Mechanism Research Of Resveratrol Improves Pyroptosis Of Vascular Endothelial Cells Induced By High Fat Diet And Palmitic Acid Via SIRT1-p66Shc-NLRP3 Pathway

Objective To study the protective effect of resveratrol on pyroptosis of endothelial cells and expression changes of related molecules in thoracic aorta and liver tissue of high fat diet-fed mice.To investigate the molecular mechanism of resveratrol in improving pyroptosis of human umbilical vein endothelial cells（HUVEC）induced by palmitic acid via SIRT1-p66Shc-NLRP3 pathway.Method1.Animal Experiment After adaptive feeding for 2 weeks,6-week-old C57BL/6 male mice were randomly divided into 3 groups: standard diet group（SCD）,high-fat diet group（HFD）,and high-fat diet + resveratrol group（HFD+RES）.The high-fat diet + resveratrol group（HFD+RES）was fed with resveratrol 400mg/kg/ day for 22 weeks.The body weight of mice were monitored during modeling,and liver tissue and thoracic aorta of mice were obtained for the follow-up experiment.2.Tissue Experiment The thoracic aorta and liver tissues of mice were embedded in paraffin and stained with HE to observe the morphological changes.Immunohistochemical assay was used to analyze protein expression and distribution of p66 Shc,SIRT1,GSDMD,NLRP3 in thoracic aorta.The protein expression levels of SIRT1,p66 Shc,NLRP3,Cysteinyl Aspartate Specific Proteinase-1（Caspase-1）,Gasdermin-D（GSDMD）,interleukin-1beta（IL-1β）,interleukin-18（IL-18）in liver tissues were detected by Western blot;The m RNA expression levels of SIRT1,p66 Shc,NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 in liver tissues were analyzed by quantitative real-time RT-PCR.3.Cell Experiment（1）Palmitic acid（PA）induced human umbilical vein endothelial cells（HUVEC）: pyroptosis of HUVEC was induced by different concentrations of palmitic acid（0 m M,0.2 m M,0.4 m M,0.6 m M,0.8 m M,1.0 m M）.CCK-8,lactate dehydrogenase（LDH）release,Western blot,flow cytometry,cell immunofluorescence and transmission electron microscopy were used to determine the concentration of PA that could induce pyroptosis of HUVEC.（2）Resveratrol ameliorated pyroptosis: HUVECs were inoculated into six-well plates and divided into control group（CON）,palmitic acid group（PA）and palmitic acid +resveratrol treatment group（PA+RES）.The protein expression levels of SIRT1,p66 Shc,NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 and ASC were detected by Western blot.The levels of pyroptosis and reactive oxygen species（ROS）were detected by flow cytometry.NLRP3/ASC co-localization and p66 Shc expression were detected by cell immunofluorescence.Lactate dehydrogenase（LDH）release level was analyzed and so on.（3）SIRT1 inhibitor EX527: The cells were divided into four groups: control group（CON）,palmitic acid group（PA）,palmitic acid and resveratrol treatment group（PA+RES）,palmitic acid + resveratrol treatment +EX527 group（PA+RES+EX527）.The protein expression levels of SIRT1,p66 Shc,NLRP3,Caspase-1,GSDMD,IL-1β,IL-18 and ASC were detected by Western blot.The levels of pyroptosis and reactive oxygen species（ROS）were detected by flow cytometry.NLRP3/ASC co-localization and p66 Shc expression levels were detected by cell immunofluorescence.Results1.Animal experiment: the results of body weight,HE staining of liver and thoracic aorta showed that the inflammatory injury model of HFD-induced obese mice was constructed successfully;Western blot and q RT-PCR results showed that SIRT1 expression was significantly decreased in HFD group,while p66 Shc,NLRP3,Caspase-1,GSDMD,IL-1β and IL-18 expression were significantly increased.Compared with HFD mice,the expression of SIRT1 in HFD+RES group was significantly increased,and the expression of p66 Shc,NLRP3,Caspase-1,GSDMD,IL-1β and IL-18 were significantly decreased.Immunohistochemical results of liver and thoracic aorta also showed the similar protein expression as Western blot results of liver tissue.2.Cell experiments:（1）The concentration of 0.2~1.0 m M palmitic acid could induce pyroptosis of HUVEC in a dose-dependent manner.Meanwhile,the protein expression of SIRT1 decreased with the increase of PA concentration,and the protein expression of p66 Shc increased with the increase of PA concentration.Based on various experimental data,the concentration of PA inducing pyroptosis of HUVEC in subsequent experiments was determined to be 0.4 m M.（2）Western blot,immunofluorescence and flow cytometry showed that resveratrol（50 μM）could improve PA induced pyroptosis of HUVEC and reduce the protein expression level of p66 Shc compared with PA group.（3）the results of Western blot,immunofluorescence,flow cytometry and other experiment showed that the addition of SIRT1 inhibitor EX527 could inhibit the improvement effect of resveratrol on PA-induced pyroptosis of HUVEC.These results suggest that resveratrol may improve the pyroptosis of HUVEC induced by PA by up-regulating SIRT1 expression.Conclusions1.Resveratrol can improve pyroptosis of thoracic aorta and liver tissues induced by high fat diet in mice,which may be related to the activation of SIRT1 and inhibition of p66 Shc and NLRP3 inflammasome.2.Palmitic acid can induce pyroptosis in HUVEC and increase the expression of pyroptosis related proteins.Resveratrol has a protective effect on pyroptosis in HUVEC induced by PA,and its mechanism may be related to SIRT1-p66Shc-NLRP3 pathway.