Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis

DING Dou; ZHANG Qi; ZENG Fu-jia; CAI Ming-xing; GAN Yuan; DONG Xiao-jun

doi:10.1007/s11655-022-3723-4

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Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis

Original Article | Updated：2023-05-25

- Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis
- Chinese Journal of Integrative Medicine Vol. 29, Issue 6, Pages: 508-516(2023)
- Affiliations：
  
  Department of Traditional Chinese Medicine, Zunyi Medical and Pharmaceutical College, Zunyi (563006), Guizhou Province, China
- Author bio：
  
  Prof. DONG Xiao-jun, E-mail: dongxiaojun8787@126.com
- Funds：
- DOI：10.1007/s11655-022-3723-4
  CLC：
- Published：2023-06，
  
  Published Online：17 October 2022，
  
  Accepted：24 May 2022
Scan for full text
DING Dou, ZHANG Qi, ZENG Fu-jia, et al. Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis. [J]. Chinese Journal of Integrative Medicine 29(6):508-516(2023)
DOI：

DING Dou, ZHANG Qi, ZENG Fu-jia, et al. Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis. [J]. Chinese Journal of Integrative Medicine 29(6):508-516(2023) DOI： 10.1007/s11655-022-3723-4.

摘要

目的:

本研究基于miR-19b-3p/RAF1轴探讨龙胆酸对类风湿性关节炎的治疗作用.

方法:

采用CCK-8法检测不同浓度龙胆酸 (0、1、5、10、50和100 mmol/L) 对MH7A细胞的生长抑制作用

并确定龙胆酸的IC50值. 采用qRT-PCR检测miR-19b-3p和RAF1 mRNA的表达. Western blotting检测RAF1、ERK1/2和p-ERK1/2蛋白表达. 采用三种方法(双荧光素酶法、qRT-PCR和western blot)验证miR-19b-3p和RAF1的靶向关系. 流式细胞术检测MH7A细胞的凋亡情况. Transwell法和划痕法测定MH7A细胞的侵袭和迁移能力.

结果:

随着龙胆酸浓度的增加

MH7A细胞的生长逐渐受到抑制. 当龙胆酸浓度超过80 mmol/L时

龙胆酸对MH7A细胞具有明显的细胞毒性

因此

GA对MH7A细胞的一半最大抑制浓度 (IC50值) 为67.019 mmol/L. 与对照组相比

龙胆酸上调miR-19b-3p表达

下调RAF1表达

抑制ERK1/2磷酸化

诱导MH7A细胞凋亡

抑制MH7A细胞侵袭和迁移(

0.05或

0.01). RAF1被确定为miR-19b-3p的靶点

能逆转龙胆酸对miR-19b-3p表达的抑制作用(

0.05或

0.01). miR-19b-3p抑制剂上调RAF1表达

促进ERK1/2磷酸化

抑制MH7A细胞凋亡

诱导MH7A细胞侵袭和迁移(

0.01).

结论:

龙胆酸通过调控miR-19b-3p/RAF1轴介导ERK通路

抑制类风湿性关节炎的发生发展.

Abstract

Objective:

To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis.

Methods:

The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells

and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1

extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay

qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells.

Results:

The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L

GA was significantly cytotoxic to MH7A cells

so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression

downregulated RAF1 expression

inhibited ERK1/2 phosphorylation

induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (

0.05 or

0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (

0.05 or

0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation

suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (

0.01).

Conclusion:

GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA.

关键词

Keywords

gentisic acidrheumatoid arthritismiR-19b-3pRAF1MAPK/ERK

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