Supplementary Material for: Green tea polyphenols alleviate kidney injury induced by di(2-ethylhexyl) phthalate in mice

Background: Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer. Studies have revealed that DEHP exposure can cause kidney damage. Green tea is among the most popular beverages in China. Green tea polyphenols (GTPs) have been proven to have therapeutic effects on organ damage induced by heavy metal exposure. However, few studies have reported on GTP relieving DEHP-induced kidney damage. Methods: C57BL/6J male mice aged 6–8 weeks were treated with distilled water (control group), 1500 mg/kg/d DEHP + corn oil (model group), 1500 mg/kg/d DEHP + corn oil + 70 mg/kg GTP (treatment group), corn oil (oil group), and 70 mg/kg GTP (GTP group) by gavage for 8 weeks, respectively. The renal function of mice and renal tissue histopathology of each group were evaluated. The renal tissues of mice in the model, treatment, and control groups were analyzed using high-throughput sequencing. We calculated the differentially expressed miRNAs and mRNAs using the limma R package, the CIBERSORT algorithm was used to predict immune infiltration, the starBase database was used to screen the miRNA–mRNA regulatory axis, and immunohistochemical analyses were performed to verify protein expression. Results: GTP alleviated the deterioration of renal function, renal inflammation and fibrosis, and mitochondrial and endoplasmic reticulum lesions induced by DEHP in mice. Differential immune infiltrations of plasma, dendritic, T, and B cells were noted between the model and treatment groups. We found that three differentially expressed miRNAs (mmu-miR-383-5p, mmu-miR-152-3p, and mmu-miR-144-3p), three differentially expressed mRNAs (Ddit4, Dusp1, and Snx18), and three differentially expressed proteins (Ddit4, Dusp1, and Snx18) played crucial roles in the miRNA–mRNA–protein regulatory axes when GTPs mitigate DEHP-induced kidney damage in mice. Conclusion: GTP can alleviate DEHP-induced kidney damage and regulate immune cell infiltration. We screened four important miRNA–mRNA–protein regulatory axes of GTP mitigating DEHP-induced kidney damage in mice.

背景：邻苯二甲酸二(2-乙基己基)酯（Di(2-ethylhexyl) phthalate, DEHP）是一种常见的塑化剂。已有研究证实，DEHP暴露可引发肾脏损伤。绿茶是国内最受欢迎的饮品之一，绿茶多酚（Green Tea Polyphenols, GTPs）已被证实对重金属暴露诱导的器官损伤具有治疗作用，但目前鲜有研究报道GTP对DEHP诱导的肾脏损伤的缓解作用。
方法：选取6~8周龄的C57BL/6J雄性小鼠，分别通过灌胃给予蒸馏水（对照组）、1500 mg/kg/d DEHP+玉米油（模型组）、1500 mg/kg/d DEHP+玉米油+70 mg/kg GTP（干预组）、玉米油（玉米油对照组）以及70 mg/kg GTP（GTP对照组），连续给药8周。随后检测各组小鼠的肾功能，并观察肾脏组织病理学变化。对模型组、干预组及对照组的小鼠肾脏组织进行高通量测序分析：采用limma R包计算差异表达的微小RNA（microRNA, miRNA）和信使RNA（messenger RNA, mRNA），利用CIBERSORT算法预测免疫浸润情况，通过starBase数据库筛选miRNA–mRNA调控轴，并通过免疫组化实验验证蛋白表达水平。
结果：GTP可缓解DEHP诱导的小鼠肾功能恶化、肾脏炎症与纤维化，以及线粒体和内质网损伤。模型组与干预组间的血浆细胞、树突状细胞、T细胞及B细胞免疫浸润存在显著差异。本研究发现，3种差异表达的微小RNA（mmu-miR-383-5p、mmu-miR-152-3p及mmu-miR-144-3p）、3种差异表达的信使RNA（Ddit4、Dusp1及Snx18）以及3种差异表达蛋白（Ddit4、Dusp1及Snx18），在GTP缓解小鼠DEHP诱导肾脏损伤的miRNA–mRNA–蛋白调控轴中发挥关键作用。
结论：GTP可减轻DEHP诱导的小鼠肾脏损伤，并调节免疫细胞浸润情况。本研究筛选出4条参与GTP缓解小鼠DEHP诱导肾脏损伤的关键miRNA–mRNA–蛋白调控轴。