Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 1. Inhibition of micro RNA miR-122-5p on lipopolysaccharide-induced myocardial injury. Wistar rats were intravenously injected with miR-122-5p antagomir, followed by lipopolysaccharide (LPS) stimulation (n = 6 rats per group). (a-b) After LPS treatment for 12 h, heart tissues were harvested for the relative expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) using real-time quantitative PCR (RT-qPCR) or western blot assay. (c) The ratio of heart weight/ body weight (HW/BW) was calculated. (d) Hematein and eosin (H&E) staining revealed the effect of miR-122-5p on LPS-induced histopathological changes in heart. (e) Levels of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were examined by enzyme-linked immunosorbent assay (ELISA). ***p < 0.001 versus control; ##p < 0.01, ###p < 0.001 versus LPS + NC antagomir.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Inhibition, Injection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 3. In vitro analysis for beneficial role of inhibiting micro RNA miR-122-5p in lipopolysaccharide (LPS)-induced apoptosis. (a-b) Rat H9c2 cells were treated with LPS for 12 h or 24 h, and the expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were assessed by real-time quantitative PCR (RT-qPCR) or western blot analysis. (c-d) H9c2 cells were transfected with NC inhibitor or miR-122-5p inhibitor for 24 h, followed by LPS treatment for another 24 h under proper culture conditions. After that, miR-122-5p and GIT1 expression levels were measured. (e) The contents of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were detected by appropriate kits. (f) Flow cytometry showed the apoptosis of LPS-stimulated myocardial cells. (g) Western blot analysis illustrated the changes of caspase-3 expression. **p < 0.01, ***p < 0.001 versus control; ++p < 0.01, ++

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 5. Potential downstream target gene of micro RNA miR-122-5p. H9c2 cells were transfected with NC mimics, miR-122-5p mimics, NC inhibitor and miR-122-5p inhibitor for 48 h. (a-b) The expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were verified by real-time quantitative PCR (RT-qPCR) assay. (c) The predicted binding sites of miR-122-5p in the 3-UTR of GIT1, and the sequence information of miR-122-5p and GIT1 (wild- or mutant- type) was displayed. (d) Luciferase assay verified the correlation between miR-122-5p and GIT1. aap < 0.01, aaap < 0.001 versus NC mimics; bbbp < 0.001 versus NC inhibitor; ddp < 0.01 versus NC mimics + GIT1 3UTR (WT).

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 6. G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency attenuates the effects of micro RNA miR-122-5p loss on myocardial injury. (a) H9c2 cells were transfected with GIT1 siRNA to downregulate GIT1 expression. (b) The cells were transfected with GIT1 siRNA and/or miR-122-5p inhibitor, and then induced by lipopolysaccharide (LPS). GIT1 expression at mRNA and protein levels was then measured using real-time quantitative PCR (RT-qPCR) or western blot. (c) Apoptosis of myocardial cells was analyzed by flow cytometry. (d) Reactive oxygen species (ROS) production was examined using flow cytometry. (e-g) The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and tumor necrosis factor alpha (TNF-α) were assessed by the enzyme-linked immunosorbent assay (ELISA) kits. XXXp < 0.001 versus NC siRNA; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 versus LPS + miR-122-5p inhibitor + NC siRNA.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1.

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Figure 7. G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency inhibits nuclear factor erythroid 2-related factor 2 (Nrf-2) activation. (a) Real-time quantitative PCR (RT-qPCR) assay was used to measure the heme oxygenase-1 (HO-1) and NAD(p)H: quinone oxidoreductase 1 (NQO-1) expression. (b) Nuclear Nrf-2 level was revealed using western blot analysis. ^^p < 0.01 versus LPS + miR-122-5p inhibitor + NC siRNA.

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: RhANP attenuates endotoxin-derived cognitive dysfunction through subdiaphragmatic vagus nerve-mediated gut microbiota–brain axis

doi: 10.1186/s12974-021-02356-z

Figure Lengend Snippet: Effects of SDV on hippocampal TrkB/BDNF signaling in LPS-challenged mice treated with rhANP. a , b Western blot analysis of phosphorylated/total tyrosine kinase receptor B ratio (p-TrkB/t-total) in the hippocampus (one-way ANOVA: F 2,27 = 5.245, P = 0.0119) and prefrontal cortex (PFC) (one-way ANOVA: F 2,27 = 0.2589, P = 0.7738) of mice treated with recombinant human ANP (rhANP) or 0.9% saline 24 h after injection of lipopolysaccharides (LPS) or 0.9% saline. c , d Western blot analysis of p-TrkB/t-total in the hippocampus (two-way ANOVA: rhANP: F 1,36 = 1.633, P = 0.2095; SDV: F 1,36 = 9.169, P = 0.0045; interaction: F 1,36 = 4.395, P = 0.0431) and PFC (two-way ANOVA: rhANP: F 1,36 = 0.1409, P = 0.7096; SDV: F 1,36 = 0.3043, P = 0.5846; interaction: F 1,36 = 0.2733, P = 0.6044) of rhANP or 0.9% saline-treated endotoxemia mice pre-subjected to SDV or sham operation. e , f Western blot analysis of BDNF in the hippocampus (one-way ANOVA: F 2,27 = 12.20, P = 0.0002) and PFC (one-way ANOVA: F 2,27 = 2.218, P = 0.1290) of mice treated with rhANP or 0.9% saline 24 h after injection of LPS or 0.9% saline. g , h Western blot analysis of BDNF in the hippocampus (two-way ANOVA: rhANP: F 1,36 = 6.305, P = 0.0167; SDV: F 1,36 = 2.378, P = 0.1318; interaction: F 1,36 = 5.767, P = 0.0216) and PFC (two-way ANOVA: rhANP: F 1,36 = 0.2615, P = 0.6122; SDV: F 1,36 = 0.1217, P = 0.7292; interaction: F 1,36 = 0.1715, P = 0.6813) of rhANP or 0.9% saline-treated endotoxemia mice pre-subjected to SDV or sham operation. Data are shown as mean ± SEM, n = 10/group. * P < 0.05, ** P < 0.01, *** P < 0.0001; N.S. not significant. SDV subdiaphragmatic vagotomy

Article Snippet: The membranes were blocked in 5% non-fat dried milk for 1 h at room temperature, and then incubated with the following primary antibodies: rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (iba-1; 1:1000, #016-20001: Wako Pure Chemical Industries, Ltd., Tokyo, Japan), rabbit monoclonal anti-IL-6 (1:1000, #12912S, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal anti-IL-17A (1:1000, #A0688, ABclonal, Wuhan, China), rabbit monoclonal anti-IFN-γ (1:1000, #A12450, ABclonal, Wuhan, China), rabbit polyclonal anti-TNF-α (1:1000, #A0277, ABclonal, Wuhan, China), rabbit monoclonal anti-inducible nitric oxide synthase (iNOS; 1:1000, #ab3523, Abcam, Cambridge, MA, USA), brain-derived neurotrophic factor (BDNF; 1:1000, #A4873, ABclonal, Wuhan, China), rabbit polyclonal phosphorylated tyrosine kinase receptor B (p-TrkB; 1:1000, #AP0423, ABclonal, Wuhan, China), rabbit polyclonal total TrkB (t-TrkB; 1:1000, #A2099, ABclonal, Wuhan, China), and mouse monoclonal anti-β-actin (1:1000, #A2319, ABclonal, Wuhan, China) overnight at 4 ℃.

Techniques: Western Blot, Recombinant, Injection