Fig 2: HMGB1-induced Drp1 activation reduces BMPR2 and Id1 expressions by triggering autophagy activation and ERK1/2/Drp1/Autophagy/BMPR2/Id1 axis mediates HMGB1-induced PASMCs proliferation and migration. A, PASMCs were transfected with Drp1 siRNA or NC siRNA for 24 h, or pre-treated with 20 µM CQ for 1 h, and then stimulated with 30 ng/mL HMGB1 for 24 h. Protein levels were examined by immunoblotting. B, PASMCs were treated with 30 ng/mL HMGB1 for 24 h, and mRNA levels of BMPR2 and Id1 were determined by qRT-PCR. C, Cells were transfected with BMPR2 siRNA or NC siRNA for 48 h. BMPR2 and Id1 protein levels were measured by immunoblotting. D, PASMCs were transfected with Drp1 siRNA or NC siRNA for 24 h, or pre-treated with 10 µM U0126 for 30 min or 20 µM CQ for 1 h, and then stimulated with 30 ng/mL HMGB1 for 24 h. Cell proliferation was evaluated using EdU incorporation assay (scale bar = 200 µm), cell migration were determined by transwell assay (scale bar = 200 µm) and wound healing assay (scale bar = 400 µm). Quantitative analysis of EdU positive cells, Transwell assay and Wound healing assay, respectively. *P < .05

Fig 3: Effect of short-term diabetes on proteins involved in mitochondrial fission/fusion: MFN2, Drp1 and OPA1. a Immunoblot of DRP1 phosphorylation from DRG total extracts of control (Ctrl) and diabetic (streptozotocin, STZ) male (M) and female (F) rats. The bar graph represents densitometric analysis and statistical evaluation of DRP1 phosphorylation over total DRP1 (DRP1P/DRP1 tot); representative western blot of phosphorylated and total DRP1 is presented above the graph. b Immunoblot of MFN2 from DRG total extracts of Ctrl and STZ M and F rats. c Immunoblot of OPA1 from DRG total extracts of Ctrl and STZ M and F rats. Representative Western blots are presented above the graphs, with the respective loading controls (GAPDH). The densitometry of each band was analyzed and the results are expressed as percentage of M Ctrl. The columns represent the mean ± SEM, n = 6 animals for each group. The effects of sex, diabetes, and the interaction sex by diabetes were analyzed using two-way ANOVA (significance: #p < 0.05) followed by Bonferroni post hoc test (significance: *p < 0.05)

Fig 4: HMGB1 stimulates PASMCs proliferation and migration, and promotes Drp1 phosphorylation, autophagy activation, BMPR2/Id1 downregulation by ERK1/2 activation. A, PASMCs were exposed in different concentrations (0-300 ng/mL) of HMGB1 for 24 h, cell viability was measured by Cell Counting Kit-8 (CCK-8) assay. B, PASMCs were exposed to 30 ng/mL HMGB1 for the indicated time (0-72 h), and cell viability was analysed using CCK-8 assay. C, PASMCs were incubated with 30 ng/mL HMGB1 for 24 h, and cell migration was detected using Transwell assay (upper panel, scale bar =200 µm) and Wound healing assay (lower panel, scale bar = 400 µm). D, p-Drp1 and t-Drp1 levels were evaluated by immunoblotting in PASMCs with different concentrations (0-300 ng/mL) of HMGB1 for 1 h. E, p-Drp1 and t-Drp1 levels were examined by immunoblotting in PASMCs with 30 ng/mL HMGB1 for indicated time (0-24 h). F, PASMCs were intervened with 30 ng/mL HMGB1 for 24 h, and Drp1 mRNA level was examined by qRT-PCR. G, HMGB1 (30 ng/mL) time-dependently activated ERK1/2 signalling pathway in PASMCs. H, PASMCs were pre-treated with 10 µM U0126 for 30 min and then stimulated by 30 ng/mL HMGB1 for 1 h, protein levels of p-Drp1 and t-Drp1 were detected by immunoblotting. Cells were pre-treated with 10 µM U0126 for 30 min and then incubated with 30 ng/mL HMGB1 for 24 h, protein levels of Beclin1, LC3B, p62, BMPR2 and Id1 were assessed by immunoblotting. *P < .05

Fig 5: BA induced Drp1-mediated mitochondrial fission via activation of AMPK pathway. (a) The expression levels of AMPKa and p-AMPKa (Thr172) were determined by western blotting. (b) A549 and H1299 cells were transfected by AMPKa lentivirus and control lentivirus and treated with or without BA (120 µM) for 48 h. The expression levels of AMPKa and p-AMPKa (Thr172) were determined by western blotting. (c) The expression levels of Drp1 and p-Drp1 (Ser616) were determined by western blotting. (d) Mitochondria morphology was determined MitoTracker red staining and detected by fluorescent microscopy (scale bar, 10 µm).