ABSTRACT
BACKGROUND: There is an inflammatory response in the lesion tissue of ischemic cerebral infarction, and the expression of miR-150-5p is significantly decreased. Whether miR-150-5p inhibits the release of inflammatory factors and alleviates the injury of ischemic cerebral infarction tissue through the Toll-like receptor-5/nuclear factor-KB pathway remains unclear. OBJECTIVE: To investigate the role and preliminary mechanism of miR-150-5p in ischemic cerebral infarction in rats. METHODS: (1) The rat models of middle cerebral artery occlusion were constructed and the rat models were divided into five groups: Control, miR-150-5p agomir, agomir control, miR-150-5p antagomir and antagomir control groups. (2) The rats in the control group was given the intracerebroventricular injection of normal saline, and the rats in the latter four groups were given the intracerebroventricular injection of miR-150-5p agomir (miR-150-5p agonist), agomir negative control, miR150-5p antagomir (miR150-5p inhibitor) and antagomir negative control, respectively. (3) After 7 days, the brain was graded by modified neurological severity score, the cerebral infarct volume was measured by MRI, and the histopathological changes were observed by hematoxylin-eosin staining. The expression levels of miR-150-5p, interleukin-6, tumor necrosis factor-a, Toll-like receptor-5 and nuclear factor-KB p65 in brain tissues were detected by qRT-PCR, ELISA and western blot assay, respectively. The target relationship between miR150-5p and Toll-like receptor-5 was verified by luciferase assay by retrieving the bioinformatics website Targetscan to predict the binding sites of miR-150-5p and Toll-like receptor-5. RESULTS AND CONCLUSION: (1) Compared with the control group, the modified neurological severity score, and levels of interleukin-6, tumor necrosis factor-a, Toll-like receptor-5 and nuclear factor-KB p65 proteins were significantly decreased in the miR-150-5p agomir group (P 0.05). (3) TargerScan website prediction results and luciferase reporter gene analysis results showed that miR-150-5p and Toll-like receptor-5 had a targeted binding site. (4) These results imply that miR-150-5p can inhibit the inflammatory signaling pathway of Toll-like receptor-5/nuclear factor-KB p65 in brain injury caused by ischemia and reduce the inflammatory response, thereby alleviating the damage of nerve function and playing a protective role.
ABSTRACT
Objective To explore the changes in expression of Klotho, an aging-suppression protein, and neutrophil gelatinase associated lipocalin ( NGAL) and their relationship with rat mesangial cells ( RMCs) cultured with high glucose in vitro, and to explore the role played by Toll-like receptor-4 (TLR4) / nuclear factor-kB(NF-kB) p65 pathways in this process. Methods Three NGAL-siRNA sequences were designed and synthesized. The effective sequence in subsequent experiments was chosen. RMCs were preincubated with pyrrolidinedithiocarbamate (PDTC)or exogenously added Klotho prior to high glucose treatment. Realtime PCR was used to analyze Klotho, TLR4, NGAL mRNA expressions. Western blot was used to observe Klotho, TLR4,NF-kB p65, NGAL,fibronectin (FN), and connective tissue growth factor ( CTGF) protein expression. ELISA assay was used to detect monocyte chemoattractant protein-1 ( MCP-1) and CXCL5 secretions. Results High glucose suppressed Klotho expression significantly(P<0. 05) and activated TLR4 / NF-kB p65 pathway. Meanwhile,the levels of NGAL,FN,CTGF, MCP-1, and CXCL5 were highly expressed ( P < 0. 01). NGAL gene silencing obviously down-regulated the increased expressions of FN, CTGF, MCP-1, and CXCL5 ( P < 0. 01). After PDTC treatment the overexpression of NGAL protein was markedly lowered(P<0. 01). In addition, Klotho treatment significantly inhibited the activity of TLR4 /NF-kB p65 pathways and down-regulated the expressions of NGAL, FN, CTGF, MCP-1 and CXCL5 stimulated by high glucose(P<0. 01). Conclusion Klotho inhibits the activity of TLR4 / NF-kB p65 pathways and thus inhibits NGAL expression in RMCs cultured with high glucose in vitro. And then it suppresses the expressions of FN, CTGF, MCP-1, and CXCL5. This provides a new basis to illustrate the protection mechanism of the anti-aging protein Klotho in diabetic nephropathy, and may provide new ideas and therapeutic targets for prevention and treatment.