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Chinese Journal of Clinical Pharmacology and Therapeutics ›› 2023, Vol. 28 ›› Issue (12): 1331-1338.doi: 10.12092/j.issn.1009-2501.2023.12.002

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Emodin reduces the injury of glomerular mesangial cells in lupus nephritis by targeting forkhead protein K2 through miR-96-5p

SHI Shanhong, LIN Weiyuan, ZHANG Jiequn, ZHENG Yanling   

  1. Department of Nephrology, Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, Fujian, China 
  • Received:2023-04-23 Revised:2023-09-15 Online:2023-12-26 Published:2023-12-21

Abstract:

AIM: To investigate the injury of emodin (EMO) in reduce of glomerular mesangial cells (MCs) in lupus nephritis by targeting forkhead protein K2 (FOXK2) through miR-96-5p. METHODS: The contents of 24 h urine protein, serum urea nitrogen (BUN) and serum creatinine (Scr) in MRL/faslpr mice (lupus nephritis group) and MRL/MPJ mice (control group) were detected. MCs were separated, purified and divided into: MCs group (MCs without any treatment), L-EMO group (MCs treated with 10 μmol/L Emodin), M-EMO group (MCs treated with 25 μmol/L Emodin), H-EMO group (MCs treated with 50 μmol/L Emodin), H-EMO+miR-96-5p-NC group (MCs treated with 50 μmol/L Emodin and transfected with miR-96-5p-NC), and H-EMO+miR-96-5p-minic group (MCs treated with 50 μmol/L Emodin and transfected with miR-96-5p-minic). Double luciferase report experiment was used to verify the targeting relationship between miR-96-5p and FOXK2. The real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-96-5p. Western blot was used to detect the expression of FOXK2 and apoptosis related proteins. The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors in MCs. cell count kit 8 (CCK-8) was used to determine the activity of MCs. Annexin-V FITC/PI double staining was used to detect apoptosis of MCs. RESULTS: Compared with the control group, 24 h urinary protein content, serum BUN and Scr levels in the lupus nephritis group were significantly increased (P<0.05). Compared with the MCs group, the miR-96-5p expression, interleukin1β (IL-1β), interleukin6 (IL-6), tumor necrosis factor-α (TNF-α), A450 value and B-lymphoblastoma-2 (Bcl-2) protein in the L-EMO group, M-EMO group and H-EMO group were significantly decreased (P<0.05), the FOXK2 level, cell apoptosis rate, Bcl-2 related X gene (Bax), aspartate specific cysteine proteinase-3 (cleaved Caspase-3) protein levels were significantly increased, respectively (P<0.05), the effect of Emodin was dose-dependent. Compared with the H-EMO group and H-EMO+miR-96-5p-NC group, H-EMO+miR-96-5p-minic group obviously increased the miR-96-5p expression, inflammatory factor levels, A450 value and Bcl-2 protein level (P<0.05), and obviously decreased FOXK2 level and cell apoptosis rate (P<0.05). CONCLUSION: EMO can reduce the injury of lupus nephritis MCs by down-regulating miR-96-5p and then up-regulating FOXK2.

Key words: emodin, miR-96-5p, forkhead box protein K2, lupus nephritis, glomerular mesangial cells

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