南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (4): 483-494.doi: 10.12122/j.issn.1673-4254.2021.04.02

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新风胶囊通过 miR-23a-3p/PTEN/PI3K/AKT/mTOR 抑制骨关节炎CD4+T与软骨细胞共培养的免疫炎症

鲍丙溪，刘 健，万 磊，张 颖，龙 琰，孙广瀚   

  • 出版日期:2021-04-20 发布日期:2021-04-29

Xinfeng capsule inhibits immune inflammation in osteoarthritis by inhibiting the miR-23a-3p/PETN/PI3K/AKT/mTOR pathway

  • Online:2021-04-20 Published:2021-04-29

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摘要： 目的 观察新风胶囊（XFC）含药血清通过调控miR-23a-3p /PTEN/PI3K/AKT/mTOR信号通路抑制骨关节炎（OA）CD4+T与软骨细胞共培养中免疫炎症反应的作用机制。方法 选取30例OA患者（OA组）及30例正常人（NC组）检测miR-23a-3p、10号染色体同源丢失性磷酸酶-张力蛋白基因（PTEN）、磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶B（AKT）、哺乳动物雷帕霉素靶蛋白（mTOR）的表达，观察临床指标：红细胞沉降率（ESR）、超敏C反应蛋白（hs-CRP）、IgA、IgG、IgM、补体 C3、补体C4的表达，并观察通路与临床指标的相关性。观察 30 例 OA 患者经 XFC 治疗后通路及临床指标的变化。分离及提取 OA-CD4 +T 细胞，transwell小室培养OA-CD4+T细胞与OA-CH，SD大鼠给药灌胃制备XFC含药血清；采用CCK8法检测OA-软骨细胞的细胞增殖；双荧光素酶报告实验分析miR-23a-3p和PTEN的靶向关系；RT-PCR技术检测共培养细胞中miR-23a-3p、PTENmRNA、PI3KmRNA、AKTmRNA、mTORmRNA 的表达；采用 ELISA 法检测 IL-1β、IL-6、IL-10、TNF-α的水平；Western blotting 检测PTEN、PI3K、AKT、p-AKT、mTOR、p-mTOR蛋白表达。结果 与NC组相比，OA组miR-23a-3p、PTEN、PI3K、AKT、IL-1β、IL-6、TNF-α表达显著上调，而PTEN、IL-10表达显著下调（P<0.01）；相关性分析表明：miR-23a-3p与IL-6呈正相关（P<0.01），PI3K与IL-10呈正相关，mTOR与IL-6、IL-10、补体C3、补体C4呈正相关（P<0.01或P<0.05）；加入XFC含药血清，miR-23a-3p及PI3K/AKT/mTOR通路被抑制，L-1β、IL-6、TNF-α表达水平下降、IL-10表达水平升高（P<0.01）；Model组miR-23a-3p、PI3K、AKT、mTOR、IL-1β、IL-6和TNF-α表达水平升高，PTEN、IL-10表达水平降低（P<0.01）；miR-23a-3p经过表达后，miR-23a-3p、PI3K、AKT、mTOR、IL-1β、IL-6和TNF-α表达水平显著升高，PTEN、IL-10表达水平显著降低（P<0.01或P<0.05）；XFC组IL-1β、IL-6、TNF-α表达降低，IL-10表达升高，miR-23a-3p、PI3K、AKT、mTOR表达下调，PTEN表达上调（P<0.01或P<0.05）。结论 XFC可通过下调miR-23a-3p的表达，抑制PTEN/PI3K/AKT/mTOR通路的活化，改善IL-1β、IL-6，IL-10和TNF-α的表达，降低OA患者炎症反应。

关键词: 骨关节炎；新风胶囊；miR-23a-3p；PTEN/PI3K/AKT/mTOR；免疫炎症反应

Abstract: Objective To observe the inhibitory effect of Xinfeng capsule (XFC) on immune inflammatory response in the co-culture system of chondrocytes and CD4+ T cells from patients with osteoarthritis (OA). Methods Thirty OA patients and 30 healthy subjects were examined for the expression of miR-23a-3p/PTEN/PI3K/AKT/mTOR. The changes in the miR-23a-3p/PTEN/PI3K/AKT/mTOR pathway and the clinical indicators of the OA patients after XFC treatment were observed. OA-CD4+T cells and OA-Chondrocytes cells were cultured in Transwel chambers, and the expressions of miR-23a-3p, PTEN, PI3K, AKT and mTOR mRNA were detected by RT-PCR; the levels of IL-1β, IL-6, IL-10 and TNF-α were detected by ELISA. The protein expressions of PTEN, PI3K, AKT, p-AKT, mTOR and p- mTOR were detected by Western blotting. Results Compared with healthy individuals, OA patients showed significantly increased expressions of miR-23a-3p, PTEN, PI3K, AKT, IL-1β, IL-6 and TNF-α and lowered expressions of PTEN and IL-10 (P<0.01). Positive correlations were found between miR-23a-3p and IL-6 and between PI3K and IL-10; mTOR was positively correlated with IL-6, IL-10, complement C3 and C4 (P<0.01 or 0.05). Intervention with XFC obviously down-regulated the expressions of IL-1β, IL-6, and TNF-α and up-regulated the expression of IL-10 (P<0.01). In the cell co-culture systems, the expressions of miR-23a-3p, PI3K, Akt, mTOR, IL-1β, IL-6 and TNF-α were up-regulated while the expressions of PTEN and IL-10 were down-regulated in the model group (P<0.01). Overexpression of miR-23a-3p significantly up-regulated the expressions of IL-1β, IL-6, TNF-α, miR-23a-3p, PI3K, AKT and mTOR and lowered the expressions of PTEN and IL-10 (P<0.01 or 0.05). The expressions of IL-1β, IL-6, TNF-α, miR-23a-3p, PI3K, AKT and mTOR were down-regulated and the expressions of IL-10 and PTEN were up-regulated in XFC-treated cells (P<0.01 or 0.05). Conclusion XFC can reduce the inflammatory response in patients with OA by down-regulating the expression of miR-23a-3p, inhibiting the activation of the PTEN/PI3K/AKT/mTOR pathway, and regulating the expression of IL-1β, IL-6, IL-10 and TNF-α.

Key words: osteoarthritis; Xinfeng capsule; miR-23a-3p; PTEN/PI3K/AKT/mTOR; immuno-inflammatory responses