目的:研究桂枝芍药知母汤(GSZD)调控PI3K/AKT/mTOR信号轴干预类风湿关节炎(rheumatoid arthritis，RA)大鼠自噬治疗RA的作用机制。方法:将60只大鼠随机分为正常组、模型组、GSZD低、中、高剂量和雷帕霉素组，除正常组外，其余各组均给予牛Ⅱ型胶原蛋白皮下注射建立RA模型，并给予相应药物干预4周。定期监测大鼠足垫厚度并进行关节炎症指数(arthritis index，AI)评分。4周后，取膝关节滑膜组织，采用HE染色观察病理形态变化;透射电镜观察滑膜细胞超微结构变化;qRT-PCR和 Western blot 分别检测 Beclin1、LC3、p62、PI3K、AKT、mTOR mRNA及蛋白表达。结果:GSZD 可有效减轻RA大鼠的足垫厚度、AI评分;透射电镜结果显示RA大鼠滑膜细胞自噬减弱，GSZD治疗后细胞自噬溶酶体和自噬小体数量增加;qRT-PCR和Western blot结果显示，与正常组比较，模型组大鼠滑膜组织Beclin1 mRNA及蛋白、LC3 mRNA及LC3Ⅱ/LC3Ⅰ表达显著降低(P＜0。01)，p62、PI3K、AKT、mTOR mRNA及蛋白表达显著升高(P＜0。01);与模型组比较，GSZD高剂量组和雷帕霉素组大鼠滑膜组织Beclin1 mRNA及蛋白、LC3 mRNA及LC3Ⅱ/LC3Ⅰ表达显著升高(P＜0。05，P＜0。01)，p62、PI3K、AKT、mTOR mRNA及蛋白表达显著降低(P＜0。05，P＜0。01)。结论:GSZD可改善RA大鼠足垫厚度和病理形态，其治疗机制可能与下调PI3K/AKT/mTOR通路，促进自噬有关。
Mechanism of Guizhi Shaoyao Zhimu Decoction on autophagy in rats with rheumatoid arthritis based upon PI3K/AKT/mTOR signaling axis
OBJECTIVE To elucidate the mechanism of Guizhi Shaoyao Zhimu Decoction(GSZD)on autophagy in a rat model of rheumatoid arthritis(RA)through regulating the signaling axis of PI3K/AKT/mTOR.METHODS Sixty rats were randomized into six groups of normal,model,GSZD low/medium/high-dose and rapamycin.Except for normal group,the other groups were subcutaneously injected with bovine type Ⅱ collagen protein for establishing a RA model.The corresponding drugs were dosed for 4 weeks.Footpad thickness was measured and arthritis index(AI)scored regularly.Four weeks later,synovial tis-sues of rat knee joint were harvested.The pathological changes of synovial tissues were observed after hematoxylin-eosin(HE)stain.Ultrastructural changes of synovial cell were observed by transmission electron microscopy.The expressions of Beclin1,LC3,p62,PI3K,AKT and mTOR mRNAs and proteins were detected by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.RESULTS GSZD could effectively alleviate footpad thickness and AI score in RA rats.The results of transmission electron microscopy indicated that synoviocytes had lower autophagy while the number of autolysosomes and autophagosomes jumped after GSZD dosing.The results of qRT-PCR and Western blot indicated that,as compared with nor-mal group,the expressions of Beclin1 mRNA and protein,LC3 mRNA and LC3Ⅱ/LC3Ⅰ were down-regulated in synovial tissues(P＜0.01)while the expressions of p62,PI3K,AKT,mTOR mRNAs and proteins rose sharply in model group(P＜0.01).As compared with model group,the expressions of Beclin1mRNA and protein,LC3mRNA and LC3Ⅱ/LC3Ⅰ spiked markedly in synovial tissues(P＜0.01,P＜0.05)while the expressions of p62,PI3K,AKT,mTOR mRNAs and proteins dropped in GSZD high-dose and rapamycin groups(P＜0.05,P＜0.01).CONCLUSION GSZD could attenuate footpad thickness and pathomor-phism of RA rats.And its therapeutic mechanism may be correlated with a down-regulation of PI3K/AKT/mTOR pathway and an acceleration of autophagy.
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