Protective Effects Of Taurine And RGD Peptides On Rat Islet Viability And Function

IntroducionDiabetes is an autoimmune disease characterized by activation of the Th1-phenotype,lymphocytic infiltration Of pancreatic islets,and programmed cell death ofβ-cells.β-cell replacement is the only way to restore euglycaemia and ameliorate the progression of diabetic complications.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.Indeed,up to 60%of theβ- cell mass undergoes apoptosis in experimental models of syngeneic PIT,and half of this loss occurs within the first 3 days after transplantation.This rate of apoptosis following PIT is 10 times higher than the rate seen in the native pancreatic islets.Several causes have been held responsible for this phenomenon,including islet death-elicited recurrence of autoimmunity,delayed vascular connection resulting in prolonged hypoxia,tissue factor production resulting in activation of coagulation,and inflammatotion.Furthermore,the quality of islet preparation might play a fundamental part in determining the outcome of the graft.Islet isolation lead to the release of proinflammary cytokines and also lead to the destruction of the islet microenvironment which exposes the islet to various forms of cellular stress and subjects the islets to a type of cellular stress that could impairβ-cell function and survival.Apoptosis of islet cells appears soon after islet isolation and primarily involves theβ-cell.For this reason,various protection factors were applied to maintain the islets activity and functions in the course of the islet preparation.Taurine (taurine TAU)the most abundant free amino acid in the body,is a free beta-amino acid that is normally present in high concentrations in many tissues of humans and animal species,taurine is known to play an important role in numerous physiological functions,including modulation of calcium levels,maintenance of osmolarity, anti-oxidation,and stabilization of membranes.Taurine can also prevent the production And release of proinflammatory cytokine.So in our experiment,the expression of proinflammary cytokines,chemotatic factor,adhesion molecule and nuclear factor(NF) is investigated by RT-PCR during islets preparion.The aim of this study is to examine the effect of taurine on in vitro proinflammary cytokines,chemotatic factor,adhesion molecule and nuclear factor(NF)release from pancreatic islets.The pancreatic cells express the integrinsα1β1,α3β1,α5β1 andα6β1,which are also known to bind argenin-glycin-aspartic acid(RGD)sequences on ECM molecules which composed of the islet basement membrane.Integrins are believed to mediate survival by activating a specific signaling pathway.In the present study,we investigate how surivival of islets of Langerhans is influenced by RGD peptides.Materials and MethodsIslet Isolation and Purification:Wistar rats(180-220g 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 collagenase V 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 27%,25%,23%,20.5%and 11%.Culture of islets:Islets were suspended in RPMI-1640 medium(Sigma-Aldrich) containing 100μg/ml penicillin,100μg/ml streptomycin and 10%FCS,and they were plated at a density of 100 islets/well.According to object,we add 0.1 mmol/l RGD peptides or 20mmol/L taurine in RPMI-1640 medium.The islets were cultured at 37℃in a humidified atmosphere of 5%CO₂ and 95%air.Islet viability assay:The effect of RGD peptides or taurine on rat islet-cell viability was measured by fluorescent microscopy,analysis.After 6h or 1 week, cultured islets were incubated with acridine orange(AO)and with ethidium bromide (EB)for 10min and examined by fluorescent microscope.Viable cells stained green, and nonviable cells were seen as crocus -stained nuclei.Insulin Secretion Assay:Cells were washed two times with HANKS' balanced salt solution and were then incubated for 2 h at 37℃with Kreb's-Hank's balanced salt solution containing 2.8 mM glucose(basal secretion),followed by 1 h at 37℃with Kreb's-Hank's balanced salt solution containing 16.7 mM glucose(stimulated secretion).The buffer was centrifuged to remove any detached cells and debris. Aliquots were stored at -20℃for subsequent insulin measurement performed by radioimmunoassay to measure Secretion Index(SI).Flow cytometry studies:Islets were cultured with RPMI-1640 medium or with RPMI-1640 medium containing RGD or containing taurine for 24h and 1w.Active caspase-9 was detected with rabbit polyclonal antibody as a dead marker and rabbit anti-phospho-Akt Ser 473 antibody was used as a survival marker.The percentage of active caspase-9-positive cells or phospho-Akt 473-positive cells was determined by flow cytometry studies to investigate the influence of the presence of the taurine and RGD peptides on the caspase-9 activity and on the phosphorylation of Akt on serine residue 473.RT-PCR:expression of TNF-α,IL-1β,NF-κB,HO-1,MCP-1,and ICAM-1 was evaluated by RT-PCR in isolated rat pancreatic islets cultured with RPMI-1640 medium and in isolated rat pancreatic islets cultured with RPMI-1640 medium containing 20mM taurine or 0.1mM RGD.Theβ-actin served as an internal control.Result600-700IEQ islet could be extracted from every rat by