| Objective To compare the changes of CHOP and Bcl-xl expression in normoal glucose tolerance (NGT) rats, impaired glueose tolerance (IGT) and IGT rats with GLP-1 agonists intervention, and to observe the ultrastructure ofβcells with transmission electron microscope, To explore protective effect and possible mechanism of GLP-1 agonists on the endoplasmic reticulum stress pathway of apoptosis in pancreaticβ-cells.Methods Forty-eight male Wistar rats in 4-5 weeks (150g-180g) of clean grade were purchased in Institute of Chinese Medicine of Shanxi. After one week adaptive feeding (five/cage),they were divided into A group (n=16) and B group (n=32) randomly. The A group rats were fed by normal diet(13.4kJ/g, which accounted for 10.2% of calories, fat and protein accounted for 23.3% respectively, carbohydrate accounted for 66.5%);The B group rats were given high-sugar and high-fat feed (21.8kJ/g, which accounted for 56.0% of calories, fat and protein accounted for 7.0% respectively, carbohydrates accounted for 37.0%) and free access to water, at room temperature controlled at 18-22℃,relative humidity 30%-70%. Two groups of rats twice a day morning and evening feeding, daily food in take for each rat to vote for 3% of body weight.When 12 weeks, do oral glucose tolerance test (OGTT):after fasting 8h, cut the tail blood and rapid blood glucose meters measure fasting blood glucose (FBG), intragastric administration with 50% glucose injection 2g/kg body weight, after 2h cut the tail blood again and measure 2h postprandial blood glucose (2hPG). Building a suceessful model was 7.8mmol/L<2hPG<11.1mmol/L and continued for more than a week.The A group, blood glucose to normal,and being setted to normoal glucose tolerance control group (NGT group, n=16). Twenty-eight rats in B group were into models, it has the making model success rate of over 87.5%, and randomly divided the B group into two groups, the impaired glucose tolerance group(IGT group,n=14).Exendin-4 interventionin group (Ex group, n=14).Select randomly eight rats in NGT group,seven rats in IGT group and Ex group respectively, weighing, and then do oral glucose tolerance test measure 2h PG. The next day,after fasting 12-14h, anesthetized rats with 5% chloral hydrate by intraperitoneal injection 0.6ml/100g body weight, Quickly remove the pancreas. A little pancreas slices were incubated with the cold fixed solution to observe by transmission electron microscopy. And then obtain a little pancreas tissues, fixed with 10% formaldehyde solution,dehydrated,embedded in paraffin,regular slices, for immunohistochemical detection the Bcl-xl expression of pancreas.and put the remaining pancreas rapidly in liquid nitrogen, after thoroughly frozen, stored in-70℃refrigerator. Measured the expression of CHOP mRNA by real time quantitative polymerase chain reaction (RT-PCR). The remaining rats in NGT group continue to give normal diet, the IGT group and Ex group were to continue to give high-sugar high-fat feeding and freedom drinking water. Ex group received Exendin-4 (Eli Lilly and Company to provide) 5ug/kg subcutaneously, twice daily, in the NGT group and the IGT group were given equal volume of saline injection. When interventing 4 weeks, weighing, and then measure 2h PG.The next day, anesthetized rats, quickly remove the pancreas, to observe theβcells by transmission electron microscopy, then obtain a little pancreas tissues, for immunohistochemical detection the Bcl-xl expression of pancreas.and measured the expression of CHOP mRNA by RT-PCR. All results were measured by(x±s), using SPSS 17.0 software package, multiple groups means were compared with single factor analysis of variance, LSD test. P≤0.05 was considered statistically significant. Then Pearson correlations were used to determine the relationship between Bcl-xl and CHOP.Results1.The changes of clinical characteristics and biochemical indices between rats in each groups Before intervention, compared with the NGT group, the body weigh of rats in the IGT group and the Ex group were higher (542.91±36.23 vs.614.32±40.43) (542.91±36.23 vs. 620.50±43.34) and the differences were also significant (both P<0.05).the 2hPG of rats were higher (6.22±0.91 vs.9.81±0.90) (6.22±0.91 vs.9.62±1.02) the differences were also significant (both P<0.05).Compared with the IGT group, the body weigh and the 2hPG of rats in the Ex group were higher (614.32±40.43 vs.620.50±43.34) (9.62±1.02 vs.9.81±0.90) but the difference was not significant (P>0.05). After intervented with Exendin-4 4 weeks, compared with the IGT group, the body weigh and the 2hPG of rats in the Ex group were decreased(636.21±37.95 