| BackgroundRecently epidemiological investigations and experimental animal studies had suggested that low birth weight was not only one of the important reasons to death and illness of perinatal baby, but also a dangerous factor of metabolic syndrome (MS) during adulthood such as hypertension and diabetes mellitus. Therefore it was a popular topic to study the mechanisms of how low birth weight can cause adulthood MS and to find an effective measure to prevent and treat MS.Recently investigations had suggested that oxidative damage had important role on the morbility of diabetes . Oxidative damage can hurt islet beta cell and induce insulin resistance. The disbanlance between oxidation and antioxidation can induce oxidative damage, then it will promote the occurrence of diabetes. Oxidative damage will hurt organizations and induce a lot of changes including lipid peroxidation of biomembrane, degeneration of protein and enzyme, damage of DNA, then cell apoptosis and disease will happen. The increasing of MDA around pancreas in diabetes indicates that oxygen free radicals play a part in diabetes. Now study is insufficient on whether oxidative damage is related with the process that low birth weight can cause MS. It is very important to investigate the mechanism of oxidative damage on the process that low birth weight can cause MS and to find an effective measure to prevent MS.Nutrient is an important regulatory factor to gene expression. Nutrient can induce the change of mRNA and protein by controlling the metabolic product or metabolic state. During the pivotal phase, islet development was sensitive to malnutrition, and malnutrition would damage the function and structure of islet. L-Arg, as an important essential amino acid during development, had been found to be applied on the clinical of IUGR. L-Arg can improve the placental function and promote the transport of amino acid. L-Arg can also promote the secretion of insulin, so it can be used on Diabetes. L-Arg can raise the activity of SOD and relieve the oxidative damage. But there was little study on the effect of L-Arg on pancreas structure and fuction.Objective(1) To make low birth weight neonatal rat model by feeding pregnant rats 10% low protein diet. To observe the changes of body weight during postnatal 2～3 months.(2) To observe the changes of blood sugar levels, serum insulin levels, pancreas insulin protein level and ultrastructure of isletβcell in the low birth weight rats during postnatal 2~3 months.(3) To study the oxidative damage state in the low birth weight rats, and to carry out the correlation investigation.(4) To study the mechanisms of why oxidative damage can change the structure and function of pancreas.(5) To study the effects and mechanisms of L-Arg on pancreas structure and fuction to low birth weight rats from several aspect such as to relieve oxidative damage, promote the synthesis and secretion of insulin, promote islet cell differentiation and decrease the apoptosis of islet.Methods(1) The building of low birth weight neonatal rat model and subgroups: eightteen male and eighteen female healthy three-month SD rats were bought. Male and female rats were put into a cage at the proportion of 1:1. The pregnant rats were divided into three groups:①the model of low birth weight neonatal rats was made by feeding 10% low protein diet to the pregnant rats of group M (M) during gestation and feeding normal protein diet during lactation;②feeding 21% normal protein diet to the pregnant rats in normal control group (C);③a part of group M were given a supplementation with L-Arg (200mg/Kg) drinking water (MT) only during 21-day lactation; Other femal rats were given running waiter. To observe the newly born member per nest, body weight of neonatal rat, IUGR incidence rate and perinatal mortality in each group.(2) Study on the changes of islet function and structure and correlative mechanisms: At postnatal day 7,21,60 or 90, rats were weighted and executed,following index were detected.①Blood were collected for detecting blood sugar and serum insulin levels. Islet beta cell secretion fuction were calculated.②Expression of insulin protein were detected by immuno-histochemical staining technique in pancreas slices.③Level of insulin protein were detected by western blot technique in pancreas.④The ultrastructural changes ofβcell were observed by electron microscopy.(3) Detecting of oxidative damage index and antioxidase level:①Serum MDA, SOD and GSH-PX level were detected.②Tissue homogenate MDA, SOD and GSH-PX level were detected in pancreas, myocardium and brain.③Correlation analysis were performed by oxidative damage condition with blood glucose level, insulin protein level and islet beta cell secretion fuction.(4) Study on the mechanism of oxidative damage on islet:①Level of NF-κB protein were detected by WB and IH technique in pancreas.②The expressions of Insulin, Bax and Bcl-2 mRNA were detected by RT-PCR technique.(5) Investigation of the change and mechanism of rats islet structure and function after treating by L-Arg:①Body weight were detected.②Blood glucose level and serum insulin level were detected. Islet beta cell secretion fuction were calculated.③Serum MDA, SOD,NO and GSH-PX level were detected.