| Background:Isletβ-cell dysfuntion and peripheral tissue insulin resistance are the two main pathogenic factors of type 2 diabetes.Increasing factors showed that isletβ-cell dysfuntion was the key link of type 2 diabetes occurrence and development.The United Kingdom prospective diabetes study(UKPDS) showed that newly diagnosed type 2 diabetes had only 50% of normal isletβ-cell function, followed byβ-cell function declined at the speed of 4% to 5% annually.Therefore,discussion of isletβ-cell injury mechanism and protection strategy has been always the hot and difficult spot of diabetes research field.Recently,many clinic trials,such as LIFE,CAPPP,SCOPE et al,suggested that angiotensin-converting enzyme inhibitor(ACEI) and angiotensin receptor blocker (ARB) can lower blood pressure,and meanwhile reduce the incidence of new-onset diabetes in high risk group.DREAM study showed that ramipril can restore the serum glucose level to normal for impaired fasting glucose(IFG) patients or impaired glucose tolerance(IGT) patients,and significantly reduce serum glucose levels after glucose loading.The newest NAVIGATOR study confirmed that based on the lifestyle intervention strickly, valsartan treatment can reduce the incidence of diabetes mellitus by 14% on IGT patients with caidivascular risk.Above-mentioned clinical studies preliminaryly suggested that there had been close relationship between type 2 diabetes and renin angiotensin system(RAS),but the specific mechanism remain not clearly.Therefore,exploring the intrisic relationship had became the hot spot of diabetes research field recently.Followed by the increasing concer about the relationship between type 2 diabetes and RAS,many researches confirmed that block RAS can reduce diabetes incidence,on the one hand RAS inhibitors improved peripheral insulin sensitivity and glucose metabolism,on the other hand RAS inhibitors played an important protective role on isletβ-cell. It had confirmed that oxidative stress was one of the most importantβ-cell dysfunction mechanism,a large amount of reactive oxygen species(ROS) production can injure P-cell function by many ways,such as influence insulin gene expression, impair insulin secretion function and inhibit proliferation, promote apoptosis.Besides,many researches indicated that there were close relationship between islet local RAS and P-cell oxidative stress damage.But there were lack of sufficient experimental evidence about the most active components of RAS-angiotensinⅡ(AngⅡ)injuredβ-cell function by oxidative stress pathway, and the specific molecular mechanism remained to be elucidate.Therefore,this reasearch selected P-cell line RIN-m cells as study object,and deeply discussed follow three aspects:(1)Observed the effect of AngⅡand pretreatment with losartan on RIN-m cells insulin gene expression,detected cellular ROS level,pancreatic duodenal homeobox factor(PDX-1) and Mus muscular v-maf musculoaponeurotic fibrosarcoma oncogene family proteinA (MafA) expression in each group,dicussed whether AngⅡinfluenced key insulin transcription factor activity by oxidative stress pathway,and then influencedβ-cell insulin gene expression;(2)Observed the effect of AngⅡand pretreatment with losartan on RIN-m cells insulin secrecion function,detected uncoupling protein 2(UCP2) expression, mitochondria transmembrane potential, cellular ATP content and Ca2+ concentration in each group,discussed whether Ang II influenced (5-cell glucose-simulated insulin secretion(GSIS) function by oxidative stress-UCP2 pathway;(3)Observed the effect of AngⅡand pretreatment with losartan on RIN-m cells proliferation, observed cells ultrasturcture by transmission electrion microscope,detected cells apoptosis, Bax/Bcl-2 expression and Caspase9,3 activity,and discussed whether AngⅡinfluenced apoptosis related signaling molecule by oxidative stress,and finally inducedβ-cell apoptosis through mitochondrion pathway.Part 1 The effect of Ang II on RIN-m cells insulin gene expression and related mechanismObejectiveTo discuss the effect of Ang II on RIN-m cells insulin gene expression and related mechanism.Methods(1)RIN-m cells cultured and activity detected:RIN-m cells were cultured with RPMI-1640 containing 10% fetal bovine serum in 37℃,5%CO2 incubator.0.4% trypan blue stained was used for detecting cells activity.