The Research Of Inducible Nitric Oxide Synthase On The Rat Islet Viability And Function

IntroductionDiabetes mellitus continues to represent a therapeutic challenge and consequently remain a substantial burden for patients and their families. Secondary diabetes complications,observed in 30%to 50%of patients by diabetes,result in poor quality of life,premature death,and considerable healthcare costs.The principal determinant of the risk of devastating diabetes complications is the total lifetime exposure to elevated blood glucose level. Currently,the only way to restore and sustain normoglycemia without the associated risk of hypoglycemia is to replace the patient's islets of Langerhans: either by the transplantation of a vascularized pancreas or by the infusion of isolated islets.Over the last two decades,prevention and reversal of secondary complications,improvement in quality of life,expansion of life span,and reduction of healthcare costs per quality-adjusted life-year have all been documented in diabetic pancreas transplant recipients.The success of pancreas transplants has demonstrated that the goals ofβ-cell replacement can be accomplished even in the presence of generalized and chronic immunosuppression.The islet transplant is the cellular transplant,which has the advantages including:safer,simpler,more convenient for the modifying in vitro, lower rate of side-effect and easier to repeat than the pancreas transplant,and has become a prospective method.The transplantation of isolated islets represents a minimal invasive approach forβ-cell replacement and recently developed protocols enhanced the short-term success rate of islet transplantation.However,there is still a lack of metabolic capacity in islet transplants in the long run which cannot even be compensated by transplantation of a massive amount of islets.This phenomenon must be mainly attributed to islet death elicited from recurrence of autoimmunity,delayed vascular connection resulting in prolonged hypoxia,and inflammatory conditions against which pancreatic islets possess no significant means of protection,Inflammatory molecules,such as cytokines like IL-1β,TNF-α,IFN-γ,and nitric oxide(NO)have the potential to damageβ-cells.They promote insulitis andβ-cell destruction in autoimmune diabetes together with nonspecific toxic molecules,such as reactive oxygen species(ROS).Furthermore,the quality of islet preparation might play a fundamental part in determining the outcome of the graft.For this reason, various protection factors were applied to maintain the islets activity and functions in the course of the islet preparation.At present,some experiments demonstrated that in the transplanted islets, inducible NO synthase(iNOS)and toxic amounts of NO were produced as a result of islets infiltration with inflammatory cells and production of proinflammatory cytokines(such as TNF-α,IL1-β),and an exclusive production of NO was deleterious for pancreasβ-cells.In our experiment,the expression and activity of iNOS was suppressed by RNAi and inducible nitric oxide synthase inhibitor aminoguanidine to prevent the islet from the damage of iNOS/NO,to discuss a new method for the long-term survival and function of islet,and improve the pancreatic islet transplantation.Materials and Methods1.Islet Isolation and Purification:Wistar rats(200-250g purchased from the experimental animal department of China medical university)were anesthetized with 10%chloral hydrate by intraperitoneal injection.Islets were isolated from the surrounding exocrine tissue by enzymatic digestion with 1.0 mg/ml collagenaseâ…¤in HANKS' balanced salt solution at 37.5±1℃for 11-15 min.At the end of digestion,double centrifuge(800r/min,4℃-8℃,2 min)and washing followed by screen by 80.Purifying islet by Ficoll purification using a modification of procedures which concentration is 25%,23%,20.5%and 11%.2.Culture and divide into groups of islets:Islets were suspended in RPMI-1640 medium(Sigma-Aldrich)containing 100μg/ml penicillin,100μg/ml streptomycin.The islets were cultured at 37℃in a humidified atmosphere of 5%CO₂ and 95%air.According to object,we divided into islets into several groups and added cytokines TNF-αand IL1-β,siRNA,and aminoguanidine respectively in RPMI-1640 medium.3.Transfection:The cultured islets were transfected with iNOS siRNA,and the siRNA work density was a 100 nmol/L.The transfection reagent RNAi-mate and iNOS-siRNA were diluted by medium without