阳和汤调节磷脂酰肌醇-3-激酶/蛋白激酶B信号通路抑制骨关节炎软骨退变的机制

Study on the Mechanism of Yanghe Decoction Regulating PI3K/AKT Signaling Pathway and Inhibiting Cartilage Degeneration in Osteoarthritis

目的:基于PI3K/AKT/BCL-2/BAX信号通路探讨阳和汤对膝骨关节炎的作用及相关机制。方法:将24只新西兰大白兔随机分为空白组、木瓜蛋白酶组、阳和汤组和塞来昔布组共四组,每组各6只。其中除空白组外均采用膝关节腔注射木瓜蛋白酶建立骨关节炎模型。在造模结束后,阳和汤组和塞来昔布组分别喂药阳和汤和西药塞来昔布治疗,1次/d,连续4周,空白组和模型组则不进行干预。采用苏木精-伊红(HE)染色观察骨关节炎兔膝关节软骨层次结构的形态变化,并行改良Mankin's评分。Western Blot和RT-qPCR分别检测软骨PI3K、AKT、BCL-2、BAX的蛋白和mRNA表达。结果:改良Mankin's评分比较,与空白对照组相比,模型组评分较空白组显著升高(P<0.01),阳和汤组和塞来昔布组较模型组显著降低,差异有统计学意义(P<0.01)。RT-qPCR与Western Blot结果显示,与空白组比较,模型组显著下调PI3K、AKT、BCL-2mRNA和蛋白的表达(P<0.01),同时上调BAX mRNA和蛋白的表达,差异有统计学意义(P<0.01);与模型组比较,阳和汤组和塞来昔布组上调了PI3K、AKT、BCL-2mRNA和蛋白的表达,差异有统计学意义(P<0.05),下调BAX mRNA和蛋白的表达,差异有统计学意义(P<0.05)。其中,模型组、阳和汤组、塞来昔布组的BCL-2/BAX蛋白表达比值显著低于空白组,差异有统计学意义(P<0.01),阳和汤组、塞来昔布组BCL-2/BAX蛋白表达比值显著高于模型组,差异有统计学意义(P<0.01)。结论:阳和汤能修复软骨损伤,抑制关节软骨退行性改变,延缓骨关节炎病程进展,其机制可能与激活PI3K/AKT信号通路,促进BCL-2的表达,抑制BAX表达,上调BCL-2/BAX值,进而抑制软骨细胞凋亡有关。

Objective:To explore the efficacy and related mechanism of Yanghe decoction on knee osteoarthritis based on PI3K/AKT/BCL2/BAX signaling pathway.Methods:24New Zealand white rabbits were randomly divided into 4groups:blank group,model group,Yanghe decoction group and celecoxib group with 6rabbits in each group.Except the blank group,the osteoarthritis model was established by injecting papain into the knee joint cavity.After modeling,the Yanghe decoction group and the celecoxib group were given Yanghe decoction and western medicine celecoxib respectively once a day for 4consecutive weeks,while the blank group and the model group were not intervened.HE staining was used to observe the morphological changes of cartilage hierarchy in the knee joint of rabbits with osteoarthritis,and Mankin's scores was modified.The protein and mRNA expressions of cartilage PI3K,AKT,BCL-2and BAX were detected by Western Blot and RT-qPCR.Results:Compared with the blank group,the Mankin's scores of the model group was significantly higher(P<0.01).Compared with the model group,the Mankin's scores of the Yanghe decoction group and the celecoxib group was significantly lower(P<0.01).RT-qPCR and Western Blot results:Compared with the blank group,the expressions of PI3K,AKT,BCL-2mRNA and protein in the model group were significantly lower(P<0.01),and the expressions of BAX mRNA and protein were significantly higher(P<0.01).Compared with the model group,the expressions of PI3K,AKT,BCL-2mRNA and protein in Yanghe decoction group and celecoxib group were significantly higher(P<0.05),and the expressions of BAX mRNA and protein were significantly lower(P<0.05).Among them,the protein expression ratio of BCL-2/BAX in the model group,Yanghe decoction group and celecoxib group was significantly lower than that in the normal group(P<0.01),and the BCL-2/BAX protein expression in the Yanghe decoction group and celecoxib group was significantly lower than that in the normal group(P<0.01).The ratio of protein expression was significantly higher than that in the model group(P<0.01).Conclusion:Yanghe decoction can repair cartilage damage,inhibit the degenerative changes of articular cartilage,and delay the progression of osteoarthritis.Its mechanism may be related to activating PI3K/AKT signaling pathway,promoting the expression of BCL-2,inhibiting the expression of BAX,and up-regulating BCL-2/BAX value,thereby inhibiting chondrocyte apoptosis.