The Protective Effect Of Anthocyanins Against Oxidative Stress-induced Pancreatic Beta-cell Injury And Its Possible Mechanism

Diabetes is a metabolic disease, possibly caused by both of polygenic inheritance and environmental factors. It is characterized by chronic hyperglycemia, mainly due to absolutely or relatively insufficient insulin release from beta-cells or insulin resistance of surrounding tissues. Islet transplantation is a promising therapy for all of type 1 and part of type 2 diabetic patients, which could regulate blood glucose manipulating physiological condition, achieve insulin independence, and delay the occurrence of diabetes-associated complications. However, during the process of islet isolation, purification and transplantation, there exists notable decrease in the number, function and viability of islet grafts. After transplantation, non-specific inflammation and immune rejection also cause the injury to the grafts. All these factors cause the situation that more than one donor are needed for one receipt to achieve insulin independence. However, the inadequate of donor exacerbates the obstacles limiting the development of islet transplantation in clinics. Improved islet isolation and better viability/function preservation before transplantation have been the keys to the evolution of this procedure.During isolation procedure, islets are exposed to the cytotoxicity of collagenase. mechanical trauma, ischemia and hypoxia, all of which contribute to the excessive production of reactive oxygen species (ROS). ROS-mediated oxidative stress plays an important role in the process of cell injury caused by these stimulations. Pancreatic beta-cells usually express low levels of antioxidant enzymes, the long-term exposure of excessive ROS will damage the intracellular proteins, membrane lipid and nucleic acid, eventually leading to apoptosis and necrosis. Some researchers found that adding some antioxidants during islet isolation and culture process could alleviate cellular injury. Nicotinamide (NA) supplementation of the processing medium during islet isolation significantly improved islet yields and protected beta-cells from cell death (both necrosis and apoptosis) induced by H2O2. Addition of glutathione in culture medium significantly reduced CCL2/MCP-1 and tissue factor (TF) production. Recently, many researchers focused on the protective role of naturally existed antioxidant, especially from plant. Some flavonoids (such as curcumin) could protect islets against cytokines-induced apoptosis; pretreatment of islets with some antioxidants could prolong the survival of graft after transplantation. Some plants themselves have hypoglycemia effect in diabetic rodent model, reduce the extent of islet loss, and promote the insulin release from the left islets.Anthocyanins, one type of flavoids, are naturally occurring water soluble polyphenolic compounds in the plant foods and widely distributed in fruits, vegetables, and pigmented cereals, imparting color (blue, purple, red, yellow etc) to plants. Anthocyanin is rich in edible berries and other colorful plants. Many studies have shown that anthocyanins exhibits an array of pharmacological properties, such as keeping the integrity of genome DNA, antioxidative. anti-inflammatory, antitumor and antiatherosclerotic activities. Berry extracts rich in anthocyanins could improve resistance of red blood cell against oxidative stress in vitro. Long-term administration of berry-derived supplements including anthocyanins could inhibit the development of the early stages of some diabetic complications without adverse drug reactions. Bollec et al suggested that bioactive anthocyanins from Cornus fruits could increase insulin release of INS-1 cells.Chinese bayberry, one of six myrica species native to China, is a fruit with high nutrition and health values due to the content of various bioactive substances such as anthocyanins, myricetin and dihydro-myricetin. Our previous study demonstrated that anthocyanins occupies the major portion of total flavonoid compounds. HPLC analysis of Chinese bayberry extract showed that cyanidin-3-O-glucoside (C3G) was the major anthocyanin component, accounting for 95% of the total anthocyanins. Chinese bayberry extracts possess notable radical scavenging activities indicated by DPPH and ABTS cation assays, which