(2) The effect of AngⅡon RIN-m cells insulin gene expression:Cells were divided into A～E five groups,A group was administered with RPMI-1640 complete medium as control group;B-E group were determined with various AngⅡat terminal concentration of 0.1,1,10,100nM.RIN-m cells insulin gene expression were detected by RT-PCR after incubation for 12h,24h or 36h.(3) The effect of pretreatment with losartan on RIN-m cells insulin gene expression:Cells were divided into thress groups,A group was control group,B group was administered with 100nMAngⅡ;1μMlosartan was administered 15 minutes before 100nMAngⅡtreatement as C group.RIN-m cells insulin gene expression were detected by RT-PCR after incubation for 24h.(4) The effect of pretreatment with losartan on RIN-m cellular ROS level:The groups were divided as part1(3).RIN-m cells were loaded by 100μM DCFH-DA fluorescent probe,and then DCF mean fluorescent indensity was detected by flow cytometry after intervention for 24h.(5) The effect of pretreatment with losartan on RIN-m cells PDX-1 and MafA expression:The group were divided as part1(3).RIN-m cells PDX-1 and MafA mRNA expression were detected by RT-PCR and protein expression were detected by Western-bloand after intervention for 24h.Results(1) RIN-m cells cultured and activity detected:RIN-m cells were elongated spindle,and refractive well.Cells activity was 93% by trypan blue stained.(2) The effect of AngⅡon RIN-m cells insulin gene expression:RIN-m cells insulin mRNA expression had no significantly different with various concentration of AngⅡfor 12h(F=0.324,P=0.858).For both 24h and 36h, insulin mRNA expression were significant difference(F=25.622,P=0.000;F=26405,P= 0.000).Except for 0.1nMAngⅡgroup,insulin mRNA expression of the other three groups significantly lower than of the control group(P<0.001).With different interference times,control group and 0.1nMAngⅡgroup had no significant difference(F=0.204,P=0.819;F=1.422, P=0.291),the insulin mRNA expression of other three groups cultured for 12h significantly higher than of the 24h and 36h(P<0.05),and there were no significant difference between 24h and 36h(P>0.05).(3) The effect of pretreatment with losartan on RIN-m cells insulin gene expression:RIN-m cells insulin mRNA expression had significantly different among control group,100nMAngⅡgroup and losartan pretreatment group(F=13.791, P=0.002).Insulin mRNA expression of the 100nMAngⅡgroup significantly lower than of the control group and losartan pretreatment group(P=0.001,0.002), and the later two groups had no significantly different(P=0.850).(4) The effect of pretreatment with losartan on RIN-m cellular ROS level: RIN-m cellular ROS level had significantly different among the three groups(F=36.693,P=0.000).ROS level of the 100nMAngⅡgroup significantly higher than of the control group and losartarn pretreatment group(P=0.000),and the later two groups had no significantly different(P=0.258).(5) The effect of pretreatment with losartan on RIN-m cell PDX-1 and MafA mRNA expression:Both RIN-m cells PDX-1 and MafA mRNA expression had significantly different among the three groups(F=4.832,P=0.038;F=5.823, P=0.024).Both PDX-1 and MafA mRNA expression of the 100nMAngⅡgroup significantly lower than of the control group and losartarn pretreatment group(P=0.016,0.046,0.010,0.030),and the later two groups had no significantly different(P=0.542;P=0.530).(6) The effect of pretreatment with losartan on RIN-m cell PDX-1 and MafA protein expression:Both RIN-m cells PDX-1 and MafA protein expression expression had significantly different among the three groups(F=9.118,P=0.007; F=16.472,P=0.001).PDX-1 and MafA protein expression of the 100nMAngⅡgroup significantly lower than of the control group and losartarn pretreatment group(P=0.004,0.006,0.000,0.001),and the later two groups had no significantly different(P=0.802;P=0.477).Conclusions(1)AngⅡinhibited isletβ-cell insulin mRNA expression in a time-dependent and dose-dependent manner;(2) AngⅡstimulated islet P-cell to produce a large amount of