Protective Mechanisms Of SIRT1 On High-fat Diet And Cholesterol-induced Inflammatory Damages In Vascular Endothelial Cells

Objective To study the effect of resveratrol on SIRT1 activation in improving endothelial inflammation and dysfunction in the thoracic aorta of high-fat diet-fed male mice;To explore the molecular mechanisms of SIRT1 improving cholesterol-induced endothelial cell toxicity.Method 1.Animal Experiment Sixty six-week-old C57 BL / 6 male mice were randomly divided into four groups after adaptive feeding for two week : The standard diet group(SCD),standard diet+resveratrol group(SCD +RES),high-fat diet group(HFD),and high-fat diet+ resveratrol group(HFD+ RES).SCD or HFD were fed with RES at 400 mg / kg / day for 22 weeks.Body weight was monitored,and serum lipid indicators TC,TG,HDL-C,n HDL-C,LDL-C,and VLDL-C were measured in each group;endothelial function indicators NO and ET-1;and inflammatory factors IL-18 and IL-1β.2.Tissue Experiment The thoracic aortic tissues of mice were dehydrated and stained with Hematoxylin &Eosin dyes to observe the morphological changes of the blood vessels;Oil red O staining was used to observe the vascular lipid deposition;Aortic endothelium-dependent diastolic experiment was used to assess endothelial function;Immunohistochemistry was used to detect the protein expression of SIRT1,MCP1,XBP1 s,e NOS;Western blot was used to detect the protein expression of SIRT1,IRE1α,p-IRE1α,p-e NOS,XBP1 s,NF-k B,p-ACC,p-AMPK,CHOP,TXNIP,NLRP3,Caspase1 in the thoracic aorta.3.Cell Experiment(1)Roles of resveratrol and EX527: MTT colorimetric assay was used to determine the experimental drug concentrations of cholesterol and resveratrol by measuring the cell rate of survival.The immunoblotting method was used to further detect the protein levels of SIRT1,p-e NOS,HMGCR,and SREBP2 in HUVEC;EX527 treatment in concentration gradients assayed by SIRT1 activity detection and Western blot was used to determine the optimal concentration of EX527,then the cells were grouped and added with EX527 or cholesterol or resveratrol,The immunoblotting method was used to further detect the protein levels of SIRT1、p-IRE1α、XBP1s、TXNIP、NLRP3、Caspase1、IL-18、IL-1β、HMGCR、SREBP2、INSIG2.(2)SIRT1 overexpression and knockdown experiment: The SIRT1 overexpression and knockdown plasmids were transfected into HUVEC,respectively.Stable transfected cell lines were selected by G418 culture for 2 weeks.Then the cells were used to detect the NLRP3 inflammasome by three classifications:(1)CON group,CHO group,CHO + RES group,EX527 group,EX527 + RES group;(2)WT group,NC group,NC + CHO group,SIRT1-OP group,SIRT1-OP + CHO Group;(3)WT group,NC group,NC + CHO group,SIRT1-KD group,SIRT1-KD + RES group.Western blot was used to detect SIRT1,p-IRE1α,p-e NOS,XBP1 s,TXNIP,NLRP3,Caspase1,IL-18,IL-1β,HMGCR,SREBP2,INSIG2 protein levels.(3)XBP1s co-immunoprecipitation test: HUVEC transfected with Empty plasmid and SIRT1 over-expression plasmid were used to select stable transfected cell lines;cholesterol interfere with endothelial cells and divided into 5 groups: WT group,NC group,NC + CHO group,SIRT1-OP group,SIRT1-OP + CHO group.The whole proteins were extracted from the cells.After adding acetylated antibodies,the mixture was shaken at 4 ℃ overnight.Acetylated-Lysine Antibody-protein complexes were captured by Protein A+G agorose beads,then Ace-XBP1 s were detected by Western blot.Results 1 Animal experiment: As compared with the SCD group,the mice in the SCD + RES group were lower in body weight;meanwhile the levels of NO,IL-1β and IL-18 were no significant changes.As compared with the SCD group,mice in the HFD group weighed more significantly;the serum levels of TC,LDL-C,HDL-C,n-HDL,ET-1,IL-1β,and IL-18 were increased significantly,and the serum level of NO was decreased significantly.As compared with the HFD group,mice in the