Ficoll purification using a modification of procedures.The DTZ stain shows purity more than 60%and islet viability more than 95%by AO/EB stain.Islets cultured with RPMI-1640 showed an Secretion Index(SI)after lw of 1.64±0.28,while islets cultured with RPMI-1640 containing taurine or RGD peptides showed an Secretion Index(SI)of 2.45±0.24, 2.28±0.16,respectively(P＜0.05).After 24 h or lw of culture,there was a decrease in dead cells in the RGD or taurine-treated islets,while the amount of dead cells in the untreated islets increased remarkably.The percentage of cells that underwent apoptosis after different treatments was analyzed by flow cytometry studies after caspase-9 immunostaining.In untreated islets that had been cultured for 1 day,a percentage of 19.66±4.66%of cells stained positively for caspase-9,which is an indication of apoptosis.In contrast,treatment with 0.1 mmol/l RGD rendered a percentage of 12.14±2.44%positive caspase-9 cells, which represents a decrease in the level of apoptosis by a factor of 8%(P＜0.05).After treatment with 20 mmol/l taurine,23.85±3.09%of cells per islet stained positively for caspase-9,which represents a decrease in the level of apoptosis by a factor of 18%,in contrast to untreated islets(41.03±4.46)after lw of culture.(P＜0.05).So we concluded that taurine and RGD peptides prevent activiation of caspase-9.Because integrin receptor activation can lead to the phosphorylation of Akt on serine residue 473,we receptor activation can lead to the phosphorylation of Akt on serine residue 473,we. determined if RGD peptides could induce Akt phosphorylation in islet cells.we determined the amount of phospho-Akt Ser 473-positive cells by flow cytometry.Only 41.70±3.25%of the cells of untreated islets stained positive for phospho-Akt Ser 473, whereas 61.05±6.03%of the cells treated with RGD peptides stained positive for phospho-Akt Ser 473(P＜0.05).After 1w of culture,we find that 49.14±6.73%of the cells treated with taurine stained positive for phospho-Akt Ser 473,whereas 31.47±4.08 of the cells of untreated islets stained positive for phospho-Akt Ser 473.We concluded that phosphorylation of Akt Ser 473 had increased through the ligation of integrins by RGD peptides.Both taurine and RGD peptides could lead to the phosphorylation of Akt Ser 473 which could inhibit apoptosis. Expression of TNF-αmRNA,IL-1βmRNA,NF-κBmRNA,HO-1mRNA,MCP-1mRNA,and ICAM-1mRNA in cultured rats islets was analyzed by RT-PCR at different time and semiquantitative analysis performed by densitometric gel scanning.We find that there are significantly expression of TNF-αmRNA,IL-1βmRNA,NF-κBmRNA,HO-1mRNA and MCP-1mRNA after 6h of culture with RPMI-1640 medium,and TNF-αmRNA,IL-1βmRNA,NF-κBmRNA,MCP-1mRNA levels were significantly decreased after 72h of culture with RPMI-1640 medium(P＜0.05),whereas HO-1mRNA level was significantly increased after 72h of culture with RPMI-1640 medium(P＜0.05).There are significantly expression of ICAM-1mRNA after 72h of culture with RPMI-1640 medium and were significantly decreased after lw of culture(P＜0.05).After 1w of culture with RPMI-1640 medium, TNF-∝mRNA,IL-1βmRNA,NF-κBmRNA,MCP-1mRNA,levels continue decreasing compared with that 6h or 72h of culture with RPMI-1640 medium(P＜0.05). HO-1mRNA level has also significant change compared with that 6h or 72h of culture with RPMI-1640 medium(P＜0.05).So we concluded that stress induced during islet isolation lead to the expression of pro-inflammatory cytokines,chemokines and intercellular adhesion molecule,such as TNF-α,IL-1β,NF-κB,HO-1,MCP-1 and ICAM-1.Along with the culture time,the expression of pro-inflammatory cytokines,chemokines and intercellular adhesion molecule decreased gradually.More ever up-regulation of HO-1 in the pancreatic islets is seen during islet isolation and overexpression of HO-1 is protective against apoptosis.To investigate the impact of taurine on the expression of pro-inflammatory cytokines,we add taurine to RPMI-1640 medium.Our results suggest that TNF-αmRNA,IL-1β3mRNA,NF-κBmRNA,MCP-1mRNA,ICAM-1mRNA levels were significantly decreased after 6h or 72h and lw of culture with RPMI-1640 medium containing taurine compared with that of culture with RPMI-1640 medium at the same time(P＜0.05).Along with the culture time,the expression of pro-inflammatory cytokines decreased significantly(P＜0.05).HO-1mRNA level was significantly increased compared with that of culture with RPMI-1640 medium at the same time(P＜0.05).After 1w of culture with RPMI-1640 medium containing taurine, HO-1mRNA level has significant change compared with that 6h or 72h of culture with RPMI-1640 medium(P＜0.05).ConclusionVrious stress responses during islet isolation result in increased expression of TNF-α,IL-1β,NF-κB,MCP-1 and ICAM-1 which lead to apoptosis of free islet and impact islet viability and function.Along with the culture time,the expression of TNF-αmRNA,IL-1βmRNA, NF-κB mRNA,MCP-1 mRNA and ICAM-1 mRNA decreased significantlyTaurine can protect pancreaticβ-cells from apoptosis by inhibiting the transcription of TNF-α,IL-1βand NF-κB or by increasing phospho-Akt activityRGD peptides can inhibit apoptosis of free islet by increasing phospho-Akt activity...