The Protective Effect Of Ursodeoxycholic Acid And 4-phenyl-acetic Acid Internal Endoplasmic Reticulum Stress In Pancreatic ¦Â Cell Apoptosis And Its Mechanism

Objects:1.To clarify the protective effect of ursodeoxycholic acid and 4-phenyl butyric acid on anti-apoptosis in pancreaticβ-cell of STZinduced diabetic rats.2.To investigate the endoplasmic reticulum stress associated apoptosis in pancreaticβ-cell of STZ-induced diabetic rats and the effects of ursodeoxycholic acid and 4-phenyl butyric acid on the endoplasmic reticulum stress.Methods:1.The preparation of STZ-induced diabetic model and the intervention of UDCA and 4-PBA:Rats(n=40)received a single injection STZ(50 mg/kg)intra-peritoneally to produce aβ-cell injury model. Weight-matched normal rats(the control group,n=10)were injected with the buffer alone.STZ-treated rats with persistent random blood glucose higher than 16.7mmol/1 for 1 week were considered as diabetic status (n=16),then divided into the following two groups randomly: STZ-induced DM group(n=8)and UDCA-treated group(n=8).UDCA (40mg/kg/d)was administered daily by intragastric intubations throughout the experimental period(30 days).The control group and the STZ-induced diabetic group without UDCA received an equivalent amount of 0.9%NaCl intragastrically.Other rats(n=22)received a single injection STZ(60 mg/kg)intraperitoneally to produce aβ-cell injury model.Weight-matched normal rats(the control group,n=10)were injected with the buffer alone.STZ-treated rats with persistent random blood glucose higher than 16.7mmol/1 for 1 week were considered as diabetic status(n=14),then divided into the following two groups randomly:STZ-induced DM group(n=7)and 4-PBA-treated group(n=7). 4-PBA(500mg/kg/d)was administered daily by intragastric intubations throughout the experimental period(20 days).During all experiments, blood glucose and weight of all rats were assessed.2.The detection of pancreaticβ-cell apoptosis:Following treatment,all rats were anaesthetized and then were killed to remove pancreas and collect the blood sample immediately.Serum insulin levels were assayed by radioimmunoassay.The morphological changes of pancreaticβ-cells apoptosis were determined by light microscope and TUNEL assay.The expressions of Bcl-2 and Bax genes mRNA in pancreas were detected by RT-PCR.3.The detection of endoplasmic reticulum stress associatedmolecules: The expressions of Bip and CHOP genes mRNA in pancreas were detected by RT-PCR and the expression of CHOP protein was detected by Western-Blot.Results:1.The establishment of DM rats model was successful and all indexs were compared between DM group and NC group:The concentration of blood glucose in two DM groups were(26.41±6.88) retool/1 and(33.71±0.77)mmol/1,higher than NC group(P＜0.01).Body weight in DM group was significantly lower compared with NC group (P＜0.01).The serum insulin level in NC group were(25.15±3.16)mIU/L and(25.27±2.51);and those in DM group were(13.52±2.98)mIU/1 and (8.10±1.63)mIU/1,but were significantly lower compared with NC group(P＜0.01).2.The protective effect of UDCA and 4-PBA on anti-apoptosis in pancreaticβ-cell of STZ-induced diabetic rats:The concentration of blood glucose in diabetic rats was gradually decreased after the UDCA and 4-PBA treatment,and at the end of the experiment those were(17.44±6.31)mmol/1 and(17.97±8.67)mmol/1,lower than DM group(P＜0.01).The serum insulin levels(19.95±3.70)mIU/L and (11.77±1.66)mIU/L rose in UDCA and 4-PBA-treated diabetic rats, but was significantly lower compared with DM group(P＜0.01).The percentage ofβ-cell apoptosis of DM group were(52.67±4.04)%and (60.29±6.05)%,as those in the UDCA and 4-PBA-treated groups were (23.00±3.6)%and(32.17±3.06)%,significantly lower than that of DM group(P＜0.01).Based on HE stained tissue sections,DM group elicited severe injury of pancreas,such as decreasing of the islets cells' numbers and diminishing of the diameter of pancreatic islet.The nuclei of islets cells were more shrunken in diabetic rat compared with NC group.It showed that the moderate expansion of islets and significantly reduced the injuries of pancreas in the UDCA and 4-PBA-treated group.The expression of Bax gene mRNA of DM group was significantly stronger than that of the control group in pancreas(P＜0.05)while the expression of Bcl-2 is lower(P＜0.05).Whereas these parameters in the groups with UDCA and 4-PBA-treated could get close to the normal.3.The expressions of ER-associated molecules gene and protein:The expressions of Bip and CHOP genes mRNA and the expressions of CHOP protein in DM group were significantly stronger than that of the control group in pancreas(P＜0.05).While these parameters in the UDCA and 4-PBA-treated groups were much lower in DM group(P＜0.01).Conclusions:1.ERS might be an essential mechanism for the apoptosis in pancreaticβ-cell of STZ-induced diabetic rats.2.UDCA and 4-PBA could protect pancreaticβ-cell from apoptosis in STZ-induced diabetes.3.UDCA and 4-PBA could protect pancreaticβ-cell from apoptosis in STZ-induced diabetes by attenuating the severity of endoplasmic reticulum stress and inhibiting the up-regulation of ERS associated molecules.4.It suggests that UDCA and 4-PBA could have potential benefits in inhibiting the development of pancreas dysfunction in diabetic patients as the adjuvant administration.