is correlated to the content of C3G. C3G was observed to exert hypolipemia and hypoglycemia effects in high-fat-diet (HFD)-feed C57BL/6 mice. Up to now, the application of anthocyanins in CBE has not been fully investigated. Whether anthocyanins has protective effect for islet has not been reported before.The present topic, supported by the National Natural Science Foundation (serial number:30872531), is to explore whether Chinese bayberry extract could protect against oxidative stress-induced beta-cell injury and its possible mechanism, and is to investigate the possible application of anthocyanins as an islet-protective agent in the field of islet transplantation. We also discuss the occurrence of autophagy in beta-cells induced by oxidative stress in vitro or after transplantation.Experimental Procedures1. The protective effect of anthocyanins to pancreatic beta-cells(1) Chinese bayberry extract preparation, HPLC analysis and antioxidant ability assessment.Chinese bayberry fruits (Biqi) were ground and extracted in methanol acidified with HCl, and concentrated through rotary evaporation. Total phenolics content was estimated using the Folin-Ciocalteau colorimetric method. Total flavonoids were determined by colorimetric method after reaction with NaNO2 and Al(NO3)3. Anthocyanin quantitation was performed by the pH differential method. HPLC analysis was performed to identify the composition. Free radical scavenging activity of fruit extracts was measured according to the DPPH method. MTT assay was performed to observe the cytotoxicity of extract to INS-1 cells.(2) The effect of anthocyanins on H2O2-induced apoptosis of pancreatic beta-cells. INS-1 cells were pre-incubated with anthocyanins followed by H2O2 stimulation. Cell viability was evaluated by MTT assay. The protective effect of anthocyains was further confirmed by trypan blue exclusion method and LDH release assay.A ROS-specific probe, DCF-DA, was used to detect intracellular ROS production in INS-1 cells after various treatments. INS-1 cells stimulated by various agents were stained with Hoechst 33258 to observe the apoptosis. Flow cytometry analysis was adopted to quantify the apoptosis and necrosis. The expression of apoptosis-related proteins (caspase-3,-9 and Bcl-2) was assessed by western blot.(3) The effect of anthocyains on H2O2-induced autophagy in pancreatic beta-cellsINS-1 cells, transfected with mRFP-GFP-LC3 plasmid, were treated with H2O2 and autophagy was evaluated by observing the formation of intracellular LC3 punctuate dots. After experimental manipulations, cells were fixed by glutaraldehyde and osmic acid, and were further observed by transmission electron microscopy. The expression of autophagic-related proteins (LC3 and BECN1) was determined by western blot to confirm the occurrence of autophagy.After transfected with BECN1 siRNA, INS-1 cells were exposed to H2O2 for 2h, and the expressions of LC3 and caspase-9 were evaluated. MTT assay and LDH release assay were performed to observe the changes of cell injury after downregulating autophagy.INS-1 cells, treated with various protocols, were stained by AO and MDC to assess the degree of lysosomes and autophagosome formation. The expression of LC3 and BECN1 were evaluated.(4) The effect of anthocyanins on cell graft at the early phase of posttransplantation beneath renal capsueINS-1 cells were transplanted under the left renal capsule of diabetic mice. Seventy-two hours later, the beta-cell grafts were harvested, snap frozen in liquid nitrogen or fixed with 4% paraformaldehyde, or were fixed in glutaraldehyde for transmission electronic microscopic analyzation.Immunohistochemistry staining for BECN1, immunofluorescence staining for LC3, TUNEL staining and transmission electronic microscopic analyzation were performed.INS-1 cells pretreated with or without anthocyanins were transplanted under renal capsule of diabetic mice. The cell grafts were harvest after 72h. Immunofluorescence for cleaved caspase-3 and LC3 and immunohistochemistry staining for BECN1 were carried out.2. Possible mechanism of anthocyanins'protective effect.RNA was extracted from INS-1 cells treated with luM anthocyanins for various time or incubated with various concentrations of anthocyanins for 12h. The mRNA level of HO-1 was determined by reverse transcription PCR and real-time