saline was administered daily to the Model and Control groups.After 4 weeks of treatment,total cholesterol（TC）,triglycerides（TG）,high-density lipoprotein-cholesterol（HDL-C）,lowdensity lipoprotein-cholesterol（LDL-C）,and triglycerides（TG）were measured spectrophotometrically in mice.The levels of low-density lipoprotein-cholesterol（LDL-C）were measured in mice by ELISA.protein-1（MCP-1）and tumor necrosis factor-α（TNF-α）levels in mice;oil red O staining and HE staining were used to observe plaque lipid area and histomorphological changes to clarify the anti-atherosclerotic effect of NGR1.Chapter 3: cellular experiments.The ox-LDL method was used to induce VSMC foaming and simulate the cell morphology in vivo;the CCK8 method was used to detect the effect of NGR1 on the proliferation ability of VSMC and determine the IC50 concentration of NGR1;Control group（without ox-LDL）,Model group,NGR1 group,NGR1+SIRT1 group and NGR1+si-SIRT1 group were set.The proliferation ability of VSMC in each group was detected by CCK8 method;the migration ability of VSMC in each group was detected by scratch assay;the cellular autophagic vesicles in each group were observed by immunofluorescence staining;the cellular lipid TC content was detected by spectrophotometric method;the levels of cellular inflammatory factors（IL-6/MCP-1/TNF-α）were detected by ELISA;the levels of target proteins in each group were detected by Western blot to detect the expression levels of target proteins in each group;Real time-PCR to detect the m RNA levels of target molecules in each group.The effect of NGR1 on VSMC proliferation,migration and autophagy was observed,and it was clear that NGR1 inhibited VSMC migration and proliferation through SIRT1/AMPK regulation of VSMC autophagy.Chapter 4: animal experiments.High-fat fed Apo E-/-mice were used to establish an atherosclerosis model.Forty modeled Apo E-/-mice were randomly and equally divided into model group（Model group）,NGR1 group,NGR1+ Sirt1 group,and NGR1+ si-Sirt1 group.To verify whether the model was successfully constructed,another 10 C57BL/6 mice（before knockdown of Apo E gene）were used as normal control group（Control group）.The mice were treated according to the conditions of each experimental group,and oil red O staining and HE staining were used to observe the changes of plaque lipid area and histomorphology in each group;the blood lipids（TC/TG/HDL-C/LDL-C）in each group were detected by spectrophotometry;the levels of inflammatory factors（IL-6/TNF-α/ MCP-1）were measured by ELISA;transmission electron microscopy was used to observe the aggregation of lipid droplets in each group and The submicroscopic structure of autophagic vesicles in each group was observed by transmission electron microscopy;the expression level of target proteins in each group was detected by Western blot;the m RNA level of target molecules in each group was detected by Real time-PCR,and it was clear that NGR1 regulates VSMC autophagy through SIRT1/AMPK to improve atherosclerosis.Results: 1.Atherosclerosis model mice were successfully constructed.In the model（Model）group of mice,the lipid（TC/TG/LDL-C）content was increased,HDLC content was decreased,the expression of inflammatory factors（IL-6/MCP-1/TNF-α）was increased,while the intra-aortic plaque lipid area was increased,the aortic wall structure was disturbed,the intima was thickened,fibrous cap and lipid necrosis were formed,convex into the lumen,and a large amount of lipids were collected in the lumen.This indicates that the atherosclerosis model mice were successfully constructed.2.NGR1 has anti-atherosclerotic effect.Compared with the Model group,the NGR1 group significantly reduced the levels of blood lipids（TC/TG/LDLC）and increased the content of HDL-C,reduced the expression of inflammatory factors IL-6/MCP-1/TNF-α,reduced the proportion of intraaortic plaque lipid area,and reduced intimal thickening and luminal lipid aggregation,indicating the anti-atherosclerotic effect of NGR1.3.In cellular assays,NGR1 was found to inhibit VSMC migration and proliferation,decreased MMP2 and MMP9 gene and protein expression,downregulate TC and IL-6/MCP-1/TNF-α content,increased LC3 II/I protein expression,attenuated P62 protein expression,enhanced VSMC autophagic vesicle fluorescence intensity and number,up-regulated SIRT1 protein expression,and up-regulated AMPK phosphorylated protein expression.Overexpression of SIRT1 enhanced NGR1 to promote VSMC autophagy,increased LC3 II/I protein expression,increased AMPK phosphorylation protein expression,attenuated P62 protein expression,inhibited VSMC migration and proliferation,and downregulated TC and IL-6/MCP-1/TNF-α content.The opposite results were observed after silencing SIRT1.It indicates that NGR1 enhances VSMC autophagy through SIRT1/AMPK to inhibit VSMC migration and proliferation,while NGR1 enhances VSMC autophagy through SIRT1/AMPK to down-regulate VSMC lipid and inflammatory factor secretion.4.NGR1 decreased VSMC signature and proliferative gene and protein α-SMA and PCNA expression,increased VSMC-associated autophagy protein LC3 II/I protein expression,attenuated P62 protein expression,and enhanced VSMC autophagy in mice.NGR1 inhibited VSMC proliferation by enhancing VSMC autophagy,and controlled the development of plaque area in mice.5.NGR1 upregulated SIRT1 and AMPK phosphorylated protein,and overexpression of SIRT1 promoted NGR1 to reduce blood lipid level and plaque lipid area,reduce inflammatory response,decrease α-SMA and PCNA gene and protein expression,increase LC3 II/I and AMPK phosphorylated protein expression,attenuate P62 gene and protein expression,and promote VSMC autophagy in mice,but This effect was significantly attenuated by silencing SIRT1.The mechanism may be that SIRT1 mediates AMPK phosphorylation to enhance VSMC autophagy,remove lipids and damaged components,resist inflammatory stress,reduce lipid levels in mice,inhibit VSMC proliferation and migration,thus controlling the development of plaques in mice and improving atherosclerotic lesions.Conclusion: NGR1 can effectively reduce the blood lipid level in atherosclerotic mice,reduce the inflammatory response in vivo,inhibit VSMC proliferation and migration in aortic plaques,and thus inhibit the development of aortic lipid plaques.The mechanism may be through the upregulation of SIRT1-mediated AMPK phosphorylation to enhance VSMC autophagy,remove lipids and damaged components,resist inflammatory stress response,reduce the blood lipid level in mice The mechanism of atherosclerotic lesion improvement may be through the upregulation of SIRT1-mediated AMPK phosphorylation to enhance VSMC autophagy,remove lipid and damaged components,resist inflammatory stress,reduce blood lipid levels in mice,and inhibit VSMC proliferation and migration to control plaque development in mice.This will provide a new therapeutic strategy and theoretical basis for the clinical treatment of atherosclerosis-related diseases.