基于网络药理学和体外实验探究石杉碱甲对急性肺损伤中肺泡上皮细胞的作用及保护机制

Effect and protective mechanism of huperzine A on alveolar epithelial cells in acute lung injury based on network pharmacology and in vitro experimentation

基于网络药理学和体外实验,探讨石杉碱甲(huperzine A,Hup A)对急性肺损伤(acute lung injury,ALI)中肺泡上皮细胞的作用及机制。首先从公共数据库查找Hup A、ALI的相关靶点,获取二者交集即为Hup A保护ALI的潜在靶点并进行富集分析;通过构建蛋白互作网络及“成分-靶点-通路”网络进而筛选Hup A保护ALI的核心靶点并进行分子对接验证;进一步采用脂多糖(lipopolysaccharide,LPS)刺激小鼠肺泡上皮细胞(MLE-12)构建细胞模型加以Hup A预处理,分别检测细胞活性、白介素-6(interleukin-6,IL-6)水平、超氧化物歧化酶(super oxide dismutase,SOD)水平、丙二醛(malondialdehyde,MDA)水平验证Hup A的保护作用,最后蛋白免疫印迹实验验证关键靶点的蛋白水平变化。网络药理学分析结果显示Hup A共有144个潜在治疗靶点,Hup A参与调节蛋白激酶活性、氧化应激、炎症反应等生物学过程和磷脂酰肌醇-3-激酶-丝苏氨酸蛋白激酶(phosphatidyl-inositol 3-kinase-serine-threonine kinase,PI3K-Akt)通路、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路等信号通路;分子对接结果显示Hup A与Akt1、Akt2和Mapk1(又称细胞外蛋白调节激酶(extracellular regulated protein kinases,Erk))均具有较好的结合活性;细胞实验显示Hup A可以改善LPS诱导的细胞活性下降(P<0.05),降低IL-6、MDA、p-Erk1/2/Erk1/2水平(P<0.05),提高SOD与p-Akt1/Akt1水平(P<0.05)。综上,Hup A可能通过调控Akt1和Mapk1保护肺泡上皮细胞进而缓解LPS诱导的ALI。

Based on network pharmacology and experimental validation,this study investigated the protective effect and mechanism of huperzine A(Hup A)on alveolar epithelial cells in acute lung injury(ALI).Firstly,the potential targets for Hup A to protect against ALI were identified by searching the public database for related targets of Hup A and ALI,and obtaining their intersection.Subsequently,the core target of Hup A protection against ALI was screened through construction of protein interaction network and"component-target-pathway"network,followed by verification through molecular docking.Furthermore,a cell model was established by stimulating mouse alveolar epithelial cells(MLE-12)with lipopolysaccharide(LPS)and pre-treating them with Hup A.The protective effect of Hup A was verified by detecting changes in cell viability,interleukin-6(IL-6)level,super oxide dismutase(SOD)level and malondialdehyde(MDA)level.Finally,Western blot test was conducted to verify alterations in protein levels of key targets.Network pharmacological analysis revealed 144 potential therapeutic targets for Hup A,implicating its involvement in the regulation of biological processes such as protein kinase activity,oxidative stress,inflammatory response,and signaling pathways including phosphatidyl-inositol 3-kinase-serine-threonine kinase(PI3K-Akt)and mitogen-activated protein kinase(MAPK).Molecular docking results demonstrated favorable binding activity of Hup A with Akt1,Akt2 and Mapk1(also known as extracellular regulated protein kinases,Erk).Cell experiments indicated that Hup A effectively ameliorated LPS-induced decline in cell viability(P<0.05),reduced levels of IL-6,MDA and p-Erk1/2/Erk1/2(P<0.05),while increasing SOD and p-Akt1/Akt1 levels(P<0.05).In conclusion,Hup A may exert a protective effect in LPS induced ALI through the regulation of Akt1 and Mapk1 targets within alveolar epithelial cells.