Résumé
Objective To analyze the effect of exogenous recombinant spherical adiponectin on the autophagy and endoplasmic reticulum stress in testis of diabetic mice induced by streptozotocin, and explore the mechanism of such protective effect to the testis of diabetic mice. Methods Forty-eight male C57BL/6 mice were randomly divided into normal control group (NC, n=8) and diabetes group (n=40). Diabetic mouse model was established by intraperitoneal injection of streptozotocin, and 24 mice were successfully established. Diabetic mice were randomly divided into diabetic group (DM, n=8), diabetic adiponectin low dose group (DLA, n=8), and diabetic adiponectin high dose group (DHA, n=8). Mice in DLA group and DHA group were given intraperitoneal injection of recombinant spherical adiponectin at dose of 5 μg/(kg·d) and 15 μg/(kg·d) respectively. Mice in NC group and DM group were given intraperitoneal injection of the same amount of normal saline. After 8 weeks of adiponectin treatment, blood samples were taken to measure fasting blood glucose with glucose oxidase method. Enzyme-linked immunosorbent assay (ELISA) was performed to determine serum testosterone, insulin, follicular stimulating hormone (FSH) and luteinizing hormone (LH). Testicular weight was measured, and the testicular index (proportion by weight of testis/body) was calculated. Sperm count and mobility were measured. Morphological changes of testicular tissues were observed by HE staining. The mRNA expressions of endoplasmic reticulum stress (GRP78, CHOP, Caspase-12) and autophagy (LC3, Beclin1 and p62) were determined by real-time PCR. Results Compared with NC group, it was found in DM group that seminiferous tubules were deformed, the number of spermatogenic cells and Sertoli's cells reduced, the number of sperm in the tubular lumen reduced, and spermatogenic cells sloughed in some tubular lumens. The body weight, testicular weight, sperm motility, sperm count, and the levels of serum insulin, testosterone, LH and FSH significantly decreased in DM group than those in NC group (P<0.05). The mRNA expression of Beclin1 and LC3 were significantly decreased (P<0.05), and of p62, GRP78, CHOP and Caspase-12 were significantly increased in the testis of DM group compared with NC group (P<0.05). Recombinant spherical adiponectin significantly reversed the changes mentioned above in the testis of diabetic mice, and the effect was better in DHA group than in DLA group. Conclusions The level of autophagy is decreased and the level of endoplasmic reticulum stress is increased in the testis of diabetic mice. Recombinant spherical adiponectin may have the protective effect on the testis of diabetic mice by regulating autophagy and endoplasmic reticulum stress, and the protective effect of adiponectin may be dose-dependent.
Résumé
In order to solve the difficucties of renaturation and immunogenicity of new bifunctional fusion protein GAD,inclusion bodies of GAD were modified by PEG-maleimide.Conformational changes of the modified GAD were compared by circular dichroism and tryptophan fluorescence spectroscopy.The biological activity was verified by oral glucose tolerance test and lipid scavenging.The results showed that PEG-maleimide completed the specific-point modification of GAD,and improved its refolding efficiency.The secondary and tertiary structures of mPEGylated GAD were consistent with that of GAD.PEG-GAD has significant hypoglycemic and lipid-lowering effects (P ＜0.001) with longer half life in vivo and lower immunogenicity (P ＜0.01).This study provides effective strategies for the development of strongly hydrophobic peptide drugs.
Résumé
AIM: To investigate the effects of globular adiponectin (gAd) on the expression of adiponectin receptor 1 (AdipoR1) in human umbilical vein endothelial cells (HUVECs), and on the apoptosis induced by advanced glycation end products (AGEs). METHODS: HUVECs were treated with the indicated concentrations of AGEs for 24 h or 48 h in vitro. The cells in gAd treatment group were pretreated with gAd for 24 h, and then were treated with AGEs for another 24 h or 48 h. Cell variability was quantified by MTT assay. Apoptotic cells were detected by flow cytometry with Annexin-FITC/PI double staining. AdipoR1 mRNA in the cells was determined by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The viability of HUVECs treated with AGEs decreased significantly as compared to the cells treated with HSA (control, P<0.05). Under the same condition of AGEs exposure, the viability of the cells treated with gAd was greatly higher than that of the cells without gAd treatment (P<0.01). The apoptotic rate of HUVECs was significantly elevated by AGEs treatment vs HSA treatment observed by Annexin V-FITC/PI double staining analysis with flow cytometry (P<0.05). Under the condition of AGEs stimulation, the apoptosis of HUVECs was decreased by pretreatment with gAd as compared to that of the cells without gAd treatment (P<0.01). Measured by quantitative real time PCR, AGEs decreased the expression level of AdipoR1 mRNA and gAd increased the expression of AdipoR1 mRNA contrarily (P<0.05). CONCLUSION: AGEs increase the apoptotic rate of HUVECs in a concentration dependent manner and gAd promotes the AdipoR1 mRNA expression.