PCR. The expression of HO-1 protein was evaluated by western blot.Primary islets were isolated from mice.100-200 islets were incubated in culture medium with or without 2uM anthocyanins for 6h. The expression of HO-1 mRNA in islets was determined by real-time PCR. Dual immunofluorescence for insulin and HO-1 of islets was performed to further observe the HO-1 expression.The expression of HO-1 in INS-1 cells was downregulated by siRNA transfection. After transfected with HO-1 or control siRNA. INS-1 cells were incubated with luM anthocyanins for 24h, then were exposed to H2O2 for 2h, MTT assay was performed to evaluate the cellular viability. The expression of caspase-3,-9 and LC3 proteins were determined.The pLNCX2-HO1 vector was constructed and transfected to INS-1 cells. INS-1 cells transfected with pLNCX2-HO1 or empty vector were exposed to H2O2. MTT assay was carried out to assess the viability between two groups. Immunofluorescence for LC3 was performed to evaluate the intracellular LC3 punctuate dot formation in both type of cells treated with H2O2.INS-1 cells were treated with various concentrations of anthocyanins for 24h, or 1uM anthocyanins for different time. The expressions of pAkt, Akt, pERK1/2, ERK1/2, pJNK and JNK protein were evaluated by western blot. After pretreated with PD98059 (ERK1/2 inhibitor) or LY294002 (PI3K inhibitor) for 1h, INS-1 cells were further incubated with anthocyanins. HO-1 protein expression was evaluated. INS-1 cells were preincubated with PD98059 or LY294002 for 1h, treated with anthocyanins for 24h, followed by H2O2 stimulation. MTT assay was performed to evaluate the viability.INS-1 cells were incubated with anthocyanins, and Immunofluorescence for Nrf2 was performed and observed under confocal microscopy. Nuclear protein fraction was extracted from cells treated as above, and immunoblotting for Nrf2 and LaminA/C was performed.Results1. The protective effect anthocyanins on pancreatic beta-cells.(1) The major portion of anthocyanins in Chinese bayberry extract is cyanidin-3-glucoside (C3G).Anthocyanin is the major portion of total flavonoids in Chinese bayberry extract. HPLC analysis of Chinese bayberry extract showed that cyanidin-3-O-glucoside (C3G) was the major anthocyanin component, accounting for more than 90% of the total anthocyanins. DPPH assay indicated the strong antioxidant capacity of CBE, and C3G is the dominant attributor. There is no cytotoxicity to INS-1 cells below the concentration of 5uM.(2) Anthocyanins alleviated H2O2-induced apoptosis and necrosis of INS-1 cellsH2O2 injured INS-1 cells in a dose-and time-dependent manner. Pretreatment of INS-1 cells with anthocyanins resulted in the prevention of cell death by H2O2. Trypan blue exclusion assay and LDH release assay further confirmed that treatment with 1 uM anthocyanins for 24h could protect bete-cells against H2O2-induced injury. FCM analysis also displayed that anthocyanins decreased H2O2-induced cell necrosis.Compared to normal cells, H2O2 caused excessive ROS production. Anthocyanins preincubation also decreased oxidative stress-induced intracellular ROS generation. FCM demonstrated anthocyanins incubation reduced the extent of both cellular apoptosis and necrosis induced by H2O2 stimulation. Treatment of INS-1 cells with 1mM H2O2 caused the activation of caspase-9 and-3, which were mitigated by pretreatment with anthocyanins in a concentration dependent manner.(3) Anthocyanins decreased H2O2-induced autophagy in pancreatic beta-cellsGFP-mRFP-LC3 fluorescence was diffusely distributed in untreated group, whereas in cells treated with H2O2 there were detectable the punctate dots of LC3 in the cytoplasm. Under TEM, cells stimulated by H2O2 showed the presence of swelling mitochondrial and some autophagosomes with cytoplasmic contents, which were rarely seen in control INS-1 cells. Western blot analysis showed that the expression of LC3B and BECN1 increased in H2O2-treated INS-1 cells in a time-dependent manner.Cells transfected with BECN1 siRNA displayed decreased expression of LC3Ⅱafter H2O2 treatment, compared with control siRNA transfection group. Downregulation of BECN1 also decreased ROS-induced activation of caspase-9. The results of MTT assay and LDH release assay demonstrated that H2O2-induced cell death was mitigated after inhibition of autophagy.Cells pretreated with anthocyanins exhibited decreased cytoplasmic AO and MDC staining. Immunoblotting analysis demonstrated decreased LC3II generation and the ratio of LC3Ⅱto LC3Ⅰin the presence of anthocyanins, compared with cells treated with H2O2 alone.