基于核因子-κB信号通路分析补肾活血祛湿中药治疗膝关节骨性关节炎的作用机制

Analysis in mechanism of tradition Chinese medicine for tonifying kidney,activating blood circulation and removing dampness in treatment of knee osteoarthritis based on nuclear factor-κB signal pathway

目的基于核因子-κb(NF-κB)信号通路分析补肾活血祛湿中药治疗膝关节骨性关节炎(KOA)的作用机制。方法对模型对照组、地塞米松组、补肾活血祛湿中药各剂量组(中药低剂量组、中药高剂量组)大鼠采用前交叉韧带横断术(ACLT)建立KOA模型。正常对照组大鼠无任何操作;地塞米松组、中药低剂量组、中药高剂量组在造模成功第1天开始给予相应剂量(依次为50、50、100 mg/kg)的药物灌胃,持续给予4周;正常对照组和模型对照组给予等体积生理盐水。实验结束后,测定大鼠缩爪热潜伏期(PWTL)、Mankin′s评分。采用酶联免疫吸附法测定血清白细胞介素-4(IL-4)、白细胞介素-1b(IL-1b)和肿瘤坏死因子-α(TNF-α)水平;采用逆转录聚合酶链式反应(RT-PCR)法及蛋白印记法测定膝关节滑膜组织微小RNA-26a(miR-26a)、NF-κB、toll样受体4(TRL4)水平。结果与正常对照组比较,模型对照组PWTL缩短,Mankin′s评分(中药低剂量组除外)、IL-1b、IL-4、TNF-α以及miR-26a、NF-κB、TRL4的mRNA、蛋白表达水平延长,差异有统计学意义(P<0.05)。与模型对照组比较,中药低剂量组、中药高剂量组、地塞米松组PWTL延长,Mankin′s评分(中药低剂量组除外)、IL-1b、IL-4、TNF-α以及miR-26a、NF-κB、TRL4的mRNA、蛋白表达水平降低,且中药高剂量组PWTL长于中药低剂量组,Mankin′s评分、IL-1b、IL-4、TNF-α以及miR-26a、NF-κB、TRL4的mRNA、蛋白表达水平低于中药低剂量组,差异均有统计学意义(P<0.05)。与地塞米松组比较,中药低剂量组PWTL缩短,Mankin′s评分、IL-1b、IL-4、TNF-α以及miR-26a、NF-κB、TRL4的mRNA、蛋白表达水平升高,差异有统计学意义(P<0.05)。与地塞米松组比较,中药高剂量组PWTL轻微缩短,Mankin′s评分、IL-1b、IL-4、TNF-α以及miR-26a、NF-κB、TRL4的mRNA、蛋白表达水平轻微升高,差异均无统计学意义(P>0.05)。结论补肾活血祛湿中药能显著抑制KOA炎症反应,其机制可能与抑制miR-26a mRNA、NF-κB mRNA、TRL4 mRNA表达水平以及抑制miR-26a/NF-κB/TRL4通路的激活有关。

Objective To analyze the mechanism of tradition Chinese medicine for tonifying kidney,activating blood circulation and removing dampness in the treatment of knee osteoarthritis(KOA)based on nuclear factor-κB(NF-κB)signal pathway.Methods KOA model was established by anterior cruciate ligament transection(ACLT)in the rats of model control group,dexamethasone group,and groups with various doses of traditional Chinese medicine for tonifying the kidney,activating blood circulation and removing dampness(low dose of traditional Chinese medicine group and high dose of traditional Chinese medicine group).No operation was given for rats in the normal control group;the dexamethasone group,the low dose of traditional Chinese medicine group and the high dose of traditional Chinese medicine group were given corresponding doses of drugs for gavage(50,50 and 100 mg/kg)on the first day,and the gavage was lasted for 4 weeks;normal control group and model control group were given equal volume of normal saline.When the experiment was over,the paw withdrawal thermal latency(PWTL)and Mankin′s score of rats were measured.Levels of serum interleukin-4(IL-4),interleukin-1b(IL-1b)and tumor necrosis factor-α(TNF-α)were measured by enzyme-linked immunosorbent assay;the levels of microRNA-26a(miR-26a),NF-κB and toll-like receptor 4(TRL4)in synovial tissues of knee joints were determined by reverse transcriptase polymerase chain reaction(RT-PCR)and western blot.Results Compared with the normal control group,PWTL in the model control group shortened significantly,while the Mankin′s score,IL-1b,IL-4,TNF-αand mRNA and protein expression levels of miR-26a,NF-κB and TRL4 prolonged significantly(P<0.05).Compared with the model control group,PWTL prolonged significantly,while the Mankin′s score(except for the low dose of traditional Chinese medicine group),IL-1b,IL-4,TNF-αand mRNA and protein expression levels of miR-26a,NF-κB and TRL4 decreased significantly in the low dose of traditional Chinese medicine group,high dose of traditional Chinese medicine group and dexamethasone group,and PWTL in high dose of traditional Chinese medicine group was significantly longer than that in the low dose of traditional Chinese medicine group,while the Mankin′s score,IL-1b,IL-4,TNF-αand mRNA and protein expression levels of miR-26a,NF-κB and TRL4 were significantly lower than those in low dose of traditional Chinese medicine group(P<0.05).Compared with dexamethasone group,PWTL shortened significantly,while the Mankin′s score,IL-1b,IL-4,TNF-αand mRNA and protein expression levels of miR-26a,NF-κB and TRL4 increased significantly in low dose of traditional Chinese medicine group(P<0.05).Compared with dexamethasone group,PWTL shortened slightly while the Mankin′s score,IL-1b,IL-4,TNF-αand mRNA and protein expression levels of miR-26a,NF-κB and TRL4 increased slightly in high dose of traditional Chinese medicine group,and there were no significant differences between two groups(P>0.05).Conclusion Tradition Chinese medicine for tonifying kidney,activating blood circulation and removing dampness can significantly inhibit the inflammatory response of KOA,and its mechanism may be related to the inhibition on expression levels of miR-26a mRNA,NF-κB mRNA and TRL4 mRNA,and inhibition on activation of miR-26a/NF-κB/TRL4 pathway.