Journal: Journal of Neuroinflammation

Article Title: Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

doi: 10.1186/s12974-023-02818-6

Figure Lengend Snippet: RVLM neuronal mitochondria were injured in SIH rats. A Representative photomicrographs of the RVLM neurons in control and SIH rats for visualization in transmission electron microscopy. Mito, mitochondria. Red arrows indicate disruption of the mitochondria with abnormal mitochondrial crests. Scale bar = 5 or 1 μm. B Representative immunoblot bands and quantitative analysis showed the expression of cytoplasmic cytochrome C (Cyt-cyto C) in the RVLM between the two groups. C Within the RVLM, AAV2:MitoEGFPmCherry infection produced punctate labeling of EGFP and mCherry, which colocalized with neuron marker NeuN. Scale bar = 20 μm. Quantitative analysis showed the degree of mitochondrial degradation by assessing the ratio value of mCherry-ONLY/total mCherry puncta. D Representative immunoblot bands and quantitative analysis showed the expression of mitochondrial respiratory chain complexes (complexes II-SDHB, III-UQCRC2, and V-ATP5A) in the RVLM of control and SIH rats. E Representative fluorescence images of reactive oxygen species (ROS) detection and statistical data of ROS production in the RVLM between the two groups. Scale bar = 100 μm. F The levels of superoxide dismutase (SOD) and catalase (CAT) were quantified by using commercial kits in each group. Data were shown as mean ± SEM and analyzed via two-tailed unpaired Student’s t -test ( B – F ). n = 3 rats per group ( B , D ). n = 6 rats per group ( F ). n = 12 slices from 6 rats, two slices per rat ( C , E ). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group

Article Snippet: The membranes were blocked with QuickBlockTM Western (Beyotime, Cat.No.P0252, China) for 1 h prior to incubation at 4 °C overnight with primary antibodies including rabbit monoclonal antibody against iNOS (1:1000, Cat.No.ab178945, Abcom, USA), rabbit monoclonal antibody against CD86 (1:1000, Cat.No.a19026, ABclonal, China), rabbit monoclonal antibody against Arg-1 (1:1000, Cat.No.A4923, ABclonal, China), mouse monoclonal antibody against TNF-α (1:1000, Cat.No.sc-52746, Santa Cruz, USA), rabbit polyclonal antibody against p-AMPK (1:500, Cat.No.AF5908, Beyotime, China), rabbit monoclonal antibody against AMPK (1:1000, Cat.No.AF1627, Beyotime, China), rabbit monoclonal antibody against Sirt3 (1:1000, Cat.No.#2627, Cell Signaling Technology, USA), rabbit polyclonal antibody against ATP5A (1:1500, Cat.No.GB113455, Servicebio, China), mouse monoclonal antibody against UQCRC2 (1:1000, Cat.No.sc-390378, Santa Cruz, USA), rabbit monoclonal antibody against SDHB (1:1000, Cat.No.AG3213, Beyotime, China), mouse monoclonal antibody against TNFR1 (1:500, Cat.No.sc-8436, Santa Cruz, USA), mouse monoclonal antibody against TNFR2 (1:500, Cat.No.sc-12750, Santa Cruz, USA), and mouse monoclonal HRP-Conjugated β-actin antibody (1:5000, Cat No.HRP-60008, Proteintech, USA).

Techniques: Control, Transmission Assay, Electron Microscopy, Disruption, Western Blot, Expressing, Infection, Produced, Labeling, Marker, Fluorescence, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

doi: 10.1186/s12974-023-02818-6

Figure Lengend Snippet: TNF-α led to neuronal mitochondrial dysfunction via downregulating the AMPK-Sirt3 pathway. A Representative immunofluorescence images and quantitative analysis showed colocalization of Sirt3 and tyrosine hydroxylase (TH)-positive neurons in the RVLM of control and SIH groups. Scale bar = 500 or 20 μm. B Representative immunoblot bands and quantitative analysis showed the expression of p-AMPK and Sirt3 in the RVLM of control and SIH rats. C Representative immunoblot bands and quantitative analysis of p-AMPK and Sirt3 expression in N2a cells treated with or without TNF-α. D Representative images and quantitative analysis showed mitochondrial membrane potential (MMP) in control, TNF-α, and TNF-α + A769662 groups. Scale bar = 50 μm. E Representative immunoblot bands and quantitative analysis showed the expression of mitochondrial respiratory chain complexes (complexes II-SDHB, III-UQCRC2, and V-ATP5A) in N2a cells with different treatments. F Representative fluorescence images of reactive oxygen species (ROS) detection and statistical data of ROS production in N2a cells with or without TNF-α treatment. Scale bar = 50 μm. G The levels of superoxide dismutase (SOD) and catalase (CAT) were quantified by using commercial kits in each group. Data were shown as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t -test ( A – C ) and one-way ANOVA followed by post hoc Bonferroni test ( D – G ). n = 12 slices from 6 rats, two slices per rat ( A ). n = 3 rats per group ( B ). n = 3 of independent cell culture preparations ( C , E ). n = 6 of independent cell culture preparations ( G ). n = 12 slices from 6 of independent cell culture preparations, two slices per independent cell culture preparation ( D , F ). *p < 0.05 vs. SIH group. # p < 0.05, ## p < 0.01 vs. TNF-α group

