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青藤碱调控NLRP3/ caspase-1通路抑制BV-2小胶质细胞焦亡及炎症的机制研究
Mechanisms of sinomenium on pyroptosis and inflammation in BV-2 microglial cells through NLRP3/ caspase-1 pathway
投稿时间:2021-05-06  修订日期:2021-09-26
DOI:
中文关键词:  青藤碱  BV-2小胶质细胞  细胞焦亡  NLRP3炎症小体  半胱氨酸天冬氨酸蛋白酶1
英文关键词:sinomenium  BV-2 microglial cells  pyroptosis  NLRP3  caspase-1
基金项目:
作者单位邮编
陈应丛 台州市立医院 318000
王国涛 台州市立医院 
徐道剑 台州市立医院 
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中文摘要:
      摘 要: 目的 探究青藤碱(sinomenium)调控NLRP3/caspase-1通路抑制BV-2小胶质细胞焦亡及炎症的机制。方法 以氧糖剥夺/复糖复氧(OGD/R)模型诱导BV-2细胞焦亡模型,采用CCK-8法检测不同浓度(0、10、20、50、100 μmol /L)的青藤碱干预BV-2小胶质细胞24h后的细胞活性,筛选药物浓度。将BV-2细胞分为正常组、OGD/R组和OGD/R+青藤碱组(20μmol/L)进行研究分组。使用微量酶标法测BV-2小胶质细胞乳酸脱氢酶(lactate dehydrogenase, LDH)活性。采用赫斯特(Hoechst)/碘化丙啶(PI)细胞染色法检测BV-2细胞中PI阳性细胞数。采用RT-PCR和Western blot方法检测青藤碱对BV-2小胶质细胞硫氧还蛋白结合蛋白(TXNIP)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶(caspase)-1、白细胞介素(interleukin, IL)-1β和IL-18的mRNA和蛋白表达水平的影响。结果 10、20μmol/L青藤碱干预24h后,OGD/R诱导的BV-2小胶质细胞活性较对照组无显著差异[(97.69±0.51)%、(96.03±1.13)%比(100.00±0.00)%,P均>0.05)], 50、100μmol/L青藤碱干预24h后,OGD诱导的BV-2小胶质细胞活性较对照组显著降低[(78.92±3.02)%、(64.12±4.55)%比(100.00±0.00)%,P均<0.05]。与正常组相比,OGD组BV-2小胶质细胞LDH相对释放量明显上升[(2.23±0.19)比(1.00±0.00),P<0.05],PI活性细胞比例显升高[(46.28±4.02)%比(3.52±0.31)%,P<0.05],TXNIP、NLRP3、caspase-1、IL-1β和IL-18 mRNA表达明显上调[TXNIP :(1.58±0.20)比(0.69±0.08),P<0.05;NLRP3:(2.32±0.18)比(0.88±0.09),P<0.05;caspase-1:(1.61±0.11)比(0.77±0.15),P<0.05;IL-1: (3.17±0.43)比(1.03±0.09),P<0.05;IL-18:(1.82±0.22)比(0.81±0.13),P<0.05]和蛋白表达水平明显上调[TXNIP:(1.26±0.13)比(0.72±0.09),P<0.05; NLRP3:(0.43±0.04)比(0.16±0.04),P<0.05; caspase-1:(1.30±0.09)比(0.58±0.07),P<0.05;IL-1β:(1.21±0.11)比(0.39±0.06),P<0.05; IL-18:(0.54±0.07)比(0.23±0.06),P<0.05]。与OGD组相比,OGD/R+青藤碱组BV-2细胞LDH相对释放量明显下降[(1.28±0.09)比(2.23±0.19),P<0.05],PI活性细胞比例显著减少[(23.08±3.46)%比(46.28±4.02)%,P<0.05],TXNIP、NLRP3、caspase-1、IL-1β和IL-18 mRNA明显下降[TXNIP :( 0.95±0.11)比(1.58±0.20),P<0.05; NLRP3:(1.26±0.12)比(2.32±0.18),P<0.05; caspase-1:(0.95±0.05)比(1.61±0.11),P<0.05; IL-1: (1.55±0.18)比(3.17±0.43),P<0.05;IL-18:(1.23±0.14)比(1.82±0.22),P>0.05]和蛋白表达水平显著下降[TXNIP :(0.78±0.04)比(1.26±0.13);NLRP3:(0.26±0.03)比(0.43±0.04); caspase-1:(0.71±0.05)比(1.30±0.09);IL-1β: (0.54±0.03)比(1.21±0.11);IL-18:(0.34±0.08)比(0.54±0.07),P均<0.05]。结论 青藤碱能通过下调NLRP3/ caspase-1通路磷酸化水平,抑制BV-2小胶质细胞焦亡及炎症反应。
英文摘要:
      Abstract: Objective To investigate the mechanisms of sinomenium on pyroptosis and inflammation in BV-2 microglial cells based on NLRP3/ caspase-1 pathway. Methods The pyroptosis model of BV-2 microglial cells was established by oxygen and glucose deprivation/recovery (OGD/R). The cell viability was detected by CCK-8 method after treated with sinomenium at different concentrations ( 0, 10, 20, 50, 100 μmol/L), respectively, and