藤黄健骨胶囊对膝骨关节炎鼠骨代谢指标及PI3K/Akt/Nrf2/HO-1信号通路的影响

Effects of Tenghuang Jiangu Capsule on Bone Metabolism and PI3K/Akt/Nrf2/HO-1 Signaling Pathway in Rats with Knee Osteoarthritis

目的 探讨藤黄健骨胶囊对膝骨关节炎鼠骨代谢指标及PI3K/Akt/Nrf2/HO-1信号通路的影响。方法 采用木瓜蛋白酶构建膝骨关节炎大鼠(KOA)模型,将造模成功的40只大鼠按随机数字表法分为模型组、阳性药物组、低剂量组和高剂量组,每组各10只。未造模的10只大鼠作为对照组。建模4周后,阳性药物组、低剂量组和高剂量组分别以美洛昔康水溶液、0.09 g/kg以及0.36 g/kg藤黄健骨胶囊干预,模型组和对照组以等量生理盐水灌胃,1次/d。给药4周后,观察比较各组大鼠Mankin评分及关节肿胀程度,酶联免疫吸附(Enzyme-linked immunosorbent assay,ELISA)检测白介素-1β(Interleukin 1β,IL-1β)、肿瘤坏死因子-α(Tumor necrosis factor alpha,TNF-α)、金属基质蛋白酶3(Metallomatrix proteinase 3,MMP3)、组织金属蛋白酶抑制因子-1(Tissue metalloproteinase inhibitor 1,TIMP-1)、骨钙素(Bone gamma-carboxyglutamic-acid-containing proteins,BGP)、抗洒石酸酸性磷酸酶(Tartrate-resistant acid phosphatase,TRACP)、I型胶原C端肽(Type I collagen carboxy-terminal peptide,CTX),Western blot法检测软骨组织PI3K/Akt/Nrf2/HO-1信号通路蛋白表达情况。结果 HE染色结果显示,对照组大鼠关节面、关节软骨和软骨细胞均正常;而模型组关节面呈不规则,关节软骨含少量软骨细胞或软骨细胞坏死,且关节软骨被纤维组织取代阳性药物组和高剂量组关节表面不规则,软骨细胞数量减少,而低剂量组显示软骨细胞中度坏死。与对照组比较,模型组Mankin评分、关节肿胀程度均升高,差异有统计学意义(P<0.05);与模型组比较,阳性药物组、高剂量组、低剂量组Mankin评分、关节肿胀程度均明显降低,差异有统计学意义(P<0.05);且各药物组Mankin评分及关节肿胀程度比较,阳性药物组<高剂量组<低剂量组,差异有统计学意义(P<0.05)。与对照组比较,模型组IL-1β、TNF-α、MMP-3、TIMP-1水平均升高,差异有统计学意义(P<0.05);与模型组比较,阳性药物组、高剂量组、低剂量组IL-1β、TNF-α、MMP-3、TIMP-1水平均明显降低,差异有统计学意义(P<0.05);且各药物组IL-1β、TNF-α、MMP-3、TIMP-1水平比较,阳性药物组<高剂量组<低剂量组,差异有统计学意义(P<0.05)。与对照组,模型组TRACP、CTX水平均升高,BGP水平降低,差异有统计学意义(P<0.05);与模型组比较,阳性药物组、高剂量组、低剂量组TRACP、CTX水平明显降低,BGP水平明显升高,差异有统计学意义(P<0.05);且各药物组TRACP、CTX水平比较,阳性药物组<高剂量组<低剂量组,BGP水平比较,阳性药物组>高剂量组>低剂量组,差异有统计学意义(P<0.05)。与对照组比较,模型组PI3K、Akt、Nrf2、HO-1蛋白水平降低,差异有统计学意义(P<0.05);与模型组比较,阳性药物组、高剂量组、低剂量组PI3K、Akt、Nrf2、HO-1蛋白水平明显升高,差异有统计学意义(P<0.05);且各药物组PI3K、Akt、Nrf2、HO-1蛋白水平比较,阳性药物组>高剂量组>低剂量组,差异有统计学意(P<0.05)。结论 藤黄健骨胶囊可显著改善膝骨关节炎鼠骨代谢水平,缓解膝骨关节炎症状,其可能机制与激活PI3K/Akt/Nrf2/HO-1信号通路有关。

