Changed Expression Of Arginase I In The Brain Of UT-B Null Mice Accompanied By Inflammatory Reactions

Objective:To research the expression of arginase I in the brain of UT-B knockout mice accompanied by inflammatory reactions and its possible mechanisms.Methods:1. In vivo experiments, distribution of arginase I of normal mouse brain was observed by immunohistochemistry; co-expression of arginase I and neurons were observed by immunofluorescence in the brain of wild-type and UT-B knockout mice; Western blot analysis were employed to assess the expressions of Arg I, inducible nitric oxide synthase (iNOS) and tumor necrosis factor-a(TNF-a) in the brain of wild-type and UT-B knockout mice.2. In vitro experiments, to imitate the accumulation of urea in UT-B knockout mouse brain, high concentrations of urea were used to stimulate the human brain microvascular endothelial cells(HBMECs). The HBMECs were incubated in high urea (25 mmol/L)or mannitol(as osmotic control) for 3,6,12,24 hours. The expression of TNF-a and iNOS were observed by Immunofluorescence. Western blot analysis were employed to assess the protein expression of TNF-a, iNOS, cycloxygenase-2(COX-2), endothelial nitric oxide synthase (eNOS), nuclear factor kappa B (NF-κB)/p65,p-p65, AMP-activated protein kinase(AMPK) and p-AMPK. Enzyme-linked immunosorbent assay (ELIS A) and chemical methods were used to detect the contents of TNF-a and NO at different times after stimulation of high concentrations of urea in human brain microvascular endothelial cells.Results:1. Arginase I is widely distributed in the mouse brain, including the cerebral cortex (except the molecular layer), hippocampus (mainly expressed in CA1, CA3 and the dentate gyrus), hypothalamus, thalamus nuclei, brainstem nucleus, cerebellum (nuclei、cortical granule cell layer and a pear-shaped cell layer), the third ventricle and the choroid plexus. Arg I-like immunoreactivity were predominantly localized to neuronal cell bodies and main dendrites of all neuronal cell types.In contrast to neurons, immunoreactivity in glial cells was less pronounced.2. The fluorescence intensity of NeuN in the brain of UT-B knockout mice was significantly decreased, compared to wild-type mice.3. The protein expression of Arg I were decreased (p<0.05), however, the protein expression of iNOS and TNF-a were significantly increased in the brain of UT-B knockout mice compared to wild-type mice (p<0.01).4. Compared with control group, the protein expression of TNF-a、COX-2、NF-κB/p-p65 and p-AMPK were significantly increased at 3 and 6 hours in HBMECs after high urea treatment (p<0.05),and iNOS were continued to increase from 3 to 24 hours (p<0.05). NO content was increased at 3 hours after high urea treatment (p<0.05).Conclusion:1. The expression of Arginase I in wild-type mice brain are widely distributed and predominantly expressed in the brain neurons, little expressed in glial cells.2. The number of neurons in the brain of UT-B knockout mice was significantly reduced, and either the arginase I, compared with wild-type mice. Moreover, the protein expression of iNOS and TNF-a were increased in the brain of UT-B knockout mice, which suggests that inflammatory response may have occurred in the brain of UT-B knockout mice.3. High concentrations of urea can induce human brain micro vascular endothelial cells inflammation, NF-κB/p65 and AMPK signaling pathway may be involved in the inflammatory reaction, which can affect energy metabolism in cells. In addition, the content of NO changes in the cells may affect the normal function of human brain microvascular endothelial cells.