Inhibition of JNK Alleviates Chronic Hypoperfusion-Related Ischemia Induces Oxidative Stress and Brain Degeneration via Nrf2/HO-1 and NF- κ B Signaling

Abstract

Cerebral ischemia is one of the leading causes of neurological disorders. The exact molecular mechanism related to chronic unilateral cerebral ischemia-induced neurodegeneration and memory deficit has not been precisely elucidated. In this study, we examined the effect of chronic ischemia on the induction of oxidative stress and c-Jun N-terminal kinase-associated detrimental effects and unveiled the inhibitory effect of specific JNK inhibitor (SP600125) on JNK-mediated brain degeneration in adult mice. Our behavioral, biochemical, and immunofluorescence studies revealed that chronic ischemic injuries sustained increased levels of oxidative stress-induced active JNK for a long time, whereas SP600125 significantly reduced the elevated level of active JNK and further regulated Nrf2/HO-1 and NF-κB signaling, which have been confirmed in vivo. Neuroinflammatory mediators and loss of neuronal cells was significantly reduced with the administration of SP600125. Ischemic brain injury caused synaptic dysfunction and memory impairment in mice. However, these were significantly improved with SP600125. On the whole, these findings suggest that elevated ROS-mediated JNK is a key mediator in chronic ischemic conditions and has a crucial role in neuroinflammation, neurodegeneration, and memory dysfunction. Our findings suggest that chronic oxidative stress associated JNK would be a potential target in time-dependent studies of chronic ischemic conditions induced brain degeneration.

Figure 1

(a) Indicating mice grouping i.e. (1.) Control (2.) Ischemic (3.) Ischemia+SP600125. (b) Showing study plan for the current research work.

Figure 2

Chronic ischemia induced the stress kinase p-JNK activation and deregulates Nrf2/HO-1 proteins. (a, b) Western blot findings of p-JNK in the different samples of the experimental groups. (c, d) Showing ROS and LPO assay results. (e, f) Immunofluorescent images representing the immunoreactivity of p-JNK (FITC; green color, DAPI; blue color) both in the cortex and hippocampal regions. (g, h) Western blot results of Nrf2/HO1, in the cortex, and hippocampus of the experimental groups. (i, j) Immunofluorescence results of Nrf2/HO1 in the experimental groups. In confocal microscopy the Magnification were kept 10x, Scale bar = 50 μm for all antibodies. In western blot assay, the β-actin was used as a loading control. Density values are relative to the control group (Rounding Rules) and expressed in arbitrary units (A.U). For western blot. The data are presented as the mean ± SEM. ^∗Significantly different from the control group; ^#Significantly different from the ischemia group. Significance =  ^∗P < 0.05, ^#P < 0.05.

Figure 3

Chronic ischemic condition increases inflammatory responses in ipsilateral cortex and hippocampus of the adult mouse. (a, b) Showing the western blot results of inflammatory mediators. (c-f) Results immunofluorescence results of inflammatory cytokines. P value = P < 0.05, ^#P < 0.05. Scale bar = 50 μm while magnification was 10x for confocal.

Figure 4

Chronic ischemic condition upregulates neuronal apoptosis and neurodegeneration. (a, b) Western blot results of apoptosis-related markers. β-Actin was used as a loading control and ^∗P < 0.05, ^#P < 0.05. Density values are expressed in arbitrary units (A.U). (c, d) Results of immunofluorescent analysis for apoptotic markers. (e) Showing FJB results while (f) Specify nissl staining results. The nissl staining result magnification was 20x. GraphPad Prism 6.0 soft was used to determine the significance level.

Figure 5

Chronic ischemic condition decreases synaptic proteins and induces memory dysfunction. (a) Represents the western blot results of synaptic markers hippocampus of adult mice. (b, c) Immunofluorescence analysis of synaptic proteins. (d, h, e, i) Indicating the water maze and Y-maze results while (f, g) specify the water maze and Y-maze trajectories, respectively.

Figure 6

Indicating the diagrammatic abstract.