| BackgroudCardiovascular disease is a kind of ischemic or hemorrhagic cerebrovascular disease caused by hyperlipidemia,atherosclerosis or other pathological changes.China Cardiovascular Disease report 2021 estimated that the total number of people suffering from cardiovascular diseases is as high as 330 million,and the mortality rate is higher than that of tumors and other diseases.Cardiovascular disease is a major public health problem threatening the health of Chinese people.The pathogenesis of cardiovascular disease is complicated and"oxidative stress damage"of endothelial cells is considered to be the main pathogenesis of cardiovascular diseases such as atherosclerosis（AS）.Recent studies demonstrated that that vascular endothelial cells are not only damaged by traditional stress factors,but also regulated via the activation of NOD-like receptor protein 3（NLRP3）inflammasome.NLRP3 inflammasome,the main pathway of innate immune response,which can be activated by perceiving a variety of risk factors.However,the activation of NLRP3inflammasome has not been fully elucidated.Researches showed that the down-expression or depletion of silent mating-type information regulation 2 homolog 3（Sirt3）triggered cardiovascular diseases including hypertension and heart disease.SIRT3,a key factor in maintaining mitochondrial homeostasis and metabolism,which exerted essential function on mitochondrial function.Moreover,NLRP3 inflammasome induce extensive inflammation through receiving mitochondrial dysfunction or metabolic dysregulation.Therefore,we speculated that the increased expression of Sirt3 could promote endothelial function via inhibiting the activation of NLRP3 inflammasome.Dietary nutrition plays an important role in the prevention and treatment of non-communicable chronic diseases such as cardiovascular diseases.A large number of studies have shown that natural plant compounds have become optimistic candidates for the prevention and treatment of metabolic diseases such as obesity,diabetes,and ca rdiovascular disease.Dihydromyricetin（DHM）is a type of natural flavonoids extracted from grape family,myricaceae,euphorbiaceae and other natural plants,and DHM contents is highest in Ampelopsis grossedentata（commonly known as"vine tea"）which conta ins about 30%of the dry mass.DHM can reduce blood glucose,blood lipid and protect liver through its strong anti-inflammatory and antioxidant effects.Recent studies suggested that DHM played a critical role in the prevention and treatment of cardiovascu lar diseases,but the specific protective mechanism is not clear.Based on the research above,we propose the following scientific hypothesis:DHM can promote SIRT3expression to inhibit the activity of NLRP3 inflammasome which in turn to alleviate vascular endothelial cell injury and the development of AS.This study will deeply reveal the mechanism of AS from the Sirt3-NLRP3 pathway and provide a new intervention strategy for the prevention and treatment of AS through diet.ObjectiveThis study focused on endothelial injury,which is the initial link of AS.Through in vivo and in vitro experiments,we confirmed the inhibitory effect of DHM on AS,revealed the effect of activation of NLRP3 inflammasome,an important signal molecule of i nnate immunity,on the occurrence of AS,and clarified the mechanism of DHM exerting endothelial cell protective effect and inhibiting the occurrence of AS by regulating Sirt3 to inhibit the activation of NLRP3 inflammasome,so as to provide scientific bas is for prevention and treatment of cardiovascular diseases from dietary approach.Methods1.The Apolipoprotein deficient(ApoE-/-)mice and wild type（WT）C57BL/6J mice were fed with high fat diet（HFD）for 12 weeks to establish AS model,and DHM was given orally according to the dose（300/500mg/kg·BW/d）.Body weight and blood glucose of mice were observed to evaluate the effects of DHM.The lipid quadruple test and glutamic transaminase（AST）and glutamic alanine transaminase（ALT）indexes were compared between the groups,and the pathological changes of atheromatous plaques in the aortic arch were observed to evaluate whether DHM had an alleviating effect on the development of AS in HFD-fed mice.The levels of tumor necrosis factorα（TNF-α）and interleukin 1β（IL-1β）in serum were detected,and the transcription and protein levels of Sirt3 and NLRP3 inflammasome in aorta were detected by q RT-PCR,Western blotting and