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. 2022 Nov 20;42(11):1726-1731.
doi: 10.12122/j.issn.1673-4254.2022.11.18.

[Cheng's Juanbi Decoction enhances autophagy in rheumatoid arthritis fibroblast-like syn-oviocytes by suppressing the PI3K/Akt/mTOR signal axis]

[Article in Chinese]
G Sun  1 , X Xu  1 , L Wan  2 , S Nan  1 , Y Wang  1 , L Zhao  1 , H Cheng  3 , K Wang  3 , Y Liu  1 , Y Fang  2 , L Sun  1 , J Zhu  1
Affiliations

[Cheng's Juanbi Decoction enhances autophagy in rheumatoid arthritis fibroblast-like syn-oviocytes by suppressing the PI3K/Akt/mTOR signal axis]

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

Abstract
in English, Chinese

Objective: To study the regulatory effect of Cheng's Juanbi Decoction (JBT) on autophagy in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and role of PI3K/Akt/mTOR signaling axis in the mechanism mediating this effect.

Methods: CCK8 assay was used to determine the optimal concentration and treatment time of JBT for inhibiting the viability of RA- FLS. The effect of freeze-dried powder of JBT, RAPA, or both on morphology of the autophagosomes in RA-FLS was observed under transmission electron microscope, and the changes in the number of autophagosomes and autolysosomes were observed with autophagy double-labeled adenovirus experiment. RT-qPCR and Western blotting were used to detect the expression levels of the related indicators.

Results: The results of CCK8 assay showed that treatment with 0.5 mg/mL JBT for 12 h produced the optimal effect for inhibiting RA-FLS viability. Observation with transmission electron microscope and the results of the autophagy double-labeled adenovirus experiment both showed the presence of a small number of autophagosomes in control RA-FLS group, and treatment with JBT significantly increased the number of autophagosomes and lowered the number of autophagolysosomes in the cells. Compared with the control cells and the cells treated with JBT or RAPA alone, the cells treated with both JBT and RAPA showed significantly decreased mRNA levels of PI3K, Akt and mTOR (P < 0.01) but without significant changes in their protein expressions (P > 0.05); the combined treatment significantly inhibited the protein expressions of p-PI3K, p-Akt, p-mTOR, and P62 (P < 0.05) and upregulated the protein expressions of Beclin-1 and LC3B (P < 0.05) in the cells.

Conclusion: JBT can inhibit the survival rate of RA-FLS and increase the level of autophagy possibly through a mechanism that down-regulates PI3K/Akt/mTOR signaling pathway.

目的: 研究程氏蠲痹汤（JBT）通过PI3K/Akt/mTOR信号轴调控类风湿关节炎滑膜成纤维细胞（RA-FLS）自噬的影响及相关机制。

方法: 实验分为4组：FLS组、FLS+JBT组、FLS+RAPA组、FLS+RAPA+JBT组。CCK8检测程氏蠲痹汤抑制RA-FLS的最佳浓度和时间，透射电镜观察细胞自噬小体的变化，自噬双标腺病毒实验观察细胞内自噬体、自噬溶酶体数量，RT-qPCR和Western blot检测相关指标表达水平。

结果: CCK8实验筛选结果表明JBT冻干粉浓度在0.5 mg/mL，培养12 h效果最佳。电镜结果与自噬双标腺病毒实验结果趋势相同，FLS组中自噬体少量存在，JBT加入能显著增加自噬体，自噬溶酶体较少。RT-qPCR结果显示，FLS+RAPA+JBT组较FLS组、FLS+JBT组、FLS+RAPA组相比，PI3K、Akt和mTOR的mRNA水平降低更明显（P＜0.01）。WB分析显示，FLS+RAPA+JBT组较FLS组、FLS+JBT组、FLS+RAPA组相比，PI3K、Akt、mTOR蛋白的水平无明显差异（P＞0.05），p-PI3K、p-Akt、p-mTOR、P62受到了明显抑制（P＜0.05）。FLS+RAPA+JBT组较FLS组、FLS+JBT组、FLS+RAPA组相比，Beclin-1、LC3B蛋白表达量明显上升（P＜0.05）。

结论: 程氏蠲痹汤可抑制RA-FLS的存活率，并可提高细胞自噬水平，其机制可能与下调PI3K/Akt/mTOR信号通路有关。

Keywords: Cheng's Juanbi Decoction; PI3K/Akt/mTOR; autophagy; fibroblast-like synoviocytes.

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Figures

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1
以25%为倍数筛选JBT最佳剂量 Determination of the optimal JBT concentration for cell treatment.
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电镜观察自噬体变化 Transmission electron microscopy for observing autophagosome changes in RA-FLS with different treatment. formula image indicates the autophagosomes, formula image the mitochondria, and formula image the rough endoplasmic reticulum. A: FLS group. B: FLS+JBT group. C: FLS+RAPA group. D: FLS+RAPA+JBT group.
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mRFP-GFP-LC3腺病毒检测自噬流情况 mRFP-GFP-LC3 adenovirus for detecting autophagic flow (Original magnification: ×600). The yellow dots indicate autophagosomes, and the red dots indicate autophagolysosomes in the merged images. A: FLS group. B: FLS+JBT group. C: FLS+RAPA group. D: FLS+RAPA+JBT group.
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PI3K/Akt/mTOR相关基因的表达 Expression of mRNAs of PI3K/Akt/mTOR-related genes. *P < 0.05, **P < 0.01 vs FLS group. ##P < 0.01 vs FLS + RAPA+JBT group.
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PI3K/Akt/mTOR通路相关蛋白表达水平 PI3K/Akt/mTOR pathway-related protein expression levels detected by Western blotting. A: FLS group. B: FLS+JBT group. C: FLS+RAPA group. D: FLS+RAPA+ JBT group. *P < 0.05, **P < 0.01 vs FLS group. #P < 0.05, ##P < 0.01 vs FLS+RAPA+JBT group.
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P62、Beclin-1、LC3B蛋白表达水平 Expression levels of P62, Beclin-1, and LC3B proteins in the FLS. A: FLS group. B: FLS+JBT group. C: FLS+RAPA group. D: FLS+RAPA+JBT group. *P < 0.05, **P < 0.01 vs FLS group; #P < 0.05, ##P < 0.01 vs FLS+RAPA+JBT group.
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