Journal: Frontiers in Cell and Developmental Biology

Article Title: TGR5 Activation Ameliorates Mitochondrial Homeostasis via Regulating the PKCδ/Drp1-HK2 Signaling in Diabetic Retinopathy

doi: 10.3389/fcell.2021.759421

Figure Lengend Snippet: Drp1 inhibits mitophagy by facilitating HK2 separation from the mitochondria. RMECs were pretreated with (A, B) INT-777 (30 μM) or (C) Mdivi-1 (10 μM) for 2 h and then exposed with or without HG (33 mM) for 3 days. RMECs were pretreated with INT-777 (30 μM) or INT-777 (30 μM) + 3-BP (10 μM) for 2 h followed by HG (33 mM) for (D, E) three or (F) 5 days. (A, C) HK2 expression in mitochondrial and cytosolic fractions was detected by Western blot. All data were presented as mean ± SD and analysed by RM ANOVA with Tukey’s HSD test. # p < 0.05 vs. control group, * p < 0.05 vs. HG group. (B) DAPI, HK2 and TOMM20 were subjected to representative immunofluorescence analysis. S (s cale bar = 25 μm. (D) Western blot analysis showed the protein expression levels of PINK1 and Parkin. (E) JC-1 dye was used to assess mitochondrial membrane potential in different groups. Quantification of the ratio of fluorescence intensity (red/green) is shown in the bar chart. S (s cale bar = 25 μm ) . (F) Cell viability was measured using the CCK8 assay. All data were presented as mean ± SD and analysed by RM ANOVA with Tukey’s HSD test. # p < 0.05 vs. control group, * p < 0.05 vs. HG group, & $ p < 0.05 vs. INT-777 group.

Article Snippet: Mdivi-1, 3-bromopyruvic acid, INT-777, 3-methyladenine, Rapamycin, BAPTA-AM, and Z-VAD (OMe)-FMK were procured from MedChemExpress (NJ, United States).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Membrane, Fluorescence, CCK-8 Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: TGR5 Activation Ameliorates Mitochondrial Homeostasis via Regulating the PKCδ/Drp1-HK2 Signaling in Diabetic Retinopathy

doi: 10.3389/fcell.2021.759421

Figure Lengend Snippet: Mdivi-1 and Rapamycin alleviate the AAV8-shTGR5-induced retinal dysfunction in vivo . (A) Experimental workflow overview of in vivo experiments. (B) TGR5 shRNA lentivirus knockdown efficiency was assessed by Western blot. (C) Rats were infused with Evans Blue dye (3%) for 2 h. Red fluorescence dots indicate retinal vascular leakage in the flat-mounted retina by fluorescence microscopy. (D) Representative images of acellular capillaries were revealed by retinal trypsin digestion assay and quantified in random fields. ( S s cale bar = 25 μm ) . (E) Representative images for haematoxylin–eosin (HE) staining in rat retinas. Retinal thickness was evaluated in sections. ( S s cale bar = 25 μm ) . All data were presented as mean ± SD and analysed by RM ANOVA with Tukey’s HSD test. # p < 0.05 vs. control group, * p < 0.05 vs. AAV8-vector group. & $ p < 0.05 vs. AAV8-shTGR5 group.

Article Snippet: Mdivi-1, 3-bromopyruvic acid, INT-777, 3-methyladenine, Rapamycin, BAPTA-AM, and Z-VAD (OMe)-FMK were procured from MedChemExpress (NJ, United States).

Techniques: In Vivo, shRNA, Knockdown, Western Blot, Fluorescence, Microscopy, Trypsin Digestion Assay, Staining, Control, Plasmid Preparation

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: P. gingivalis promotes Drp1 phosphorylation and mitochondrial translocation in HAECs. (A, B) Representative confocal images of mitochondrial morphology in P. gingivalis -infected HAECs (MOI = 100 for 24 h) following Mdivi-1 (50 μM) treatment. Mitochondrial length in each group was calculated by Image J. Scale bars =10 μm. ( n = 3). (C, D) p -Drp1(ser616), Drp1, OPA1, Fis1 protein levels in HAECs at 24 h after P. gingivalis infection (MOI = 100) and quantitative bar chart. ( n = 3). (E, F) Relative protein expression of p -Drp1(ser616) in cytoplasmic and mitochondrial fractions of HAECs and quantitative bar chart. ( n = 3). (G, H) Representative immunofluorescence images and statistics analysis showing the co-location of mitochondria (red) and p -Drp1 (green) in HAECs. Scale bars: 20 μm (original) and 2 μm (Zoom). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. ns = not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Phospho-proteomics, Translocation Assay, Confocal, Morphology, Infection, Expressing, Immunofluorescence, Analysis

