目的 利用網(wǎng)絡藥理學和實驗驗證探討蒲黃治療脊髓損傷的分子機制。方法 使用TCMSP數(shù)據(jù)庫及文獻資料篩選蒲黃的有效成分及其對應靶點，使用DrugBank、OMIM、TTD、GeneCard數(shù)據(jù)庫獲得脊髓損傷相關靶點。Cytoscape構建蒲黃抗脊髓損傷靶點蛋白相互作用（PPI）網(wǎng)絡圖及“藥物–化合物–靶點”網(wǎng)絡圖，根據(jù)網(wǎng)絡圖中各靶點的拓撲學參數(shù)篩選出蒲黃抗脊髓損傷關鍵靶點及關鍵成分。使用DAVID數(shù)據(jù)庫對蒲黃抗脊髓損傷靶點進行基因本體論（GO）功能和京都基因與基因組百科全書（KEGG）富集分析。采用Autodock Vina軟件對關鍵化合物與關鍵靶點蛋白進行分子對接驗證。動物實驗觀察蒲黃對脊髓損傷大鼠后肢運動神經(jīng)功能的影響及對關鍵靶點蛋白mRNA表達的影響。結果 共篩選出花生四烯酸、異鼠李素、β谷甾醇、山柰酚、棕櫚酸睪酮、山柰酚-3-O-α-L-鼠李糖基（1→2）-β-D-葡萄糖苷、槲皮素、異鼠李素-3-O-新橙皮苷、香蒲新苷等活性成分。PPI網(wǎng)絡分析顯示，腫瘤壞死因子（TNF）、白細胞介素（IL）-6、蛋白激酶B（AKT1）、IL-1B、胱天蛋白酶3（CASP3）等蛋白可能在脊髓損傷的治療中起關鍵作用。蒲黃抗脊髓損傷靶點的GO分析顯示生物過程主要涉及對脂多糖的反應、對細菌源性分子的反應、細胞對化學應激的反應、活性氧代謝過程的調控過程等。KEGG富集分析顯示富集顯著性較高的通路主要有TNF信號通路、TLR信號通路、MAPK信號通路等。分子對接顯示關鍵活性成分與關鍵靶蛋白分子之間親和力良好。動物實驗結果表明，與模型組相比，蒲黃能有效促進脊髓損傷大鼠模型動物的后肢運動恢復，降低TNF、IL-6、IL-1B、CASP3 mRNA表達，升高AKT1 mRNA表達（P＜0.05、0.01）。結論 蒲黃中槲皮素、山柰酚、異鼠李素、β-谷甾醇等活性成分可能通過作用于TNF、IL-6、AKT1、IL-1B、CASP3等靶點，進而影響炎癥及凋亡相關的通路發(fā)揮治療脊髓損傷的作用。

Objective To explore the molecular mechanism of Typhae Pollen in treatment of spinal cord injury by using network pharmacology and validation experiments. Methods TCMSP database and literature data were used to screen the active components and corresponding targets of Typhae Pollen. DrugBank, OMIM, TTD, and GeneCard databases were used to obtain the targets of spinal cord injury. Cytoscape constructed PPI network diagram and drug-compound-target network diagram, and screened the key targets and components of Typhae Pollen against cord injury according to topological parameters of each target in the network diagram. DAVID database was used to analyze the GO function and KEGG enrichment of the anti-spinal cord injury targets of Typhae Pollen. Autodock Vina software was used for molecular docking verification of key compounds and key target proteins. Animal experiments were conducted to observe the effect of Typhae Pollen on the motor nerve function of hind limbs and the expression of key target protein mRNA in rats with spinal cord injury. Results A total of arachidonic acid, isorhamnetin, β sitosterol, kaempferol, testosterone palmitate, kaempferol-3-O-α-L-rhamnosyl(1→2)-β-D-glucoside_qt, quercetin, isorhamnetin-3-O-neohesperidoside, ctyphloside and other active ingredients were screened. PPI network analysis showed that TNF, IL-6, AKT1, IL-1B, CASP3 and other proteins may play a key role in the treatment of spinal cord injury. Gene ontology (GO) analysis of the anti-spinal cord injury targets of Typhae Pollen showed that the biological processes mainly involve the reaction to lipopolysaccharide, the reaction to bacterial molecules, the cellular response to chemical stress, and the regulation of reactive oxygen metabolism. The enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that the pathways with high enrichment significance mainly included TNF signaling pathway, TLR signaling pathway, and MAPK signaling pathway. Molecular docking showed that the affinity between key active components and key target protein molecules was good. Animal experimental results showed that compared with the model group, Typhae Pollen could effectively promote the recovery of hind limb movement, decrease the mRNA expression of TNF, IL-6, IL-1B and CASP3, and increase the mRNA expression of AKT1 in rats with spinal cord injury (P < 0.05, 0.01). Conclusion Quercetin, kaempferol, isorhamnetin, β-sitosterol and other active ingredients in Typhae Pollen may affect inflammatory and apoptosis-related pathways by acting on TNF, IL-6, AKT1, IL-1B, CASP3 and other targets.

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