ROS,and induced P-cell oxidative stress damage;(3) AngⅡdown-regulated islet P-cell key transcription factor PDX-1 and MafA both mRNA and protein expression;(4) Pretreatment with losartan can antagonize the effect of AngⅡ:promote insulin mRNA expression,decrease celluar ROS level,up-regulate PDX-1 and MafA both mRNA and protein expression;(5) AngⅡmaybe down-regulate isletβ-cell key transcription factor PDX-1 and MafA expression by oxidative stress pathway,and then inhibit insulin gene expression; pretreatment with losartan can antagonize the effect of AngⅡ,and have protective function forβ-cell at the aspect of insulin gene expression.Part 2 The effect of AngⅡon RIN-m cells insulin secretion and related mechanismObejectiveTo discuss the effect of AngⅡon RIN-m cells insulin secretion and related mechanism.Methods(1) The effect of AngⅡon RIN-m cells insulin secretion:Cells were divided into A～E five groups as part 1(2). After incubation for 12h,24h or 36h, RIN-m cells were simulated with KRBH contained 3.3mM or 16.7mM glucose respectly for lh,radio-immunity assay method was used to detect insulin secretion.(2) The effect of pretreatment with losartan on RIN-m cells insulin secretion: Cells were divided into thress groups as part1 (3).After incubation for 24h, RIN-m cells insulin secretion was detected by RIA method,and the operation procedure as the same as above.(3) The effect of pretreatment with losartan on RIN-m cells UCP2 mRNA and protein expression:The groups were divided as partl(3).RIN-m cells UCP2 mRNA expression was detected by RT-PCR and protein expression was detected by Western-blot after intervention for 24h.(4) The effect of pretreatment with losartan on RIN-m cells mitochondria membrance potential:The groups were divided as part1(3).RIN-m cells were loaded by 5μg/ml rhodamine 123 fluorescent probe,and then mean fluorescent intensity was detected by flow cytometry after intervention for 24h.(5) The effect of pretreatment with losartan on RIN-m cellular ATP content: The groups were divided as part1(3).Per hole was administed 100μl Celltiter-GloTM mixture after intervention for 24h,fluorescent light was detected by luminometer.(6) The effect of pretreatment with losartan on RIN-m cellular Ca2+ concentration:The group were divided as part1(3).RIN-m cells were loaded by 5μM Fluo-3AM,and then Fluo-3 mean fluorescent intensity was detected by flow cytometry after intervention for 24h.Results(1) The effect of AngⅡon RIN-m cells basal insulin secretion:RIN-m cells basal insulin secretion had no significantly different with various concentration of AngⅡfor 12h(F=0.029,P=0.998).For both 24h and 36h,basal insulin secretion had significantly different(F=5.862, P=0.011;F=12.468, P=0.001).Except for 0.1nMAngⅡand 0.1nMAngⅡgroup,basal insulin secretion of the other two groups significantly lower than of the control group for 24h(P<0.05).Except for 0.1nMAngⅡgroup,basal insulin secretion of the other three groups significantly lower than of the control group for 36h(P<0.05).With different interference times,control group and 0.1nMAngⅡgroup had no significantly different (F=0.029,P=0.971;F=1.422,P=0.291),basal insulin secretion of the other three groups cultured for 12h significantly higher than of the 24h and 36h(P<0.05),and there were no significant difference between 24h and 36h(P> 0.05).(2) The effect of pretreatment with losartan on RIN-m cells GSIS:RIN-m cells GSIS had no significantly different with various concentration of AngⅡfor 12h(F=2.335,P=0.126).For both 24h and 36h,GSIS had significantly different (F=15.639,P=0.000;F=17.916,P=0.000).Except for 0.1nMAngⅡgroup,GSIS of the other three groups significantly lower than of the control group for 24h(P<0.05).GSIS of the other four groups significantly lower than of the control group for 36h(P<0.05).With different interference times,except of control group,GSIS of the other four groups cultured for 12h significantly higher than of the 24h and 36h(P<0.05),and there were no significant difference between 24h and 36h(P>0.05).