基于TLR4/MyD88/NF-κB信号通路探讨左归丸、右归丸影响骨关节炎模型的作用机制研究  

Study on Mechanism of Zuogui Pill(左归丸)and Yougui Pill(右归丸)on Osteoarthritis Model Based on TLR4/MyD88/NF-κB

作　　者：金连峰[1] 顾炜[1] 刘子闻 马楠 JIN Lianfeng;GU Wei;LIU Ziwen;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》2023年第11期28-33,I0011,共7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项目(201602482);沈阳市科技计划项目(18013083);辽宁中医药大学自然科学类重点项目(2021LZY052)。

摘　　要：目的利用细胞、分子生物学技术,采用蛋白免疫印迹(Western blot,WB)、实时荧光定量反转录聚合酶链反应(Real-time PCR,RT-PCR)、酶联免疫吸附测定(Enzyme linked immunosorbent assay,Elisa)检测方法,观察左归丸与右归丸对大鼠膝骨关节炎(Knee osteoarthritis,KOA)软骨细胞、韧带组织的作用机制及细胞凋亡的影响,并讨论左归丸组与右归丸治疗膝骨关节炎的作用机制。方法选取8月龄的无特定病原体(Specific pathogen free,SPF)级别Wistar大鼠40只(250±20 g),雌鼠与雄鼠各20只,使用数字随机法分组,分为正常组、模型对照组、左归丸组、右归丸组、硫酸氨基葡萄糖组,每组各8只大鼠(雌雄各4只),造模方法为膝关节腔注射0.1 mL浓度为10%的木瓜蛋白酶溶液,每隔2 d造模1次,共进行3次造模,每次造模后驱赶大鼠30 min,用此方法对模型对照组、左归丸组、右归丸组、硫酸氨基葡萄糖组大鼠进行模型复制。模型复制成功后分别对5组大鼠进行相应干预。连续治疗4周后,所有大鼠取腹主动脉血后处死,Elisa法检测各组大鼠血清白细胞介素(IL)-1β表达水平,取出膝关节软骨及韧带组织,RT-PCR法与WB法检测软骨及韧带组织中toll样受体4(Toll-like receptor 4,TLR4)、IκB激酶(IKK)蛋白及mRNA的表达。剩余膝关节组织HE染色检测软骨组织情况。采用SPSS 22.0软件包对检测结果进行方差分析,P<0.05差异有统计学意义。结果(1)膝关节病理切片结果:经左、右归丸治疗后KOA炎症减轻。(2)RT-PCR检测结果:软骨和韧带组织中,较模型组对比左归丸组与右归丸组TLR4、IKK表达含量明显减少(P<0.05)。(3)WB检测结果:软骨和韧带组织中,较模型组对比左归丸组与右归丸组TLR4、IKK蛋白含量明显降低(P<0.05)。(4)Elisa法检测结果:各组大鼠血清中,左归丸组与右归丸组IL-1β表达水平明显降低(P<0.05)。结论左归丸与右归丸可通过抑制TLR4/髓样分化因子88(Myeloid differeObjective To observe the mechanism of Zuogui Pilll(左归丸)group and Yougui Pill(右归丸)group on chondrocytes and ligament tissue of rats with knee osteoarthritis and the effect of apoptosis by using cellular and molecular biological techniques and real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR),Western blot(WB)and enzyme linked immunosorbent assay(ELISA).The mechanism of Zuogui Pill group and Yougui Pill group in treating knee osteoarthritis was discussed.Methods Forty 8-month-old SPF grade Wistar rats[(250±20)g],20 female and 20 male,were randomly divided into normal group,model control group,Zuogui Pill group,Yougui Pill group and glucosamine sulfate group,with 8 rats in each group(4 male and 4 female).The modeling method was to inject 0.1 mL of Papain solution with a concentration of 10%into the knee joint cavity,and the model was made every two days,three times in total.The rats were driven out for 30 minutes after each modeling,and the rats in the model control group,Zuogui Pill group,Yougui Pill group and glucosamine sulfate group were replicated with this method.After the successful replication of the model,the corresponding intervention was carried out on the rats of the five groups.After 4 weeks of continuous treatment,all rats were killed after abdominal aortic blood was taken out,and serum interleukin-1β(IL-1β)was detected by ELISA.The expression levels of Toll-like receptor 4(TLR4)and IκB kinase(IKK)protein in the cartilage and ligament of knee joint were detected by RT-PCR and Western blot.The cartilage tissue was detected by HE staining in the remaining knee joint tissues.SPSS 22.0 software package was used to analyze the variance of the test results,with statistical significance(P<0.05).Results(1)The results of pathological section of knee joint:The symptoms of knee osteoarthritis were alleviated after treatment with Zuogui Pill and Yougui Pill.(2)RT-PCR results:Compared with those of the model group,the expressions of TLR4 and IKK in the cartila

关 键 词：左归丸 右归丸 骨关节炎 TLR4/MyD88/NF-κB 信号通路 

分 类 号：R285.5[医药卫生—中药学]