To investigate the mechanism of renal ischemia-reperfusion injury (IRI) via the regulation of N6-methyl-adenosine (m6A) and relevant genes, IRI was induced in Sprague Dawley rats and the urine and serum creatinine levels and tissue structure changes were observed. m6A and METTL3 protein levels were assessed via dot blotting and western blotting, respectively. The hypoxia/reoxygenation (H/R) cell model was constructed using NRK-52E cells, and METTL3 protein levels were assessed. METTL3 was inhibited to observe its impact on NRK-52E cell apoptosis and m6A expression in H/R processes. Methylated RNA immunoprecipitation (MeRIP) sequencing was conducted, followed by MeRIP-qRT-PCR and qRT-PCR validation. Our results indicated that urine and serum creatinine levels increased and that renal injury and cell apoptosis were both observed in IRI model. In additon, m6A expression increased in the IRI model, and METTL3 protein levels significantly increased in the IRI and H/R models. When METTL3 was inhibited, the m6A levels were accordingly decreased and cell apoptosis was suppressed in the H/R in vitro model. Based on MeRIP sequencing, tfap2a, cyp1b1, and foxd1 were significantly differentially expressed, as was m6A, which is involved in the negative regulation of cell proliferation and kidney development. We confirmed that foxd1 mRNA and its methylation levels contributed to IRI and H/R.

Background Circular RNA (circRNA) is involved in the process of acute kidney injury (AKI), but only a few circRNAs have been reported. In the study, we investigated a new circRNA and its association with AKI. Methods An AKI model was established in Sprague-Dawley rats, followed by serum creatinine and urea nitrogen tests measured by a biochemical analyzer. The pathological changes and apoptosis in the renal tissue were detected by Hematoxylin and Eosin, and TUNEL staining. Then, circRNA expression in AKI was determined by quantitative real-time-PCR (qRT-PCR). NRK-52E cells were induced with hypoxia/reoxygenation (H/R) as in vitro models and the circ-Snrk level was tested by qRT-PCR. The effects of circ-Snrk in H/R-induced NRK-52E cells were assessed by flow cytometry, western blot, and enzyme-linked immunosorbent assay. Finally, RNA sequencing and western blot analysis were used to validate the mRNA profile and pathways involved in circ-Snrk knockdown in H/R-induced NRK-52E. Results A reliable AKI rat model and H/R cell model were established. qRT-PCR demonstrated that circ-Snrk level was upregulated in AKI left kidney tissue and NRK-52E cells with H/R treatment. Circ-Snrk knockdown inhibited apoptosis of NRK-52E cells and secretion of inflammatory factors (IL-6 and TNF-α). RNA sequencing showed that the mRNA profile changed after inhibition of circ-Snrk and differential expression of mRNA mainly enriched various signaling pathways, including MAPK signaling pathway. Furthermore, western blot indicated that circ-Snrk knockdown could inhibit the activation of p-JNK and p-38 transcription factors. Conclusions Circ-Snrk is involved in AKI development and associated with the MAPK signaling pathway in AKI.

Background: Ferroptosis is a new form of regulated cell death characterized by the accumulation of iron-dependent lipid peroxides and membrane damages. Recent studies have identified an important role for cancer cell ferroptosis in antitumor therapy. On the other hand, polyphyllin I (PPI) has been reported to exert antitumor effects on some types of cancers. However, it remains unknown whether or not PPI regulates cancer cell ferroptosis.Methods: Two types of human gastric cancer cells (AGS and MKN-45) were used to establish tumor xenograft models in nude mice that were treated with polyphyllin I (PPI) to observe tumor growth, while cells also were cultured for in vitro studies. Ferroptosis, based on the intracellular ROS/lipid ROS production and accumulation of ferrous ions, was detected using a fluorescence microscope and flow cytometer, while the expression of NRF2/FTH1 was measured using Western blotting assays.Results: Here we found that PPI inhibited the gastric cancer growth in vivo and in vitro while increasing the intracellular reactive oxygen species (ROS)/lipid peroxides and ferrous ions in the gastric cancer cells. PPI also decreased the levels of nuclear factor erythroid 2-related factor 2 (NRF2) and ferritin heavy chain 1 (FTH1) in gastric cancer cells in vitro. Moreover, liproxstain-1, an inhibitor of cell ferroptosis, mostly reversed the cell ferroptosis and tumor growth arrest induced by PPI. Finally, the effects of PPI on cancer cell ferroptosis were diminished by the overexpression of NRF2.Conclusion: For the first time, our results have demonstrated that PPI exerts its antitumor activity on the gastric cancer by, at least partially, inducing cancer cell ferroptosis via regulating NRF2/FTH1 pathway. These findings may be implicated for clinical replacement therapy of the gastric cancer.

