基于PI3K/Akt/mTOR通路探究马钱子总生物碱对类风湿性关节炎大鼠的作用及机制

Effect and Mechanism of Total Alkaloids from Strychnos nux-vomica on Rheumatoid Arthritis Rats Based on PI3K/Akt/mTOR Pathway

目的:分析马钱子总生物碱对类风湿性关节炎(RA)大鼠的作用及对磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶点(mTOR)通路的影响。方法:50只Wistar大鼠随机分为对照组、RA组、RA+马钱子总生物碱组(100 mg·kg^(-1))、RA+PI3K抑制剂组(20 mg·mL^(-1)LY294002)、RA+马钱子总生物碱+PI3K激活剂组[100 mg·kg^(-1)马钱子总生物碱+20μg·kg^(-1)胰岛素样生长因子1(IGF-1)],除对照组外,其余各组均构建RA模型并根据剂量给药,计算每只大鼠关节评分,测定大鼠血清抗瓜氨酸化蛋白抗体(ACPA)、丙二醛(MDA)水平;苏木精-伊红(HE)染色观察评估大鼠膝关节滑膜组织病理,比较各组Mankin评分、炎细胞浸润数、软骨厚度;测定滑膜组织白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)、核转录因子(NF)-κB受体活化因子配体(RANKL)水平;蛋白免疫印迹(Western blot)测定大鼠滑膜组织PI3K/Akt/mTOR通路相关蛋白表达。结果:1)对照组大鼠关节软骨组织结构正常,表面光滑完整,腔隙内组织有良好的完整软骨细胞;RA组关节表面广泛侵蚀,形态无规则,软骨细胞明显丢失,炎性细胞大量浸润,关节软骨厚度低,细胞间基质显著丢失,滑膜中单核炎性细胞浸润可见;RA+马钱子总生物碱组、RA+PI3K抑制剂组呈现保护性体征,关节表面侵蚀降低,炎性细胞浸润减轻,关节软骨厚度增加;与RA+马钱子总生物碱组相比,RA+马钱子总生物碱+PI3K激活剂组关节软骨损伤加重。2)与对照组相比,RA组大鼠关节炎症评分、血清ACPA及MDA水平、Mankin评分、炎细胞浸润数、滑膜组织IL-1β、IL-6、TNF-α、RANKL升高(P<0.05),软骨厚度降低(P<0.05);与RA组相比,RA+马钱子总生物碱组、RA+PI3K抑制剂组大鼠关节炎症评分、血清ACPA及MDA水平、Mankin评分、炎细胞浸润数、滑膜组织IL-1β、IL-6、TNF-α、RANKL降低(P<0.05),软骨厚度升高(P<0.05);与RA+马钱子总生物碱组相比,RA+马钱子总生物碱+PI3K激活剂组大鼠关节炎症评分、血清ACPA及MDA水平、Mankin评分、炎细胞浸润数、滑膜组织IL-1β、IL-6、TNF-α、RANKL升高(P<0.05),软骨厚度降低(P<0.05)。3)与对照组相比,RA组大鼠滑膜组织磷酸化(p)-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR升高(P<0.05);与RA组相比,RA+马钱子总生物碱组、RA+PI3K抑制剂组大鼠滑膜组织p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR降低(P<0.05);与RA+马钱子总生物碱组相比,RA+马钱子总生物碱+PI3K激活剂组大鼠滑膜组织p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR升高(P<0.05)。结论:马钱子总生物碱可能通过抑制PI3K/Akt/mTOR通路缓解RA大鼠炎症。

Objective:To analyze the effect of total alkaloids from Strychnos nux-vomica on rheumatoid arthritis(RA)rats and its impact on phosphoinositide 3-kinases(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway.Methods:Fifty Wistar rats were randomly divided into control group,RA group,RA+S.nux-vomica total alkaloids group(100 mg·kg^(-1)),RA+PI3K inhibitor group(20 mg·mL^(-1) LY294002),and RA+S.nux-vomica total alkaloids+PI3K activator group[100 mg·kg^(-1) total alkaloids from S.nux-vomica+20μg·kg^(-1) insulin-like growth factor 1(IGF-1)].In addition to the control group,RA models were established in all other groups and administration was performed according to the dose.The joint score of each rat was calculated,and the levels of serum anti-citrullinated protein antibody(ACPA)and malondialdehyde(MDA)were measured.Hematoxylin-eosin(HE)staining was applied to observe the histopathology of synovium of knee joint in rats,Mankin score,number of inflammatory cell infiltrated,and cartilage thickness were compared among different groups.The levels of interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α)and receptor activator of nuclear factor-κB ligand(RANKL)in synovial tissue were determined.Western blot was used to determine the ratio of PI3K/Akt/mTOR pathway related proteins in rat synovium.Results:In the control group,the structure of articular cartilage tissue was normal,and the surface was smooth and complete,with good,intact chondrocytes in the cavity.In RA group,the joint surface was extensively eroded,with irregular morphology,obvious loss of chondrocytes,massive infiltration of inflammatory cells,low thickness of articular cartilage,significant loss of intercellular matrix,and visible infiltration of mononuclear inflammatory cells in synovium.The RA+S.nux-vomica total alkaloids group and RA+PI3K inhibitor group showed protective signs,that is,decreased joint surface erosion,reduced inflammatory cell infiltration,and increased articular cartilage thickness.Compared with the condition in the RA+S.nux-vomica total alkaloids group,the injury of articular cartilage in RA+S.nux-vomica total alkaloids+PI3K activator group was aggravated.Compared with the control group,the RA group had elevated arthritis score,serum ACPA and MDA,Mankin score,number of inflammatory cell infiltrated,and IL-1β,IL-6,TNF-α,and RANKL in synovial tissue(P<0.05),while decreased cartilage thickness(P<0.05).Compared with the RA group,the RA+S.nux-vomica total alkaloids group and the RA+PI3K inhibitor group presented reduced serum ACPA and MDA,Mankin score,number of inflammatory cell infiltrated,and IL-1β,IL-6,TNF-α,and RANKL in synovial tissue(P<0.05),while increased cartilage thickness(P<0.05).The RA+S.nux-vomica total alkaloids+PI3K activator group was higher than the RA+S.nux-vomica total alkaloids group in serum ACPA and MDA,Mankin score,number of inflammatory cell infiltrated,and IL-1β,IL-6,TNF-α,and RANK in synovial tissue(P<0.05),while lower in cartilage thickness(P<0.05).In terms of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR in synovial tissue,the RA group was higher than the control group(P<0.05);the RA+S.nux-vomica total alkaloids group and the RA+PI3K inhibitor group were lower than the RA group(P<0.05);the RA+S.nux-vomica total alkaloids+PI3K activator group was higher than the RA+S.nux-vomica total alkaloids group(P<0.05).Conclusion:Total alkaloids from S.nux-vomica may alleviate inflammation in RA rats by inhibiting PI3K/Akt/mTOR pathway.