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

摘要 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。

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

ABSTRACT 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)-1β and IL-18 were detected by Western blot. The levels of IL-1β and 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-1β and IL-18 protein and IL-1β and 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-1β and IL-18 protein and IL-1β and 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-1β and IL-18 protein and IL-1β and 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-1β and IL-18 protein and IL-1β and 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.