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: Mdivi-1 rescues P. gingivalis -induced mitochondrial and endothelial dysfunction. HAECs was pretreated with or without Mdivi-1 (50 μM) and then infected with P. gingivalis for 24 h (MOI = 100). (A, B) JC-1, MitoSOX staining assay and quantitative bar chart. Scale bars = 20 μm. ( n = 3). (C) ATP production in HAECs. ( n = 3). (D, E) TUNEL staining, tube formation assay and quantitative bar chart. Scale bars = 200 μm. ( n = 3). (F, G) Apoptotic ratio in HAECs with quantitative data. ( n = 3). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Infection, Staining, TUNEL Assay, Tube Formation Assay, Mitosox Assay

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: P. gingivalis facilitates Drp1-dependent mPTP opening in endothelial cells. HAECs was pretreated with or without Mdivi-1 (50 μM) and then infected with P. gingivalis for 24 h (MOI = 100). (A, B) Representative immunoblots and quantification of CytC levels in cytosolic and mitochondrial fractions of HAECs p . ( n = 3). (C, D) Calcein staining assay and quantitative bar chart. Scale bars = 20 μm. ( n = 3). HAECs was pretreated with or without CsA (1 μM) and then infected with P. gingivalis for 24 h (MOI = 100). (E, F) JC-1, MitoSOX staining assay and quantitative bar chart. Scale bars = 20 μm. ( n = 3). (G) ATP production in HAECs. ( n = 3). (H, I) Tube formation assay and quantitative bar chart. Scale bars = 200 μm. ( n = 3). (J, K) Apoptotic ratio in HAECs with quantitative data. ( n = 3). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Infection, Western Blot, Staining, Tube Formation Assay, Immunofluorescence, FACS

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: Drp1-dependent VDAC1 oligomerization is required for P. gingivalis induced mitochondrial dysfunction. HAECs was pretreated with or without Mdivi-1 (50 μM) and then infected with P. gingivalis for 24 h (MOI = 100). (A, B) Representative immunofluorescence images and statistics analysis showing the co-location of VDAC1 (red) and p -Drp1 (green) in HAECs. Scale bars: 20 μm (original) and 2 μm (Zoom). (C) The interaction of p -Drp1 and VDAC1 was validated using Co-immunoprecipitation assays. ( n = 3). (D, E) Immunoblot of VDAC1 cross-linking in HAECs pretreated with Mdivi-1 (50 μM) or VBIT-4 (20 μM) and quantitative analysis of oligomers. ( n = 3). (F, G) Calcein, MitoSOX staining assay and quantitative bar chart. Scale bars = 20 μm. ( n = 3) (H, I) Tube formation assay and quantitative bar chart. Scale bars = 200 μm. ( n = 3). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. * P < 0.05. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Infection, Immunofluorescence, Analysis, Co-Immunoprecipitation Assay, Western Blot, Staining, Tube Formation Assay

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: P. gingivalis triggers HK2 dissociation from VDAC1. HAECs were pretreated with Mdivi-1 (50 μM) or VBIT-4 (20 μM), followed by infection with P. gingivalis for 24 h. (MOI = 100). (A, B) Representative immunofluorescence images and statistics analysis showing the co-location of VDAC1 (red) and HK2 (green) in HAECs. Scale bars: 20 μm (original) and 5 μm (Zoom). (C) The interaction of VDAC1 and HK2 was validated using Co-immunoprecipitation assays. ( n = 3). (D, E) Representative immunoblots and quantification of HK2 levels in cytosolic and mitochondrial fractions of HAECs ( n = 3). (F, G) Representative immunofluorescence images and statistics analysis showing the co-location of mitochondria (red) and HK2 (green) in HAECs. Scale bars: 20 μm (original) and 5 μm (Zoom). ( n = 3). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. ns = not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Infection, Immunofluorescence, Analysis, Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: Drp1-mediated mPTP opening contributes to P. gingivalis -induced endothelial dysfunction in vivo. (A) CD31 immunofluorescence of aorta tissue sections, scale bars = 100 μm. ( n = 6). (B) TUNEL stained aorta tissue sections, scale bars = 20 μm. ( n = 6). (C) MitoSOX stained aorta tissue sections, scale bars = 20 μm. ( n = 6). (D) Quantitative bar chartof (A)–(C). (E) Concentration–response curves of mice aortic rings after exposure to Ach or SNP treatments, from Vehicle-treated mice, P. gingivalis -infected mice, CsA (20 mg/kg) treated P. gingivalis infected mice and Mdivi-1 (30 mg/kg) treated P. gingivalis infected mice aorta tissue sections. ( n = 3). (F) Proposed molecular mechanism by which P. gingivalis induces endothelial dysfunction via Drp1-HK2-mPTP axis. Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. ns = not significant. ** P < 0.01. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: In Vivo, Immunofluorescence, Tissue, TUNEL Assay, Staining, Concentration Assay, Infection