Sun G, Xu X, Wan L, Nan S, Wang Y, Zhao L, Cheng H, Wang K, Liu Y, Fang Y, Sun L, Zhu J
School of Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, China.
First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Nov 20;42(11):1726-1731. doi: 10.12122/j.issn.1673-4254.2022.11.18.
To study the regulatory effect of 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.
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.
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 ( < 0.01) but without significant changes in their protein expressions ( > 0.05); the combined treatment significantly inhibited the protein expressions of p-PI3K, p-Akt, p-mTOR, and P62 ( < 0.05) and upregulated the protein expressions of Beclin-1 and LC3B ( < 0.05) in the cells.
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.
研究健脾补肾汤（JBT）对类风湿关节炎成纤维样滑膜细胞（RA-FLS）自噬的调控作用及PI3K/Akt/mTOR信号轴在介导该作用机制中的作用。
采用CCK8法检测JBT抑制RA-FLS活力的最佳浓度和作用时间。透射电镜观察JBT冻干粉、雷帕霉素（RAPA）或二者联合对RA-FLS自噬体形态的影响，自噬双标腺病毒实验观察自噬体和自溶酶体数量变化。采用RT-qPCR和蛋白质免疫印迹法检测相关指标的表达水平。
CCK8法结果显示，0.5 mg/mL JBT作用12 h对抑制RA-FLS活力效果最佳。透射电镜观察及自噬双标腺病毒实验结果均显示，对照RA-FLS组细胞中有少量自噬体，JBT处理显著增加细胞中自噬体数量并减少自溶酶体数量。与对照细胞及单独用JBT或RAPA处理的细胞相比，JBT与RAPA联合处理的细胞PI3K、Akt和mTOR的mRNA水平显著降低（P<0.01），但其蛋白表达无显著变化（P>0.05）；联合处理显著抑制细胞中p-PI3K、p-Akt、p-mTOR和P62的蛋白表达（P<0.05），上调Beclin-1和LC3B的蛋白表达（P<0.05）。
JBT可能通过下调PI3K/Akt/mTOR信号通路抑制RA-FLS的存活率并提高自噬水平。