(4) Anthocyanins pretreatment improved the graft's tolerance in beta-cell transplantation model.INS-1 cells were transplanted under left renal subcapsue.72h later, the graft was resected. Beta-cell grafts were confirmed by immunohistochemical staining for insulin. TEM examination showed that autophagic vacuolization was observed in transplanted cells. Notable immunopositive for BECN1 and detectable the punctate dots of LC3 in grafts further supported the occurrence of autophagy at the early phase of transplantation. In addition, TUNEL positive staining verified that some cells underwent apoptosis.Beta-cell grafts pretreated with anthocyanins for 24h displayed less positive for active caspase-3, BECN1 and decreased extent of punctate dots of LC3.2. The possible mechanism of anthocyanins'protective effect to beta-cells(1) Anthocyanins increased HO-1 expression in beta-cell lines and isletsINS-1 cells treated with anthocyanins resulted in a dose-dependent increment in HO-1 mRNA(12h) and protein expression(24h). In Beta-TC-6 cells, a mouse insulinoma cell line, anthocyanins also cause the upregulation of HO-1 in a dose dependent manner.Real-time PCR analysis demonstrated that the expression of HO-1 was significantly upregulated in primary islets treated with anthocyanins (6.2±1.9 fold increase compared to control, p<0.01). Immunocytochemical analysis of HO-1 in islets further supported this observation.(2) Increased HO-1 expression contributed to the protective effect of anthocyaninsCompared to control siRNA transfection group, INS-1 cells transfected with HO-1 siRNA displayed more notable cell death after H2O2 stimulation. Reducing HO-1 expression by siRNA attenuated the protective effect of anthocyanidins against H2O2-induced cytotoxity, as reflected by MTT assay. Additionally, downregulating HO-1 in INS-1 cells displayed decreased of pro-caspase-3 and-9, but increased expression of cleaved caspase-9 and LC3II protein.Overexpression of HO-1 in INS-1 cells reduced H2O2-induced injury. Similarly, immunofluorescence of MAP LC3 indicated that INS-1 overexpressing HO-1 displayed decreased autophagic cell death.(3) Anthocyanins upregulate HO-1 expression via ERK1/2 and PI3K/Akt pathwaysThe results showed that anthocyanins dose-and time-dependently induced the activation of Akt and ERK1/2 via induction of phosphorylation. Inhibition of PI3K/Akt and ERK1/2 signaling with LY294002 and PD98059 attenuated the anthocyanins-induced HO-1 expression. The results of MTT assay showed that pre-incubating with PD98059 or LY294002 compromised the protective effect of anthocyanins compared with anthocyanins treating alone.(4) Anthocyanins induced the nuclear translocation of transcription factor Nrf2Compared to control group, INS-1 cells treated with anthocyanins displayed more notable Nrf2 accumulation in nucleus. Cellular protein of nuclear fraction was extracted, and immunoblotting for Nrf2 demonstrated that anthocyanins increased the expression of Nrf2 in nuleus. Conclusions:1. Cyanidin-3-O-glucoside (C3G) was indentified as a major anthocyanin component, which accounted for 95% of the total anthocyanins in Chinese bayberry fruit.2. Anthocyanins pre-incubation decreased oxidative stress-induced intracellular ROS generation, and reduced ROS-mediated apoptosis and necrosis of beta cells.3. Besides of apoptosis and necrosis, H2O2 exposure caused autophagic cell death in beta-cells and pretreated with anthocyanins attenuated such type of cell death.4. Under various stimulations, cell grafts underwent apoptosis and autophagy at the early phase of posttransplantation. Anthocyanins pretreatment improved the graft's tolerance in beta-cell transplantation model.5. Anthocyanins could time-and dose-dependently upregulate HO-1 expression in beta-cells. Overexpression of HO-1 decreased H2O2-induced apoptosis and autophagy, contributing to the protective effect of anthocyanins. Anthocyanins increased the HO-1 expression via ERK1/2 and PI3K/Akt pathways. Additionally, anthocyanins could induce transcription factor Nrf2 translocating into nucleus to increase its transcriptive activity.