基于蛋白质组学探究三七总皂苷对2型糖尿病小鼠肝脏的保护作用

Proteomics study the protective effects of Panax notoginsenosides on liver in mice with type 2 diabetes mellitus

肝脏是机体糖脂代谢的主要器官,持续性高血糖是导致肝损伤的常见原因。三七总皂苷(Panax notoginsenosides, PNS)是三七中的主要活性成分,具有抗炎、抗氧化等作用。本研究拟采用定量蛋白质组学结合实验验证探究PNS对2型糖尿病(type 2 diabetes mellitus, T2DM)小鼠肝损伤的保护作用及其潜在机制。所有动物实验经川北医学院实验动物伦理委员会批准(批准号:NSMC2022023)。采用苏木精-伊红染色(hematoxylin-eosin staining, H&E)和透射电镜分析PNS对T2DM小鼠肝脏结构及形态的影响;通过脱氧核苷酸末端转移酶介导的dUTP缺口末端标记(TdT-mediated dUTP nick-end labeling, TUNEL)染色分析PNS对T2DM小鼠肝细胞凋亡的影响;采用活性氧(reactive oxygen species, ROS)和丙二醛(malonaldehyde, MDA)试剂盒探究PNS对T2DM小鼠肝脏氧化损伤的影响作用。基于定量蛋白质组学探究T2DM和T2DM+PNS组小鼠肝脏蛋白质表达谱变化。利用倍数变化及显著性水平分析T2DM和T2DM+PNS组间差异表达蛋白;采用GeneAnalytics数据库通路富集分析;使用Metascape数据库基因本体分析;基于STRING数据库构建蛋白-蛋白互作网络;采用Western blot检测蛋白表达水平。结果表明, PNS能够改善T2DM小鼠肝脏结构,抑制肝细胞凋亡,改善线粒体和内质网形态。蛋白质组数据显示,489种基因在T2DM小鼠肝脏中表达显著改变, PNS治疗后42种基因表达改变且向正常组转归。通路富集表明,T2DM组固醇激素生物合成、AMP依赖蛋白激酶(adenosine 5′-monophosphate-activated protein kinase, AMPK)通路、氧化压力、胰岛素信号、磷脂酰肌醇通路、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)介导的炎症、胰岛素抵抗及mTOR信号异常改变, PNS能够激活AMPK、TNF-α、凋亡及胰岛素通路。Western blot表明, PNS抑制Bax、Grp78和Chop的表达,降低cleaved casp6/casp6比值,增加pAMPKα、HO-1和核内Nrf2的表达,提示PNS可能通过激活AMPK/Nrf2/HO-1信号通路减轻氧化应激和内质网应激,抑制细胞凋亡进而发挥对糖尿病小鼠的肝脏保护作用。

Liver is the main organ of glucose and lipid metabolism,and persistent hyperglycemia is a common cause of liver injury.Panax notoginsenosides(PNS)is the main active ingredient in Panax notoginseng,which have anti-inflammatory and antioxidant effects.In this study,quantitative proteomics combined with experimental verification was used to explore the protective effect of PNS on liver injury in type 2 diabetes mellitus(T2DM)mice and its potential mechanism.All experiments were approved by the Ethical Committee Experimental Animal Center of North Sichuan Medical College(NSMC2022023).Hematoxylin-eosin(H&E)staining and transmission electron microscopy were used to observe the effect of PNS on the histopathological changes of liver in T2DM mice.TdT-mediated dUTP Nick-end labeling(TUNEL)staining was used to analyze the effect of PNS on hepatocyte apoptosis in T2DM mice.Reactive oxygen species(ROS)and malonaldehyde(MDA)kits were used to detect the effect of PNS on oxidative damage of liver in T2DM mice.Subsequently,proteomics profiling of mice in T2DM and T2DM+PNS groups were investigated based on quantitative proteomics.Differentially expressed proteins were screened out according to fold change and significance level in T2DM and T2DM+PNS groups,respectively.Pathway enrichment analysis of these differential proteins was done using GeneAnalytics database.Gene ontology analysis was conducted by Metascape database.Protein-protein interaction networks were constructed based on STRING database.Western blot was used to detect protein expression.These results showed that PNS could improve liver abnormalities,inhibit hepatocyte apoptosis,and improve the morphology of mitochondria and endoplasmic reticulum in T2DM mice.Proteome data demonstrated that 489 genes expression changed significantly in liver of T2DM mice compared with normal,and 42 ones were significantly reversed after PNS treatment and returned to normal levels.Pathway analysis showed that sterol hormone biosynthesis,adenosine 5′-monophosphate-activated protein kinase(AMPK)signaling pathway,oxidative stress,insulin signaling,phosphatidylinositol pathway,tumor necrosis factor-α(TNF-α)mediated inflammation,insulin resistance,and mTOR signaling pathway exhibited notable changes based on pathway enrichment ratio and significance level.It is worth noting that PNS could improve the abnormal changes of AMPK,TNF-α,apoptosis and insulin pathways.Western blot manifested that PNS inhibit the expression of Bax,Grp78 and Chop,reduce ratio of cleaved casp6/casp6,increase the levels of pAMPKα,HO-1 and Nu-Nrf2 in the liver of T2DM mice.These results suggested that PNS may play protective roles in the liver of T2DM mice by inhibiting apoptosis via activating AMPK/Nrf2/HO-1 signaling pathway,alleviating oxidative stress and endoplasmic reticulum stress.