Fig 2: BTG3 siRNA transfection abolishes the miR-18a-5p inhibitor influences on inflammatory cytokine release in HFLSs. After transfection, the secretion of inflammatory cytokines, such as (a) TNF-a, (b) IL-1β, and (c) IL-6, and MMPs including MMP1 (d) and MMP3 (e) were determined via ELISAs in different groups. **P < 0.01 vs. inhibitor control; ##P < 0.01 vs. miR-18a-5p inhibitor + control siRNA. miR, microRNA; BTG3, B-cell translocation gene 3. Experiments were repeated for three times.

Fig 3: MSC-CM attenuates LX-2 activation. (a) qRT-PCR analysis of mRNA expression levels for COL1A1 and TGFβ-1 in LX-2 cultured alone (designated as LX-2 control), LX-2 activated with 10 ng/mL TGFβ1 (denoted as LX-2 + TGFβ-1) and treated with MSC-CM following TGFβ-1 activation (labled as LX-2 + TGFβ-1 + MSC-CM). (b) Immunofluorescence micrographs were captured to visualized the cells, with green fluorescence denoting collagen expression and blue fluorescence for nuclear staining. (c) The protein concentrations of pre-collagen and TGFβ-1 assessed by ELISA. (d) Quantification of MMP1 and TIMP1 protein measured by ELISA. All values were presented as mean ± SD, obtained from triplicate experiments. Statistical significance was as follows: *P < 0.05, **P < 0.01, and ***P < 0.001

Fig 4: Rescue experiments demonstrated that GEM overexpression induces macrophage senescence via the SIRT3‐mitochondrial pathway. Representative SA‑β‑gal staining images (A) and quantification of SA‑β‑gal–positive cells (B) in THP‐1‐M and MH‑S macrophages with GEM overexpression and/or treatment with the SIRT3 activator 1 illustrate that GEM‑induced senescence is attenuated by SIRT3 activation. (C) Bar plots showing the percentage of S‑phase cells in MH‑S under the same conditions. Heatmaps summarizing the changes in CDKN1A (Cdkn1a), CDKN2A (Cdkn2a), SOD2 (Sod2), and SIRT3 (Sirt3) expression in THP1‑M (D) and MH‑S (E) cells following GEM overexpression and/or SIRT3 activator 1 treatment. (F–H) Western blot analysis and densitometric quantification of SIRT3, SOD2, P16, and P21 protein levels under these conditions. Original blots can be found in Supporting Information S2. Flow cytometric measurement of mitochondrial ROS (mitoROS) (I) and quantification of the mean fluorescence intensity (J) revealed that SIRT3 activation reduces GEM‑induced oxidative stress. (K) Heatmap of MMP1 and IL‐1B levels in THP‐1‑M culture supernatants, reflecting changes in SASP‑related cytokine secretion. Flow cytometric analysis of the mitochondrial membrane potential via JC‑1 staining in THP‐1‑M and MH‑S (L) cells and quantification of the red/green fluorescence ratio (M), together with MitoTracker‑based assessment of mitochondrial quality (N) and corresponding quantification (O), demonstrated that SIRT3 activation mitigated GEM‑induced mitochondrial dysfunction. All the cell experiments were performed with three independent biological replicates. Groups that do not share a letter differ significantly (p < 0.05), whereas groups sharing a letter are not significantly different. SA‑β‑gal, senescence‑associated β‑galactosidase; ROS, reactive oxygen species.