HFD + RES group have significantly lower weight;the serum levels of TC,LDL-C,IL-1β,and IL-18 were significantly reduced,meanwhile the serum NO levels were significantly increased,but the serum TG and ET-1 levels were not significantly different.2 Tissue experiment: When compared with the SCD group,there were no significant differences in all the results of H&E staining,immunohistochemistry,oil red O staining,and Western blot in the SCD + RES group.The thoracic aorta of the mice in the HFD group was thickened,shown as infiltration of inflammatory cells and obvious lipid deposition,meanwhile endothelium-dependent vasodilation in the thoracic aorta was severely impaired.The protein expression of SIRT1 and e NOS were decreased,meanwhile the protein expression of XBP1 s and MCP1 were increased by immunohistochemical analysis.The protein expression of SIRT1,p-ACC,p-AMPK,p-IRE1α,p-e NOS were decreased,meanwhile the protein expression of XBP1 s,NF-k B,CHOP,TXNIP,NLRP3 and caspase1 protein levels were increased obviously.As compared with the HFD group,the HFD + RES mice had reduced inflammation and lipid deposition;improved endothelium-dependent vasodilation of thoracic aorta;and all reverted protein expressions.3 cell experiments:(1)Compared with the CON group,SIRT1 protein expressions in the CHO group and EX527 group were reduced,p-IRE1α,XBP1 s,TXNIP,NLRP3,Caspase1,IL-18,IL-1β and HMGCR,SREBP2,INSIG2 protein levels in CHO group were increased;compared with CHO group and EX527 group,SIRT1 protein expressions were increased in CHO + RES group and EX527 + RES group,meanwhile p-IRE1α,XBP1 s,TXNIP,NLRP3,Caspase1,IL-18,IL-1β and the HMGCR,SREBP2,and INSIG2 protein expressions in the CHO + RES group were decreased.(2)There was no significant difference in the indicators of the WT group and the NC group.Compared with the NC group,the protein expressions of INSIG2,IL-18,and pro-Caspase1 in the SIRT1-OP group were significantly reduced,and the protein expressions of SIRT1 and p-e NOS were significantly increased.IRE1α,XBP1 s,TXNIP,NLRP3,Caspase1,IL-1β,HMGCR,and SREBP2 protein levels were not significantly changed.Compared with the NC + CHO group,There had higher SIRT1,p-e NOS protein levels,and lower p-IRE1α,XBP1 s,TXNIP,NLRP3,Caspase1,IL-18,IL-1β,HMGCR,SREBP2,and INSIG2 protein levels in SIRT1-OP + CHO group.(3)There was no significant difference in the indicators of the WT group and the NC group.Compared with the NC group,SIRT1-KD group had significantly lower SIRT1,p-e NOS protein expressions,and higher p-IRE1α,XBP1 s,TXNIP,NLRP3,Caspase1,IL-18,IL-1β,HMGCR,SREBP2,INSIG2 protein levels.Compared with SIRT1-KD group,SIRT1 and p-e NOS protein levels were increased significantly,p-IRE1α,XBP1 s,TXNIP,NLRP3,Caspase1,IL-18,IL-1β,HMGCR,SREBP2,INSIG2 protein levels were significantly reduced in SIRT1-KD + RES group.(4)The results of co-immunoprecipitation showed that the protein expression of Ace-XBP1 s between the WT group and NC group was no significant difference.Compared with the NC group,the level of Ace-XBP1 s in the CHO group increased significantly,and the level of Ace-XBP1 s in the SIRT1-OP group decreased significantly.Compared with the CHO group,the protein expression of Ace-XBP1 s in the SIRT1-OP + CHO group was significantly reduced.Conclusions 1 Resveratrol improves hypercholesterolemia,lipid ectopic deposition,and endothelial dysfunction in thoracic aorta caused by high-fat diet feeding in C57BL/6 male mice.The mechanism may be related to reduced ER stress and NLRP3 inflammasome activation mediated by increased SIRT1 expression.2 Cholesterol can induce XBP1 s and TXNIP expression,activate NLRP3 inflammasome in HUVEC via inhibiting SIRT1 expression.SIRT1 overexpression deacetylate XBP1 s,thus reducing endoplasmic reticulum stress,inhibiting the formation of TXNIP/NLRP3,so decreasing secretion of inflammation factors.