丹参酮ⅡA通过调控SIRT6/NF-κB/NLRP3信号通路减轻TNF-α诱导的人脐静脉内皮细胞损伤OA
Tanshinone ⅡA attenuates TNF-α-induced human umbilical vein endothe-lial cell injury via regulation of SIRT6/NF-κB/NLRP3 signaling pathway
目的:探讨丹参酮ⅡA(TSN)抑制肿瘤坏死因子α(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)损伤的分子机制.方法:采用20 μg/L TNF-α刺激HUVECs 24 h构建细胞损伤模型,并给予不同浓度TSN进行干预.实验分为:对照(control)组,模型(TNF-α)组,低、中、高剂量(5、10和20 μmol/L)TSN(TNF-α+TSN-L、TNF-α+TSN-M和TNF-α+TSN-H)组,阴性对照siRNA组,沉默信息调节因子6(SIRT6)siRNA(siSIRT6)组,TNF-α+TSN(20 μmol/L)+siSIRT6组,实验均独立重复5次(n=5).通过CCK-8法评价细胞活力,采用试剂盒测定乳酸脱氢酶(LDH)释放水平以反映细胞膜损伤.利用免疫荧光观察核因子κB(NF-κB)p65的核转位情况.RT-qPCR检测SIRT6、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)及炎症相关黏附分子[血管细胞黏附分子1(VCAM-1)和细胞间黏附分子1(ICAM-1)]的mRNA表达;Western blot分析SIRT6和NLRP3蛋白水平;采用酶活性试剂盒测定SIRT6活性.ELISA检测细胞上清中白细胞介素1β(IL-1β)的含量.结果:与模型组相比,5、10和20 μmol/L TSN处理均能显著减轻TNF-α诱导的HUVECs活力降低,减少LDH释放,并下调ICAM-1和VCAM-1的mRNA表达及IL-1β的分泌水平(P<0.05).同时,TSN处理显著抑制了TNF-α引起的SIRT6 mRNA和蛋白表达及酶活性的下降(P<0.05).功能实验表明,沉默SIRT6削弱了TSN对内皮细胞的保护作用,并消除了其对NF-κB-NLRP3信号通路的调控效应(P<0.05).结论:丹参酮ⅡA通过激活SIRT6并抑制NF-κB-NLRP3信号通路,减少TNF-α诱导的HUVECs体外炎症损伤.
AIM:To investigate the protective effects of tanshinone ⅡA(TSN)against tumor necrosis factor-α(TNF-α)-induced injury in human umbilical vein endothelial cells(HUVECs)and the underlying molecular mechanisms.METHODS:An in vitro endothelial injury model was established by stimulating HUVECs with 20 μg/L TNF-α for 24 h,followed by treatment with different concentrations of TSN.The cells were divided into the following groups:control,TNF-α,low-,middle-and high-dose(5,10 and 20 μmol/L)TSN(TNF-α+TSN-L/M/H),negative control siRNA,silent infor-mation regualtor 6(SIRT6)siRNA(siSIRT6),and siSIRT6+TNF-α+TSN(20 μmol/L)groups.All experiments were in-dependently repeated 5 times(n=5).Cell viability was assessed by the CCK-8 assay,and lactate dehydrogenase(LDH)release was measured to evaluate cytotoxicity and membrane damage.Nuclear translocation of nuclear factor-κB(NF-κB)p65 was examined by immunofluorescence.The mRNA expression of SIRT6,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)was determined by RT-qPCR,whereas the protein levels of SIRT6 and NLRP3 were analyzed by Western blot.The SIRT6 enzymatic activity was measured using a commercial assay kit,and interleukin-1β(IL-1β)secretion was quan-tified by ELISA.RESULTS:Compared with TNF-α group,TSN treatment at 5,10 and 20 μmol/L significantly amelio-rated the cell viability,reduced LDH release,suppressed the mRNA expression of ICAM-1 and VCAM-1,and decreased IL-1β secretion(P<0.05).Moreover,TSN markedly reversed the TNF-α-induced reductions in SIRT6 mRNA expres-sion,protein expresssion,and enzymatic activity(P<0.05).Functional experiments demonstrated that SIRT6 knockdown attenuated the protective effects of TSN on endothelial cells and abolished its regulatory effects on NF-κB/NLRP3 signaling pathway(P<0.05).CONCLUSION:Treatment with TSN alleviates TNF-α-induced inflammatory injury in HUVECs by activating SIRT6 and subsequently suppressing NF-κB/NLRP3 signaling pathway.
罗柠;彭美奇;石璨;危丽琴;林传钟;曾晓晖;方健
广州市花都区人民医院药学部,广东 广州 510800广州医科大学药学院,广东 广州 511436广州医科大学药学院,广东 广州 511436广州市花都区人民医院药学部,广东 广州 510800广州市花都区人民医院药学部,广东 广州 510800广州市花都区人民医院药学部,广东 广州 510800广州市花都区人民医院药学部,广东 广州 510800
医药卫生
丹参酮ⅡA血管内皮炎症肿瘤坏死因子αSIRT6/NF-κB/NLRP3信号通路
tanshinone ⅡAvascular endothelial inflammationtumor necrosis factor-αSIRT6/NLRP3/NF-κB signaling pathway
《中国病理生理杂志》 2026 (9)
1829-1837,9
广东省中医药局科研项目(No.20241252)
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