Fig 2: JNK silencing alleviated HR injury by regulating mitochondrial dysfunction and inhibiting apoptosis, and RCAN1, JNK, and Mff all bound together.A siRNA against JNK and si-NC were transfected into cells. The expression level of JNK was monitored via Western blotting. (n = 5) B An IF assay for p-JNK, and DAPI was used to tag the nucleus. Scale bar, 10 μm. n = 3. C The renal tubular epithelial cell line HK-2 was used with HR, and siRNA against JNK and si-NC were transfected into cells. The expression levels of Pro-caspase-3, Cle-caspase-3, Pro-caspase-9, Cle-caspase-9, Bax, and Bcl-2 were detected by Western blotting (n = 5). D Mitochondrial morphology of HK-2 was assessed by MitoTrackerTM Deep Red staining, and the average length of mitochondria was measured. Scale bar, 10 μm n = 3. E Western blotting was performed to analyze the expression of p-Drp1S616, Drp1, p-Mff, Mff, Fis1, Mfn1, Mfn2, and Opa1 in HK-2 cells (n = 5). F Mitochondrial function was measured with an ATP assay kit (n = 4). G–I Representative Co-IP analysis of RCAN1, JNK, and Mff in HK-2 cells under HR injury (n = 3). J–L The co-localization of RCAN1 and p-JNK, Mff, and JNK, as well as Mff and RCAN1, were detected by IF, and DAPI was used to tag the nucleus. Scale bar, 10 μm. n = 3. *p < 0.05, **p < 0.01.

Fig 3: Calcium chelator BAPTA-AM protects chondrocytes against iron overload-induced mitochondrial damage. (A) Representative fluorescence images of mitochondria and local amplification images of the selected area. Chondrocytes were treated with 100 µm FAC in the presence or absence of 10 µm of the calcium chelator BAPTA-AM and the morphology of the mitochondria was visualized using Mito-Tracker Green staining. Scale bars, 25 µm. (B) Chondrocytes were treated with 100 µm FAC or 10 ng/ml IL-1 β in the presence or absence of 10 µm of the calcium chelator BAPTA-AM. Western blotting was conducted to examine the protein expression levels of MFF, DRP1 and FIS1. (C) Densitometric analysis of MFF, DRP1 and FIS1 protein expression normalized to β-actin. Data are presented as mean ± SD. *P<0.05; ***P<0.001. MFF, mitochondrial fission factor; DRP1, dynamin-related protein 1; FIS1, mitochondrial fission 1 protein; FAC, ferric ammonium citrate; BAPTA-AM, BAPTA acetoxymethyl ester; Ctrl, control group; FAC + BA, FAC + BAPTA-AM group.

Fig 4: Berberine balanced mitochondrial fission and fusion in high glucose-induced H9C2 cells. Representative western blot results and quantification of Mfn1 and Mfn2 (A), OPA1 (B), and MFF (C). Each western blot was conducted for three times. D: Representative western blot results and quantification of Drp1 and Drp1-p616. Each western blot was conducted for three times. E: Representative confocal images of immunofluorescence of Drp1 and mitochondria. Mitochondria were stained with mitotracker. Nuclei were stained with DAPI. Scale bar is 10 μm. ∗∗P < 0.01 vs. Ctrl; ∗∗∗P < 0.001 vs. Ctrl; ##P < 0.01 vs. HG.

Fig 5: Berberine-inhibited mitochondrial fission can attenuate hypertrophy. A: Representative confocal images of mitochondria of H9C2 cells stained with mitotracker. Scale bar is 10 μm. B: Representative western-blot results and quantification of Mfn1 and Mfn2 (B), OPA1 (C), and MFF (D) when treated with Mdivi-1. Each western blot was conducted for three times. E: Representative western blot results and quantification of Drp1 and Drp1-p616. Each western blot was conducted for three times. F: Representative confocal images of H9C2 cells stained with FITC-labeled phalloidin to show its area. Scale bar is 10 μm. G: Quantification of H9C2 cell area in each group. For each group, n = 50–55 cells were quantified. H: Relative mRNA level of myocardium hypertrophy biomarkers quantified by real-time PCR. n = 3 for each group, each sample were measured in three replications. ∗∗P < 0.01 vs. Ctrl; ∗∗∗P < 0.001 vs. Ctrl; ∗∗∗∗P < 0.0001 vs. Ctrl; #P < 0.05 vs. HG; ##P < 0.01 vs. HG;###P < 0.001 vs. HG.