Supplementary Material for: Diosgenin improves lipid metabolism in diabetic nephropathy via regulation of miR-148b-3p/DNMT1/FOXO1 axis

Background: The progression of diabetic nephropathy (DN) is closely associated with lipid accumulation. Diosgenin (Dio) plays a beneficial role in the lipid metabolism associated with multiple diseases. Thus, the mechanism underlying Dio’s function in DN associated with aberrant lipid accumulation warrants further investigation.
Methods: To model DN in vitro, HK-2 cells were treated with high glucose (HG) and palmitic acid. Cell viability was evaluated using MTT assay. The triglyceride (TG) content in HK-2 cells was measured using a commercial assay kit. The formation of lipid droplets in HK-2 cells was observed using Oil Red O staining. The expression levels of mRNA and protein were detected using RT-qPCR and western blot, respectively. The DNA methylation of FOXO1 was assessed using MSP. The interaction between DNMT1 and the FOXO1 promoter was confirmed by ChIP assay.
Results: Dio treatment reduced TG levels and lipid droplet formation in HK-2 cells co-treated with HG and palmitic acid. Simultaneously, the levels of miR-148b-3p and FOXO1 were increased by Dio, while Dio decreased the expression levels of DNMT1 and SREBP-2. Meanwhile, miR-148b-3p can bind to DNMT1, which in turn inhibits the expression of FOXO1 by mediating the DNA methylation of FOXO1. In addition, FOXO1 negatively regulates the expression of SREBP-2 by interacting with the SREBP-2 promoter. MiR-148b-3p inhibition or silencing of FOXO1 abolished the inhibitory effect of Dio on TG production and lipid droplet formation. This effect was further exacerbated by the downregulation of DNMT1. FOXO1 overexpression may counteract the promotive effects of miR-148b-3p inhibitor on lipid accumulation.
Conclusion: Dio treatment reduced TG production and lipid droplet formation in HK-2 cells during the progression of DN by modulating the miR-148b-3p/DNMT1/FOXO1/SREBP-2 axis. This finding provides new evidence supporting the therapeutic potential of Dio for DN.

背景：糖尿病肾病（DN）的进展与脂质积累密切相关。 Diosgenin（Dio）在多种疾病相关的脂质代谢中发挥着有益作用。因此，Dio在糖尿病肾病中与异常脂质积累相关的功能机制值得进一步研究。
方法：为了在体外模拟DN，对HK-2细胞进行了高糖（HG）和棕榈酸的联合处理。通过MTT试验评估细胞活力。使用商业试剂盒测量HK-2细胞中的甘油三酯（TG）含量。利用Oil Red O染色观察HK-2细胞中脂滴的形成。分别使用RT-qPCR和蛋白质印迹法检测mRNA和蛋白的表达水平。通过甲基化特异性PCR（MSP）评估FOXO1的DNA甲基化。通过ChIP试验证实DNMT1与FOXO1启动子的相互作用。
结果：Dio处理降低了与HG和棕榈酸共处理的HK-2细胞中的TG水平和脂滴形成。同时，Dio增加了miR-148b-3p和FOXO1的水平，而Dio降低了DNMT1和SREBP-2的表达水平。同时，miR-148b-3p可以与DNMT1结合，进而通过介导FOXO1的DNA甲基化来抑制FOXO1的表达。此外，FOXO1通过与SREBP-2启动子相互作用，负调节SREBP-2的表达。miR-148b-3p的抑制或FOXO1的沉默消除了Dio对TG产生和脂滴形成的抑制作用。这一效应通过DNMT1的下调进一步加剧。FOXO1过表达可能抵消了miR-148b-3p抑制剂对脂质积累的促进作用。
结论：Dio处理通过调节miR-148b-3p/DNMT1/FOXO1/SREBP-2轴，降低了HK-2细胞在糖尿病肾病进展过程中的TG产生和脂滴形成。这一发现为Dio在糖尿病肾病治疗中的潜在疗效提供了新的证据。