Renal fibrosis underlies all forms of end-stage kidney disease. Endophilin A2 (EndoA2) plays a role in nephrotic syndrome; however, its effect on renal fibrosis remains unknown. Here, we demonstrate that EndoA2 protects against kidney interstitial fibrosis via the transforming growth factor-β (TGF-β)/Smad signaling pathway. Mouse kidneys with fibrosis or kidney biopsy specimens from patients with fibrotic nephropathy had lower levels of EndoA2 protein expression than that in kidneys without fibrosis. In vivo overexpression of EndoA2 with the endophilin A2 transgene (EndoA2 Tg ) notably prevented renal fibrosis, decreased the protein expression of profibrotic molecules, suppressed tubular injury, and reduced apoptotic tubular cells in the obstructed kidney cortex of mice with unilateral ureteral obstruction (UUO). In vivo and in vitro overexpression of EndoA2 markedly inhibited UUO-or TGF-β1induced phosphorylation of Smad2/3 and tubular epithelial cells dedifferentiation. Furthermore, EndoA2 was coimmunoprecipitated with the type II TGF-β receptor (TβRII), thus inhibiting the binding of the type I TGF-β receptor (TβRI) to TβRII. These findings indicate that EndoA2 mitigates renal fibrosis, at least partially, via modulating the TGF-β/ Smad signaling. Targeting EndoA2 may be a new potential therapeutic strategy for treatment of renal fibrosis.

Objective Inflammation of the surrounding environment is a major reason causing loss or injury of oligodendrocyte precursor cells (OPCs) in myelin-associated diseases. Lipopolysaccharide-activated microglia can release various inflammatory factors such as tumor necrosis factor-α (TNF-α). One of the ways of OPC death is necroptosis, which can be triggered by TNF-α, a death receptor ligand, by activating receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL) signaling pathway. This study investigated whether inhibiting microglia ferroptosis can decrease TNF-α release to alleviate OPC necroptosis. Methods Lipopolysaccharide and Fer-1 stimulate BV2 cells. The expressions of GPX4 and TNF-α were detected by western blot and quantitative real-time PCR; malondialdehyde, glutathione, iron, and reactive oxygen species were measured by the assay kits. After lipopolysaccharide stimulation of BV2 cells, the supernatant was taken to culture OPC. The protein expression levels of RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL were detected by western blot. Results Lipopolysaccharide administration could induce ferroptosis in microglia by decreasing ferroptosis marker GPX4, while ferroptosis inhibitor Fer-1 could significantly increase GPX4 level. Fer-1 prevented oxidative stress and iron concentration elevation and alleviated mitochondrial damage in lipopolysaccharide-induced BV2 cells. The results revealed that Fer-1 downregulated the release of lipopolysaccharide-induced TNF-α in microglia and attenuated OPC necroptosis by significantly decreasing the expression levels of RIPK1, p-RIPK1, MLKL, p-MLKL, RIPK3, and p-RIPK3. Conclusion Fer-1 may be a potential agent for inhibiting inflammation and treating myelin-related diseases.