目的 探讨糖络宁通过高血糖大鼠血清外泌体抑制细胞凋亡的机制。方法 SD大鼠分为对照组、模型组和糖络宁（10.9 g/kg）组。除对照组外，其余组大鼠ip链脲佐菌素诱导高血糖大鼠模型。给药组大鼠ig药物，对照组和模型组大鼠ig等体积蒸馏水，1次/d，连续12周。给药结束后，收集大鼠血清，采用差异离心法提取血清外泌体，使用大鼠雪旺细胞RSC96内化外泌体，将RSC96细胞分别与对照组、模型组、糖络宁组的大鼠血清外泌体共培养24 h。采用CCK-8法测定RSC96细胞活力；采用Western blotting法测定RSC96细胞中肌醇酶1α（inositol-requiring enzyme 1α，IRE1α）、磷酸化IRE1α（p-IRE1α）和蛋白质二硫键异构酶（protein disulfide isomerase A6，PDIA6）蛋白表达；采用qRT-PCR法测定外泌体中miR-322 mRNA表达，RSC96细胞中miR-322、IRE1α及IRE1依赖性衰解（IRE1-dependent decay，RIDD）底物CD59、蛋白激酶D2（protein kinase D2，PKD2）、A类清道夫受体（scavenger receptor class A，SCARA）、蛋白水解酶D （peptidase D，PEPD） mRNA表达。结果 与对照组比较，模型组血清外泌体数量显著增加（P<0.01），外泌体及RSC96细胞中miR-322 mRNA水平显著降低（P<0.05、0.01），RSC96细胞活力显著降低（P<0.01），RSC96细胞IRE1α蛋白和mRNA表达水平显著升高（P<0.01），PDIA6蛋白表达水平显著降低（P<0.01），RIDD底物CD59、PKD2、SCARA和PEPD mRNA表达水平显著降低（P<0.01）；与模型组比较，糖络宁组血清外泌体数量明显降低（P<0.05），外泌体及RSC96细胞中miR-322 mRNA水平显著升高（P<0.05、0.01），RSC96细胞活力显著增加（P<0.05），RSC96细胞IRE1α蛋白和mRNA表达水平显著降低（P<0.05），PDIA6蛋白表达水平显著升高（P<0.01），RIDD底物CD59、PKD2、SCARA和PEPD mRNA表达水平显著升高（P<0.05、0.01）。结论 糖络宁通过增加高血糖大鼠血清外泌体中miR-322 mRNA水平，从而抑制IRE1α-RIDD诱导的细胞凋亡。

Objective To explore the mechanism of Tangluoning (糖络宁) on inhibiting cell apoptosis through serum exosomes in hyperglycemic rats. Methods SD rats were divided into control group, model group, and Tangluoning (10.9 g/kg) group. Except for the control group, rats in the other groups were ip streptozotocin to induce hyperglycemia rat model. Rats in administration group were ig drugs, rats in control group and model group were ig equal volumes of distilled water, once a day for 12 weeks. After administration, serum of rats was collected, the serum exosomes were extracted by differential centrifugation, and RSC96 cells were used to internalize the exosomes. RSC96 cells were co-cultured with serum exosomes of control group, model group, and Tangluoning group for 24 h. CCK-8 method was used to determine the viability of RSC96 cells. Western blotting was used to determine the expressions of inositol-requiring enzyme 1α (IRE1α), phosphorylated IRE1α (p-IRE1α), and protein disulfide isomerase (PDIA6) in RSC96 cells. qRT-PCR was used to measure the expression of miR-322 mRNA in exosomes, miR-322, IRE1α and IRE1-dependent decay (RIDD) substrate such as CD59, protein kinase D2 (PKD2), scavenger receptor class A (SCARA), protein hydrolase D (PEPD) mRNA expressions in RSC96 cells. Results Compared with control group, the number of serum exosomes in model group was significantly increased (𝑃 < 0.01); The level of miR-322 mRNA in exosomes and RSC96 cells was significantly reduced (𝑃 < 0.05, 0.01); The viability of RSC96 cells was significantly reduced (𝑃 < 0.01); IRE1α protein and mRNA expressions in RSC96 cells were significantly increased (𝑃 < 0.01); PDIA6 expression was significantly reduced (𝑃 < 0.01); RIDD substrates such as CD59, PKD2, SCARA and PEPD mRNA levels were significantly reduced (𝑃 < 0.01). Compared with model group, the number of serum exosomes in Tangluoning group was significantly reduced (𝑃 < 0.05); The mRNA level of miR-322 in exosomes and RSC96 cells was significantly increased (𝑃 < 0.05, 0.01); The viability of RSC96 cells was significantly increased (𝑃 < 0.05); IRE1α protein and mRNA levels in RSC96 cells were significantly reduced (𝑃 < 0.05); PDIA6 expression was significantly increased (𝑃 < 0.01); RIDD substrates such as CD59, PKD2, SCARA and PEPD mRNA levels were significantly increased (𝑃 < 0.05, 0.01). Conclusion Tangluoning can inhibit the apoptosis induced by IRE1α-RIDD through increasing miR-322 mRNA level in serum exosomes of hyperglycemic rats.

R285.5

北京市自然科学基金资助项目（7194268）