新止骨增生丸基于miR-146a对TGF-β/SMAD信号转导通路下TGF-β1、SMAD4表达的影响及治疗骨性关节炎的作用机制研究  

Effect of New Zhigu Zengsheng Pill(新止骨增生丸)on Expressions of TGF-β1 and SMAD4 under TGF-β/SMAD Signal Transduction Pathway Based on miR-146a and Mechanism of Action in Treatment of Osteoarthritis

作　　者：金连峰[1] 顾炜[1] 马楠 JIN Lianfeng;GU Wei;MA Nan(Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning,China;Liaoning University of Traditional Chinese Medicine,Shenyang 110847,Liaoning,China)

机构地区：[1]辽宁中医药大学附属医院,辽宁沈阳110032 [2]辽宁中医药大学,辽宁沈阳110847

出　　处：《中华中医药学刊》2022年第2期141-145,I0030,共6页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(81574050);辽宁省自然科学基金(201602482);沈阳市科技计划(18013083)。

摘　　要：目的利用细胞、分子生物学技术,采用实时荧光定量反转录聚合酶链反应(Real-time PCR,RT-PCR)、蛋白免疫印迹(Western Blot,WB)检测方法,观察新止骨增生丸对大鼠膝骨关节炎软骨细胞的作用机制及细胞凋亡的影响,并讨论新止骨增生丸治疗膝骨关节炎的作用机制。方法SPF级6月龄大鼠24只,随机分为两组,空白组6只;模型复制组18只。模型复制组大鼠采用前后交叉韧带离断术复制骨性关节炎大鼠模型,模型复制成功后,将改组大鼠再次随机分为3组:模型组(6只),中药组(6只),西药组(6只)。连续治疗6周后,取整个膝关节,分离软骨组织。膝关节病理切片HE染色法观察各组软骨细胞状态,采用RT-PCR法检测各组大鼠膝关节中miR-146a的表达水平,采用WB法检测各组大鼠膝关节软骨组织中SMAD4和TGF-β1表达,采用SPSS 16.0软件包对RT-PCR法和WB法检测结果进行方差分析,P<0.05有统计学意义。结果(1)膝关节病理切片结果:经新止骨增生丸治疗后KOA症状减轻。(2)RT-PCR检测结果:模型组较空白组的miR-146a表达明显上调(P<0.05);中药组较模型组的miR-146a表达明显上调(P<0.05);西药组较模型组对比无统计学意义(P>0.05)。(3)WB检测结果:模型组较空白组的SMAD4、TGF-β1表达水平明显上调(P<0.05);中药组较模型组SMAD4、TGF-β1蛋白表达水平均呈现明显上调趋势(P<0.05)。结论新止骨增生丸能够通过miR-146a对TGF-β/SMAD信号通路的调控,达到治疗膝骨性关节炎的效果。Objective By use of cell and molecular biology techniques,real-time fluorescent quantitative reverse transcription polymerase chain reaction(Real-time PCR,RT-PCR)and protein immunoblot(Western Blot,WB)detection method,to observe the mechanism of New Zhigu Zengsheng Pill(新止骨增生丸)on knee osteoarthritis cartilage cells of rats and the effect of cell apoptosis,and discuss the mechanism of New Zhigu Zengsheng Pill in the treatment of knee osteoarthritis.Methods Twenty-four SPF 6 months old rats were randomly divided into two groups and 6 blank groups.Model replication group had 18 rats.The rats in the model replication group were replicated the rat model of osteoarthritis by anterior and posterior cruciate ligament dissection.After successful replication of the model,the reconstituted rats were randomly divided into 3 groups again:model group(6 rats),Traditional Chinese medicine group(6 rats)and Western medicine group(6 rats).After 6 weeks of continuous treatment,the whole knee was taken out and the cartilage tissue was separated.The expression levels of miR-146 a in the knee joint of rats in each group were detected by RT-PCR,the expression levels of SMAD4 and TGF-β1 in the knee joint cartilage of rats in each group were detected by WB,and the results of RT-PCR and WB were analyzed by SPSS 16.0 software package,and the value of P less than 0.05 was statistically significant.Results(1)Pathological section of the knee:KOA symptoms were alleviated after treatment with New Zhigu Zengsheng Pill.(2)RT-PCR detection results:The miR-146 a expression was significantly up-regulated in the model group compared with that of the blank group(P<0.05).The expression of miR-146 a in the traditional Chinese medicine group was significantly up-regulated compared with that of the model group(P<0.05).There was no statistically significant difference between the western medicine group and the model group(P>0.05).(3)Western Blot results:The expression levels of SMAD4 and TGF-β1 in the model group were significantly up-regulate

关 键 词：新止骨增生丸 膝骨关节炎 TGF-Β/SMAD 信号通路 

分 类 号：R285.5[医药卫生—中药学] R274.984.3[医药卫生—中医学]