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Objective:To investigate the inhibitory effect of melatonin on pyroptosis of lens epithelium cells (HLECs) induced by hydrogen peroxide (H 2O 2) and its mechanism. Methods:The cultured HLECs were divided into normal control group, model control group, melatonin group, vitamin E group, and vitamin E solvent group.Cells in melatonin group, vitamin E group and vitamin E solvent group were pre-cultured with 1×10 -6 mol/L melatonin, 100 μmol/L vitamin E or equal volume of vitamin E solvent, then cultured with 100 μmol/L H 2O 2, respectively, and the cells in the normal control group and model control group were cultured with normal condition or 100 μmol/L H 2O 2, respectively.The HLECs transfected with nuclear factor erythroid 2-related factor 2 short hairpin RNA (shNrf2) or shNrf2 negtive control lentivirus and following with 100 μmol/L H 2O 2 treatment were served as shNrf2 group and shNrf2 negative control group, respectively; and the transfected cells treated with 1×10 -6 mol/L melatonin and subsequent 100 μmol/L H 2O 2 treatment were served as melatonin+ shNrf2 group and melatonin+ shNrf2 negative control group.The activity of lactic dehydrogenase (LDH) and the expression levels of interleukin (IL)-1β and IL-18 in the culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA) kit.The concentration of reactive oxygen species (ROS) was assessed by flow cytometry.The expression level of Nrf2, NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1 p20 and GSDMD-N proteins were evaluated by Western blot. Results:Compared with model control group, the activity of LDH and the concentrations of IL-1β and IL-18 were significantly decreased in melatonin group and vitamin E group, showing statistically significant differences (all at P<0.05). The ROS fluorescence intensities were 13 040.67±1 550.66 and 12 593.67±1 677.06 in melatonin group and vitamin E group, respectively, which were significantly lower than 18 310.33±1 248.01 in model control group (both at P<0.05). The relative expression levels of Nrf2 protein were 4.24±0.44 and 3.73±0.38 in melatonin group and vitamin E group, respectively, which were significantly higher than 2.28±0.34 in model control group, and the relative expression levels of NLRP3, ASC, caspase-1 p20 and GSDMD-N in melatonin group and vitamin E group were significantly decreased than model control group (all at P<0.05). The relative expression level of Nrf2 protein in shNrf2 group and melatonin+ shNrf2 group was significantly reduced, and the expression levels of LDH, IL-1β, IL-18, ROS content, NLRP3, ASC, caspase-1 p20 and GSDMD-N were significantly increased in comparison with shNrf2 negative control group and melatonin+ shNrf2 negative control group, respectively (all at P<0.05). Conclusions:Melatonin can inhibit the release of NLRP3 inflammasome by activating Nrf2, and has an inhibitory effect on the pyroptosis of HLECs.
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Objective:To investigate the protective effect of asiatic acid (AA) on blood-retinal barrier (BRB) in diabetic rats and its possible mechanism.Methods:Ninety-six healthy 8-week-old male SD rats were randomly divided into normal control group, diabetes group, low-dose AA group and high-dose AA group, with 24 rats in each group.Intraperitoneal injection of streptozocin (STZ) was used to establish diabetes model.One month after the establishment of the model, the low-dose AA group and the high-dose AA group were given intragastrical administration of 37.5 mg/kg AA and 75.0 mg/kg AA, respectively, once a day according to grouping.The normal control group and the diabetes group were administrated with the same amount of 0.5% sodium carboxymethyl cellulose.The body weight of the rats were weighted at week 0, 1, 2, 3, 4 after intragastrical administration.Blood was taken from the tail vein and the blood glucose level was measured.The retina was obtained one month following the administration.Pathological changes of the rats retina were detected by hematoxylin-eosin (HE) staining.Evan's blue quantitative method was used to detect the damage of blood-retinal barrier (BRB). Immunofluorescence staining was performed to detect the distribution of Occludin, Notch1, Jagged canonical Notch ligand 1 (JAG1) and Delta like canonical Notch ligand 4 (DLL4) in retina.The mRNA and protein expressive levels of Occludin, Notch1, JAG1 and DLL4 were detected by Real-time PCR and Western blot.The