| Background:Type 1 diabetes mellitus (T1DM) is an autoimmune disease that usually occurs in childhood, adolescence, or young adulthood. A person’s beta cells, which produce insulin in islets of the pancreas, are destroyed by their immune cells in T1DM. Even when well controlled, T1DM may still result in serious secondary complications including cardiovascular disease, retinopathy, neuropathy and nephropathy, and some may be life-threatening. Pancreatic islet transplantation offers a promising therapeutic option for T1DM because this therapy can prevent the long-term complications associated with this disease. However, significant obstacles to the widespread implementation of islet transplantation remain unsolved. One of these obstacles is the progressive loss and dysfunction of islets during isolation and transplantation procedures, which consequently requires a large amount of donor islets. Oxidative stress is one of the major contributors of islet injury and dysfunction during islet isolation and transplantation procedures.Chinese bayberry, one of six Myrica species native to China, is a fruit with high nutrition and health values. This fruit is rich in anthocyanin, and cyanidin-3-O-glucoside (C3G) is identified as the most abundant component of these anthocyanin (12,13). Our previous studies have demonstrated that C3G possesses high antioxidant capacities. We demonstrated that C3G promoted pancreatic β cells survival and enhanced their function both in vitro and in vivo. Recently, we showed that C3G could enhance the viability of mouse islets and improve their function after transplantation under the kidney capsule or into the portal vein of syngeneic mouse recipients. However, whether C3G could enhance the function of neonatal porcine islets (NPI) still remains unclear, which we believe is of great importance to the future clinical practice of islet transplantation. Because the availability of suitable donor organs is always limited but the demand for this procedure is still increasing, there is a particularly high need for new sources of islets for transplantation. By consensus, pigs are identified as ideal candidate as an alternative organ source for reasons that they mature rapidly, have organs relatively similar in size and physiologic capacity as those found in humans and their insulin is biologically active in humans. In addition, the underlying mechanisms whereby C3G protects the islets are still not well investigated.Therefore, in this study, we determined whether treatment of neotatal porcine islets xenografts with an antioxidant C3G could enhance their function after transplantation under the kidney capsule and investigated the underlying mechanism of protection.Methods:Neonatal porcine islets are harvested and isolated from three-day-old Duroc cross-neonatal pigs. After cultured for 7-day in vivo, the islets were treated with various concentrations of C3G (0,0.1,0.5,1.0 and 5.0 μM), and the toxicity of C3G on NPI was determined by Trypan blue exclusion staining and flow cytometry analysis. The gene (HO-1, Bcl-2 and Survivin) and protein (insulin and glucagon) expressions of C3G-treated or -untreated islets were evaluated by qPCR and immunohistochemistry analysis, respectively. The effects of C3G on extracellular signal-regulated kinasel/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K/Akt) signaling pathways, as well as the protein expressions of nuclear erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were assessed by western blotting.In the in vivo study, the C3G-treated (1.0μM) or -untreated porcine islets (1000 and 2000 IEQ) were transplanted under the kidney capsule of diabetic B6ragr1-/- mice. After transplantation, the mice were fed with or without C3G (1.0 μM) in their drinking water, and their blood glucose levels were monitored for more than 15 weeks post-transplantation. Two weeks after the mice achieved normal blood glucose levels, an intraperitoneal glucose tolerance test (IPGTT) was performed. At the end of the study (15 weeks post-transplantation), all recipients underwent a survival nephrectomy of the graft-bearing kidney to confirm that the islet transplant was responsible for their normoglycemic state. The gene (HO-1, Bcl-2 and survivin) and protein (insulin and glucagon) expressions of C3G-treated or -untreated grafts were also determined by qPCR and immunohistochemistry analysis, respectively.Results:The Trypan blue staining showed that islets treated with C3G had slightly higher viability compared to the untreated islets, but no statistical significance was found except in islets treated with 1.0 μM C3G, which also showed the highest cell viability. Treatment of islets with 5.0 μM C3G had similar viability observed in the untreated islets, indicating that high C3G concentration might not be beneficial to the islet cells. Flow cytometry analysis showed the same trend with Trypan blue staining, however, there was no statistically significant difference between the groups compared. Insulin and glucagon positive cells were detected in all islets and no significant differences were found. However, the staining showed that islets treated with H2O2 without C3G pre-incubation had disrupted membrane and there were no intact islets detected. In contrast, islets treated with C3G before H2O2 treatment showed intact structure, suggesting that C3G has potent protective property in maintaining cellular integrity of the NPI.In the in vivo study, the islets were transplanted under the kidney capsule of diabetic B6 ragl-/- mice. All mice that received 1,000 IEQ achieved normoglycemia. Recipients of 2,000 IEQ NPI treated with C3G alone or in combination with C3G supplemented drinking water showed significantly lower blood glucose levels at 5 to 11 weeks post-transplantation compared to recipients of untreated islets. In addition, these mice also achieved normoglycemia much earlier than those mice that were transplanted with untreated islets (10,8 and 12 weeks, respectively). When mice were challenged with glucose, the blood glucose levels of mice that received islets cultured with C3G alone or in addition to drinking water supplemented with C3G decreased significantly faster than what was observed in recipients of untreated islets. All recipients of 2,000 IEQ responded well to the glucose challenge and there were no significant differences found among the three groups. The blood glucose levels of all mice increased dramatically after removal of the kidney bearing the islet graft, indicating that the islet transplant was responsible for maintaining the normal blood glucose levels. All NPI xenografts stained strongly positive for insulin and glucagon at 15 weeks post-transplantation.Significantly higher HO-1 gene expression compared to untreated islets was observed in vitro. However, no significant difference was found in term of the anti-apoptotic Bcl-2 and survivin gene espression. Grafts from mice that received 1,000 IEQ showed no significant differences in the levels of HO-1, Bcl-2 and survivin gene expression. However, the HO-1 gene expression in grafts of mice that received 2,000 IEQ treated with C3G with or without addition of C3G in the drinking water was significantly higher when compared to those detected in untreated islet grafts. No significant difference was found in the gene expression of Bcl-2 and survivin in the three groups, similar to what we observed in the islets prior to transplantation. Western blot analysis showed that C3G induced the phosphorylation of ERKl/2 and PI3K/Akt at a concentration of 0.5 μM or more, and the phosphorylation of ERKl/2 and PI3K/Akt reached maximum at 6 h to 12 h. We also found that C3G significantly elevated the level of nuclear Nrf2 and the subsequent HO-1 induction at a concentration of 0.5 and 1.0μM.Conclusion:Our results demonstrated that C3G could enhance viability of NPI and provide substantial promotion of their function after transplantation under the kidney capsule of diabetic mice. The protection of C3G was through the activation of ERK1/2 and PI3K/Akt signaling pathway, which induced the nuclear translocation of Nrf2 and the subsequent HO-1 protein elevation. |
PDF Full Text Request