the appropriate drug concentration was selected. The BV-2 microglial cells were divided into the normal group, OGD/R group and OGD/R +sinomenium group(20 μmol/L). The elevation of dehydrogenase(LDH) relative activity and amount of propidium iodide (PI) positive cells was applied to detect the pyroptosis level of BV-2 cells. Thioredoxin-interacting protein(TXNIP), NOD-like receptor family pyrin domain containing 3(NLRP3), caspase-1, interleukin-1β(IL-1β) and interleukin-18(IL-18) mRNA and protein expressions were detected by real-time PCR and Western blot, respectively. Results After the treatment of sinomenium at 10 and 20 μmol / L for 24 hours,the cell viability of OGD/R-induced BV-2 cells showed no significant difference compared with the control group[(97.69±0.51)%、(96.03±1.13)% vs. (100.00±0.00)%,P>0.05)]. While after the treatment of sinomenium at 10 and 20 μmol / L for 24 hours,the cell viability of OGD/R-induced BV-2 cells decreased significantly[(78.92±3.02)%、(64.12±4.55)% vs. (100.00±0.00)%,P<0.05]. Compared with the normal group, after established by OGD/R, LDH relative activity was increased notibly[(2.23±0.19) vs. (1.00±0.00),P<0.05], the percent of PI positive cells was suppressed significantly[(46.28±4.02)% vs. (3.52±0.31)%,P<0.05], and the mRNA[TXNIP :(1.58±0.20) vs. (0.69±0.08), P<0.05; NLRP3:(2.32±0.18) vs. (0.88±0.09),P<0.05; caspase-1:(1.61±0.11) vs. (0.77±0.15),P<0.05; IL-1: (3.17±0.43) vs. (1.03±0.09),P<0.05;IL-18:(1.82±0.22) vs. (0.81±0.13),P<0.05] and protein [TXNIP :(1.58±0.20) vs. (0.69±0.08),P<0.05; NLRP3:(2.32±0.18) vs. (0.88±0.09),P<0.05; caspase-1:(1.61±0.11) vs. (0.77±0.15),P<0.05; IL-1: (3.17±0.43) vs. (1.03±0.09),P<0.05;IL-18:(1.82±0.22) vs. (0.81±0.13),P<0.05] expressions of TXNIP, NLRP3, caspase-1, IL-1β and IL-18 were decreased significantly. Compared with OGD/R group, after the treatment of sinomenium, LDH relative activity was decreased significantly[(1.28±0.09) vs. (2.23±0.19),P<0.05] , the percent of PI positive cells was reduced visibly[(1.28±0.09) vs. (2.23±0.19),P<0.05] , and the mRNA [TXNIP:( 0.95±0.11) vs. (1.58±0.20),P<0.05; NLRP3:(1.26±0.12)vs.(2.32±0.18),P<0.05; caspase-1:(0.95±0.05) vs. (1.61±0.11),P<0.05; IL-1: (1.55±0.18) vs. (3.17±0.43),P<0.05;IL-18:(1.23±0.14) vs. (1.82±0.22),P>0.05] and protein[TXNIP :(0.78±0.04) vs. (1.26±0.13);NLRP3:(0.26±0.03) vs. (0.43±0.04);caspase-1:(0.71±0.05) vs. (1.30±0.09);IL-1β: (0.54±0.03) vs. (1.21±0.11);IL-18:(0.34±0.08) vs. (0.54±0.07), all P<0.05] expressions of TXNIP, NLRP3, caspase-1, IL-1β and IL-18 were suppressed significantly. Conclusion Sinomenium can down-regulate pyroptosis and inflammation of BV-2 microglial cells by inhibiting the NLRP3/ caspase-1 pathway.
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