Paeoniflorin binds to VEGFR2 to restore autophagy and inhibit apoptosis for podocyte protection in diabetic kidney disease through PI3K-AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) was found to exhibit renal protection from diabetic kidney disease (DKD) in previous trials, but its specific mechanism remains to be elucidated. AIM OF THE STUDY: This study furtherly explored the specific mechanism of PF in protect podocyte injury in DKD. MATERIALS AND METHODS: We observed the effects of PF on renal tissue and podocytes in DKD by constructing the vitro and vivo models after measuring the pharmacokinetic characteristics of PF. Target proteins of PF were found through target prediction, and verified by molecular docking, CESTA, and SPR, and then furtherly explored the downstream regulation mechanism related to podocyte autophagy and apoptosis by network prediction and co-immunoprecipitation. Finally, by using the target protein inhibitor in vivo and knocking down the target protein gene in vitro, it was verified that PF played a role in regulating autophagy and apoptosis through the target protein in diabetic nephropathy. RESULTS: This study found that in STZ-induced mice model, PF could improve the renal biochemical and pathological damage and podocyte injure (p < 0.05), upregulate autophagy activity (p < 0.05), but inhibit apoptosis (p < 0.01). Vascular endothelial growth factor receptor 2 (VEGFR2), predicted as the target of PF, directly bind with PF reflected by molecular docking and surface plasmon resonance detection. Animal studies demonstrated that VEGFR2 inhibitors have a protective effect similar to that of PF on DKD. Network prediction and co-immunoprecipitation further confirmed that VEGFR2 was able to bind PIK3CA to regulate PI3K-AKT signaling pathway. Furthermore, PF downregulated the phosphorylation of PI3K and AKT (p < 0.05). In vitro, similarly to autophagy inhibitors, PF was also found to improve podocyte markers (p < 0.05) and autophagy activity (p < 0.05), decrease caspase 3 protein (p < 0.05) and further inhibited VEGFR2-PI3K-AKT activity (p < 0.05). Finally, the results of VEGFR2 knockdown were similar to the effect of PF in HG-stimulated podocytes. CONCLUSION: In conclusion, PF restores autophagy and inhibits apoptosis by targeting the VEGFR2-mediated PI3K-AKT pathway to improve renal injury in DKD, that provided a theoretical basis for PF treatment in DKD.

Paeoniflorin directly binds to TNFR1 to regulate podocyte necroptosis in diabetic kidney disease.

Necroptosis was elevated in both tubulointerstitial and glomerular renal tissue in patients with diabetic kidney disease (DKD), and was most pronounced on glomerulus in the stage with macroalbuminuria. This study further explored whether paeoniflorin (PF) could affect podocyte necroptosis to protect kidney injure in vivo and in vitro. Our study firstly verified that there are obvious necroptosis-related changes in the glomeruli of DKD through bioinformatics analysis combined with clinicopathological data. STZ-induced mouse diabetes model and high-glucose induced podocyte injury model were used to evaluate the renoprotection, podocyte injury protection and necroptosis regulation of PF in DKD. Subsequently, the target protein-TNFR1 that PF acted on podocytes was found by computer target prediction, and then molecular docking and Surface plasmon resonance (SPR) experiments were performed to verify that PF had the ability to directly bind to TNFR1 protein. Finally, knockdown of TNFR1 on podocytes in vitro verified that PF mainly regulated the programmed necrosis of podocytes induced by high glucose through TNFR1. In conclusion, PF can directly bind and promote the degradation of TNFR1 in podocytes and then regulate the RIPK1/RIPK3 signaling pathway to affect necroptosis, thus preventing podocyte injury in DKD. Thus, TNFR1 may be used as a new potential target to treat DKD.

Identification of Genes Reveals the Mechanism of Cell Ferroptosis in Diabetic Nephropathy.

Aims/Introduction: Diabetic nephropathy (DN) is one of the main complications of diabetes. Genomics may reveal the essential pathogenesis of DN. We analyzed datasets to search for key genes to explore pathological mechanisms of DN. Materials and Methods: In this study, weighted gene co-expression network analysis (WGCNA) was used to divide the differential expression genes (DEGs) from GSE142025 into different modules, and enrichment pathway analysis was conducted for each module to find key genes related to cell death pathway. Then, verification was carried out through network and histopathology. Finally, the regulatory mechanisms of key gene expression, including transcription factors (TFs), miRNA and E3 ligases related to ubiquitination, were predicted through website prediction and then miRNA results were validated using GSE51674 dataset. Results: The results of WGCNA and enrichment pathway analysis indicated that ferroptosis had significantly occurred in advanced DN (AND) group. Analysis of DEGs indicated that the occurrence and development of ferroptosis are mainly through ALOX15-mediated lipid metabolism pathway, which was found in all intrinsic cells of the glomerulus detected by IHC and IF staining. Moreover, network predictions were used for searching ALOX15-related TFs and ubiquitination. Meanwhile, the network predictions combining with other dataset furtherly discovered miRNAs which regulated ALOX15 expression. This study showed that the levels of mmu-miR-142-3p increased in DN mice kidney tissues, compared with the NC group. Conclusion: Ferroptosis existed in glomerular intrinsic cells of ADN group and its potential key candidate gene was ALOX15 which may be regulated by miR-142 and miRNA-650, TFs (CREBBP, EP300, HDAC1, MTA1, SPI1, STAT6) and E3 ligases related to ubiquitination (PML, ZMIZ1, MARCHF1, MARCHF3, MARCHF8, MARCHF11).

Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-ß1/Smad3 pathway in vivo and in vitro.

Diabetes nephropathy (DN) is characterized by abnormal interactions between kidney-infiltrating macrophages and glomerular mesangial cells. Recently, a novel cell-cell communication mediated by exosomes has gained attention. Exosomes are membrane-bound vesicles that contain a variety of molecules such as proteins, lipids, DNA, mRNA, and microRNAs. Exosomes play an important role in the pathogenesis of DN. In this study, we show that high glucose (HG) led to increased excretion of exosomes from macrophages. Mesangial cells took up exosomes in vitro, which resulted in the activation and proliferation of mesangial cells and the secretion of extracellular matrix and inflammatory cytokines. In addition, C57BL/6 mice injected with exosomes from HG-treated macrophages showed morphologic and functional changes. We then showed that exosomes from HG-treated TGF-ß1 knockdown macrophages induced less extracellular matrix and fewer inflammatory factors in mesangial cells compared with vector control. Our findings suggest that TGF-ß1 mRNA in exosomes serves a role between macrophages and mesangial cells by activating the TGF-ß1/ mothers against decapentaplegic homolog 3 pathway.-Zhu, Q.-J., Zhu, M., Xu, M.-X., Meng, X.-M., Wu, Y.-G. Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-ß1/Smad3 pathway in vivo and in vitro.

Selection and Validation of Reference Genes for mRNA Expression by Quantitative Real-Time PCR Analysis in Neolamarckia cadamba.

Neolamarckia cadamba is an economically-important fast-growing tree species in South China and Southeast Asia. As a prerequisite first step for future gene expression studies, we have identified and characterized a series of stable reference genes that can be used as controls for quantitative real time PCR (qRT-PCR) expression analysis in this study. The expression stability of 15 candidate reference genes in various tissues and mature leaves under different conditions was evaluated using four different algorithms, i.e., geNorm, NormFinder, BestKeeper and RefFinder. Our results showed that SAMDC was the most stable of the selected reference genes across the set of all samples, mature leaves at different photosynthetic cycles and under drought stress, whereas RPL10A had the most stable expression in various tissues. PGK and RPS25 were considered the most suitable reference for mature leaves at different developmental stages and under cold treatment, respectively. Additionally, the gene expression profiles of sucrose transporter 4 (NcSUT4), and 9-cis-epoxycarotenoid dioxygenase 3 (NcNCED3) were used to confirm the validity of candidate reference genes. Collectively, our study is the first report to validate the optimal reference genes for normalization under various conditions in N. cadamba and will benefit the future discovery of gene function in this species.

Hepatoprotective effect of peony total glucosides and the underlying mechanisms in diabetic rats.

CONTEXT: Total glucosides of peony (TGP), compounds extracted from the dried roots of Paeonia lactiflora Pall, have been reported to have anti-inflammatory and antioxidative activities. However, the protective effect of TGP on liver injury and the underlying mechanisms remains unknown in diabetic rats. OBJECTIVES: Current study investigates prevention of liver injury by TGP in diabetic rats and its mechanism was related to the inhibition of endoplasmic reticulum stress (ERS). MATERIALS AND METHODS: Fifty adult male rats were randomly divided into: Normal group, diabetic group, TGP (50, 100 and 200 mg/kg/day) treatment groups (n = 10 per group). At the end of the 8th week, the liver was removed for biochemical and histological examinations. RESULTS: Compared with the diabetic group, administration of TGP at doses of 50, 100 and 200 mg/kg significantly prevented the increase of hepatic fibrosis score (ED50 139.4 mg/kg). Compared with diabetic group, TGP at doses of 50, 100 and 200 mg/kg showed an inhibition on the increased macrophage infiltration. MCP-1 and TNF-α mRNA and protein expression were significantly increased in diabetic group compared with normal group; TGP administration caused significant reduction of high levels of MCP-1 and TNF-α mRNA as well as protein levels. Also, TGP at all doses showed an inhibition on the increased GRP78 levels, p-Perk levels and p-Eif2α levels in liver from diabetic group. DISCUSSION AND CONCLUSIONS: Our results indicate that TGP has potential as a treatment for diabetic liver injury attenuating liver lipid accumulation and inflammation as well as ERS induced by diabetic condition.