Genipin ameliorates diabetic retinopathy via the HIF-1α and AGEs-RAGE pathways.

BACKGROUND: Traditional Chinese medicine (TCM) is useful in disease treatment and prevention. Genipin is an active TCM compound used to treat diabetic retinopathy (DR). In this study, a network pharmacology (NP)-based approach was employed to investigate the therapeutic mechanisms underlying genipin administration in DR. METHODS: The potential targets of DR were identified using the gene expression omnibus (GEO) database. TCM database screening and NP were used to predict the potential active targets and pathways of genipin in DR. Cell viability was tested in vitro to determine the effects of different doses of glucose and genipin on Human Retinal Microvascular Endothelial Cells (hRMECs). CCK-8, CCK-F, colony formation, CellTiter-Lum, Annexin V-FITC, wound healing, Transwell, tube-forming, reactive oxygen species (ROS), and other assay kits were used to detect the effects of genipin on hRMECs during high levels of glucose. In vivo, a streptozotocin (STZ)-mouse intraocular genipin injection (IOI.) model was used to explore the effects of genipin on diabetes-induced retinal dysfunction. Western blotting was performed to identify the cytokines involved in proliferation, apoptosis, angiogenesis, ROS, and inflammation. The protein expression of the AKT/ PI3K/ HIF-1α and AGEs/ RAGE pathways was also examined. RESULTS: Approximately 14 types of TCM, and nearly 300 active ingredients, including genipin, were identified. The NP approach successfully identified the HIF-1α and AGEs-RAGE pathways, with the EGR1 and UCP2 genes, as key targets of genipin in DR. In the in vitro and in vivo models, we discovered that high glucose increased cell proliferation, apoptosis, angiogenesis, ROS, and inflammation. However, genipin application regulated cell proliferation and apoptosis, inhibited angiogenesis, and reduced ROS and inflammation in the HRMECs exposed to high glucose. Furthermore, the retinal thickness in the genipin-treated group was lower than that in the untreated group. AKT/ PI3K/ HIF-1α and AGEs/ RAGE signaling was increased by high glucose levels; however, genipin treatment decreased AKT/ PI3K and AGEs/ RAGE pathway expressions. Genipin also increased HIF-1α phosphorylation, oxidative phosphorylation of ATP synthesis, lipid peroxidation, and the upregulation of oxidoreductase. Genipin was found to protect HG-induced hRMECs and the retina of STZ-mice, based on; 1 the inhibition of UCP2 and Glut1 decreased intracellular glucose, and glycosylation; 2 the increased presence of HIF-1α, which increased oxidative phosphorylation and decreased substrate phosphorylation; 3 the increase in oxidative phosphorylation from ATP synthesis increased lipid peroxidation and oxidoreductase activity, and; 4 the parallel effect of phosphorylation and glycosylation on vascular endothelial growth factor (VEGF), MMP9, and Scg3. CONCLUSION: Based on NP, we demonstrated the potential targets and pathways of genipin in the treatment of DR and confirmed its effective molecular mechanism in vitro and in vivo. Genipin protects cells and tissues from high glucose levels by regulating phosphorylation and glycosylation. The activation of the HIF-1α pathway can also be used to treat DR. Our study provides new insights into the key genes and pathways associated with the prognosis and pathogenesis of DR.

Gpx4, Selenov, and Txnrd3 Are Three Most Testis-Abundant Selenogenes Resistant to Dietary Selenium Concentrations and Actively Expressed During Reproductive Ages in Rats.

Almost all selenogenes are expressed in the testis, and those have the highest and constant expressions will be the primary candidates for functional analysis of selenium (Se) in male reproduction. This study aimed to profile the mRNA expressions of the testis-abundant selenogenes of rat models in responses to growth and dietary Se concentrations. Forty-eight weaning SD male rats were fed Se deficient basal diet (BD) for 5 weeks and then randomly grouped (n = 12/group) for being fed BD or BD plus 0.25, 3, or 5 mg Se/kg for 4 more weeks before sacrifice. Abundances of selenogenomic mRNAs in the liver and testis were determined with relative qPCR and those of the testis-abundant selenogenes in 13 kinds of tissues were assayed with a molecular beacon-based qPCR. Spatiotemporal expressions of rat selenogenome were also analyzed with the RNA-Seq transcriptomic data published by NCBI. mRNA abundances of glutathione peroxidase 4 (Gpx4), nuclear Gpx4 (nGpx4), selenoprotein V (Selenov), and thioredoxin reductase 3 (Txnrd3) in the testis were significantly higher than that in any other tissues (P < 0.05). Moreover, testicular mRNA abundances of Gpx4, Selenov, and Txnrd3 were not affected by levels of dietary Se supplementation (P > 0.05), and much higher at 6-21 weeks old than at 2 and 104 weeks old (P < 0.05). The result showed that Gpx4, Selenov, and Txnrd3 were most highly expressed in the testis of rats especially at reproductive ages and resistant to the impact of dietary Se levels, which suggested their specific importance in male reproduction.

Dietary Selenium Deficiency or Excess Reduces Sperm Quality and Testicular mRNA Abundance of Nuclear Glutathione Peroxidase 4 in Rats.

Background: Glutathione peroxidase (GPX) 4 and selenoprotein P (SELENOP) are abundant, and several variants are expressed in the testis.Objective: We determined the effects of dietary selenium deficiency or excess on sperm quality and expressions of GPX4 and SELENOP variants in rat testis and liver.Methods: After weaning, male Sprague-Dawley rats were fed a Se-deficient basal diet (BD) for 5 wk until they were 9 wk old [mean ± SEM body weight (BW) = 256 ± 5 g]. They were then fed the BD diet alone (deficient) or with 0.25 (adequate), 3 (excess), or 5 (excess) mg Se/kg for 4 wk. Testis, liver, blood, and semen were collected to assay for selenoprotein mRNA and protein abundances, selenium concentration, GPX activity, 8-hydroxy-deoxyguanosine concentration, and sperm quality.Results: Dietary selenium supplementations elevated (P < 0.05) tissue selenium concentrations and GPX activities. Compared with those fed BD + 0.25 mg Se/kg, rats fed BD showed lower (P < 0.05) BW gain (86%) and sperm density (57%) but higher (P < 0.05) plasma 8-hydroxy-deoxyguanosine concentrations (189%), and nonprogressive sperm motility (4.4-fold). Likewise, rats fed BD + 5 mg Se/kg had (P = 0.06) lower BW gain and higher (1.9-fold) sperm deformity rates than those in the selenium-adequate group. Compared with the selenium-adequate group, dietary selenium deficiency (BD) or excess (BD + 3 or 5 mg Se/kg) resulted in 45-77% lower (P < 0.05) nuclear Gpx4 (nGpx4) mRNA abundance in the testis. Rats fed BD had lower (P < 0.05) mRNA levels of 2 Selenop variants in both testis and liver than those in the other groups. Testicular SELENOP was 155-170% higher (P < 0.05) in rats fed BD + 5 mg Se/kg and hepatic c/mGPX4 was 13-15% lower (P < 0.05) in rats fed BD than in the other groups.Conclusions: The mRNA abundance of rat testicular nGPX4 responded to dietary selenium concentrations in similar ways to sperm parameters and may be used as a sensitive marker to assess appropriate Se status for male function.