④Expression of insulin, NF-κB and PDX-1 protein level were detected by immuno-histochemical staining technique and western blot technique in pancreas .⑤Expression of insulin, Bax and Bcl-2 mRNA were detected by RT-PCR in pancreas.⑥The ultrastructural changes ofβcell were observed by electron microscopy.(6) Statistics analysis: All data were statisticed by the SPSS-13.0. Differences between groups were evaluated using t test, rate comparison between groups were evaluated using x~2 test, correlation analysis were performed by Bivariate Correlations and Pearson correlation coefficient were calculated. P value<0.05 was considered significant.Results(1) The building of low birth weight neonatal rat model: the offspring in group M got low birth weight and more offspring Could be called IUGR rat. But there were no obvious changes in the newborn member per nest and perinatal mortality between group M and C. Body weights in group M were less than which in group C, but they were equal at postnatal 3 month.(2) Study on the changes of islet function and structure and correlative mechanisms:①Hyperglycemia were occurred in group M at postnatal 3 months.②The levels of serum insulin had no significant difference between two groups at every stage. But the islet beta cell secretetion functions in group M were decreased at postnatal 2 months.③The level of insulin protein in group M were decreased at each stage.④The quantity of endocrinal grains inβcell were decreased in group M at postnatal 2 months than in group C, and more pale-granules and degranulations ofβcell were found in group M than in group C.(3) Detecting of oxidative damage index and antioxidase level:①The serum MDA level in group M was more than which in group C, the serum SOD and GSH-PX level in group M was less than which in group C.②The changes of tissue homogenate MDA, SOD and GSH-PX level were similar in pancreas, myocardium and brain. (3)MDA levels were positively correlated with hyperglycemia, negatively correlated with insulin protein level and the islet beta cell secretetion functions.(4) Study on the mechanism of oxidative damage on islet:①The level of NF-κB protein in group M were increased.②Expressions of insulin and Bcl-2 mRNA were increased in group M than in group C, the changes of Bax mRNA were contrary.(5) Investigation of the change of rats growth and oxidative damage after treating by L-Arg:①The body weight of rats in group MT was higher than which in group M.②The serum MDA level in group MT was less than which in group M, The change of SOD was opposite. The serum NO level in group MT was higher than which in group M. (6) Investigation of the change and mechanism of rats islet structure and function after treating by L-Arg:①The islet beta cell Secretion function was raised than which in group M.②The insulin protein level in group MT was higher than which in group M.③The PDX-1 protein level in group MT was higher than which in group MT.④The insulin mRNA expression in group MT was higher than which in group M.⑤L-Arg could up-regulate the expression of Bcl-2 mRNA and down-regulate the expression of Bax mRNA.⑥The quantity of endocrinal grains inβcell were increased in group MT than which in group M at postnatal 2 months, and less pale-granules and degranulations ofβcell were found in group MT than in group M.Conclusion(1) Feeding 10% low protein diet to the pregnant rat was an effective method to make low birth weight neonatal rat model.(2) The low birth weight rats had appeared hyperglycemia and body weight increasing at postnatal 3 months. It had the prone to developing MS.(3) The islet beta cell secretion function in group M was degraded at postnatal 2 and 3 months, the insulin protein and mRNA levels in group M were decreased. These findings indicated that the insulin synthesis and secretion function was disturbed in group M. The ultrastructures of islet had damaged in low birth weight rats.(4) The oxidative damage condition was raised and antioxidase level was decreased in group M. MDA level was correlated with hyperglycemia, insulin synthesis and secretion. Investigation results indicated that oxidative damage played important role on the process which low birth weight rats may occur hyperglycemia and the reduction of insulin secretion, synthesis.(5) NF-κB protein levels were increased in group M. The expression of Bax mRNA was increased and the expression of Bcl-2 mRNA was decreased in group M. These investigate results indicated that oxidative stress may change the structure and function of pancreas by the effect to NF- K B protein and Bcl-2/Bax balance.(6) L-Arg supplement can promote the growth and development of low birth weight rats.(7) L-Arg supplement can relieve oxidative damage by raising the level of SOD and raising the level of NO. Investigating results indicate that L-Arg can improve the islet beta cell secretion function by relieving oxidative damage.(8) L-Arg supplement can improve the structure and endocrine function of islet by multiple mechanisms such as inhibiting the expression of NF-κB, lessening oxidative damage, inhibiting islet apoptosis by changing Bcl-2/Bax balance and up-regulating the expression of PDX-1 mRNA.
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