| Research significance:Metabolic syndrome(MS)is a public health problem that has attracted worldwide attention and a prelude to cardiovascular and cerebrovascular diseases.Vascular endothelial injury is the initial link.Therefore,it is very important to find a method to alleviate or even reverse the vascular endothelial injury induced by MS.Recent studies have shown that the endothelial injury that induced by MS is closely related to autophagy,which can induce vascular endothelial injury.Our previous experimental results showed that Chaiqi Decoction could effectively alleviate the vascular endothelial injury in MS.Our further analysis showed that the improvement effects related with the NF-kB signaling pathway,which correlated with mTOR autophagy pathway.In order to verify whether Chaiqi Decoction can improve the vascular endothelial injury induced by MS via up-regulating cell autophagy,this study investigated the effect of Chaiqi Decoction on the expression of Beclin-1,LC3,p62 and other related molecules when it improved the vascular endothelial injury induced by MS.Our results revealed the mechanism of Caiqi Decoction in the prevention and treatment of metabolic syndrome and its related vascular complications,provided a theoretical basis for the clinical application of Chaiqi Decoction and enriched the theoryof TCM prevention and treatment of MSResearch purpose:To clarify the mechanism of Chaiqi Decoction in improving vascular endothelial injury induced by MS via up-regulating cell autophagy.Research methods:(1)SD male rats were fed a—diet high in salt,fat and sugar to produce an animal Model of vascular damage induced by MS.Physiological methods combined with radioimmunoassay were used to detect the indicators of MS.HE staining,Masson staining and oil red O staining combined with Elisa,PCR and WB were used to detected the contents of NO and ET-1 in serum and tissues to evaluate the vascular endothelial injury.(2)SD male rats were fed a diet high in salt,fat and sugar to create an animal Model of vascular injury in MS.The contents of LC3B,p62,Beclin-1,mTOR,p-mTOR,AMPK and p-AMPK in the tissues were determined by WB.The morphology of autophagosomes and autolysosomes were observed by transmission electron microscope(TEM).The expression of LC3B was observed by laser confocal microscope.(3)HUVECs were cultured with oligosaccharide and palmitate to produce a vascular injury cell Model of MS,and CCK8 was used to detect cell activity.The contents of NO and ET-1 in cellular supernatant and cells were determined by Elisa,PCR and WB.(4)HUVECs were cultured with oligosaccharide and palmitate to produce a vascular injury cell Model of MS.LC3B,p62,Beclin-1,mTOR,p-mTOR,AMPK and p-AMPK in cells were detected by WB.The morphology of autophagosomes and autolysosomes were observed by TEM.GFP-mRFP-LC3 adenovirus transfection and DALGreen-DAPRed double staining were used to observe the expression of autophagosomes and autolysosomes.Research results:(1)According to the results of physiological indicators,serum biochemistry,tissue sections,Elisa,WB and PCR,the body weight,Lee’s index,body fat rate,SBP,blood glucose and lipid levels of the rats in the Model group were significantly higher than those in the Control group after 8 weeks of high salt,fat and sugar feeding,and the vascular endothelial injury was severer.After 8 weeks of gavage,Chaiqi Decoction could significantly reverse the abnormal indicators of MS,significantly reduce the intima thickness,collagen fiber,lipid drop area and the serum et-1 content.It could also increase the content of NO,the phosphorylation of eNOS and the expression of eNOS mRNA,and reduce the expression of ET-1 protein and mRNA in tissues.In summary,our study suggested that the animal Models of vascular injury with MS could be created by 8 weeks of high salt,fat and sugar feeding,and Chaiqi Decoction could improve the vascular injury in these Model rats with MS.(2)TEM,immunofluorescence,and WB results showed that the autophagosomes,autolysosomes,autophagy related protein LC3 and the autophagy upstream protein Beclin-1 expression of Chaiqi Decoction group in tissue were significantly higher than those of Control group and Model group.p62,an autophagy substrate protein,negatively correlated with autophagy.We found its expression in tissue was significantly lower than that in the Model group,which was similar to Rapamycin,an autophagy activator.In the exploration of upstream signaling factors of autophagy,Chaiqi Decoction could significantly reduce the phosphorylation of mTOR and improve the phosphorylation of AMPK compared with the Model group.In conclusion,Chaiqi decoction can up-regulate the level of autophagy in tissues to improve vascular endothelial injury in MS.This effect may be related to the reduction of phosphorylation of mTOR and the increase of phosphorylation of AMPK.(3)CCK8 results showed that the d7 10%24h Chaiqi Decoction serum significantly improved cell viability compared with d7 10%24h blank serum,with statistically significant difference(P<0.05),and the cell viability ratio of this concentration Chaiqi Decoction serum was closest to 100%(99.41%).The results of WB,Elisa and PCR suggested that the Chaiqi Decoction serum could reduce the content of ET-1 in cellular supernatant,increase the content of NO,reduce the expression of et-1 protein and mRNA in cells,increase the phosphorylation of eNOS and the expression of eNOS mRNA.To sum up,Chaiqi Decoction could reduce the cell damage of HUVECs caused by high-sugar high-fat culture.(4)TEM,GFP-mRFP-LC3 adenovirus transfection,and WB results showed that the autophagosomes,autolysosomes,autophagy related protein LC3 and the autophagy upstream protein Beclin-1 expression of Chaiqi Decoction group in cells were significantly higher than those of Control group and Model group.p62,an autophagy substrate protein,negatively correlated with autophagy.We found its expression in cells was significantly lower than that in the Model group,which was similar to Rapamycin,an autophagy activator.DALGreen-DAPRed double staining indicated that the expression of autophagosomes and autolysosomes increased by serum containing Chaiqi Decoction at 12h,18h,24h and Oh were significantly different,which was time-dependent.In the exploration of upstream signaling factors of autophagy,Chaiqi Decoction could significantly reduce the phosphorylation of mTOR and improve the phosphorylation of AMPK compared with the Model group.Chaiqi Decoction can up-regulate the level of autophagy in cells to improve vascular endothelial injury in MS.This effect may be related to the reduction of phosphorylation of mTOR and the increase of phosphorylation of AMPK.Research conclusions:(1)The Animal Models of vascular injury with MS could be created by 8 weeks of high salt,fat and sugar feeding,and Chaiqi Decoction could improve the vascular injury in these Model rats with MS.(2)Chaiqi Decoction could up-regulate the autophagy level of thoracic aorta in rats with MS,which may be related to the AMPK/mTOR pathway(3)Chaiqi Decoction could alleviate the cell damage of HUVECs in high-sugar high-fat culture.(4)Chaiqi decoction could gradually up-regulate the autophagy level of HUVECs cells in high-sugar high-fat culture with the increase of time,which may be related to the AMPK/mTOR pathway.(5)Combined with previous studies,the efficacy of Chaiqi Decoction to improve the abnormalities of the metabolic syndrome and vascular endothelial damage may be multi-targeted,which is related to the "holistic concept" of traditional Chinese medicine,suggesting that the clinical prevention and treatment of metabolic syndrome by traditional Chinese medicine should focus on the "acquired constitution" and the theory of spleen. |
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