vs.555.95±52.00) (10.06±0.85 vs.6.76±1.61) the differences were significant (both P<0.05).Compared with the Ex group before intervented, the levels were decreased(620.50±43.34 vs.555.95±52.00) (9.62±1.02 vs.6.76±1.61) the differences were significant (both P<0.05).Compared with the NGT group the levels were higher(533.89±44.08 vs.555.95±52.00)(6.45±1.47 vs.6.76±1.61)but the difference was not significant (both P> 0.05).2.The expression of BCL-XL in pancreas with eath groups Before intervention,compared with the NGT group, the expression of BCL-XL with rats in the IGT group and the Ex group were decreased(49.27±2.14 vs.30.65±1.46)(49.27±2.14 vs.30.85±2.47) and the differences were significant (both P<0.05).Compared with the IGT group, the expression of BCL-XL with rats in the Ex group was higher (30.65±1.46 vs.30.85±2.47) but the difference was not significant(P>0.05). After intervented with Exendin-4 4 weeks, compared with the IGT group, the expression of BCL-XL in the Ex group was higher (30.11±2.20 vs.41.81±2.99) the differences was significant (P<0.05).Compared with the Ex group before intervented, the levels was also higher (30.85±2.47 vs.41.81±2.99) the differences were significant (P<0.05). Compared with the NGT group the level was decresed (50.85±1.70 vs.41.81±2.99) the difference was significant(P<0.05).3. The expression of CHOP in pancreas with eath groups Before intervention, compared with the NGT group, the expression of CHOP with rats in the IGT group and the Ex group were increased(0.15±0.07 vs.1.08±0.19) (0.15±0.07 vs.1.12±0.21) and the differences were significant (both P<0.05).Compared with the IGT group, the expression of CHOP with rats in the Ex group was higher (1.08±0.19 vs.1.12±0.21) but the difference was not significant(P> 0.05). After intervented with Exendin-4 4 weeks, compared with the IGT and Ex group, the expression of CHOP in the Ex group was decreased (1.11±0.32 vs.0.46±0.17) (1.12±0.21 vs. 0.46±0.17) the differences was significant (both P<0.05).Compared with the NGT group the level was incresed (0.19±0.09 vs.0.46±0.17) the difference was significant(P<0.05).4. The ultrastructural changes of isletβcells in each groups Under the transmission electron microscope, before intervention, pancreaticβcell morphology in NGT group generally normal, present the full structure,The cell nucleu were globular with clear nucleoles, chromatin uniformly distributed,In the cytoplasm it has a massive of rough endoplasmic reaticulums with normal structure,and mitochondria were distributed in the cytoplasm. The cytoplasm contains abundent secretory granules. In IGT group, the decrease of cell volume could be observed and the shape of nucleus is irregular, nuclei have become pyknotic and nucleolus disappears, the rough endoplasmic reticula expanded and degranulstion, it appeared mitochondria swelling and remarkable decrease in the secretory granules. In the Ex group brfore intervented, the shape of nucleus is irregular, nuclei have become pyknotic and nucleolus disappears, the rough endoplasmic reticula expanded and degranulstion, it appeared mitochondria swelling and remarkable decrease in the secretory granules. After intervented with Exendin-4 4 weeks, the ultrastructural changes of islet (3 cells in NGT group is similar to the NGT group before intervented. And the IGT group is similar to it before intervented. In the Ex group after intervention the shape of nucleus is regular and the chromatin distributed evenly,compared with the Ex group before intervention and the IGT group, the rough endoplasmic reticula is remarkably improved, only a few mitochondria showed varying degrees of vacuolar degeneration and the secretory granule increased obviously.5. The expression of CHOP and the expression of BCL-XL in pancreas was significantly correlated in rats with impaired glucose tolorence (r=-0.89, P<0.01).Conclusions1. In rats with impaired glucose tolerance, the expression of CHOP with rats was increased, the expression of BCL-XL with rats was decreased, endoplasmic reticulum stress has happened in pancreaticβcells,2. In the stage of IGT, the decrease ofβcells volume could be observed and the shape of nucleus is irregular, nuclei have become pyknotic and nucleolus disappears, the rough endoplasmic reticula expanded and degranulstion, it appeared mitochondria swelling and remarkable decrease in the secretory granules, apoptoticβcells were observed.3. Exendin-4 inhibits the CHOP signaling pathways, to reduce endoplasmic reticulum stress response, and ultimately inhibit the endoplasmic reticulum-mediated apoptosis in pancreaticβcells.
|
PDF Full Text Request