基于PI3K/Akt信号通路探讨祛风骨痛巴布膏对寒湿痹阻型骨关节炎模型大鼠的干预作用

Intervention Effect of Qufeng Gutong Babu Ointment on Rat Model of Osteoarthritis with Cold-dampness Obstruction Based on PI3K/Akt Signal Pathway

目的:探讨祛风骨痛巴布膏对寒湿痹阻型骨关节炎(OA)大鼠的干预作用,并初步阐明其作用机制。方法:将SD雄性大鼠分成6组,为正常组、模型组、活血止痛膏组(1.26 cm^(2)·d^(-1))、祛风骨痛巴布膏低、中、高剂量组(75、150、300 mg·d^(-1))。通过关节腔注射木瓜蛋白酶联合L-半胱氨酸制备OA模型,造模第2天开始施加气候因素,建立寒湿痹阻型OA大鼠病证结合动物模型。标准VonFrey纤维评价机械痛阈值;双后肢负重差评分及关节功能评分;苏木素-伊红(HE)染色和番红固绿染色观察大鼠膝关节组织病理变化和软骨退变;免疫组化法(IHC)检测白细胞介素(IL)-1β、白细胞介素(IL)-8、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶-9(MMP-9)和组织蛋白酶K(CTSK)的表达;蛋白免疫印迹法(Western blot)检测磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、T细胞的核因子1(NFATc1)、MMP-9和CTSK的蛋白表达水平。结果:与正常组比较,模型组在造模后出现了显著的机械痛敏反应(P<0.01),双后肢负重差和关节功能评分明显升高(P<0.05,P<0.01)。与模型组比较,祛风骨痛巴布膏高剂量组和活血止痛膏组均可明显降低大鼠机械痛敏反应、负重差和关节功能评分(P<0.05,P<0.01),且祛风骨痛巴布膏中剂量也可一定程度改善关节功能,大鼠膝关节软骨退变明显减轻(P<0.05,P<0.01);祛风骨痛巴布膏、活血止痛膏均在一定程度上抑制OA大鼠关节软骨中IL-1β、IL-8、TNF-α、MMP-9、CTAK、PI3K、p-Akt、Akt等相关蛋白表达(P<0.05,P<0.01)。结论:祛风骨痛巴布膏通过抑制寒湿痹阻型OA模型大鼠关节软骨中PI3K/Akt信号通路进而抑制炎性因子和基质金属蛋白酶的释放,最终减弱局部软骨退变,改善关节功能。

Objective:To investigate the intervention effect of Qufeng Gutong Babu ointment(QFGT)on rats with osteoarthritis(OA)with cold-dampness obstruction,and preliminarily clarify its mechanism.Method:SD male rats were divided into 6 groups,namely,the blank group,model group,positive control drug Huoxue Zhitong ointment(HXZTG)group(1.26 cm^(2)·d^(-1)),and low,medium,and high-dose QFGT group(75,150,300 mg·d^(-1)).OA model was prepared by joint cavity injection of papain and L-cysteine.On the second day of modeling,climate factors were applied to establish an animal model of combination of disease and syndrome of OA rats with cold-dampness obstruction.Standard VonFrey fiber was used to evaluate the threshold of mechanical pain.Weight bearing difference score and joint function score of both hind limbs were recorded.Hematoxylin-eosin(HE)staining and safranine fixation green staining were used to observe the pathological changes and cartilage degeneration of rat knee joint.Immunohistochemistry(IHC)was used to detect the expression of interleukin-1β(IL-1β),interleukin-8(IL-8),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-9(MMP-9),and cathepsin K(CTSK).Western blot was used to detect the protein expression of kinase B(Akt),phosphorylated protein kinase B(p-Akt),phosphatidylinositol 3-kinase(PI3K),nuclear factor 1(NFATc1),MMP-9,and CTSK in T cells.Result:Compared with the normal group,the model group showed significant mechanical pain sensitivity reaction after modeling(P<0.01),and the weight bearing difference of both hind limbs and joint function score were significantly increased(P<0.05,P<0.01).Compared with the model group,both the high-dose QFGT group and the HXZTG group significantly reduced the mechanical pain sensitivity,weight difference,and joint function score of rats(P<0.05,P<0.01),and the medium-dose QFGT group also improved the joint function to a certain extent,and the degeneration of the knee joint cartilage of rats was significantly reduced(P<0.05,P<0.01).QFGT and HXZTG both inhibited the protein expression of IL-1β,IL-8,TNF-α,MMP-9,CTAK,PI3K,p-Akt,Akt,and other related proteins in articular cartilage of rats with OA to a certain extent(P<0.05,P<0.01).Conclusion:QFGT can inhibit the release of inflammatory factors and matrix metalloproteinases by inhibiting the PI3K/Akt signal pathway in articular articular cartilage of rats with OA with cold-dampness obstruction,thus ultimately weakening local cartilage degeneration and improving joint function.