Effects And Mechanisms Of Ingredients Of Xingnaojing Injection For Traumatic Brain Injury Based On Integrative Analysis

Objective: Xingnaojing injection is an injection of the main components of traditional Chinese medicine Angong Niuhuang Pills,and it has been widely used in Chinese medicine since it can effectively alleviate brain injury.We aim to explore the active ingredients and molecular mechanisms of XNJI in TBI.Methods:Part 1: The chemical ingredients were acquisited from literature and TCMSP.The ADME parameter-based virtual algorithm was utilized to further identify bioactive components.The targets of screened compounds and TBI were retrieved using public databases,then intersected to obtain Venn diagram of hub targets.The network was constructed and visualized by Cytoscape software.Besides,protein-protein interaction(PPI)network was created and worked on online STRING11.0 according to hub proteins.Based on the information obtained from the DAVID bioinformatics resource database,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were investigated and visualized by R language.Then we screen out the potential active monomers including muscone,borneol,camphor and curdione,and obtain the apoptosis-related signaling pathways that may be affected by Xingnaojing injection.Subsequently,combined with real-time fluorescence quantitative(Q-PCR)analysis,the neuroprotective effects of muscone,borneol,camphor and curdione on craniocerebral trauma rats were preliminarily explored.We separated 78 adult male SD rats around 300 g into 2 groups,group 1 and group 2.Group 1 was divided into 6subgroups,namely sham-24 h subgroup,sham-72 h subgroup,sham-168 h subgroup,TBI-24 h subgroup,TBI-72 h subgroup,TBI-168 h subgroup;group 2 was divided into 7subgroups,sham subgroup,TBI subgroup,XNJI subgroup,muscone subgroup,borneol subgroup,camphor subgroup,curdione subgroup each with 6 rats.The TBI group adopted CCI(controlled cortical impact),while the sham group only opened the bone window and did not attack.The brains of all rats were taken at 24 h,72h and 168 h after operation.The dose of the XNJI group was 10 ml/kg/d.The dose of the muscone borneol,camphor,curdione group was 0.54 mg/kg/d,8mg/kg/d,0.7 mg/kg/d,0.1mg/kd/d respectively,and the brain was taken 168 hours after surgery.Part two:18 rats were randomly divided into 3 groups,each with 6 rats,namely sham,TBI,TBI+muscone groups.All rats were sampled at 168 h postoperatively.The daily injection dose of TBI+muscone was 0.54 mg/kg muscone injection.The TBI and sham groups were injected with a certain dose of saline.These rats were used for TUNEL neuronal apoptosis detection and Western Blot detection of p-p65,p65,p-PI3K,PI3K,p-Akt1(Thr308),Akt1,Bax,Bcl-2,Caspase-3,p53 expression Level,to explore the mechanism of PI3K/Akt1 pathway in the neuroprotective effect of anti-apoptosis.Result:Part1:1.1 The systematic network pharmacology analysis model was established.A total of 173 targets and 35 potential Xingnaojing injection compounds were identified.STRING software analysis illustrates the interaction between protein and protein network,and muscone plays an important role in Xingnaojing.Enrichment analysis revealed the key signaling pathways in the potential protein targets of these components,such as PI3K/AKT1,NF-kB and p53 signaling pathways.In addition,central proteins(CASP3,BCL2L1 and CASP8)are also involved in apoptosis and are associated with PI3K/AKT,NF-kB and p53 signaling pathways.1.2 Changes in apoptosis-related m RNA at different time points There was no significant difference at 24 hours,72 hours and 168 hours in the sham group for the levels of Bax,Bcl-2,Caspase-3,and p53 respectively(P>0.05),and then the three subgroups of brain trauma increased with time Significant changes occurred after prolonging,reaching the highest level at 168 hours and there was a significant difference compared with the 24-hour group(P<0.05).That is,the time point after the CCI was selected through the preliminary experiment was 168 h.1.3 The effects of Xingnaojing and its active ingredients muscone、borneol、camphor、curdione on apoptosisThe results showed that Xingnaojing injection can significantly reduce the levels of Bax Bcl-2,Caspase-3,and p53 m RNA after CCI.In the TBI+muscone group,muscone can exert a neuroprotective effect similar to that of Xingnaojing,but there was no significant impact on apoptosis-related m RNA for rest of ingredients.2.1 Muscone inhibited neuronal cell apoptosis The apoptosis of rat cortical neurons at 168 hours after CCI attack was significantly increased,but it was inhibited by muscone(P<0.05).2.2 Muscone can relieve the expression of apoptosis-related molecules In the TBI group,the expression levels of Bax,Bcl-2,and Caspase-3 were significantly higher than those in the sham group,which could be alleviated by muscone.2.3 Muscone can regulate the activity and expression of PI3K and AKT1 In the TBI group,PI3K as well as AKT1,and their phosphorylation expression levels increased(P<0.05).However,the expression of TBI+muscone was suppressed compared to TBI group.2.4 Muscone can inhibit the activity and expression of NF-kB In the TBI group,the expression level of NF-kB(p65)and its phosphorylation(p-p65)increased(P<0.05).Compared with the TBI group,the expression of p65 and p-p65 was suppressed.2.5 Muscone can reduce p53 expression After the CCI attack,the p53 level of the traumatic brain injury group was higher than that of the sham operation group(P<0.05),but the increase trendency can be alleviated by muscone.Conclusion:This is the first time that muscone has been used to verify the neuroprotective mechanism of a rat brain injury(CCI)model.The results showed that the muscone in XNJI may significantly attenuate neuronal apoptosis through the PI3K/Akt1/NF-kB/P53 pathway.To improve nerve and nerve damage,network pharmacology provides a new method for studying the identification of potential active ingredients in Xingnaojing.