immunohistochemical methods.To explore whether DHM can improve the activation of mouse NLRP3 inflammasome induced by HFD in vivo.2.A hyperlipidemic injury model of human umbilical vein endothelial cells（HUVECs）was established by palmitic acid（PA）.The cytotoxicity was detected by cell viability and lactate dehydrogenase（LDH）release rate,to determine the appropriate intervention concentration of DHM.The effect of DHM on the secretion of HUVECs was evaluated by detecting intercellular adhesion molecule-1（ICAM-1）and intervascular adhesion molecule-1（VCAM-1）in HUVECs.The effect of DHM on the activation of NLRP3 inflammasome was evaluated by detecting IL-1βand detecting the gene transcription and protein expression of NLRP3 inflammasome in HUVECs by q RT-PCR,Western blotting and immunofluorescence.3.Based on the vitro injury model,the effect of DHM on mitochondrial homeostasis was evaluated by detecting mitochondrial function through the Seahorse mitochondrial stress test assay and the JC-1 mitochondrial membrane potential fluorescence probe assay.Sirt3transcription and protein expression in HUVECs were detected by q RT-PCR and immunoblotting to evaluate the effect of DHM on Sirt3 expression.Using Sirt3overexpression plasmids,Sirt3-specific inhibitors,and siRNA interference techniques,the role of Sirt3 in DHM regulating NLRP3 inflammatory vesicle activity and thus attenuating endothelial cell injury was clarified.Results1.DHM reduced the body weight of ApoE-/-mice induced by HFD,improved glucose tolerance,decreased the levels of serum lipids,AST and ALT,reduced the area of AS plaque and inhibited the activation of NLRP3 inflammasome in mice.DHM reduced serum triglyceride（TG）,total cholesterol（TCH）,and low-density lipoprotein（LDL-C）levels and increased high-density lipoprotein（HDL-C）levels in ApoE-/-mice compared to HFD-fed ApoE-/-mice.DHM decreased serum IL-1βand TNF-αlevels in HFD-fed ApoE-/-mice and reduced the development of AS plaques,reduced the size of the core area of plaque necrosis,and increased plaque collagen content in HFD-fed ApoE-/-mice.1β,IL-18,NF-κB and TNF-α.DHM upregulated the expression of Sirt3 mRNA and protein in HFD-induced mouse arterial tissues.2.DHM inhibited the decrease of vascular endothelial cell viability and the increase of LDH release rate induced by PA,decreased the secretion of ICAM-1 and VCAM-1,and inhibited the activation of NLRP3 inflammasome.In a model of PA-induced hyperlipidemia in endothelial cells,PA induced a decrease in cell viability,an increase in the rate of LDH release and growth in ICAM-1 and VCAM-1 secretion,and DHM intervention restored endothelial cell viability,reduced LDH release and decreased ICAM-1 and VCAM-1 level.PA-induced decreases in endothelial cell NLRP3,caspase-1 p20,IL-1β,IL-18,and NF-κB mRNA and protein expression levels were significantly increased.DHM intervention inhibited PA-induced activation of the above NLRP3 inflammatory vesicle-associated factors.3.DHM can promote the expression of Sirt3 in endothelial cells,maintain mitochondrial homeostasis,inhibit the activation of NLRP3 inflammasome and reduce endothelial cell injury.Using the injury model in vitro,DHM was found to reduce PA-induced mitochondrial stress,inhibit the decrease in mitochondrial membrane potential,and maintain mitochondrial homeostasis.It was also found that DHM upregulated PA-induced Sirt3 mRNA and protein expression levels in vascular endothelial cells.Further study revealed that transfection with Sirt3 overexpression plasmid enhanced the inhibitory effect of DHM on NLRP3 inflammatory vesicles and the protective effect on endothelial cell viability and LDH release rate.In contrast,treatment with Sirt3-specific inhibitor3-TYP and transfection with Sirt3 siRNA weakened the inhibitory effect of DHM on NLRP3inflammatory vesicles and the protective effect on endothelial cell viability and LDH release rate.And inhibition of LDH release rate.ConclusionDHM reduced the injury of vascular endothelial cells induced by PA through inhibiting the activation of NLRP3 inflammasome via promoting the expression of Sirt3,which in turn to prevent the occurrence of AS.This study suggested that Sirt3-NLRP3 may be a new target for the prevention and treatment of AS and provides an important scientific basis for the application of DHM in the prevention and treatment of cardiovascular diseases. |
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