八宝丹抑制TNF-α诱导C2C12成肌细胞Atrogin-1表达的研究

Inhibitory Effect of Babaodan on Expression of Atrogin-1 in C2C12 Myoblasts Induced by TNF-α

目的研究八宝丹(BBD)对肿瘤坏死因子-α(TNF-α)诱导的C2C12成肌细胞中核因子κB(NF-κB)通路及下游通路UPS系统中萎缩相关基因1(Atrogin-1)的作用。方法①筛选BBD干预浓度实验:取对数生长期C2C12成肌细胞分别予0、0.125、0.25、0.5、0.75、1 mg/mL不同浓度BBD干预48 h,采用MTT法检测细胞活力;②筛选TNF-α刺激细胞的浓度和时间实验:取对数生长期C2C12成肌细胞,分别予0、1、10、50 ng/mL TNF-α蛋白液刺激C2C12成肌细胞,Westernblot法检测p-P65、Atrogin-1蛋白表达以筛选出TNF-α刺激细胞的最佳浓度;再根据TNF-α刺激细胞的最佳浓度干预C2C12成肌细胞0 min、8 min、15 min、30 min、1 h、4 h,West⁃ernblot法检测p-P65、Atrogin-1蛋白筛选出TNF-α刺激细胞的最佳时间;③BBD干预实验:将C2C12成肌细胞随机分为空白对照组、药物对照组、模型组、八宝丹组,药物对照组和八宝丹组分别加入BBD预处理2 h,再根据筛选的TNF-α刺激细胞最佳浓度和时间,于模型组和八宝丹组中加入TNF-α刺激细胞,Westernblot法检测p-P65、Atrogin-1的蛋白表达水平。结果①筛选BBD干预浓度实验结果表明,不同浓度BBD对C2C12成肌细胞活力无影响(P>0.05),根据本课题组前期研究结果,最终选取0.75 mg/mL作为BBD干预细胞浓度;②筛选TNF-α刺激细胞的浓度和时间实验结果表明,10 ng/mL TNF-α刺激C2C12成肌细胞8 min后p-P65蛋白表达明显升高(P<0.05),10 ng/mLTNF-α刺激C2C12成肌细胞4 h后Atrogin-1蛋白表达水平明显升高(P<0.01);③BBD干预实验结果表明,与空白对照组比较,模型组p-P65、Atrogin-1蛋白表达显著上调(P<0.05,P<0.01);与模型组比较,八宝丹组p-P65、Atrogin-1蛋白表达显著下调(P<0.05,P<0.01)。结论BBD可抑制TNF-α诱导的NF-κB信号通路中NF-κB的磷酸化水平及下游通路Atrogin-1蛋白表达。

Objective:To study the effect of Babaodan(BBD)on NF-κB pathway and atrophy gene 1(Atrogin-1)of UPS system in C2C12 myoblasts induced by tumor necrosis factorα(TNFα).Methods:(1)MTT assay was used to detect the cell viability of C2C12 myoblasts pretreated with various concentrations of BBD(0,0.125,0.25,0.5,0.75,1 mg/mL)for 48 hours to confirm optimum BBD concentrations.(2)The Logarithmic growth phase cells was treated with different concentrations of TNF-α(0,1,10,50 ng/mL)at different time intervals.Western blot analysis was used to detect the protein expression levels of p-P65 and Atrogin-1 to confirm the optimal concentration and time of TNFαtreatment.(3)C2C12 myoblasts were randomly divided into blank control group,drug control group,model group and BBD group.The drug control group and BBD group were pretreated with BBD for 2 hours respec‐tively.The model group and BBD group were induced with TNF-αof the screened optimal concentration and time.Western blot anal‐ysis was used to detect the protein expression levels of p-P65 and Atrogin-1.Results:(1)Different concentrations of BBD had no sig‐nificant effect on C2C12 myoblasts viability(P>0.05).Based on research basis and results of MTT,0.75 mg/mL was selected as the BBD intervention concentration.(2)The protein expression of p-P65 was obviously increased at 8 minutes following 10 ng/mL TNFαstimulation(P<0.05),whereas the protein expression of Atrogin-1 was obviously increased at 4 hours following 10 ng/mL TNF-αstimulation(P<0.05).(3)C2C12 myoblasts pretreated with BBD(0.75 mg/mL)significantly attenuated the TNF-α-induced activations of p-P65 and Atrogin-1(P<0.05,P<0.01).Conclusion:BBD can inhibit the phosphorylation level of NF-κB in NF-κB signaling path‐way and the protein expression of Atrogin1 in downstream pathway in C2C12 myoblasts induced by TNF-α.