Article Snippet: The membranes were blocked with QuickBlockTM Western (Beyotime, Cat.No.P0252, China) for 1 h prior to incubation at 4 °C overnight with primary antibodies including rabbit monoclonal antibody against iNOS (1:1000, Cat.No.ab178945, Abcom, USA), rabbit monoclonal antibody against CD86 (1:1000, Cat.No.a19026, ABclonal, China), rabbit monoclonal antibody against Arg-1 (1:1000, Cat.No.A4923, ABclonal, China), mouse monoclonal antibody against TNF-α (1:1000, Cat.No.sc-52746, Santa Cruz, USA), rabbit polyclonal antibody against p-AMPK (1:500, Cat.No.AF5908, Beyotime, China), rabbit monoclonal antibody against AMPK (1:1000, Cat.No.AF1627, Beyotime, China), rabbit monoclonal antibody against Sirt3 (1:1000, Cat.No.#2627, Cell Signaling Technology, USA), rabbit polyclonal antibody against ATP5A (1:1500, Cat.No.GB113455, Servicebio, China), mouse monoclonal antibody against UQCRC2 (1:1000, Cat.No.sc-390378, Santa Cruz, USA), rabbit monoclonal antibody against SDHB (1:1000, Cat.No.AG3213, Beyotime, China), mouse monoclonal antibody against TNFR1 (1:500, Cat.No.sc-8436, Santa Cruz, USA), mouse monoclonal antibody against TNFR2 (1:500, Cat.No.sc-12750, Santa Cruz, USA), and mouse monoclonal HRP-Conjugated β-actin antibody (1:5000, Cat No.HRP-60008, Proteintech, USA).

Techniques: Immunofluorescence, Control, Western Blot, Expressing, Membrane, Fluorescence, Two Tailed Test, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

doi: 10.1186/s12974-023-02818-6

Figure Lengend Snippet: Blockage of TNF-α rescued mitochondrial injury in the RVLM neurons of SIH rats. The expression of A TNFR1 and B TNFR2 was determined by Western blot and RT-qPCR assays in the RVLM of control + vehicle, SIH + vehicle, and SIH + R7050 rats. C Representative immunoblot bands and quantitative analysis showed the expression of p-AMPK and Sirt3 in the RVLM between the three groups. D Representative images of MitoEGFPmCherry labeling in the RVLM of different groups, and quantitative analysis of the degree of mitochondrial injury was evaluated as the ratio value of mCherry-ONLY/total mCherry puncta. Scale bar = 10 μm. E Representative immunoblot bands and quantitative analysis of mitochondrial respiratory chain complexes (complexes II-SDHB, III-UQCRC2, and V-ATP5A) in the RVLM in control + vehicle, SIH + vehicle, and SIH + R7050 groups. F Representative fluorescence images of reactive oxygen species (ROS) detection and statistical data of ROS production in the RVLM between the three groups. Scale bar = 100 μm. G The levels of superoxide dismutase (SOD) and catalase (CAT) were quantified by using commercial kits in each group. Data were shown as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by post hoc Bonferroni test ( A – G ). n = 3 rats per group ( A , C , E ). n = 6 rats per group ( B , G ). n = 12 slices from 6 rats, two slices per rat ( D , F ). *p < 0.05, **p < 0.01, ***p < 0.001 vs. SIH + Vehicle group. ns means nonsignificant vs. SIH + Vehicle group

Article Snippet: The membranes were blocked with QuickBlockTM Western (Beyotime, Cat.No.P0252, China) for 1 h prior to incubation at 4 °C overnight with primary antibodies including rabbit monoclonal antibody against iNOS (1:1000, Cat.No.ab178945, Abcom, USA), rabbit monoclonal antibody against CD86 (1:1000, Cat.No.a19026, ABclonal, China), rabbit monoclonal antibody against Arg-1 (1:1000, Cat.No.A4923, ABclonal, China), mouse monoclonal antibody against TNF-α (1:1000, Cat.No.sc-52746, Santa Cruz, USA), rabbit polyclonal antibody against p-AMPK (1:500, Cat.No.AF5908, Beyotime, China), rabbit monoclonal antibody against AMPK (1:1000, Cat.No.AF1627, Beyotime, China), rabbit monoclonal antibody against Sirt3 (1:1000, Cat.No.#2627, Cell Signaling Technology, USA), rabbit polyclonal antibody against ATP5A (1:1500, Cat.No.GB113455, Servicebio, China), mouse monoclonal antibody against UQCRC2 (1:1000, Cat.No.sc-390378, Santa Cruz, USA), rabbit monoclonal antibody against SDHB (1:1000, Cat.No.AG3213, Beyotime, China), mouse monoclonal antibody against TNFR1 (1:500, Cat.No.sc-8436, Santa Cruz, USA), mouse monoclonal antibody against TNFR2 (1:500, Cat.No.sc-12750, Santa Cruz, USA), and mouse monoclonal HRP-Conjugated β-actin antibody (1:5000, Cat No.HRP-60008, Proteintech, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Labeling, Fluorescence