serum or antibiotic,and then mixed to form a siRNA and RNAi-mate mixture.The mixture was added to islets medium,and cultured at 37℃in a humidified atmosphere of 5%CO₂ and 95%air for 8 hours.Then we continued to culture the islets in medium with serum and antibiotic for 24 hours.4.Analysis of mRNA:Expression of iNOS mRNA was evaluated by RT-PCR in isolated rat pancreatic islets cultured in RPMI-1640 medium to detect the effect of RNAi,and the expression of apoptosis correlated gene,Bax,Fas were analysised meanwhile.GAPDH served as an internal control.5.Expression of iNOS in cultured islets was evaluated by Western-blot to detect the effect of RNAi,and theβ-actin served as an internal control.6.Nitrate reductase detected nutrient fluid nitric oxide level and NOS Test Kit detected islets iNOS activity.7.Examination to the viability of the islets by AO/EB stain:After isolation, purification,and culture,the islets were incubated with acridine orange(AO)and ethidium bromide(EB)for 10min,and examined by fluorescent microscope. Viable cells stained green,and nonviable cells were seen as orange-stained nuclei.8.The examination to the function of the islets: Insulin secretion index assay:Islets were washed twice with Kreb's-Hank's balanced salt solution(containing 10mmol/L HEPES and 0.25%BSA)and counted 10IEQ islets.The islets were cultured in the low density glucose (2.8mmol/L)Kreb' S-Hank' balanced salt solution for 2 hours(basal secretion), then cultured in the highly concentrated glucose(16.7mmol/L)Kreb' S-Hank' for 1hour(stimulated secretion),and then collected the 2h and the 3h nutrient fluid.The buffer was centrifuged to remove any detached cells and debris. Aliquots were stored at -20℃for subsequent insulin measurement performed by radioimmunoassay to calculate the insulin secretion index(SI). SI=3h insulin content(high sugar environment)/2h insulin content(low sugar environment).9.Statisticis analysis:All the datas were presented as mean±SD,and P＜0.05 was considered having statistical significance.Result1.By Ficoll purification using a modification of procedures,500-600IEQ islets could be extracted from every rat.The DTZ stain showed the islets purity was more than 60%,and the viability exceeded 95%by AO/EB stain.2.The effect of RNAi:After stimulus with cytokines IL-1βand TNF-α,the expression of iNOS on cultured islets enhanced significantly,whereas RNAi could inhibit the stimulus,attenuate the expression of iNOS,and restrain the synthesis of iNOS protein.3.The NO level of nutrient fluid and the iNOS activity of islets tissue:With regard to fresh islets,the activity of iNOS in islet tissue and concentration of NO in medium were low level.However,with the treatment of cytokines IL-1βand TNF-αfor 24 hours,the level of iNOS and NO increased remarkably.When we added aminoguanidine together with cytokines IL-1βand TNF-αto islets medium,the iNOS activity of islets tissue was suppressed significantly,and the NO level of nutrient fluid decreased obviously.4.Viability of the islets:The AO/EB stain showed the viability of fresh islets exceeded 95%,while the mass mortality appeared when the islets were cocultured with cytokines IL-1βand TNF-α.We found that eiher RNAi or iNOS inhibitor aminoguanidine could improve the survival of islets cultured with cytokines,according to AO/EB stain.5.Apoptosis of the islets:The expression of Bax and Fas ascended distinctly on islets cultured with cytokines IL-1βand TNF-αfor 24 hours,and descended in group of RNAi+cytokines.6.Function of islets:The basal insulin excretion and insulin secretion index were satisfying on fresh islets,but decreased strikingly when the islets cocultured with cytokines IL-1βand TNF-αfor 24 hours,which suggested the islets encountered harmfulness.When treatment with RNAi or aminoguanidine, the islets cultured with cytokines excreted more insulin and the SI was higher.Conclusion1.Inducible NO synthase(iNOS)was produced generously as a result of stimulus of cytokines(such as TNF-α,IL1-1β),and an exclusive production of NO was deleterious for pancreasβ-cells.2.In our experiment,the damage from cytokines to islets mediated by iNOS/NO could be suppressed by RNA interfere(RNAi),which leaded to favorable function and survival of islets.3.The inducible nitric oxide synthase inhibitor aminoguanidine,could prevent the islet from the damage of iNOS/NO,alleviate the impairment of cytokines to islets,ameliorate the survival and function of islets.