(3) The effect of pretreatment with losartan on RIN-m cells insulin secretion: Both RIN-m cells basal insulin secretion and GSIS had significantly different among control group,100nMAngⅡgroup and losartan pretreatment group (F=28.785,P=0.000; F=63.341,P=0.000).Both basal insulin secretion and GSIS of the 1 OOnMAngⅡgroup significantly higher than of the control group and losartarn pretreatment group(P<0.05),and the later two groups had no significantly different(P=0.611;P=0.399).(4) The effect of pretreatment with losartan on RIN-m cell UCP2 mRNA and protein expression:Both RIN-m cells UCP2 mRNA and protein expression had significantly different among the three groups(F=694.903,P=0.000;F=117.157, P=0.000).Both UCP2 mRNA and protein expression of the 100nMAngⅡgroup significantly higher than of the control group and losartarn pretreatment group (P=0.000),and the later two groups had no significantly different(P=0.181, 0.153).(5) The effect of pretreatment with losartan on RIN-m cells mitochondria membrance potential:RIN-m cells mitochondria membrance potential had significantly different among the three groups(F=361.163,P=0.000). Mitochondria membrance potential of the 100nMAngⅡgroup significantly lower than of the control group and losartarn pretreatment group(P=0.000),and the later two groups had no significantly different(P=0.107).(6) The effect of pretreatment with losartan on RIN-m cellular ATP content:RIN-m cellular ATP content had significantly different among the three groups(F=10.094,P=0.001).Cellular ATP content of the 100nMAngⅡgroup significantly lower than of the control group and losartarn pretreatment group(P= 0.000;P=0.001),and the later two groups had no significantly different(P=0.634).(7) The effect of pretreatment with losartan on RIN-m cellular Ca2+ concentration:RIN-m cellular Ca2+ concentration had significantly different among the three groups(F=7.506,P=0.003).Cellular Ca2+ concentration of the 100nMAngⅡgroup significantly lower than of the control group and losartarn pretreatment group(P=0.001;P=0.020),and the later two groups had no significantly different(P=0.198).Conclusions(1) AngⅡinhibited isletβ-cell basal insulin secretion and GSIS in a time-dependent and dose-dependent manner;(2) AngⅡup-regulated isletβ-cell both UCP2 mRNA and protein expression, decreased mitochondria membrance potentia, cellular ATP content and Ca2+ concentration;(3) Pretreatment with losartan can antagonize the effect of AngⅡ:restore basal insulin secretion and GSIS,increase mitochondria membrance potentia, cellular ATP content and Ca2+ concentration;(4) AngⅡmaybe down-regulat P-cell insulin mRNA expression and then impaired basal insulin secretion;AngⅡmaybe up-regulateβ-cell UCP2 expression by oxidative stress pathway,and then impair GSIS;pretreatment with losartan can antagonize the effect of AngⅡ,and have protective function forβ-cell at the aspect of insulin secretion.Part 3 The effect of AngⅡon RIN-m cells proliferation,apoptosis and related mechanismObejectiveTo discuss the effect of AngⅡon RIN-m cells proliferation,apoptosis and related mechanism.Methods(1) The effect of AngⅡon RIN-m cells proliferation:Cells were divided into five groups as partl(2).MTT mothod was used to detect RIN-m cells proliferation after incubation for 24h,36h or 48h.(2) The effect of pretreatment with losartan on RIN-m cells proliferation:Cells were divided into thress groups as part1 (3).MTT method was used to detect RIN-m cells proliferation aftre incubation for 48h.(3) Ultramicrostructure was observed by transmission electron microscope:The groups were divided as part1(3).After intervention for 48h,RIN-m cells were glutaraldehyde fixed,alcohol dehydrated,oxidation propylene soaked, resin embedd,ultramicrotome cutted,finally observed by transmission electron microscope for ultramicrostructure.(4) The effect of pretreatment with losartan on RIN-m cells apoptosis:The groups were divided as part1 (3).RIN-m cells were staied by AnnexinV-FITC/PI, and flow cytometry was used to detect apoptosis ratio.(5) The effect of pretreatment with losartan on RIN-m cells Bcl-2 and Bax expression:The groups were divided as part1(3).RIN-m cells Bcl-2 and Bax mRNA expression were dectected by RT-PCR and protein expression were detected by Western-blot after intervention for 48h.