糖络宁通过外泌体调控IRE1α诱导细胞凋亡的作用机制研究

Research on the Mechanism of Tangluoning Regulating IRE1α-induced Apoptosis via Exosomes

目的:探讨糖络宁(以下简称TLN)通过外泌体对雪旺细胞(schwann cells,SCs)IRE1α诱导的细胞凋亡的调控作用。方法:将30只SD大鼠分为空白组、模型组和糖络宁组,每组10只。除空白组外,其余各组大鼠腹腔注射链脲佐菌素(streptozotocin,STZ)诱导糖尿病模型。空白组及模型组灌胃给予蒸馏水,TLN组灌胃给予糖络宁溶液,12周后处死大鼠并收集血清。差速离心法分离血清中的外泌体,将SCs分为3组并分别与3组大鼠血清外泌体共培养。收集SCs,提取细胞中的总蛋白和总RNA,采用Western blot法测定TRAF2、p-JNK、Bcl-2、Bax、Cyto C和cleaved-Caspase-3的蛋白表达,采用RT-PCR法测定ASK1和Caspase-9的mRNA表达。结果:与空白组比较,模型组TRAF2、p-JNK、Bax、Cyto C和cleaved-Caspase-3的蛋白表达显著升高,Bcl-2的蛋白表达显著降低,ASK1和Caspase-9的mRNA表达显著升高(P<0.01);与模型组比较,TLN组TRAF2、p-JNK、Bax、Cyto C和cleaved-Caspase-3的蛋白表达显著降低,Bcl-2的蛋白表达显著升高,ASK1和Caspase-9的mRNA表达显著降低(P<0.01)。结论:糖络宁能够通过外泌体抑制IRE1α-TRAF2-ASK1复合体,进而抑制JNK的磷酸化,然后分别抑制Bax表达,促进Bcl-2表达,以及抑制Cyto C、Caspase-9和Caspase-3的表达,从而抑制细胞凋亡。

Objective:To discuss the regulatory effects of Tangluoning(TLN)on IRE1α-induced apoptosis of schwann cells(SCs)via exosomes.Methods:Thirty SD rats were allocated to the blank group,the model group and TLN group,ten rats in each group.Except for the blank group,the rats in the other groups accepted intraperitoneal injection of STZ to induce DM models.The blank group and the model group were drenched with distilled water,and TLN group TLN solution,to collect the serum when the rats were sacrificed after 12 weeks.Differential centrifugation method was used to separate the exosomes from the serum,SCs were assigned to three groups,and cultured with serum exosomes of the rats in three groups.To extract total protein and total RNA from cells after collecting SCs,Western blot method was adopted to detect the expressions of TRAF2,p-JNK,Bcl-2,Bax,Cyto C and cleaved-Caspase-3 protein,RT-PCR was applied to detect the expressions of ASK1 and Caspase-9 mRNA.Results:Compared with the blank group,the expressions of TRAF2,p-JNK,Bax,Cyto C and cleaved-Caspase-3 protein raised apparently,the expression of Bcl-2 protein decreased noticeably,the expressions of ASK1 and Caspase-9 mRNA heightened remarkably(P<0.01);compared with the model group,the expressions of TRAF2,p-JNK,Bax,Cyto C and cleaved-Caspase-3 protein decreased in TLN group noticeably,the expressions of Bcl-2 protein lifted distinctly,the levels of ASK1 and Caspase-9 mRNA decreased obviously(P<0.01).Conclusion:TLN could inhibit IRE1α-TRAF2-ASK1 compound through exosomes,and then restrain the phosphorylation of JNK,inhibit the expressions of Bax,promote the levels of Bcl-2,inhibit the expressions of Cyto C,Caspase-9 and Caspase-3,thereby reversing cellular apoptosis.