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目的:观察独活寄生汤含药血清抗膝骨性关节炎(KOA)大鼠关节软骨细胞凋亡、内质网应激相关蛋白葡萄糖调节蛋白78(GRP78),CCAAT/增强子结合蛋白同源蛋白(CHOP)及衰老蛋白组蛋白调节因子(HIRA),染色质组装和调节复合因子1a(ASF1a)的影响。方法:采用Hulth法建立KOA模型大鼠,模型成功后采用酶原消化法培养正常软骨细胞与KOA软骨细胞;另将40只大鼠分为独活寄生汤低、中、高剂量组(0.85,1.7,3.4 g·kg~(-1))及阳性药组(硫酸氨基葡萄糖胶囊3 g·kg~(-1)),灌胃14 d后处死取血清;将培养细胞分为空白组、模型组、独活寄生汤含药血清低、中、高剂量组(对应剂量组大鼠血清培养)及阳性药组(阳性药组大鼠血清培养);采用活性检测试剂盒(CCK-8)法检测各组软骨细胞增值率;采用TUNEL法检测各组软骨细胞凋亡情况;蛋白质免疫印迹(Western blot)检测各组软骨细胞中ASF1a,HIRA,GRP78及CHOP蛋白的表达。结果:与空白组软骨细胞比较,模型组细胞增殖抑制率明显升高,与模型组细胞比较,独活寄生汤含药血清低、中、高剂量组及阳性药组软骨细胞增殖抑制率降低(P<0.05);与空白组软骨细胞比较,模型组细胞凋亡数量明显增加(P<0.05),与模型组软骨细胞比较,独活寄生汤低、中、高剂量组及阳性药组细胞凋亡数量明显降低(P<0.05);与空白组软骨细胞比较,模型组细胞中ASF1a,HIRA,GRP78及CHOP蛋白表达明显升高(P<0.05),与模型组软骨细胞比较,独活寄生汤低、中、高剂量组及阳性药组细胞中ASF1a,HIRA,GRP78及CHOP蛋白表达降低(P<0.05)。结论:独活寄生汤含药血清可通过减少KOA软骨细胞内质网应激相关因子GRP78,CHOP及衰老相关因子ASF1a,HIRA的表达,从而起到抑制KOA软骨细胞衰老、凋亡,促进软骨细胞再生的作用,进而起到治疗KOA的目的。
OBJECTIVE: To observe the apoptosis of articular chondrocytes induced by anti-knee osteoarthritis (KOA) in rats with drug-containing serums of Duhuo Shisheng Tang, the expression of endoplasmic reticulum stress related protein glucose regulated protein 78 (GRP78), CCAAT / enhancer binding protein (CHOP) and senescent histone regulators (HIRA), chromatin assembly and regulatory factor 1a (ASF1a). Methods: KOA model rats were established by Hulth method. After the successful model, normal chondrocytes and KOA chondrocytes were cultured by zymogen. Forty rats were divided into two groups: low, medium and high dose of live alone soup (0.85,1.7 , 3.4 g · kg ~ (-1)) and the positive drug group (3 g · kg ~ (-1) glucosamine sulfate capsules). The cells were sacrificed after 14 days and the cells were sacrificed. The cultured cells were divided into blank group, model group (CCK-8) method was used to detect the expression levels of IL-6, IL-6, IL-6, IL- Group chondrocytes proliferation rate. The apoptosis of chondrocytes in each group was detected by TUNEL method. The expressions of ASF1a, HIRA, GRP78 and CHOP protein in chondrocytes were detected by Western blot. Results: Compared with the blank group, the inhibition rate of proliferation in the model group was significantly higher than that in the blank group (P <0.05). Compared with the model group, the inhibitory rate of proliferation of the chondrocytes in the low, medium, <0.05). Compared with the chondrocytes in the blank group, the number of apoptotic cells in the model group increased significantly (P <0.05). Compared with the chondrocytes in the model group, (P <0.05). Compared with the chondrocytes of the blank group, the expressions of ASF1a, HIRA, GRP78 and CHOP in the model group were significantly increased (P <0.05). Compared with the chondrocytes in the model group, , High dose group and positive drug group ASF1a, HIRA, GRP78 and CHOP protein expression decreased (P <0.05). Conclusion: The serum of Daohuayisheng decoction can reduce the senescence and apoptosis of KOA chondrocytes and promote the regeneration of chondrocytes by decreasing the expression of endoplasmic reticulum stress-related factors GRP78, CHOP and aging-related factors ASF1a and HIRA in KOA chondrocytes Role, and then play the purpose of treatment of KOA.