番茄素通过激活转录因子EB减轻脂多糖诱导的神经细胞损伤

Tomatidine attenuates lipopolysaccharide-induced nerve cell injury via transcription factor EB

目的:探讨番茄素(tomatidine,TA)在脂多糖(lipopolysaccharide,LPS)介导的神经细胞损伤中的作用及其调节机制。方法:体外培养人神经母细胞瘤细胞(SH-SY5Y细胞),用LPS诱导神经炎症模型,分为对照(control,CON)组、LPS组和LPS+TA组。其中LPS组细胞加入5μg/mL LPS处理24 h,建立炎症模型;LPS+TA组先加入5μmol/L番茄素处理24 h后再加入5μg/mL LPS共培养24 h。CCK-8法检测细胞活力;实时荧光定量PCR(RTqPCR)法检测炎症因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和白细胞介素1β(interleukin-1β,IL-1β)的mRNA表达;Western blot检测转录因子EB(tuanscription factor EB,TFEB)、p-TFEB、P62和微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)蛋白的表达;免疫荧光检测TFEB的定位和cleaved caspase-3的表达;流式细胞术检测细胞凋亡率。结果:(1)与CON组相比,LPS引起细胞炎症因子IL-1β和TNF-αmRNA水平显著上升(P<0.05),细胞凋亡率和p-TFEB表达水平显著上升(P<0.01),而P62、LC3-II/LC3-I和TFEB蛋白表达水平显著下降(P<0.05),且TFEB主要定位于胞质。(2)与LPS组相比,番茄素处理显著降低p-TFEB蛋白的表达水平(P<0.01),增加TFEB蛋白的表达水平(P<0.01),并促进TFEB蛋白移入细胞核;番茄素处理后LC3-II/LC3-I蛋白表达水平显著上升(P<0.05),P62蛋白表达水平有下降趋势,但差异无统计学意义,细胞凋亡率显著降低(P<0.01);此外,番茄素处理后细胞炎症因子IL-1βmRNA水平有下降趋势,但差异无统计学意义,TNF-αmRNA水平则显著下降(P<0.01)。结论:番茄素通过激活转录因子EB缓解LPS引起的SH-SY5Y细胞自噬功能障碍,减轻炎症反应并抑制细胞凋亡。

AIM:To explore the effect of tomatidine(TA)on lipopolysaccharide(LPS)-induced nerve cell injury and the underlying mechanism.METHODS:The neuroinflammation model was induced by treating SH-SY5Y cells with LPS.These cells were divided into control(CON),LPS,and LPS+TA groups.The LPS group was treated with 5μg/mL LPS for 24 h to establish an inflammatory model.The LPS+TA group was first treated with 5μmol/L tomatidine for 24 h and then co-cultured with 5μg/mL LPS for 24 h.Cell viability was detected using the CCK-8 assay.RT-qPCR was used to detect the mRNA expression of inflammatory factors tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β).The protein expression of transcription factor EB(TFEB),p-TFEB,P62,and microtubule-associated protein 1 light chain 3(LC3)expression was detected through Western blot.TFEB localization and cleaved caspase-3 expression were detected through immunofluorescence.The cell apoptosis rate was detected through flow cytometry.RESULTS:(1)Compared with the CON group,the LPS group exhibited significant increases in IL-1βand TNF-αmRNA levels(P<0.05),the cell apoptosis rate,and the p-TFEB level(P<0.01).By contrast,P62,LC3-II/LC3-I,and TFEB protein ex‐pression levels decreased significantly(P<0.05),and TFEB was mainly localized in the cytoplasm.(2)Compared with the LPS group,tomatidine treatment significantly decreased the p-TFEB protein expression level(P<0.01),increased the TFEB protein expression level(P<0.01),and promoted the TFEB protein to migrate into the nucleus.After treatment of tomatidine,the LC3-II/LC3-I protein expression level significantly increased(P<0.05),and the cell apoptosis rate signifi‐cantly decreased(P<0.01).In addition,the TNF-αmRNA level significantly decreased after tomatidine treatment(P<0.01).CONCLUSION:Tomatidine improves autophagy dysfunction,inflammatory reaction,and cell apoptosis induced by LPS via activating the transcription factor EB.