Vitamin D Protected Against Traumatic Brain Injury Via Modulating The TLR4/MyD88/NF-κB Pathway

Objective:We attempted to investigate the effects of vitamin D on tissue damage and functional impairments in the hippocampus after traumatic brain injury(TBI).The expression of TLR4 in the hippocampal CA1 region of TBI rats and the regulation of TLR4 on neuroinflammation were observed in TBI rat model.It was confirmed whether vitamin D may regulate TLR4/My D88/NF-κB signaling pathway to improve microglia activation and neuroinflammatory response after TBI,further facilitate prognosis of brain injury,which will reveal the molecular mechanism of neuroprotective effects of vitamin D administration,and provide theoretical basis for clinical treatment of TBI.Methods:(1)In vivo experiment: Adult male SD rats were divided into Sham group,TBI group and TBI+VD group according to random number method,30 animals in each group.A rat TBI model was established using a controlled cortical impact method,and rats were intraperitoneally injected with VD(1μg/kg)post-injury.The degree of neurological impairment,motor coordination ability and spatial learning and memory ability of rats were observed by modified Neurological function score(m NSS),rotarod test and Morris water maze test.Secondary brain injury of TBI rats was evaluated by measuring the water content of brain tissue and of blood-brain barrier(BBB)permeability.HE staining and Nissl staining of brain tissue was used to observe the survival of neurons at 3 days post-TBI.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX)and malondialdehyde(MDA),as well as the contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and IL-6in hippocampus tissues were determined by enzyme-linked immunosorbent assay(ELISA).In vitro experiment: Hippocampal neurons of newborn SD rats were extracted,cultured and identified in vitro.Cultured neurons were randomly divided into Control group,LPS group and LPS+VD group).Primary cultured hippocampal neurons were incubated with 1 μg/ml LPS and treated with active vitamin D.MTT colorimetric assay was used to detect the survival rate of hippocampal neurons in each group,and lactate dehydrogenase(LDH)kit was used to detect LDH content in cell culture supernatant.(2)TLR4 sh RNA was injected into the lateral ventricle of rats to silence the expression level of TLR4.Adult male SD rats were randomly divided into Sham group,TBI group,TLR4 sh RNA group and control sh RNA group.15 ul TLR4 sh RNA or control sh RNA was intracerebroventricularly injected 48 h before TBI model establishment.Real-time PCR and Western blot were used to detect the m RNA and protein expression of TLR4 in the hippocampus of each group of rats,and the co-localization neuron specific marker Neu N and astrocyte specific protein GFAP was observed by double mmunofluorescence staining.HE staining,brain water content and m NSS score were used to evaluate the protective effects of TLR4 inhibition in TBI rats.ELISA was used to detect the protein contents of neuroinflammatory factors TNF-α,IL-1β and IL-6 in hippocampus of rats in each group.Western blot was used to detect autophagy-related indicators l C-3II /LC-3I ratio and Beclin-1 protein expression in hippocampal CA1 region to evaluate the effect of TLR4 inhibition on neuronal autophagic activation in hippocampal CA1 region.Western blot analysis of GFAP protein was used to observe the effect of TLR4 inhibition on TBI-induced astrocyte activation.(3)Adult male SD rats were randomly divided into four groups: Sham operation Sham group,TBI group,vitamin D treatment group(TBI+VD group),and vitamin D combined with TLR4 agonist group(TBI+VD+LPS group).TLR4 agonist LPS(50μg/kg)was intraperitoneously injected 24 h before TBI model preparation.The protein expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor88(My D88),nuclear NF-κB and cytoplasmic NF-κB in the hippocampus were detected by Western blot analysis.Real-time PCR was used to detect m RNA levels of pro-inflammatory factor(TNF-α,IL-1β)and M1 microglia marker inducible nitric oxide synthase(i NOS),as well as anti-inflammatory factor(IL-4,IL-10)and M2 microglia marker Arginase-1(Arg-1)in rat hippocampus.The m NSS score and Morris water maze test were used to observe the degree of neurological impairment in rats,and the damage of brain tissue was assessed by the detection of brain tissue water content and