Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Background: In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. Methods: The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. Results: RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination–proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. Conclusion RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Objective:To observe the effects of RasGRP4 gene deletion on the structure and function of the retina in diabetic mice, and to explore the mechanism of RasGRP4 in diabetic retinopathy (DR) by transcriptome sequencing in conjunction with bioinformatics analysis. Methods:A total of 12 male C57BL/6J mice were divided into normal group, diabetic group (DM group), with 6 mice in each group. Six male RasGRP4 knockout mice were uesd as RasGRP4 knockout diabetic group (DM-KO group). Mice in the DM group and DM-KO group were fed with high-fat diet combined with intraperitoneal injection of streptozotocin to establish diabetic model and body weight and blood glucose were monitored regularly. Three months after modeling, optical coherence tomography was used to detect the retinal thickness and ganglion cell layer thickness. Electroretinography was used to detect the function of the retina in mice under dark-adapted conditions. Total RNA was extracted from the retinas of mice in DM group and DM-KO group, and transcriptomic sequencing was performed to screen differentially expressed genes (DEG). Core genes were screened using MCODE and Cytohubba plug-ins of Cytoscape v3.8.2 software. At the same time, the functional enrichment analysis of gene samples (GO) of the selected DEG was performed. The mRNA relative expression levels of interleukin-8, transforming growth factor-β (TGF-β), interferon-γ (IFN-γ), NOd-like receptor thermal protein domain protein 3 (NLRP3), Caspase-1 and IL-1β in each group were detected by real-time quantitative polymerase chain reaction. t test was used to compare the two groups. One-way analysis of variance was used to compare the three groups. Results:Compared with the DM group, there was no significant difference in blood glucose and body weight in the DM-KO group with the extension of high-fat diet ( t=0.12, 2.02, 0.22, 0.10, 0.59, 0.41, 1.35, 0.31, 1.12, 1.58, 1.47, 1.20, 1.24, 0.39, 0.66, 0.14; P>0.05). The retinal thickness and ganglion cell layer thickness of mice in the three groups were significantly reduced in the DM group compared with the normal group, while DM-KO was significantly increased compared with the DM group, and the differences were statistically significant ( F=30.43, 7.81; P<0.000 1, 0.01). Comparison of a-wave and b-wave amplitudes among the three groups showed that the DM group was significantly lower than the normal group, while the DM-KO was significantly higher than the DM group, and the differences were statistically significant ( F=16.46, 35.58; P<0.001, 0.000 1). Compared with the DM group, 184 differential genes (DEG) were screened in the DM-KO group, among which 39 up-regulated and 145 down-regulated genes were detected, respectively. The results of the MCODE plug-in analysis showed that Col1a2, Fbln1, Fbn1, Col6a3, Fmod, Ogn, Tgfb, Mfap4, Vcan, Nid2, and Col18a1 were core genes in the DEG. Cytohubba plug-in analysis showed that Col1a2, Mrc1, Cd47, Fbn1, Cybb, Cd163, Fbln1, Fmod, Adgre1, and Col6a3 were the core genes in DEG. The results of the GO functional enrichment analysis showed that DEG was mainly involved in hemoglobin complexes, MHC class Ⅱ protein complex, apical plasma membrane, inflammasome complex, immunological synapse, response to bacterium, inflammatory response, immune system processe, response to hypoxia, and cell adhesion were significantly enriched. Comparison of mRNA relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1 and IL-1β in the three groups showed that the DM group was significantly higher than the normal group, while the DM-KO was significantly lower than the DM group, with statistical significance ( F=12.43, 15.41, 70.09, 29.04, 11.79, 41.28; P<0.01). Conclusion:RasGRP4 deficiency plays a therapeutic role in the development of DR through inhibition of inflammatory factor secretion and NLRP 3 inflammasome pathway activation.

