Supplementary Material for: Dihydroquercetin/Taxifolin inhibits METTL3/m6A/PIM2 expression by suppressing RIPK2 phosphorylation mediated NF-κB p65 signaling pathway to alleviates renal ischemia-reperfusion injury in mice

There is a close relationship between oxidative stress and renal ischemia-reperfusion injury (RIRI). Dihydroquercetin (DHQ, also konown as Taxifolin) can reduce kidney damage, and suppress cellular damage caused by oxidative stress. This study aimed to investigated the effects and underlying mechanisms of DHQ on RIRI. Firstly, hypoxia/reoxygenation (H/R) treated HK-2 cells were used for RIRI in vitro experiments, and the dosage of DHQ was selected by observing cell viability and lactate dehydrogenase (LDH) release. The protective effects of DHQ were evaluated by detecting cell apoptosis, reactive oxygen species (ROS) levels, and oxidative stress indicators levels. Our data suggested that DHQ inhibited cell apoptosis, ROS levels, and oxidative stress of H/R treated HK-2 cells. PIM2 regulates cell survival and proliferation, and H/R treatment up-regulated PIM2 levels. Over-expression of PIM2 partly reversed the effects of DHQ on protecting HK-2 cells after H/R induction, and promoted RIPK2 phosphorylation mediated NF-κB p65 signaling pathway. Additionally, METTL3 is an N6-methyladenosine (m6A) writer, and DHQ suppressed its expression in H/R treated HK-2 cells. METTL3 was enriched in PIM2 mRNA by m6A modification and stabilized the expression of PIM2. As for in vivo experiments, DHQ also inhibited the PIM2 level in mice kidney tissues and alleviated the kidney damage caused by RIRI. These effects were manifested in the reduction of renal injury markers, the reduction of oxidative stress levels, and the results of renal tissue HE staining. DHQ also suppressed PIM2 mediated RIPK2 phosphorylation mediated NF-κB p65 signaling pathway in renal tissues. Taken together, DHQ inhibited PIM2 levels by suppressing METTL3 mediated m6A modification to protect HK-2 cells from H/R induced injury, and this may be related to the regulation of PIM2 on RIPK2 phosphorylation mediated NF-κB p65 signaling pathway. This study suggested that DHQ may be a promising drug to treat RIRI, and may be useful for finding effective therapeutic targets in RIRI.