Investigations On The Role Of LncRNA Vof16 In TBI Rats And Its Relationship With Therapeutic Efficacy Of Lutong Granules Protecting TBI

Objective:This study aimed to investigate on the role of lncRNA vof16 in TBI rats and its relationship with therapeutic efficacy of Lutong Granules protecting TBI.Methods:1.Adult male Sprague-Dawley(SD)rats were randomly divided into sham operation group(sham),high dose of Lutong Granules s group(LTG),medium dose of Lutong Granules group(LTZ)and normal saline group(NS).The TBI rat model was established to explore the protective effect and possible mechanism of Lutong Granules on TBI rats.Firstly,the neurological severity score(NSS)was used to evaluate the nerve injury of rats,then the motor function and the learning and memory ability was tested by behavioral tests including Rotating Rod test and Morris water maze test.The degree of brain tissue damage of rats was diagnosed by MRI examination.Hematoxylin-eosin(HE)staining and Nissl staining were used to observe the pathological changes of rats after brain injury.TUNEL/NeuN double labeling immunostaining was used to detect the apoptosis of neurons in each group.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect expression changes of TNF-α,IL-1β,IL-10,caspase 3,Bax and Bcl-2 in the injured cortex of rats in each group 1 day after TBI.2.LncRNA microarray was used to detect the expression of lncRNAs in rat brain 1 days after TBI injury.The relative expression of the screened genes was detected by qRT-PCR.Then,the target genes of the selected genes were predicted and analyzed by bioinformatics.3.LncRNA vof16+/-and lncRNA vof16-ORF rats were constructed and divided into Sham,NS,lncRNA vof16+/-TBI and lncRNA vof16-ORF TBI groups to further explore the function of lncRNA vof16 in rat brain injury.NSS was used to evaluate the nerve injury of rats in each group,and then the motor function of rats was tested by behavioral tests including wire suspension,rotating rod and Morris water maze tests.The pathological changes of rats after brain injury were observed by HE staining and Nissl staining.4.TBI rats were divided into sham group,wild-type rat group(WT),wild-type rat treated with Lutong Granules(WT-LT),lncRNA vofl6+/-TBI group and lncRNA vof16+/-treated with Lutong Granules(lncRNA vof16+/--LT),so as to further explore the relationship between the therapeutic efficacy of Lutong Granules in the treatment of brain injury and expression variation of lncRNA vof16.NSS score,Morris water maze and Rotating rod tests were performed on rats in each group to evaluate the effect of Lutong Granules on the severity of nerve injury and learning and memory ability of TBI rats.HE staining and Nissl staining were used to observe the pathological changes of rats in each group after brain injury.5.LncRNA vof16 plays an important role in the neuroprotective mechanism of Lutong Granules.In order to further explore its regulatory mechanism,we applied network pharmacology-based analysis and screened out seven traditional Chinese medicine components of Lutong Granules.Disease targets of TBI were obtained on Genecards,which were then intersected with drug targets.Then,the potential targets of Lutong Granules in the treatment of TBI were analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).At last,the above common targets were further intersected with the target genes of vof16 to find the downstream crucial genes.6.West blotting(WB)test was used to detect the protein expression of the crucial downstream gene IL-10 in the cerebral cortex of rats in each group.Results:1.Through behavioral test and morphological test experiment,it was found that different doses of Lutong Granules injection can reduce brain damage in TBI rats,promote the recovery of motor function and improve the learning and memory ability of rats,while the therapeutic effect of high concentration(4g/kg)of Lutong Granules seems much more prominent.QRT-PCR reveled that Lutong Granules treatment prominently decreased the expression of IL-1β,TNFα,Bax and caspase3.2.In view of the expression changes of lncRNAs in brain tissues after TBI in rats,we screened 9 differentially expressed lncRNAs.The expression of lncRNA vof16 was validated by qRT-PCR,which was consistent with the results of bioinformatics prediction.Target prediction and bioinformatics analysis on lncRNA vof16 revealed that the target genes of lncRNA vof16 were related to neuroinflammatory pathways.3.As revealed,lncRNA vof16+/-significantly aggravated the pathological changes of neurons as well as the neurobehavior after TBI injury,while lncRNA vof16-ORF improved the learning and memory,spatial memory and working memory abilities of TBI rats along with neuronal damage.4.Lutong Granules improved the learning and memory ability,spatial memory ability and working memory ability of TBI rats,and Lutong Granules treatment alleviated the neuronal injury and neurological dysfunctions caused by LncRNA vof16+/-.5.By taking the drug disease target intersection,we obtained a total of 87 overlapping genes between disease-related genes and targets of active compound of Lutong Granules.The analysis of differentially expressed genes by GO and KEGG showed that Lutong Granules is very crucial in regulating inflammatory and immune response.We also intersected the target genes from network pharmacology with vof16 regulatory genes,and obtained the crucial gene IL-10.6.After lncRNA vof16 knockdown,the protein expression level of IL-10 decreased significantly.However,after treatment with Lutong Granules,the protein expression level of IL-10 increased prominently.ConclusionsLutong Granules exerts a significant protective effect on TBI brain injury,which can significantly alleviate the brain damage of TBI rats,inhibit the apoptosis of cells and neurons,promote the recovery of motor function and improve the learning and memory ability of rats.The therapeutic mechanism of Lutong Granules may be associated with mediating lncRNA vof16-targeted regulation of anti-inflammatory factor IL10.Lutong Granules play a neuroprotective role by regulating inflammatory response,and reduce the acute and chronic injury caused by TBI.