| Objective: IUGR(Intrauterine Growth Retardation),a kind of pathophysiological process,means the stature of fetus below the standard under the intervention of gene.IUGR may increase the risk of obesity;insulin resistance or metabolic syndrome of type 2 diabetes mellitus in adulthood.The “thrifty phenotype hypothesis” was accepted generally to explain the phenomenon.However,the detail mechanisms were unclear.The malnutrition of fetus and neonates may result in dysplasia of islet β cells and injury of islet function.The varying of anatomical structure and function leads to happiness of T2 DM in adulthood.The islet β cells obtain the intact function during the rebuilding process in 2-3 weeks after birth,when the multiplication level of islet β cells would be changed by stimulation of drugs.Osteoprotegerin(OPG)was widely researched for the important role in metabolism of bone.OPG has close relations between islet β cells.A large number studies have shown that the PI3K/AKT pathway plays an vital role in proliferation of islet β cells,and could be stimulated by OPG.FoxO1 was expressed in the islet β cells,and phosphorylation plays an important regulatory role in nucleoplasmic transfer of FoxO1.PI3K/AKT pathways activated could promote FoxO1 protein phosphorylation,prompting FoxO1 nuclear output and block shall be a signal to prevent FoxO1 to return to the nuclear,leads to the change of FoxO1 transcription activity,the changes in the body cell proliferation differentiation and apoptosis and oxidative stress resistance play an important role,its role in the change of islet β cell proliferation and function,which has also been demonstrated by many scholars.In conclusion,this research compared the expression level of OPG in the pancreas of IUGR rats,and there was any difference in normal rats.Furthermore,it was clear that the exogenous supplemental OPG of the IUGR rats during the reconstruction of islet cells can effect the proliferation of islet cells.The role of OPG in promoting the signaling pathway of PI3K/AKT/FoxO1 in the process of cell proliferation of islets was also discussed,and the mechanism of OPG to effect the proliferation of islet cells was clarified.Methods: The IUGR model of rat was established using the “whole-process low-protein diet” method.The critical periods of pancreas development,neonatal stage(1 day),infancy stage(1 week),juvenile stage(3 weeks),adult stage(12 weeks),were selected as the key point of research.The weight of rats and its pancreas were measured after sampling.The fast biochemical glucose meter was applied to measure blood glucose,the fasting insulin was determined by ELISA method.The islet morphology was observed by HE staining.The proliferation of islet β cell was observed by Ki67 and insulin immunofluorescence double staining.OPG protein and mRNA expression in the pancreas was detected through Westen Blot immunohistochemical Real-time PCR.The rats,including healthy and IUGR samples,were divided in three groups in random.Group 1 could be called IUGR+OPG group,which was the first intraperitoneal recombinant human OPG in 24 hours after birth,with a dose of 3ug/kg,one time per day,until it used for materials or three weeks after birth.Group 2 was IUGR group,which the first intraperitoneal injection was the same as that of the recombinant human OPG solvent in the first 24 hours after the birth,one time per day,until it used for materials or three weeks after birth.Group 3 was CON group,which intraperitoneal injection for the first time in 24 hours was the same as that of the recombinant human OPG solvent,with the same amount of water used for injection,one time per day,until it was used for material or 3 weeks after birth.The weight of body,weight of pancreas,fasting blood glucose levels and fasting insulin levels of rats in each group were measured in the key point of 1 week,3 weeks and 12 weeks.The proliferation of islet β cells was also observed.Firstly,to clearify the mechanism of islet β cell proliferation,the pancreas of healthy and IUGR rats of 12 weeks after birth were studied.The expressions of AKT,p-AKT,FoxO1,p-FoxO1,FoxO1 within the cytoplasm and nucleus FoxO1 expression levels were detected by Westen Blot method.Additionally,the mechanism was discussed further on the cellular level,based on INS-1 cells.The cells were divided into four groups: CON group(INS-1 cells cultured normally),OPG group(adding recombinant human OPG to INS-1 cells cultured normally),LY group(adding LY294002 to INS-1 cells cultured normally),and LY+OPG group(adding both recombinant human OPG and LY29400 to INS-1 cells cultured normally).The cell proliferation was detected by application of CCK-8,and cycle and apoptosis of cells were detected by application of flow cytometry technology after drug intervention in 48 hours.The expression of Cyclin D1 and Cyclin E,the expression of apoptosis related proteins bcl2 and bax,the expressions of AKT,p-AKT,FoxO1,p-FoxO1,FoxO1 within the cytoplasm and nucleus FoxO1 expression levels were detected by Westen Blot method.The experimental results were statistical analyzed by SPSS21.0 software.The measurement data was expressed as mean value ±standard deviation(x ± s).Independent sample t test was used for comparison between the two groups,and one-way anova was used for multiple groups.The correlation analysis of double variables was established by the Penrson relative analysing