目的 探討新橙皮苷治療對創(chuàng)傷性腦損傷大鼠的影響其潛在機制。方法 借助TCMSP、PharmMapper、OMIM、GeneCards等數據庫并結合文獻資料補充,獲取藥物、疾病相關靶點,通過STRING數據庫構建新橙皮苷治療與創(chuàng)傷性腦損傷疾病靶點蛋白質相互作用(PPI)網絡,利用Cytoscape軟件對PPI網絡進行拓撲分析,篩選得到核心靶點,基于DAVID數據庫進行基因本體(GO)功能與京都基因與基因組百科全書(KEGG)富集分析,并通過Autodock Vina軟件模擬新橙皮苷與關鍵靶點蛋白可能的對接結果。通過Feeney's法構建SD大鼠創(chuàng)傷性腦損傷模型,造模后進行改良大鼠神經功能缺損嚴重程度評分(mNSS);通過尼氏染色觀察神經元病理學改變,利用TUNEL染色觀察神經元凋亡情況,ELISA法測定腦組織損傷灶炎癥因子白細胞介素(IL)-6、IL-1β、腫瘤壞死因子-α(TNF-α)表達水平,qRT-PCR法檢測腦組織損傷灶血管內皮生長因子(VEGF)、酪氨酸蛋白激酶(SRC)、蛋白激酶B1(Akt1) mRNA表達水平。結果 共獲取得到藥物-疾病交集靶點85個,篩選得到核心靶點10個,主要包括基質金屬蛋白酶9(MMP9)、表皮生長因子受體(EGFR)、絲裂原活化蛋白激酶(MAPK)8、IL-6、SRC、TNF、Akt1等。通過KEGG通路分析結果顯示,關鍵靶點可能集中在VEGF信號通路、TNF通路、Rap1信號通路等信號通路。新橙皮苷與核心靶點MMP9、EGFR、MAPK8、IL-6、TNF、Akt1等均有較好的親和力。動物實驗發(fā)現,新橙皮苷治療改善了創(chuàng)傷性腦損傷大鼠的神經功能,減少神經細胞損傷,抑制細胞凋亡(P<0.05),新橙皮苷治療使大鼠腦組織中炎癥因子IL-6、IL-1β、TNF-α表達水平明顯下調(P<0.05),VEGF、SRC、Akt1 mRNA表達水平顯著上調(P<0.05)。結論 新橙皮苷可能作用于MAPK8、IL-6、SRC、TNF、Akt1等核心靶點,通過VEGF信號通路、TNF通路等多條信號通路發(fā)揮治療創(chuàng)傷性腦損傷的作用,具有多靶點、多通路的特點。

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-α were 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-1β and TNF-α in 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.

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河南省醫(yī)學科技攻關計劃項目（212102310708）；河南省神經修復重點實驗室開放課題（HNSJXF-2021-001）