Xuefu Zhuyu Decoction Treating Traumatic Brain Injury Through The Non-coding RNA Regulation

Background and Objective: Traumatic brain injury(TBI)is a complex injury with a multi-faceted recovery process,with high mortality and disability.Researchers have focused on the understanding of the pathobiology of cellular and molecular changes and injury mechanisms after TBI.Physicians have considered that blood stasis is a pathophysiological process throughout the whole course of TBI.Therefore,activating blood circulation and removing stasis is an effective treatment for TBI.Xuefu Zhuyu Decoction(XFZYD)is recorded in Yilin Gaicuo by Qingren Wang.The evidenced-based investigations demonstrated that XFZYD had exhibited neuroprotective activities in TBI.However,the underlying mechanisms are unclear.Long non-coding RNAs(lnc RNAs)are demonstrated to be involved in central nervous system(CNS)disorders.However,the roles of lnc RNA in neurological deficits post-TBI are poorly understood.The present study depicted the microarray’s lnc RNA and messenger RNA(m RNA)profiles at 14 days in TBI mice hippocampi.We aimed to investigate the novel therapeutic targets of XFZYD on the function of TBI and explore new ideas for traditional Chinese medicine.Methods: C57BL/6 mice were randomly divided into three groups:Sham group,TBI group and XFZYD group.Modified neurological severity score,corner turn test,weight change,Morris water maze,HE staining,Nissl’s,Evans blue,and water content were used to assess the neurological outcome.The expression profiles of lnc RNA and m RNA in the hippocampus of the three groups were investigated by microarray.The altered lnc RNA and m RNA profiles in the TBI mice could be identified by the comparative analysis of the lnc RNA and m RNA data between sham and TBI groups.Then,bioinformatics analysis was used to indicate the potential regulatory functions of lnc RNA and m RNA in TBI.Moreover,we employed quantitative real-time polymerase chain reaction(q RT-PCR)to validate the results.Besides,through conjoint analysis of lnc RNA and m RNA data of the three groups,we could find out the lnc RNA and m RNA as the therapeutic targets of XFZYD in TBI treatment.Additionally,bioinformatics analysis was used to construct lnc RNA-mi RNA-m RNA networks.q RT-PCR was applied to validate the reliability of the lnc RNA,mi RNA,and m RNA.In vitro experiment demonstrated that ENSMUST00000180908,which is regulated by XFZYD,could alleviate neuroinflammation in LPS-stimulated BV2 microglial cells through mi R-466h-5p/Gabbr2 axis.The expressions of i NOS and COX2 were detected by western blot.Inflammatory cytokine expression levels were determined by ELISA assay.The interaction between ENSMUST00000180908,micro RNA(mi RNA),and its target gene was validated by luciferase reporter assay.Results: A total of 264 differentially expressed lnc RNAs and 232 expressed m RNAs were identified(fold change > 1.5 and P-value < 0.05).Altered genes were enriched in inflammation,immune response,bloodbrain barrier,glutamatergic neurological effects,and neuroactive ligandreceptor,which may be associated with TBI-induced pathophysiologic changes in the long-term neurological deficits.The lnc RNAs-m RNAs coexpression network was generated for 74 lnc RNA-m RNA pairs,most of which are positive correlations.The lnc RNA-mi RNA-m RNA interaction network included 12 lnc RNA,59 mi RNAs,and 25 m RNAs.Numerous significantly altered lnc RNA and m RNA in mice hippocampi were enriched in inflammation and immune response.Furthermore,these dysregulated lnc RNA and m RNAs may be promising therapeutic targets to overcome obstacles in long-term recovery following TBI.The m NSS and morris water maze indicated that XFZYD notably improved neurological deficits and cognitive function after TBI.H&E staining and Nissl staining demonstrated that XFZYD suppressed damage and neuronal loss.Interestingly,157 lnc RNAs and 96 m RNAs were distinctly regulated by XFZYD.The bioinformatics analysis showed that XFZYD-related m RNAs could contribute to signaling transduction,immune response,and axon guidance.The lnc RNA-mi RNA-m RNA interaction network included4 lnc RNAs,39 mi RNAs,and 18 m RNAs.The results of q RT-PCR indicated that ENSMUST00000180908(P < 0.01)and Gabbr2(P < 0.01)were downregulated in TBI and upregulated in XFZYD(ENSMUST00000180908,P < 0.01;Gabbr2,P < 0.05).The expression of mi R-466h-5p was upregulated in TBI(P < 0.01)and downregulated in XFZYD(P < 0.01).The research provides new insight into XFZYD’s therapeutic targets in treating TBI.LPS treatment suppressed ENSMUST00000180908 expression in BV-2 cells.Overexpression of ENSMUST00000180908 inhibited LPS-induced BV-2 microglial cell activation and decreased the expression of inflammatory cytokine.ENSMUST00000180908 function as a competing endogenous RNA(ce RNA)by sponging mi R-466h-5p.In LPS-stimulated BV-2 cells,ENSMUST00000180908 exerted its inhibitory function via negatively modulating mi R-466h-5p expression.Moreover,we identified that Gabbr2 was a direct target of mi R-466h-5p.Dual-luciferase reporter assay further validated the relationship among ENSMUST00000180908,mi R-466h-5p,and Gabbr2.Conclusion: 1.The lnc RNA and m RNAs profiles are significantly altered in the hippocampus at the subacute stage of TBI 2.The expression of lnc RNA and m RNA is regulated by XFZYD at the subacute stage of TBI.3.lnc RNA-ENSMUST00000180908 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through mi R-466h-5p/Gabbr2 axis.