(6) The effect of pretreatment with losartan on RIN-m cells Caspasae 9,3 activity:The groups were divided as part1(3).RIN-m cells Caspasae9,3 activity were detected by spectrophotometric method after intervention for 48h.Results(1) The effect of AngⅡon RIN-m cells proliferation activity:RIN-m cells proliferation activity had significantly differen with various concentration of AngⅡfor 24h,36h or 48h(F=288.043,P=0.000;F=198.079,P=0.000;F=201.157, P=0.000).Except for control and 0.1nMAngⅡgroup,there were significant difference between every two groups for 24h(P<0.05),and there were significant difference between every two group for 36h and 48h(P<0.05).With different interference times,except for control group,the other four groups cell proliferation activity cultured for 24h significantly higher than of the 36h and 48h(P<0.05),and there were no significant difference between 36h and 48h(P> 0.05).(2) The effect of pretreatment with losartan on RIN-m cells proliferation activity:RIN-m cells proliferation activity had significantly different among control group,100nMAngⅡgroup and losartan pretreatment group(F=896.424, P=0.000).100nMAngⅡgroup proliferation activity significantly lower than of the control group and losartarn pretreatment group(P=0.000),and the later two groups had no significantly different(P=0.459). (3) Ultramicrostructure was observed by transmission electron microscope:The ultarmicrostructure of control group cells had normal size,clear structure, prominent nucleoli,uniform chromatin and normal organelles;the ultarmicrostructure of the 100nMAngⅡgroup showed morphological changes of apoptosis: cytoplasmic condensation,nuclear condensation and fragmentation, chromatin condensation and margination,cytoplasm with vacuolization, mitochondrial swelling,cristae significant reduced,electron lucent zone formation;the ultarmicrostructure of the losartan pretreatment group showed normal morphology and structure.(4) The effect of pretreatment with losartan on RIN-m cells apoptosis ratio: RIN-m cells apoptosis ratio had significantly different among the three groups(F=101.298,P=0.000).Apoptosis ratio of the 100nMAngⅡgroup significantly higher than of the control group and losartarn pretreatment group(P=0.000),and the later two groups had no significantly different(P=0.220).(5) The effect of pretreatment with losartan on RIN-m cells Bcl-2,Bax mRNA and protein expression:RIN-m cells Bcl-2 mRNA and protein expression had no significantly different among the three groups(F=2.180,P=0.169; F=0.622,P=434.795);Bax mRNA and protein expression had significantly different among the three groups(F=57.535,P=0.000;F=434.795,P=0.000).Both Bax mRNA and protein expression of the 100nMAngⅡgroup significantly higher than of the control group and losartarn pretreatment group(P=0.000),and the later two groups had no significantly different(P=0.225;P=0.108).(6) The effect of pretreatment with losartan on RIN-m cells Caspasae 9,3 activity.RIN-m cells Caspase9 and 3 activity had significantly different among the three groups(F=5.108,P=0.012;F=5.401,P=0.009).The Caspase 9 and 3 activity of the 100nMAngⅡgroup significantly higher than of the control group and losartarn pretreatment group (P=0.026,P=0.004; P=0.025,P=0.003), and the later two groups had no significantly different(P=0.220;P=0.420).Conclusions(1) AngⅡinhibited isletβ-cell proliferation activity in a time-dependent and dose-dependent manner;(2) AngⅡinduced typical apoptosic morphology of isletβ-cell,and promoted apoptosis;(3) AngⅡhad no effect on Bcl-2 expression,but up-regulated Bax mRNA and protein expression,increased Bax/Bcl-2 ratio,promoted Caspase 9,3 activity;(4) Pretreatment with losartan can antagonize the effect of AngⅡ:promoteβ-cell proliferation,inhibitβ-cell apoptosis,down-regulated Bax expression,decrease Bax/Bcl-2 ratio,inhibit Caspase 9,3 activity;(5) AngⅡmaybe up-regulat apoptosis gene expression and activate Caspase by oxidative stress,and then induceβ-cell apoptosis by mitochodrion pathway; pretreatment with losartan can antagonize the effect of AngⅡ,and have protective function forβ-cell at the aspect of proliferation and apoptosis.
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