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

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Chinese Journal of Integrative Medicine Aims and scope Submit manuscript

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 (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<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 (P<0.01).

Conclusion

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

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Authors and Affiliations

  1. Department of Traditional Chinese Medicine, Zunyi Medical and Pharmaceutical College, Zunyi, Guizhou Province, 563006, China

    Dou Ding, Qi Zhang, Fu-jia Zeng, Ming-xing Cai, Yuan Gan & Xiao-jun Dong

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  1. Dou Ding
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  2. Qi Zhang
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  3. Fu-jia Zeng
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  4. Ming-xing Cai
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  5. Yuan Gan
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  6. Xiao-jun Dong
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Contributions

Ding D wrote the paper, Dong XJ applied for funds, Zhang Q designed the experiments, Zeng FJ finished the experiments, Cai MX analyzed the data, Gan Y submitted the manuscript, and all authors approved the submitted manuscript.

Corresponding author

Correspondence to Xiao-jun Dong.

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Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Supported by a grant from Zunyi Science and Technology Bureau, China (No. [2019]194)

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Ding, D., Zhang, Q., Zeng, Fj. et al. Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis. Chin. J. Integr. Med. 29, 508–516 (2023). https://doi.org/10.1007/s11655-022-3723-4

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  • DOI: https://doi.org/10.1007/s11655-022-3723-4

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