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目的:观察独活寄生汤含药血清对膝骨性关节炎(KOA)大鼠关节软骨细胞代谢、骨形成蛋白-7(BMP-7)及沉默信息调节因子相关酶1(SIRT1)的影响,为独活寄生汤的临床应用补充理论依据。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))及硫酸氨基葡萄糖组(硫酸氨基葡萄糖胶囊0.3 g·kg~(-1)),灌胃14 d后处死取血清;将培养细胞分为正常组、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)及硫酸氨基葡萄糖胶囊组(硫酸氨基葡萄糖组大鼠血清培养);采用结晶紫法检测各组软骨细胞增殖能力;采用荧光原位末端转移酶标记法(TUNEL)法检测各组软骨细胞凋亡情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测各组软骨细胞中基质金属蛋白酶-13(MMP-13),MMP-3,Ⅱ型胶原蛋白(ColⅡ),聚集蛋白聚糖(Aggrecan),BMP-7及SIRT1 mRNA的表达;蛋白质印迹法(Western blot)检测各组软骨细胞中MMP-13,MMP-3,ColⅡ,Aggrecan,BMP-7及SIRT1的表达。结果:(1)与正常组软骨细胞比较,模型组细胞增殖能力明显降低,与模型组细胞比较,独活寄生汤含药血清低、中、高剂量组及硫酸氨基葡萄糖组软骨细胞增殖能力提高(P<0.01);(2)与正常组软骨细胞比较,模型组细胞凋亡数量明显增加,与模型组软骨细胞比较,独活寄生汤低、中、高剂量组及硫酸氨基葡萄糖组细胞凋亡数量明显降低(P<0.01);(3)与正常组软骨细胞比较,模型组细胞MMP-13,MMP-3表达升高,ColⅡ,Aggrecan,BMP-7及SIRT1下降(P<0.01);与模型组比较,低剂量组细胞中MMP-13,MMP-3 mRNA及蛋白表达无明显变化;硫酸氨基葡萄糖组及中、高剂量组细胞中MMP-13,MMP-3 mRNA及蛋白表达明显下降,且剂量越高上述蛋白及mRNA表达越低;硫酸氨基葡萄糖组及低、中、高剂量组细胞中ColⅡ,Aggrecan,BMP-7及SIRT1表达升高,且随剂量的增高而升高(P<0.01)。结论:独活寄生汤含药血清可通过增加KOA软骨细胞内BMP-7及SIRT1的表达,增加KOA软骨细胞合成代谢、抑制分解代谢,从而起到抑制KOA软骨细胞凋亡,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 effect of serum of Dafu Xie Sheng Tang on metabolism of articular chondrocytes, bone morphogenetic protein-7 (BMP-7) and SIRT1 in knee osteoarthritis (KOA) rats. Clinical application of independence and living parasitic soup supplementary theoretical basis. Methods: KOA model rats were established by Hulth method. After the successful model, KOA chondrocytes were cultured by zymogen. Forty rats were divided into low, middle and high dosage groups (0.85,1.7,3.4 g · kg -1), and glucosamine sulfate group (glucosamine sulfate 0.3 g · kg -1). The mice were sacrificed after 14 days of operation. The cultured cells were divided into normal group, model group, The serum containing chondrocytes in the low, medium and high dose groups (serum in the corresponding dose group) and glucosamine sulfate group (serum in the glucosamine sulfate group) were detected by crystal violet method; The apoptosis of chondrocytes in each group was detected by TUNEL method. The expression of matrix metalloproteinase-13 (MMP-13) in each group was detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) MMP-3, ColⅡ, Aggrecan, BMP-7 and SIRT1 mRNA were detected by Western blot. The expressions of MMP-13 and MMP- 3, ColⅡ, Aggrecan, BMP-7 and SIRT1 expression. Results: (1) Compared with normal group, the proliferation of model group was significantly lower than that of normal group. Compared with model group, the proliferation ability of chondrocytes in low, medium, high dose group and DDP group increased P <0.01). (2) Compared with normal chondrocytes, the number of apoptotic cells in model group increased significantly. Compared with chondrocytes in model group, the number of apoptotic cells in low, middle, high dose and independent groups (P <0.01). (3) Compared with the normal group, the expression of MMP-13 and MMP-3 in the model group increased, while ColⅡ, Aggrecan, BMP-7 and SIRT1 decreased The mRNA and protein expressions of MMP-13 and MMP-3 in the low-dose group were not significantly different. The expressions of MMP-13 and MMP-3 mRNA and protein were significantly decreased in the cells treated with glucosamine sulfate and medium- The higher the dose was, the lower the expression of the above protein and mRNA was. The expression of ColⅡ, Aggrecan, BMP-7 and SIRT1 in glucosamine sulfate group and low, medium and high dose groups increased and increased with increasing dose (P <0.01 ). CONCLUSION: The drug serum of Dovestroemia Decoction can increase the expression of BMP-7 and SIRT1 in KOA chondrocytes and increase the anabolism and catabolism of KOA chondrocytes, thereby inhibiting the apoptosis of KOA chondrocytes and promoting the regeneration of chondrocytes. , And then play the purpose of treatment of KOA.