Objective To investigate the effects of Tenghuang Jiangu Capsule on bone metabolism and PI3K/Akt/Nrf2/HO-1 signaling pathway in rats with knee osteoarthritis(KOA).Methods A rat model of KOA was constructed with papain,and 40 successfully modeled rats were randomly divided into model group,positive drug group,Tenghuang Jiangu Capsule low-dose group and high-dose group according to random number table method,with 10 rats in each group.At the same time,10 untreated rats were as assigned to control group.After 4 weeks of modeling,the rats in positive drug group,Tenghuang Jiangu Capsule low-dose group and high-dose group were treated with meloxicam aqueous solution,0.09 g/kg and 0.36 g/kg Tenghuang Jiangu Capsule,respectively,and the rats in model group and control group were administrated with the same amount of normal saline once a day.After 4 weeks of administration,Mankin scores and joint swelling were compared among all groups.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of interleukin1β(IL-1β),tumor necrosis factor α(TNF-α),metallomatrix proteinase 3(MMP3),tissue metalloproteinase inhibitor 1(TIMP-1),osteocalcin(BGP),tartrate-resistant acid phosphatase(TRACP)and C-terminal telopeptide of type I collagen(CTX)in the five groups.The protein expression of PI3K/Akt/Nrf2/HO-1 signaling pathway in cartilage tissues was detected by Western blot.Results HE staining showed that the articular surface,articular cartilage and chondrocytes were normal in the control group.In the model group,the articular surface was irregular,with a small amount of chondrocytes or chondrocyte necrosis,and the articular cartilage was replaced by fiber tissue.The articular surface was irregular and the number of chondrocytes was reduced in the positive drug group and the Tenghuang Jiangu Capsule high-dose group,while the Tenghuang Jiangu Capsule low-dose group showed moderate chondrocyte necrosis.Compared with control group,Mankin score and joint swelling degree were significantly increased in model group(P<0.05).Compared with model group,Mankin score and joint swelling degree were significantly decreased in positive drug group,Tenghuang Jiangu Capsule low-dose group and high-dose group(P<0.05).The comparison of Mankin score and joint swelling degree among different drug treatment groups showed the positive drug grouP< Tenghuang Jiangu Capsule high-dose grouP< Tenghuang Jiangu Capsule low-dose group,and the difference was statistically significant(P<0.05).Compared with control group,the levels of IL-1β,TNF-α,MMP-3 and TIMP-1 in model group were significantly increased(P<0.05).Compared with model group,the levels of IL-1β,TNF-α,MMP-3 and TIMP-1 in positive drug group,Tenghuang Jiangu Capsule high-dose group and low-dose group were significantly decreased(P<0.05).The comparison of levels of IL-1β,TNF-α,MMP-3 and TIMP-1 among all drug treatment groups showed the positive drug grouP<Tenghuang Jiangu Capsule high-dose grouP<Tenghuang Jiangu Capsule low-dose group,and the difference was statistically significant(P<0.05).Compared with control group,TRACP and CTX levels in model group were significantly increased,while BGP levels were significantly decreased(P<0.05).Compared with model group,TRACP and CTX levels in positive drug group,Tenghuang Jiangu Capsule high-dose group and low-dose group were significantly decreased,while BGP levels were significantly increased(P<0.05).The comparison of TRACP and CTX levels among all drug treatment groups showed positive drug grouP<Tenghuang Jiangu Capsule high-dose grouP<Tenghuang Jiangu Capsule low-dose group.The comparison of BGP level showed positive drug group>Tenghuang Jiangu Capsule high-dose group>Tenghuang Jiangu Capsule low-dose group,and the difference was statistically significant(P<0.05).Compared with control group,the protein levels of PI3K,Akt,Nrf2 and HO-1 in model group were significantly decreased(P<0.05).Compared with model group,the protein levels of PI3K,Akt,Nrf2 and HO-1 in positive drug group,Tenghuang Jiangu Capsule high-dose group and low-dose group were significantly increased(P<0.05).Moreover,the protein levels of PI3K,Akt,Nrf2 and HO-1 among all drug treatment groups were compared,and the protein levels were higher in positive drug group than those in Tenghuang Jiangu Capsule high-dose group,which were higher than those in Tenghuang Jiangu Capsule low-dose group,with statistical significant difference(P<0.05).Conclusion Tenghuang Jiangu Capsule can significantly improve bone metabolism and alleviate the symptoms of KOA in rats,which may be related to the activation of PI3K/Akt/Nrf2/HO-1 signaling pathway.