基于miR-19b-3p/RAF1轴研究龙胆酸对类风湿性关节炎的作用机制

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

doi:10.1007/s11655-022-3723-4

Your Location：

Home >

Browse articles >

基于miR-19b-3p/RAF1轴研究龙胆酸对类风湿性关节炎的作用机制

Original Article | Updated：2023-05-25

- 基于miR-19b-3p/RAF1轴研究龙胆酸对类风湿性关节炎的作用机制
- Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis
- 中国结合医学杂志（英文版） 2023年29卷第6期页码：508-516
- 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：
  
  a grant from Zunyi Science and Technology Bureau, China([2019]194)
- DOI：10.1007/s11655-022-3723-4
  中图分类号：
- 纸质出版日期：2023-06，
  
  网络出版日期：2022-10-17，
  
  录用日期：2022-05-24
Scan for full text
基于miR-19b-3p/RAF1轴研究龙胆酸对类风湿性关节炎的作用机制[J]. 中国结合医学杂志（英文版）, 2023,29(6):508-516.

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, 2023,29(6):508-516.
基于miR-19b-3p/RAF1轴研究龙胆酸对类风湿性关节炎的作用机制[J]. 中国结合医学杂志（英文版）, 2023,29(6):508-516. DOI： 10.1007/s11655-022-3723-4.

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, 2023,29(6):508-516. 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

references

Kong XY, Wen CP. On research progress of Western and Chinese medicine treatment on pre-rheumatoid arthritis. Chin J Integr Med 2019; 25:643-647.

Yang M, Guo MY, Luo Y, Yun MD, Yan J, Liu T, et al. Effect of artemisia annua extract on treating active rheumatoid arthritis: a randomized controlled trial. Chin J Integr Med 2017; 23:496-503.

Rezaeepoor M, Pourjafar M, Tahamoli-Roudsari A, Basiri Z, Hajilooi M, Solgi G. Altered expression of microRNAs may predict therapeutic response in rheumatoid arthritis patients. Int Immunopharmacol 2020; 83:106404.

Tavasolian F, Hosseini AZ, Soudi S, Naderi M. miRNA-146a improves immunomodulatory effects of MSC-derived exosomes in rheumatoid arthritis. Curr Gene Ther 2020; 20:297-312.

Lenert A, Fardo DW. Detecting novel microRNAs in rheumatoid arthritis with gene-based association testing. Clin Exp Rheumatol 2017; 35:586-592.

Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK. Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients. Arthritis Res Ther 2008; 10: R101.

Dong L, Wang X, Tan J, Li H, Qian W, Chen J, et al. Decreased expression of microRNA-21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis. J Cell Mol Med 2014; 18:2213-2224.

Wang Y, Zheng F, Gao G, Yan S, Zhang L, Wang L, et al. MiR-548a-3p regulates inflammatory response via TLR4/NF-κB signaling pathway in rheumatoid arthritis. J Cell Biochem 2018. DOI: 10.1002/jcb.26659http://doi.org/10.1002/jcb.26659

Jin S, Chen H, Li Y, Zhong H, Sun W, Wang J, et al. Maresin 1 improves the Treg/Th17 imbalance in rheumatoid arthritis through miR-21. Ann Rheum Dis 2018; 77:1644-1652.

Romo-García MF, Bastian Y, Zapata-Zuñiga M, Macías-Segura N, Castillo-Ortiz JD, Lara-Ramírez EE, et al. Identification of putative miRNA biomarkers in early rheumatoid arthritis by genome-wide microarray profiling: a pilot study. Gene 2019; 720:144081.

Nishikawa M, Myoui A, Tomita T, Takahi K, Nampei A, Yoshikawa H. Prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogen-activated protein kinase inhibitor FR167653. Arthritis Rheum 2003; 48:2670-2681.

Rubbert-Roth A. New kinase inhibitors. Z Rheumatol 2012; 71:479-484.

Joshi R, Gangabhagirathi R, Venu S, Adhikari S, Mukherjee T. Antioxidant activity and free radical scavenging reactions of gentisic acid: in-vitro and pulse radiolysis studies. Free Radic Res 2012; 46:11-20.

Han X, Guo J, Gao Y, Zhan J, You Y, Huang W. Gentisic acid prevents diet-induced obesity in mice by accelerating the thermogenesis of brown adipose tissue. Food Funct 2021; 12:1262-1270.

