ABSTRACT
Objective To clarify the protective effect of nrsodeoxycholic acid ( UDCA ) on endoplasmic reticulum stress-mediated apoptosis in pancreatic β-cell of streptozotocin ( STZ )-induced diabetic rats.Methods Rats( n =40) received a single injection STZ( 50 mg/kg) intra-peritoneally and formed a β-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 16.7 mmol/L for 1 week were considered as diabetic status( n=14 ),then divided randomly into STZ-induced diabetes mellitus ( DM ) group ( n =7 ) and UDCA-treated DM group ( n =7 ).UDCA (40 mg· kg- 1,d-1 ) was administered daily by intragastric intubations throughout the experimental period (30 d).During the entire experiment,blood glucose in all rats was assessed.By the end of the experiment,all rats were sacrificed with the pancreas removed and the blood sample collected immediately.Fasting insulin levels were assayed by radioimmunoassay.The morphological changes of pancreatic β-cells apoptosis were determined by TUNEL assay.RNA in pancreas was abstracted and microarray containing 89 pieces of apoptosis related genes was applied.The related gene expressions were detected by RT-PCR and Western blot.Results The concentration of blood glucose in diabetic rats was gradually decreased after UDCA treatment,but at the end of the experiment it was still higher than that in the normal control group.The treatment with UDCA raised the fasting insulin level in diabetic rats,but this concentration was significantly lower as compared to the control group.Based on TUNEL stained tissue sections,the percentage of β-cell apoptosis of UDCA-treated DM group was significantly lower than that of STZ-induced diabetic group(P＜0.05 ).Among 89 genes,42 genes up-regulated and 46 genes down-regulated in diabetic rats,some of which were ameliorated by UDCA treatment.The expressions of Caspase-3,Bax,Bip,and CHOP mRNA in pancreas of DM group were significantly up-regulated as compared with those in the control group ( P ＜ 0.05 ) ; while the expression of Bcl-2 mRNA was markedly down-regulated (P＜0.05 ).However,these parameters in the U DCA-treated animals showed a marked improvement.Conclusion Ursodeoxycholic acid seems to protect pancreatic β-cell from apoptosis in STZ-induced diabetes by attenuating the severity of endoplasmic reticulum stress.
ABSTRACT
Objective To detect the signaling pathways involved in aldosterone (ALDO)induced mesangial cell (MC) proliferation. Methods The incorporation of 3H-thymidine (3H-TdR)and cell count were used as the measure of mesangial cell (MC) proliferation. Reactive oxygen species (ROS) production was determined by DCFDA fluorescence. Epidermal growth factor receptor (EGFR) activation was assayed by Western blotting. Results ALDO induced MC proliferation.When incubation with 100 nmol/L ALDO for 24 h, the 3H-TdR incorporation and cell number increased by 2.63- and 2.15-fold, respectively. Mineralocorticoid receptor (MR) antagonist EPLE almost completely blocked ALDO-induced MC proliferation (P<0.01), however, glucocorticoid receptor (GR) antagonist RU-486 had no effect on MC proliferation. ALDO increased intracellular ROS production in cultured human MCs. When incubation with ALDO (100 nmol/L) for 60 min,ROS production increased by 2.14-fold. ALDO-induced ROS generation was completely blocked by EPLE as well as mitochondrial complex Ⅰ inhibitor rotenone (P<0.01=, NADPH oxidase inhibitors diphenyleneiodonium sulfate (DPI) and apocynin inhibited ALDO-induced ROS production by 30%to 35% (P<0.05=. In contrast, inhibitors of other oxidant-producing enzymes, including allopurinol,indomethacin, nordihydroguiaretic acid, ketoconazole and G-nitro-L-arginine methyl ester (L-NAME)had no effect on ALDO-induced ROS production. Antioxidant N-acetyl-L-cysteine (NAC) and ROT inhibited ALDO-induced MC proliferation by 75% to 80%, whereas the inhibition of NADPH oxidase inhibitor apocynin and DPI on ALDO-induced MC proliferation was 25% to 30%. ALDO induced EGFR transactivation. When incubation with 100 nmol/L ALDO for 60 min, EGFR phosphorylation was increased by 4.95-fold, which was completely inhibited by EPLE and antioxidant NAC (P<0.01=. NAC and EGFR antagonist AG1478 significantly blocked ALDO-induced MC proliferation (P<0.01=. Conclusions ALDO-induced MC proliferation is mediated by ROS-dependent EGFR transactivation. ALDO-stimulated ROS is mainly generated by mitochondria.
ABSTRACT
Objective To observe the expression of Rock Ⅰand its functional activation in renal tissue from a rat UUO model, and to investigate its role in the progression of renal interstitial fibrosis(RIF). Methods Expression of RockI mRNA and protein were examined by RT PCR and Western blotting, respectively.The phosphorylation of MBS(binding subunit of myosin phosphatase)—a substrate of Rock Ⅰwas detected by Western blotting and defined,as the mark of functional activation of the kinase. Results (1)The expression of Rock ⅠmRNA was increased before the onset of RIF(the 3rd day after the experiment) (F=15 18,P