Objective:To observe the effects of RasGRP4 gene deletion on the structure and function of the retina in diabetic mice, and to explore the mechanism of RasGRP4 in diabetic retinopathy (DR) by transcriptome sequencing in conjunction with bioinformatics analysis. Methods:A total of 12 male C57BL/6J mice were divided into normal group, diabetic group (DM group), with 6 mice in each group. Six male RasGRP4 knockout mice were uesd as RasGRP4 knockout diabetic group (DM-KO group). Mice in the DM group and DM-KO group were fed with high-fat diet combined with intraperitoneal injection of streptozotocin to establish diabetic model and body weight and blood glucose were monitored regularly. Three months after modeling, optical coherence tomography was used to detect the retinal thickness and ganglion cell layer thickness. Electroretinography was used to detect the function of the retina in mice under dark-adapted conditions. Total RNA was extracted from the retinas of mice in DM group and DM-KO group, and transcriptomic sequencing was performed to screen differentially expressed genes (DEG). Core genes were screened using MCODE and Cytohubba plug-ins of Cytoscape v3.8.2 software. At the same time, the functional enrichment analysis of gene samples (GO) of the selected DEG was performed. The mRNA relative expression levels of interleukin-8, transforming growth factor-β (TGF-β), interferon-γ (IFN-γ), NOd-like receptor thermal protein domain protein 3 (NLRP3), Caspase-1 and IL-1β in each group were detected by real-time quantitative polymerase chain reaction. t test was used to compare the two groups. One-way analysis of variance was used to compare the three groups. Results:Compared with the DM group, there was no significant difference in blood glucose and body weight in the DM-KO group with the extension of high-fat diet ( t=0.12, 2.02, 0.22, 0.10, 0.59, 0.41, 1.35, 0.31, 1.12, 1.58, 1.47, 1.20, 1.24, 0.39, 0.66, 0.14; P>0.05). The retinal thickness and ganglion cell layer thickness of mice in the three groups were significantly reduced in the DM group compared with the normal group, while DM-KO was significantly increased compared with the DM group, and the differences were statistically significant ( F=30.43, 7.81; P<0.000 1, 0.01). Comparison of a-wave and b-wave amplitudes among the three groups showed that the DM group was significantly lower than the normal group, while the DM-KO was significantly higher than the DM group, and the differences were statistically significant ( F=16.46, 35.58; P<0.001, 0.000 1). Compared with the DM group, 184 differential genes (DEG) were screened in the DM-KO group, among which 39 up-regulated and 145 down-regulated genes were detected, respectively. The results of the MCODE plug-in analysis showed that Col1a2, Fbln1, Fbn1, Col6a3, Fmod, Ogn, Tgfb, Mfap4, Vcan, Nid2, and Col18a1 were core genes in the DEG. Cytohubba plug-in analysis showed that Col1a2, Mrc1, Cd47, Fbn1, Cybb, Cd163, Fbln1, Fmod, Adgre1, and Col6a3 were the core genes in DEG. The results of the GO functional enrichment analysis showed that DEG was mainly involved in hemoglobin complexes, MHC class Ⅱ protein complex, apical plasma membrane, inflammasome complex, immunological synapse, response to bacterium, inflammatory response, immune system processe, response to hypoxia, and cell adhesion were significantly enriched. Comparison of mRNA relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1 and IL-1β in the three groups showed that the DM group was significantly higher than the normal group, while the DM-KO was significantly lower than the DM group, with statistical significance ( F=12.43, 15.41, 70.09, 29.04, 11.79, 41.28; P<0.01). Conclusion:RasGRP4 deficiency plays a therapeutic role in the development of DR through inhibition of inflammatory factor secretion and NLRP 3 inflammasome pathway activation.

Objective:To evaluate the effect of canagliflozin on intrarenal fat content and oxygenation in newly-diagnosed type 2 diabetes patients.Methods:Twenty-three newly-diagnosed type 2 diabetes patients were divided into canagliflozin( n=11) and glimepiride control( n=12) groups .Both groups received MRI scanning with Dixon MRI and BOLD MRI sequence to assess patients′ intrarenal fat content, oxygenation level before treatment and 24 weeks after treatment. Fasting blood glucose, glycosylated hemoglobin, blood uric acid, blood lipids, blood pressure, weight, and other metabolic index were also tested before and after treatment. Furthermore, the relationship between body mass index(BMI) and intrarenal fat content and the correlation between changes in intrarenal fat content and improvement in renal hypoxia were analyzed. Results:No significant differences were found in baseline age, body weight, fasting blood glucose, glycosylated hemoglobin, blood lipid, and serum uric acid between the two groups. There was no significant difference in fasting blood glucose, glycosylated hemoglobin, cholesterol(CHO), low-density lipoprotein-cholesterol(LDL-C), and triglycerides(TG) levels in both groups after 12 and 24 weeks of treatment. The decrease in body weight, blood uric acid level, and diastolic blood pressure from baseline in the canagliflozin group was greater than those in the control group( P<0.05). Two groups of patients with type 2 diabetes at baseline had no obvious difference in intrarenal fat content, and the patients′ BMI showed no obvious correlation with degree of intrarenal fat accumulation. Canagliflozin treatment for 24 weeks could reduce intrarenal fat content, which was higher than that of control group. The R2 * values of renal cortex and medulla in the canagliflozin group decreased from baseline by 19.22% and 22.63% respectively( P<0.05), whereas no significant difference was seen in the glimepiride control group. The decrease of intrarenal fat content in the canagliflozin group was related to the improvement of renal cortex and medulla oxygenation. Conclusion:Canagliflozin can reduce intrarenal fat accumulation and improve renal cortical hypoxia in newly diagnosed type 2 diabetes patients with normal renal function.

