独活寄生汤含药血清对膝骨性关节炎大鼠关节软骨细胞凋亡及GRP78，CHOP，HIRA及ASFla表达的影响

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(P0.05);与空白组软骨细胞比较,模型组细胞凋亡数量明显增加(P0.05),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(P0.05);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(P0.05),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(P0.05)。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。

Effect of Serum Containing Duhuo Jisheng Tang on Expressions of GRP78, CHOP, HIRA and ASFla and Apoptosis and in Articular Chondrocytes of Rats with Knee Osteoarthritis

Objective: To observe the effect of serum containing Duhuo Jisheng Tang for rat knee osteoarthritis (KOA) on articular chondrocyte apoptosis, endoplasmic reticulum stress related proteins glucoseregulated protein 78(GRP78),CCAAT/enhace-binding protein homologous protein(CHOP) and anti-aging proteins HIRA, ASF1a. Method: Hulth method was adopted to establish the rat KOA model. After successful modeling, enzyme digestion method was applied in primary culture of normal chondrocytes and KOA cartilage cells; Forty rats were divided into for groups: low, middle and high-dose groups(0.85, 1.7, 3.4g·kg^-1) and positive control group (glucosamine sulfate, 3 g·kg^-1). After 14 days of intragastric administration, serum was collected. Cultured cells were divided into control group, model group, Duhuo Jisheng Tang-containing serum groups (high, middle and low-dose groups) and positive control group; cell counting kit-8(CCK-8)method was used to test the cell proliferation of each group; TUNEL method was used to measure the cell apoptosis of each group. The expressions of ASF1a, HIRA, GRP78 and CHOP in chondrocytes were detected by Western blot. Result: Compared with the control group, the inhibition rate of cell proliferation was significantly increased in the model group; compared with the model group, the proliferation inhibition rates of chondrocytes in the low, middle, high-dose Duhuo Jisheng Tang groups and the positive control group were significantly decreased (P<0.05); compared with the control group, the number of apoptotic cells in the model group was significantly increased; compared with the model group, the apoptotic rates of chondrocytes in the low, middle, high-dose Duhuo Jisheng Tang groups and the positive control group were significantly decreased (P<0.05). Compared with the control group, the expressions of ASF1a, HIRA, GRP78 and CHOP in the model group were significantly higher than those in the control group; and compared with the model group, the expressions of ASF1a, HIRA, GRP78 and CHOP in chondrocytes of the low, middle, high-dose Duhuo Jisheng Tang groups and the positive control group were significantly decreased (P<0.05). Conclusion: The serum containing Duhuo Jisheng Tang could reduce the expressions of GRP78, CHOP and ASF1a, HIRA in KOA chondrocytes, inhibit the aging and apoptosis of KOA chondrocytes, and promote the regeneration of chondrocytes, so as to treat KOA.