BBB integrity.Results:(1)In vivo experiment: Compared with Sham group,m NSS score was increased,latency to fall from rotating rod is shorter,escape latency is prolonged,time spent in the platform quadrant and the number of crossing platform is significantly reduced in the TBI group.However,Vitamin D treatment significantly improved sensorimotor dysfunctions,motor imbalance and cognitive impairments in rats subjected to TBI.In addition,increased brain water content and blood-brain barrier permeability,hippocampal morphology damage,the up-regualation of oxidative markers MDA content,the down-regualation of antioxidant enzymes SOD and GSH-PX level,and an increase of inflammatory cytokines TNF-α,IL-1β and IL-6 release.Compared with TBI group,vitamin D treatment in TBI+VD group significantly inhibited cerebral edema and blood-brain barrier disruption,alleviated hippocampal tissue damage,oxidative stress injury and neuroinflammatory response,finally contributing to the recovery of secondary brain injury after TBI.In vitro experiments: LPS stimulation could induce morphological damage of cultured primary hippocampal neurons,inhibit neuronal activity and up-regulate the content of LDH in cell culture supernatant compared to the control group.Compared with the LPS group,vitamin D treatment significantly enhanced hippocampal neuron activity and inhibited cytotoxicity,ultimately promoting the survival of injured neurons caused by LPS in vitro.(2)In the TBI rat model,endogenous TLR4 protein showed a significant higher expression in the injured hippocampus,and localized in hippocampal neurons and astrocytes in rats.The intracerebroventricular injection of TLR4 sh RNA significantly inhibited the m RNA and protein expression of TLR4 in the hippocampus in rats,confirming the transfection efficiency of TLR4 sh RNA.Compared with TBI group,m NSS score and brain water content were decreased,and hippocampal tissue injury was improved in the TLR4 sh RNA group.In the study of mechanism,compared with Sham group,the ratio of LC-3II/LC-3I,the expression levels of Beclin-1 and GFAP protein were significantly increased in the hippocampus of TBI rats,revealing that the activation of autophagy and astrocyte proliferation were initiated by brain injury.Inhibition of TLR4 expression could significantly reduce LC-3II/LC-3I ratio,Beclin-1protein levels and GFAP protein expression to repress TBI-induced autophagy activation and astrocyte proliferation of rat hippocampal neurons,which plays an important role in the pathological process of TBI disease.(3)The protein expression of TLR4,My D88 and nuclear NF-κB p65 were increased and cytoplasmic NF-κB p65 level was decreased in the hippocampus of TBI rat model,which activated the TLR4/My D88/NF-κB signaling pathway.However,vitamin D supplementation significantly down-regulated the protein expression of TLR4,My D88 and nuclear NF-κB p65,but up-regulated the protein expression of NF-κB p65 in the cytoplasm,finally inhibiting the activation of TLR4/My D88/NF-κB signaling pathway and antagonizing the hippocampal neuroinflammation.Furthermore,vitamin D treatment could reverse the up-regulation of TNF-α,IL-1βand i NOS expression and the down-regulation of IL-4,IL-10 and Arg-1 expression in hippocampal tissue induced by TBI,promoting microglial M2-type polarization.However,TLR4 activator LPS combined with vitamin D therapy showed that TLR4 activator could partially block the regulation of vitamin D on microglia polarization and neuroinflammatory response.In addition,TLR4 activator could partially abolished the protective effects of vitamin D on promoting brain tissue and restoring neurocognitive dysfunction in rat models of TBI.Conclusion:Combination in vivo animal experiments and in vitro cell experiments revealed that vitamin D treatment might effectively inhibit secondary brain injury,improve neurological deficits and cognitive dysfunctions,attenuate oxidative stress injury and neuroinflammatory response in the hippocampus,and promoting the survival of hippocampal neurons in vivo and in vitro,representing significant therapeutic benefits following TBI.TLR4 mediated-hippocampal neuronal autophagy and astrocytes activation participated in hippocampal tissue and function repair after TBI.Vitamin D plays a neuroprotective role in microglia polarization and neuroinflammation involving in TLR4/My D88/NF-κB signaling pathway.