Objective To explore the impacts of Danhong injection on physiological and biochemical indicators in malnourished mice at physiological low doses, evaluate its safety, and test the practical value of safety re-evaluation of Traditional Chinese Medicinal ( TCM) injections. Methods A total of 32 ICR mice during growth period were selected to set up corn deficient nutrition mice model. Mice were assigned into the normal control group (given 0. 9% saline), Danhong injection at low, medium and high dosages (0. 2, 0. 4 and 0. 6 mL) groups (n=8 in each group);Mice were administered with respective medications intraperitoneally for 7 consecutive days. Blood samples were taken and mice were executed on the 8th day. All 9 kinds of organ or tissue were obtained completely, to measure related physiological and serum biochemical parameters. The safety of Danhong injection was evaluated by using Benefit and Damage Index - General Score ( BDI-GS ) system. Results The Danhong injection showed only slight damages on major organs or tissues, the BDI values were all above 0. 85, and the GS values were all above 9. 0;BDI values for Danhong injection at different dosages were all above 1. 0 for spleen and pancreas, showing better replenishing and healthy effects, and the differences were of statistical significance compared with the normal control group (P<0. 05 or P<0. 01). Meanwhile, it exerted obviously hypoglycemic effect. Conclusion Danhong injection is of rather low risk under physiological dosages, and therefore is safe to use. The mal-nutrition model combined with the BDI-GS system may be developed as a novel approach for safety re-evaluation of TCM injection in clinic.

Objective To investigate the radiosensitization of artesunate on nude mouse transplanted with HeLa cells,and to explore its possible mechanisms.Methods HeLa cells were inoculated into the nude mice to establish tumor model.Mice were randomly divided into 4 groups as blank control,artesunate group,radiation group and artesunate + radiation group when average volume of tumor were about 5 mm × 5 mm× 5 mm.During the term of treatment,the volume of tumors were measured every 2days.After 14 days treatment,the mice were killed and tumor tissues were harvested for flow cytometry to detect the alteration of cell cycle.Meanwhile,the pathological change of the tumor tissue was observed with HE staining method,and the change of expression of cycle regulatory protein Cyclin B1,Cdc2 and Wee1 were detected by Western blot.Results The growth of tumor was significantly inhibited by artesunate combined with radiation and its inhibition rate was 72.34％.Flow cytometry results showed that the percent of cells in G1 phase increased and G2 phase decreased in the artesunate + radiation group compared with those in irradiation group ( t =4.41,4.12,P ＜ 0.05 ).The expression level of Cyclin B1 was obviously increased while that of Wee1 decreased in the artesunate + radiation compared with irradiation group.There was no difference in the expression of Cdc2 among the four groups.Conclusions Artesunate can dramatically increase the radiosensitivity of transplanted tumor of HeLa cells.The possible mechanism might be related to the decreasing G2 phase by regulating the expression of Cyclin B1 and Wee1.

Rhesus retinal vascular endothelial cell line RF/6A cells were treated with human insulin. Cell proliferation, migration, and lumen formation, as well as the expressions of vascular endothelial growth factor-A ( VEGF-A ), VEGF-A receptors, and phosphorylated receptors were measured. Insulin promoted RF/6A cell proliferation, migration, and lumen formation ( all P＜0. 01 ). Insulin increased the expression of VEGF-A mRNA and improved its protein activity ( all P＜0. 05 ), and promoted the expression of VEGFR2 mRNA and its phosphorylation ( both P＜0. 01 ). There was no significant difference in the expression of VEGFR1 mRNA among the groups ( P＞0. 05 ).