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. 2021 Apr 20;41(4):483-494.
doi: 10.12122/j.issn.1673-4254.2021.04.02.

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

[Article in Chinese]
B Bao  1 , J Liu  1 , L Wan  1 , Y Zhang  1 , Y Long  1 , G Sun  1
Affiliations

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

[Article in Chinese]
B Bao et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To observe the inhibitory effect of Xinfeng capsule (XFC) on immune inflammatory response in the coculture system of chondrocytes and CD4+ T cells from patients with osteoarthritis (OA).

Objective: 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.

Objective: 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).

Objective: 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.

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。

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

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Figures

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1
OA患者通路相关指标的表达水平 Expression level of pathway-related indexes in patients with OA.
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OA患者细胞因子的表达水平 Expression level of cytokines in patients with OA.
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OA患者通路指标与相关指标的相关性分析 Correlation analysis of pathway indexes and related indexes in patients with OA.
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治疗前后通路指标的变化 Changes of pathway indexes before and after treatment.
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治疗前后细胞因子的变化 Changes of cytokines before and after treatment.
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hsa-miR-23a-3p与h-PTEN-3UTR靶位点结合示意图 Schematic diagram of binding of hsa-miR-23a-3p and H-PTEN-3UTR target sites.
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双荧光素酶报告基因检测hsa-miR-23a-3p与h-PTEN-3UTR相互作用 Interaction between hsa-miR-23a-3p and H-PTEN-3UTR was detected by dual luciferase reporter genes. ***P < 0.001.
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不同剂量含药血清在不同时间点对OA-CD4+T细胞联合OA-CH的影响 Effects of different doses (0.2、0.4、0.8 mg/100 g) of drug-containing serum on OA-CD4+T cells combined with OA-CH at different time points. **P < 0.01 vs the low-dose group during the same time.
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L-1β、IL-6、IL-10、TNF-α表达水平 Expression levels of IL-1β, IL-6, IL-10, and TNF-α. **P < 0.01 vs the control1、control2、model group, ##P < 0.01 the control1.
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miR-23a-3p及PI3K/AKT/mTOR通路的表达水平 Expression levels of miR-23a-3p and PI3K/AKT/mTOR pathway. **P < 0.01 vs control1, control2, and model group; ##P < 0.01 vs control1.
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IL-1β、IL-6、IL-10、TNF-α表达水平 Expression levels of IL-1β, IL-6, IL-10, and TNF-α. **P < 0.01, *P < 0.05 vs Model、Mimics-NC、mimics group; ##P < 0.01, #P < 0.05 vs Model group.
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过表达miR-23a-3p及PI3K/AKT/mTOR通路的表达水平 Expression levels of miR-23a-3p and PI3K/AKT/mTOR pathway. **P < 0.01, *P < 0.05 vs Model, Mimics-NC, and mimics groups; ##P < 0.01, #P < 0.05. **P < 0.01 vs Model group.
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PTEN/PI3K/AKT/mTOR通路蛋白表达水平 Expression levels of PTEN/PI3K/ Akt/mTOR pathway related proteins. The latter group was compared to the former group, **P < 0.01, *P < 0.05; ##P < 0.01, #P < 0.05 vs the model group.
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