Xinfeng capsule regulates pyroptosis and ameliorates cardiac fibrosis in rheumatoid arthritis through lncRNA GAS5 and TLR4/ROS/miR-21 pathways.

Figures

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  Figure 1.. Optimal concentration and duration for LPS and XFC drug-containing serum. (A) Optimal concentration and duration for LPS. (B) Optimal concentration and duration for XFC drug-containing serum.

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  Figure 2.. Target binding interaction between GAS5 and miR-21. (A) The binding sites between GAS5 and miR-21 are predicted by Starbase. (B) Detection results for luciferase activity. GAS5-WT + miR-21 Compared to GAS5-WT + NC: ***P < 0.0001.

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  Figure 3.. Effects of GAS5 overexpression and knockdown on miR-21, TLR4, and myocardial fibrosis. (A) The expression levels of GAS5 and miR-21, and the mRNA levels of TLR4, NF-κB p65, NLRP3, TGF-β1, MMP-9, and CTGF were measured by RT-qPCR. (B and C) TLR4, NF-κB p65, NLRP3, TGF-β1, MMP-9, and CTGF were detected by western blotting. NC, normal control group; OE-NC, negative control overexpression group; OE-GAS5, GAS5 overexpression group; KD-NC, interference negative control group; KD-GAS5, GAS5 interference group. Compared with the NC group, *P < 0.05, **P < 0.001, and ***P < 0.0001.

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  Figure 4.. GAS5 and TLR4/ROS/miR-21 pathways regulate myocardial fibrosis. (A and B) Flow cytometry analysis of ROS production. The red portion represents the entire cell population, while the blue portion represents the cell population labeled with ROS, and the ratio of these portions indicates the level of ROS expression. (C) Levels of proinflammatory cytokines were measured by ELISA. (D) The expression levels of GAS5 and miR-21 and the mRNA levels of TLR4, NLRP3, NF-κB p65, GSDMD, caspase-1, TGF-β1, MMP-9, and CTGF were assessed by RT-qPCR. (E and F) Protein levels of TLR4, NLRP3, NF-κB p65, GSDMD, caspase-1, TGF-β1, MMP-9, and CTGF were measured by Western blotting. (G) Changes in the protein levels of TLR4, NLRP3, and NF-κB p65 were assessed by immunofluorescence analysis (scale bar = 50 μm). NC, normal control group; LPS, 20 µg/mL LPS for 24 h group; LPS + EV, LPS + lentivirus empty control group; LPS + GAS5, LPS + GAS5 overexpression group; LPS + miR-21-inhibitor, LPS + miR-21 inhibitor group; LPS + GAS5 + NSA, LPS + GAS5 overexpression + NSA inhibitor group. Compared with the LPS group: *P < 0.05, **P < 0.001, and ***P < 0.0001; Compared with the LPS + GAS5 group: #P < 0.05, ##P <…

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  Figure 5.. XFC-containing serum improved myocardial fibrosis by regulating the GAS5 and TLR4/ROS/miR-21 pathway. (A and B) Changes in ROS levels were measured using flow cytometry. (C) LDH levels were measured using an LDH assay kit. (D) Inflammatory factors were assayed by ELISA. (E) GAS5, miR-21, and TLR4 levels were assessed by RT-qPCR. (F and G) The protein levels of TLR4, NLRP3, NF-κB p65, GSDMD, caspase-1, CTGF, MMP-9, and TGF-β1 were measured by western blotting. (H) Pyroptosis was evaluated by electron microscopy; the red arrow points to pyroptotic bodies. (I) Immunofluorescence was used to quantify changes in TLR4, NLRP3, and NF-κB p65 protein levels (scale bar = 50 μm). NC, normal control group; LPS, 20 µg/mL LPS for 24 h group; LPS + XFC, 10% XFC + 20 µg/mL LPS for 24 h group. Compared with the LPS group: *P < 0.05, **P < 0.001, and ***P < 0.0001.