基于网络药理学、分子对接技术及实验验证探讨新橙皮苷治疗创伤性脑损伤作用机制

Mechanism of neohesperidin in treatment of traumatic brain injury based on network pharmacology,molecular docking technique and animal experiments

目的探讨新橙皮苷治疗对创伤性脑损伤大鼠的影响其潜在机制。方法借助TCMSP、PharmMapper、OMIM、GeneCards等数据库并结合文献资料补充,获取药物、疾病相关靶点,通过STRING数据库构建新橙皮苷治疗与创伤性脑损伤疾病靶点蛋白质相互作用(PPI)网络,利用Cytoscape软件对PPI网络进行拓扑分析,筛选得到核心靶点,基于DAVID数据库进行基因本体(GO)功能与京都基因与基因组百科全书(KEGG)富集分析,并通过Autodock Vina软件模拟新橙皮苷与关键靶点蛋白可能的对接结果。通过Feeney’s法构建SD大鼠创伤性脑损伤模型,造模后进行改良大鼠神经功能缺损严重程度评分(mNSS);通过尼氏染色观察神经元病理学改变,利用TUNEL染色观察神经元凋亡情况,ELISA法测定脑组织损伤灶炎症因子白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)表达水平,qRT-PCR法检测脑组织损伤灶血管内皮生长因子(VEGF)、酪氨酸蛋白激酶(SRC)、蛋白激酶B1(Akt1)mRNA表达水平。结果共获取得到药物–疾病交集靶点85个,筛选得到核心靶点10个,主要包括基质金属蛋白酶9(MMP9)、表皮生长因子受体(EGFR)、丝裂原活化蛋白激酶(MAPK)8、IL-6、SRC、TNF、Akt1等。通过KEGG通路分析结果显示,关键靶点可能集中在VEGF信号通路、TNF通路、Rap1信号通路等信号通路。新橙皮苷与核心靶点MMP9、EGFR、MAPK8、IL-6、TNF、Akt1等均有较好的亲和力。动物实验发现,新橙皮苷治疗改善了创伤性脑损伤大鼠的神经功能,减少神经细胞损伤,抑制细胞凋亡(P<0.05),新橙皮苷治疗使大鼠脑组织中炎症因子IL-6、IL-1β、TNF-α表达水平明显下调(P<0.05),VEGF、SRC、Akt1mRNA表达水平显著上调(P<0.05)。结论新橙皮苷可能作用于MAPK8、IL-6、SRC、TNF、Akt1等核心靶点,通过VEGF信号通路、TNF通路等多条信号通路发挥治疗创伤性脑损伤的作用,具有多靶点、多通路的特点。

Objective To study the effect of neohesperidin on traumatic brain injury in rats and explore its potential mechanism.Methods To obtain relevant targets of drugs and diseases by TCMSP,PharmMapper,OMIM,GeneCards and other databases and supplemented with literature.Construct the target PPI network between neohesperidin therapy and traumatic brain injury disease through STRING database.GO function and KEGG enrichment were analyzed based on the DAVID database,and the possible docking results of neohesperidin with key target proteins were simulated by Autodock Vina software.The model of traumatic brain injury in SD rats was established by Feeney's method,and the modified neurological impairment severity score(mNSS)was performed after the model.The pathological changes of neurons were observed by Nishi staining,the apoptosis of neurons was observed by TUNEL staining,and the expression levels of inflammatory factors IL-6,IL-1β,and TNF-αwere measured by ELISA.The mRNA expression levels of VEGF,SRC,and Akt1 were detected by qRT-PCR.Results A total of 85 drug-disease intersection targets were obtained,and 10 core targets were screened,including MMP9,EGFR,MAPK8,IL-6,SRC,TNF,Akt1,etc.KEGG pathway analysis showed that the key targets may focus on the VEGF signaling pathway,TNF signaling pathway,Rap1 signaling pathway and other signaling pathways.Neohesperidin has a good affinity with the core targets MMP9,EGFR,MAPK8,IL-6,TNF,Akt1,etc.Animal experiments showed that neohesperidin treatment improved nerve function,reduced nerve cell injury and inhibited apoptosis(P<0.05),and neohesperidin treatment significantly decreased the expression levels of inflammatory factors IL-6,IL-1βand TNF-αin rat brain tissue(P<0.05).The mRNA expression levels of VEGF,SRC,and Akt1 were significantly up-regulated(P<0.05).Conclusion Neohesperidin may act on MAPK8,IL-6,SRC,TNF,Akt1,and other core targets,and play a role in the treatment of traumatic brain injury through multiple signaling pathways such as VEGF signaling pathway and TNF pathway,which has the characteristics of multi-target and multi-pathway.