基于网络药理学和SIRT1/PGC-1α信号通路探究青光安Ⅱ号方的视神经保护作用

Protective effects of Qingguang'anⅡFormula on the optic nerve based on network pharmacology and SIRT1/PGC-1αsignaling pathway

目的基于网络药理学和实验研究探讨青光安Ⅱ号方对青光眼视神经的保护作用机制。方法通过TCMSP数据库筛选青光安Ⅱ号方成分靶点,在GeneCards、Disgenet、CTD数据库挖掘青光眼相关靶点,进而筛选青光安Ⅱ号方作用于青光眼的靶点;制作蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络取其交集,并通过GO分析和KEGG富集分析。建立自发性慢性高眼压DBA/2J青光眼小鼠模型,将C57BL/6J小鼠设置为空白组(等体积蒸馏水),DBA/2J小鼠随机分为模型组(等体积蒸馏水)、益脉康组[0.31 g/(kg·d)]、青光安Ⅱ号方低浓度组[0.85 g/(kg·d)]、青光安Ⅱ号方中浓度组[1.7 g/(kg·d)]、青光安Ⅱ号方高浓度组[3.4 g/(kg·d)],每组8只,每日灌胃1次。干预4周后,触式眼压笔监测小鼠眼压;HE染色观察小鼠视网膜形态结构;Western blot检测沉默信息调节因子-1(silent information regulator type-1,SIRT1)、过氧化物酶体增殖物激活受体γ共激活因子-1α(peroxisome proliferationactivated receptor-γ-coactivator 1α,PGC-1α)的蛋白表达水平;qRT-PCR检测SIRT1、PGC-1α的mRNA表达水平。结果从青光安Ⅱ号方共筛选得到101个活性成分和245个相关靶点,2412个青光眼疾病相关基因靶点;药物-活性成分-靶点相互作用最强的5个靶点分别是前列腺素内过氧化物合成酶2、核受体共激活因子2、胃蛋白酶原Ⅱ、前列腺素内过氧化物合成酶1以及过氧化物酶体增殖物激活受体γ(peroxisome proliferative activated receptor gamma,PPARG);PPI网络显示较强的靶点是SIRT1、PPARG;GO分析和KEGG富集分析得到细胞衰老、IL-17等信号通路。与给药前相比,给药后用药组眼压显著降低(P<0.01)。给药后,与空白组相比,模型组眼压显著升高(P<0.01),视网膜中SIRT1、PGC-1αmRNA表达量和蛋白表达量均显著降低(P<0.01);与模型组相比,用药组眼压显著降低(P<0.01),SIRT1、PGC-1αmRNA表达量和蛋白表达量均显著升高(P<0.01)。与益脉康组和青光安Ⅱ号方低浓度组相比,青光安Ⅱ号方中、高浓度组SIRT1、PGC-1αmRNA表达量和SIRT1蛋白表达量显著升高(P<0.01);与青光安Ⅱ号方低浓度组相比,青光安Ⅱ号方高浓度组PGC-1α蛋白表达量均显著上升(P<0.01)。与青光安Ⅱ号方中浓度组相比,青光安Ⅱ号方高浓度组PGC-1αm RNA表达量和蛋白表达量显著升高(P<0.01)。结论青光安Ⅱ号方可有效调控SIRT1/PGC-1α信号通路,抑制RGC的丢失,主要在氧化应激、细胞衰老等方面对青光眼视神经发挥保护作用。

Objective To explore the mechanism of protective action of Qingguang'anⅡFormula(QGAFⅡ)on optic nerve in glaucoma based on network pharmacology and experimental research.Methods Targets of QGAFⅡcomponents were screened through the TCMSP database,and glaucoma-related targets were mined from GeneCards,Disgenet,and CTD databases.Then the targets through which QGAFⅡacts on glaucoma were identified.A protein-protein interaction(PPI)network was constructed using the intersection of these targets,and GO and KEGG pathway analyses were performed on them.A spontaneous chronic high intraocular pressure(IOP)DBA/2J glaucoma mouse model was established.C57BL/6J mice were set as the blank group(an equal volume of distilled water),and DBA/2J mice were randomize into the model group(an equal volume of distilled water),Yimaikang(YMK)group[0.31 g/(kg·d)],and low-[0.85 g/(kg·d)],medium-[1.7 g/(kg·d)],and high-concentration[3.4 g/(kg·d)]QGAFⅡgroups.Each group consisted of eight mice,and they were administered by gavage once daily.After 4 weeks of intervention,the IOP of mice was monitored by a contact tonometer;HE staining was used to observe the morphological structure of the mouse retina;Western blot was used to examine the protein expression levels of silent information regulator type-1(SIRT1)and peroxisome proliferator-activated receptor-γ-coactivator 1α(PGC-1α);qRT-PCR was used to check the mRNA expression levels of SIRT1 and PGC-1α.Results A total of 101 active ingredients and 245 related targets were screened from QGAFⅡ.Additionally,2,412 disease-related gene targets for glaucoma were identified.The five targets with the strongest drug-active ingredient-target interactions were prostaglandin-endoperoxide synthase 2,nuclear receptor coactivator 2,pepsinogenⅡ,prostaglandin-endoperoxide synthase 1,and peroxisome proliferator activated receptor gamma(PPARG).The PPI network showed that the stronger targets were SIRT1 and PPARG.Signaling pathways such as oxidative stress,cellular senescence,and IL-17 were obtained by GO and KEGG enrichment analyses.Compared with before administration,the IOP of the treatment groups significantly decreased after administration(P<0.01).After administration,compared with the blank group,the model group showed a significant increase in IOP(P<0.01),and the mRNA and protein expression levels of SIRT1 and PGC-1αin the retina were significantly reduced(P<0.01).Compared with the model group,the IOP in the treatment groups showed a significant decrease(P<0.01),while the mRNA and protein expression levels of SIRT1 and PGC-1αsignificantly increased(P<0.01).Compared with the YMK group and the low-concentration QGAFⅡgroup,the mRNA expression levels of SIRT1 and PGC-1αand the protein expression level of SIRT1 significantly increased in the medium-and high-concentration QGAFⅡgroups(P<0.01);compared with the low-concentration QGAFⅡgroup,the protein expression level of PGC-1αin the high-concentration QGAFⅡgroup significantly increased(P<0.01);compared with the medium-concentration QGAFⅡgroup,the mRNA and protein expression levels of PGC-1αsignificantly increased in the high-concentration QGAFⅡgroup(P<0.01).Conclusion QGAFⅡcan effectively regulate the SIRT1/PGC-1αsignaling pathway,and inhibit the loss of retinal ganglion cells(RGCs).It mainly exerts protective effects on the optic nerve in glaucoma by targeting oxidative stress and cellular senescence.