Journal: Acta Pharmaceutica Sinica. B

Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

doi: 10.1016/j.apsb.2020.05.002

Figure Lengend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with their controls. The numbers at the bottom of the bars indicate the number of repeats or mice in the group.

Article Snippet: After blocking by 5% milk, PVDF membranes were incubated with primary antibodies at 4 °C overnight such as rat anti-IL-4 antibody (1:500, Abcam, ab11524), mouse monoclonal anti-KCa3.1 antibody (1:250, Alomone, ALM-051), rabbit anti-Nav1.1 antibody (1:250, Alomone, ASC-001), rabbit GABA A R α 6 Polyclonal Antibody (1:200, Alomone, AGA-004), mouse anti- β -actin antibody (1:5000, Abcam) and mouse anti-GAPDH antibody (1:5000, Abcam).

Techniques: Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

doi: 10.1016/j.apsb.2020.05.002

Figure Lengend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

Article Snippet: After blocking by 5% milk, PVDF membranes were incubated with primary antibodies at 4 °C overnight such as rat anti-IL-4 antibody (1:500, Abcam, ab11524), mouse monoclonal anti-KCa3.1 antibody (1:250, Alomone, ALM-051), rabbit anti-Nav1.1 antibody (1:250, Alomone, ASC-001), rabbit GABA A R α 6 Polyclonal Antibody (1:200, Alomone, AGA-004), mouse anti- β -actin antibody (1:5000, Abcam) and mouse anti-GAPDH antibody (1:5000, Abcam).

Techniques: Binding Assay, Protein-Protein interactions, Membrane