CUL1基因对MPP+诱导的SH-SY5Y细胞存活和NLRP3炎症体通路的影响

Effects of CUL1 Gene on MPP+-induced SH-SY5Y Cell Survival and NLRP3 Inflammasome Pathway

目的:探究Cullin1(CUL1)基因对1-甲基-4-苯基吡啶离子(MPP+)诱导的SH-SY5Y细胞存活和核苷酸结合寡聚化结构域样受体3(NLRP3)炎症体通路的影响。方法:(1)将SH-SY5Y细胞分为NC组、NC-sh组、CUL1-sh组、NC-OE组和CUL1-OE组。使用Lipofectamine 2000试剂对细胞转染相应的慢病毒。(2)将SH-SY5Y细胞分为Control组、MPP+组和MPP++CUL1-OE组。MPP+组和MPP++CUL1-OE组细胞使用1 mmol/L的MPP+处理48 h,Control组细胞正常培养。通过MTT法检测细胞增殖,通过Annexin V-FITC/PI双染色法和TUNEL染色法检测细胞凋亡,通过qRT-PCR检测CUL1的mRNA水平,通过Western blot检测CUL1、NLRP3、凋亡相关斑点样蛋白(ASC)、cleaved caspase-1、白细胞介素(IL)-1β和IL-18蛋白水平。通过ELISA法检测细胞培养上清液中IL-1β和IL-18水平。结果:(1)与NC组和NC-sh组比较,CUL1-sh组CUL1的mRNA和蛋白相对表达量降低,相对细胞活力降低,Annexin V-FITC/PI阳性率和TUNEL阳性率升高,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平升高(P<0.05)。与NC组和NC-OE组比较,CUL1-OE组CUL1的mRNA和蛋白相对表达量升高,相对细胞活力升高,Annexin V-FITC/PI阳性率和TUNEL阳性率降低,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平降低(P<0.05)。(2)与Control组比较,MPP+组CUL1的mRNA和蛋白相对表达量降低,相对细胞活力降低,Annexin V-FITC/PI阳性率和TUNEL阳性率升高,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平升高(P<0.05)。与MPP+组比较,MPP++CUL1-OE组CUL1的mRNA和蛋白相对表达量升高,相对细胞活力升高,Annexin V-FITC/PI阳性率和TUNEL阳性率降低,NLRP3、ASC、cleaved caspase-1、IL-1β和IL-18蛋白相对表达量以及细胞培养上清液中IL-1β和IL-18水平降低(P<0.05)。结论:CUL1可能通过抑制NLRP3炎症体激活促进MPP+诱导的SH-SY5Y细胞存活。

Objective:To reveal the effect of Cullin1(CUL1)gene on 1-Methyl-4-phenylpyridinium ion(MPP+)-induced SH-SY5Y cell survival and NLR family,pyrin domain-containing 3(NLRP3)inflammasome pathway.Methods:(1)SH-SY5Y cells were divided into NC group,NC-sh group,CUL1-sh group,NC-OE group and CUL1-OE group.The cells were transfected with the corresponding lentivirus using Lipofectamine 2000 reagent.(2)SH-SY5Y cells were divided into control group,MPP+group and MPP++CUL1-OE group.The MPP+group and MPP++CUL1-OE group cells were treated with 1 mmol/L MPP+for 48 hours,while the control group cells were cultured normally.Cell proliferation was detected by MTT method.Apoptosis was detected by Annexin V-FITC/PI double staining and TUNEL staining.The level of CUL1 mRNA was detected by qRT-PCR.The protein levels of CUL1,NLRP3,apoptosis-related spot-like protein(ASC),cleaved caspase-1,interleukin(IL)-1βand IL-18 were detected by Western blot.The levels of IL-1βand IL-18 in cell culture supernatant were detected by ELISA.Results:(1)Compared with NC group and NC-sh group,the relative expression of CUL1 mRNA and protein decreased,the relative cell viability decreased,the Annexin V-FITC/PI positive rate and the positive rate of TUNEL increased,the relative expression of NLRP3,ASC,cleaved caspase-1,IL-1βand IL-18 protein and IL-1βand IL-18 levels in cell culture supernatant increased in CUL1-sh group(P<0.05).Compared with NC group and NC-OE group,the relative expression of CUL1 mRNA and protein increased,the relative cell viability increased,the Annexin V-FITC/PI positive rate and the positive rate of TUNEL decreased,the relative expression of NLRP3,ASC,cleaved caspase-1,IL-1βand IL-18 protein and IL-1βand IL-18 levels in cell culture supernatant decreased in CUL1-OE group(P<0.05).(2)Compared with control group,the relative expression of CUL1 mRNA and protein decreased,the relative cell viability decreased,the Annexin V-FITC/PI positive rate and the positive rate of TUNEL increased,the relative expression of NLRP3,ASC,cleaved caspase-1,IL-1βand IL-18 protein and IL-1βand IL-18 levels in cell culture supernatant increased in MPP+group(P<0.05).Compared with MPP+group,the relative expression of CUL1 mRNA and protein increased,the relative cell viability increased,the Annexin V-FITC/PI positive rate and the positive rate of TUNEL decreased,the relative expression of NLRP3,ASC,cleaved caspase-1,IL-1βand IL-18 protein and IL-1βand IL-18 levels in cell culture supernatant decreased in MPP++CUL1-OE group(P<0.05).Conclusion:CUL1 may promote the survival of SH-SY5Y cells induced by MPP+by inhibiting the activation of NLRP3 inflammasome.