Roseman S, Dorfman A. Effect of gentisic acid and related compounds on bovine testicular hyaluronidase. J Biol Chem 1952; 199:345-355.

Zhang Q, Peng W, Wei S, Wei D, Li R, Liu J, et al. Guizhi-Shaoyao-Zhimu Decoction possesses anti-arthritic effects on type Ⅱ collagen-induced arthritis in rats via suppression of inflammatory reactions, inhibition of invasion & migration and induction of apoptosis in synovial fibroblasts. Biomed Pharmacother 2019; 118:109367.

Shen Y, Teng L, Qu Y, Liu J, Zhu X, Chen S, et al. Anti-proliferation and anti-inflammation effects of corilagin in rheumatoid arthritis by downregulating NF-κB and MAPK signaling pathways. J Ethnopharmacol 2022; 284:114791.

Wang J, Yan S, Yang J, Lu H, Xu D, Wang Z. Non-coding RNAs in rheumatoid arthritis: from bench to bedside. Front Immunol 2019; 10:3129.

McInnes IB, Schett G. Pathogenetic insights from the treatment of rheumatoid arthritis. Lancet 2017; 389:2328-2337.

Kim M, Kim J, Shin YK, Kim KY. Gentisic acid stimulates keratinocyte proliferation through ERK1/2 phosphorylation. Int J Med Sci 2020; 17:626-631.

Abedi F, Razavi BM, Hosseinzadeh H. A review on gentisic acid as a plant derived phenolic acid and metabolite of aspirin: comprehensive pharmacology, toxicology, and some pharmaceutical aspects. Phytother Res 2020; 34:729-741.

Evangelatos G, Fragoulis GE, Koulouri V, Lambrou GI. MicroRNAs in rheumatoid arthritis: from pathogenesis to clinical impact. Autoimmun Rev 2019; 18:102391.

Metzinger-Le Meuth V, Burtey S, Maitrias P, Massy ZA, Metzinger L. microRNAs in the pathophysiology of CKD-MBD: biomarkers and innovative drugs. Biochim Biophys Acta Mol Basis Dis 2017; 1863:337-345.

Chen JQ, Papp G, Szodoray P, Zeher M. The role of microRNAs in the pathogenesis of autoimmune diseases. Autoimmun Rev 2016; 15:1171-1180.

Chen XM, Zhao Y, Wu XD, Wang MJ, Yu H, Lu JJ, et al. Novel findings from determination of common expressed plasma exosomal microRNAs in patients with psoriatic arthritis, psoriasis vulgaris, rheumatoid arthritis, and gouty arthritis. Discov Med 2019; 28:47-68.

Malemud CJ. Negative regulators of JAK/STAT signaling in rheumatoid arthritis and osteoarthritis. Int J Mol Sci 2017; 18:484.

Lin K, Su HY, Jiang LF, Chu TG, Li Z, Chen XL, et al. Influences of miR-320a on proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis through targeting MAPK-ERK1/2. Eur Rev Med Pharmacol Sci 2019; 23:1907-1914.

Lechuga CG, Simón-Carrasco L, Jacob HK, Drosten M. Genetic validation of cell proliferation via ras-independent activation of the RAf/MEK/ERK pathway. Methods Mol Biol 2017; 1487:269-276.

Li R, Cai L, Tang WJ, Lei C, Hu CM, Yu F. Apoptotic effect of geniposide on fibroblast-like synoviocytes in rats with adjuvant-induced arthritis via inhibiting ERK signal pathway in vitro. Inflammation 2016; 39:30-38.

More>

浏览量

Downloads

CSCD

文章被引用时，请邮件提醒。

Submit

工具集

关联资源

Isorhamnetin Downregulates MMP2 and MMP9 to Inhibit Development of Rheumatoid Arthritis through SRC/ERK/CREB Pathway

Prevalence and Factors Associated with Concomitant Chinese Medicine Use by Rheumatoid Arthritis Patients in A Multi-Ethnic Asian Population

Inokosterone Is a Potential Drug Target of Estrogen Receptor 1 in Rheumatoid Arthritis Patients:Analysis from Active Ingredient of Cyathula Officinalis

Deciphering Potential Correlations between New Biomarkers and Pattern Classification in Chinese Medicine by Bioinformatics:Two Examples of Rheumatoid Arthritis*

Chinese Herbal Formula Ermiao Powder (二妙散) Regulates Cholinergic Anti-inflammatory Pathway in Rats with Rheumatoid Arthritis