山豆根水提物对类风湿关节炎骨破坏及PI3K/Akt信号通路的影响

Effect of Aqueous Extract of Sophorae Tonkinensis Radix et Rhizoma on Bone Destruction and PI3K/Akt Signaling Pathway in Rheumatoid Arthritis

目的:观察山豆根水提物对类风湿关节炎(RA)的干预影响,并基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路研究其抗RA骨破坏的作用及机制。方法:采用高效液相色谱(HPLC)测定山豆根水提物中主要活性物质的含量,应用Ⅱ型胶原诱导法建立类风湿关节炎模型(CIA)小鼠,设正常组、模型组、甲氨蝶呤组(0.5 mg·kg^(-1))、山豆根水提物低、高剂量组(100、200 mg·kg^(-1))。观察CIA小鼠关节红肿畸形表现及关节炎临床积分;苏木素-伊红(HE)染色观察小鼠关节组织病理改变;微计算机断层扫描技术(Micro-CT)检测小鼠关节骨破坏程度;抗酒石酸酸性磷酸酶(TRAP)染色观察小鼠关节组织中破骨细胞形成情况;蛋白免疫印迹法(Western blot)检测小鼠关节组织中PI3K/Akt信号通路关键蛋白表达。结果:与正常组比较,模型组小鼠关节红肿畸形程度、关节炎临床积分、滑膜增生及炎性细胞浸润程度显著升高(P<0.01),关节组织骨破坏程度、破骨细胞形成数目及基质金属蛋白酶-9(MMP-9)、组织蛋白酶K(CTSK)、活化T细胞核因子1蛋白(NFATc1)、PI3K和磷酸化(p)-Akt蛋白表达均显著升高(P<0.01)。与模型组比较,山豆根水提物各剂量组能明显改善CIA小鼠关节畸形程度及关节炎临床积分(P<0.05,P<0.01),缓解小鼠关节组织病理改变(P<0.01),且降低小鼠关节骨破坏程度和破骨细胞形成情况(P<0.05,P<0.01),此外还显著下调小鼠关节组织中PI3K/Akt信号通路相关蛋白表达(P<0.01)。结论:山豆根水提物能够显著延缓RA炎症反应,尤其抑制骨破坏,其作用机制可能与下调PI3K/Akt信号通路有关。

Objective:To observe the effect of aqueous extract of Sophorae Tonkinensis Radix et Rhizoma(STRR)on rheumatoid arthritis(RA)and to explore the anti-bone destruction mechanism based on phosphatidylinositol 3-kinase(PI3K)/serine/threonine-protein kinase(Akt) pathway.Method:Highperformance liquid chromatography(HPLC)was used to determine the content of main active components in aqueous extract of STRR,and type Ⅱ collagen to induce RA(CIA)in mice.The blank group,model group,methotrexate(MTX)group(0.5 mg·kg^(-1)),and low-dose(100 mg·kg^(-1))and high-dose(200 mg·kg^(-1))STRR aqueous extract groups were designed.Joint swelling was observed and clinical scores of CIA mice were calculated.Pathological changes of mouse joints were observed based on hematoxylin and eosin(HE)staining,and Micro-CT was performed to monitor joint destruction.TRAP staining was used to observe osteoclast formation in mouse joint,and Western blot to detect the expression of key proteins in PI3K/Akt signaling pathway in mouse joint tissue.Result:The model group demonstrated higher degree of joint swelling,clinical scores of CIA,and degrees of synovial hyperplasia,inflammatory cell infiltration(P<0.01),and joint destruction,more osteoclasts,and higher levels of matrix metallopeptidase-9(MMP-9),cathepsin K(CTSK),nuclear factor of activated T-cells cytoplasmic 1(NFATc1),PI3K,and phosphorylated-Akt(p-Akt)proteins than the blank group(P<0.01).Compared with the model group,the low-dose and high-dose aqueous extract of STRR alleviated joint swelling,reduced the clinical scores of CIA mice(P<0.05,P<0.01),relieved the pathological changes of joint tissue(P<0.01)and joint destruction,decreased osteoclasts(P<0.05,P<0.01),and lowered the levels of PI3K/Akt signaling pathway-related proteins in joint tissue of mice(P<0.01).Conclusion:The aqueous extract of STRR can significantly delay the inflammatory response of RA and especially inhibit bone destruction by down-regulating the PI3K/Akt signaling pathway.