method.Results: 1.The birth weight of rats in IUGR group was lower than which in CON group obviously,during 3 weeks from birth.The catch-up growth of rats in IUGR group appeared from suckling period.The weight has no obvious difference,at 12 weeks from birth,between the rats of IUGR group and ones in CON group.The pancreas weight and the ratio of pancreas weight/body weight of rats in IUGR group was lower than ones in CON group generally at each key point.IUGR rats appeared hyperinsulinemia and insulin resistance at 12 weeks after birth.2.It has been shown from HE coloration results that the volume of islet β cell aggregate of rats in IUGR group were lower than ones in CON group.It has been shown from Ki67 and insulin immunofluorescence double staining results that the proliferation of islet β cell of rats in IUGR group were lower than ones in CON group.3.The Westen Blot and immunohistochemical results shown that the OPG protein expression of rats in IUGR group were obviously lower than ones in CON group,on any testing point.The Real-time PCR results shown that the OPGmRNA expression of rats in IUGR group were obviously lower than ones in CON group,on any testing point.4.It was seen from the results of correlation analysis that the OPG protein and mRNA level showing positive correlations with the weight of pancreas.5.The weight of IUGR rats injected recombinant human OPG have no vital difference from the ones common IUGR rats.However,the ratio of pancreas weight/body weight of rats rejected OPG was larger than ones of common IUGR rats.The insulin level and insulin resistance index of rats in IUGR+OPG group were lower than IUGR group at 12 weeks after birth.6.The islet β cells proliferation of rats in IUGR group was lower than ones in CON group,at 1 week,3 weeks and 12 weeks after birth.Comparing to the rats in IUGR group,the islet β cells proliferation of rats in IUGR+OPG group were improved obviously.7.In the 12 weeks after birth,the expression of p-AKT and p-FoxO1 protein in the pancreatic tissues of IUGR rats decreased significantly compared to the normal control group,and the expression of FoxO1 protein in the cytoplasm was significantly reduced,while the expression of FoxO1 in the nucleus was also increased.After treating by recombinant human OPG,in IUGR rats pancreatic tissue,the level of phosphorylation of AKT and FoxO1 were significantly improved.The protein expression of FoxO1 in the cytoplasm was increased,but decreased in nucleus.It was revealed that the injecting recombinant human OPG leads to increasing of AKT and FoxO1 protein phosphorylation and decreasing of active FoxO1 protein.The islet β cell proliferation was further promoted.8.The cell proliferation in test of cells was detected by CCK-8 method.It was shown that the proliferation of INS-1 cell would be promoted by recombined human OPG,but restrained by LY294002.At the same time,recombinant human OPG and LY294002 stimulate cells to inhibit OPG-induced cell proliferation.Further to detect the cell cycle by flow cytometry,the results suggested that compared with normal control group,group OPG G0/G1 phase ratio decreases,rising rates of S phase and G2/M phase,LY group on the contrary,LY + OPG proliferation index is lower than the OPG group.The apoptosis detection results found that OPG group of apoptosis decreased,apoptosis in LY group was obviously increased,the proportion of apoptosis in LY + OPG group was obviously higher than that in OPG group.The mechanism of islet β cells proliferation promoted by OPG was discussed finally on the level of cells.This study found that the expression of p-akt and p-foxo1 protein in OPG group was higher than which in CON group,and the expression of FoxO1 protein in the cytoplasm was increased,the expression of FoxO1 was decreased in the nucleus.On the contrary,the expression of p-AKT and p-FoxO1 protein decreased significantly,and FoxO1 protein expression decreased significantly in cytoplasm,the expression of FoxO1 was significantly increased in the nucleus.In LY+OPG group,LY294002 Y dampened by OPG promote FoxO1 and AKT phosphorylation levels,make its phosphorylation levels decrease in the OPG group,and suppresses the FoxO1 expression in the cytoplasm,promoting expression in the nucleus.It is inferred that OPG could transfer the FoxO1 from the nucleus by activating the PI3K/AKT/FoxO1 pathway,and the cell proliferation was further improved.Conclusion: 1.In this study,the IUGR rats model was established successfully through low protein diet throughout pregnancy.The rats in IUGR group appeared many symptoms as slow growth and development,loss of weight,pancreas weight losing,the proliferation level of islet β cells decreased and insulin resistance at 12 weeks after birth.2.OPG was expressed in both normal group and IUGR group.The levels of OPG protein and mRNA in the islet of IUGR group were lower than ones in the normal group,from neonatal period to adult stage.3.The recombinant human OPG was used to promote pancreatic development and islet cell proliferation,and improved insulin resistance.4.OPG could promote the proliferation of ins-1 cells and promote the smooth transition of cell cycle from G0/G1 phase to S phase and G2/M phase,and inhibit cell apoptosis.5.The process of OPG promoting cell proliferation requires the involvement of PI3K/AKT/FoxO1 signaling pathway. |
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