基于高内涵分析技术的新型PPARα/γ双重激动剂C333H和P633H的肝细胞毒性初步研究

Cytotoxicity of PPARα/γ dual agonists C333H and P633H on HepG2 cells using high content analysis

目的基于建立的高内涵细胞多参数毒性分析方法,观察新型PPARα/γ双重激动剂C333H和P633H的HepG2肝细胞毒性,并初步探查其可能的损伤机制。方法在HepG2人肝癌细胞上,采用高内涵分析(HCA)技术,进行C333H和P633H的细胞毒性(细胞数量、核固缩、膜通透性、线粒体膜电位和细胞色素C),以及氧化应激和DNA损伤的多参数分析。结果 MTT法测得C333H和P633H抑制HepG2细胞增殖的IC50分别为(161.57±15.29)μmol/L和(101.08±17.58)μmol/L。HCA研究表明C333H在10～100μmol/L,P633H在3～30μmol/L显著降低膜通透性和线粒体膜电位;C333H在30～300μmol/L、P633H在10～100μmol/L浓度依赖性地降低细胞数量。至高浓度时,300μmol/L C333H和100μmol/L P633H进一步显著降低线粒体膜电位和细胞数量,显著增加核DNA及细胞色素C含量,显著减少核面积,此浓度下膜通透性和超氧化物歧化酶显著增加。结论 C333H在低于100μmol/L,P633H在低于30μmol/L时对HepG2肝细胞无明显的毒性反应;线粒体损伤、膜完整性破坏是两化合物引起细胞毒性反应的主要机制,其中线粒体损伤为毒性反应早期敏感性检测指标。此外,HCA法与MTT法在细胞增殖作用的分析上具有较好的一致性。

Objective To observe the hepatotoxicity of novel PPARα/γ dual agonists C333H and P633H on HepG2 cells using high content analysis（HCA）,and to explore the characteristics of toxicity.Methods Multi-parameter analysis of cytotoxicity（cell number,pyknosis,membrane permeability,mitochondrial membrane potential and cytochrome C）,oxidative stress and DNA damage of C333H and P633H was carried out on the human hepatoma cell line HepG2 cells by using HCA.Results The IC_50 of C333H and P633H on inhibiting the proliferation of HepG2 cells was（161.57±15.29） μmol/L and（101.08±17.58）μmol/L,respectively.HCA indicated that the membrane permeability and mitochondrial membrane potential were significantly decreased at the concentration of 10 to 100 μmol/L of C333H,and at 3 to 30 μmol/L of P633H.Reduction of the cell number in a concentration-dependent manner was observed in C333H（30 to 300 μmol/L） and P633H（10 to 100 μmol/L）.300 μmol/L of C333H and 100 μmol/L of P633H led to a lower mitochondrial membrane potential and cell number,increased the content of nuclear DNA and cytochrome C,decreased the nuclear area.Meanwhile,the membrane permeability and manganese-containing superoxide dismutase were also significantly increased at this concentration.Conclusion The results indicated that C333H（100 μmol/L） and P633H（30 μmol/L） show no significant cytotoxicity on HepG2 cells.The damage to the mitochondrial and to the membrane integrity is the main mechanism of cytotoxicity,the former being an early sensitive index.Additionally,HCA and MTT methods show better consistency in the analysis of the impact on cellular proliferation.