study protocol was approved by a Scientific Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2020-KY-228). The use and care of animals complied with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health and the 3R rules.Results:At 4 weeks after intragastrical administration, the body weight of the high-dose AA group was significantly higher than that of the diabetes group, and the blood glucose values were significantly lower in the high-dose AA group and the low-dose AA group in comparison with the diabetes group (all at P<0.05). The cells were arranged orderly with clear layered structure in the normal control group.In the diabetes group, the retina was thicker than that of the normal control group, with a thicker outer nuclear layer, disordered cell arrangement and unclear layered structure.Compared with the diabetes group, the total retinal thickness and structure were obviously improved in the low-dose AA group and the high-dose AA group.Evan's blue leakage in retina was (3.07±1.30), (13.73±3.88), (9.57±2.69) and (6.55±1.61)ng/mg in the normal control group, the diabetes group, the low-dose AA group and the high-dose AA group, respectively.There was a significant difference in leakage of Evan's blue among the four groups ( F=18.50, P<0.01), among which the leakage of Evan's blue dye in the high-dose AA group was significantly lower than that of the diabetes group ( P<0.01). Compared with the diabetes group, there was significantly higher relative expression level of Occludin protein and significantly lower relative expression levels of Notch1, JAG1 and DLL4 proteins in the other three groups (all at P<0.05). The relative expression level of Occludin protein was significantly higher and the relative expression levels of Notch1, JAG1 and DLL4 proteins were significantly lower in the high-dose AA group than those in the low-dose AA group (all at P<0.05). Compared with the normal control group, the Occludin mRNA expression level was significantly decreased and the expression levels of Notch1, JAG1 and DLL4 mRNA were significantly increased in the diabetes group and low-dose AA group (all at P<0.01). The Occludin mRNA expression level was higher and the Notch1 mRNA expression level was lower in the high-dose AA group than those in the diabetes group and the low-dose AA group, and the expression levels of JAG1 and DLL4 mRNA were lower in the high-dose AA group in comparison with the diabetes group, and the differences were statistically significant (all at P<0.05). Conclusions:Asiatic acid might play a protective role on BRB in diabetic rats by inhibiting Notch1 signaling pathway.
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Objective To determine the role of liver function test in the preoperative diagnosis of concomitant asymptomatic common bile duct (CBD)stone in patients with cholecystolithiasis.Methods A retrospective study was conducted from January 2012 to October 2013 at the Department of Hepatobiliary Surgery,the Affiliated Hospital of Zunyi Medical College on 426 patients who were operated for cholecystolithiasis.According to whether they had abnormal liver function,these patients were divided into the CBD stone group (n =44) and the cholecystolithiasis group (n =382).The values of the different components of liver functions test such as ALT,AST,AKP,GGT,TBIL and DBIL in diagnosing CBD stone were statistically analyzed.Results This study involved 426 patients,159 men and 231 women,with a mean age of (50.96 ± 12.93) years.44 patients with both CBD stone and cholecystolithiasis.The rates of abnormal liver function were 77.27％ (34/44) vs 4.45％ (17/382) in CBD stone group vs cholecystolithiasis group.The difference was significant (x2 =198.54 ; P =0.000).In logistic regression analysis,only elevated serum level of GGT (OR =10.067 ; P =0.007) remained as an independent predictor of CBD stone.On ROC (receiver operating characteristic) curves analysis,the area under the curve was 0.881.With a cut-off point for GGT at 84.5 U/L,there was a sensitivity of 72.2 per cent,specificity of 96.1 per cent,and positive and negative predictive values of 76.3 per cent and 96.2 per cent respectively.Conclusion Our study showed that liver function,especially GGT,was a predictor of CBD stone.
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Diabetic retinopathy (DR) is one of common causes of vision loss.Recent years,progress has been made on the signal pathway in DR which provides more theoretical basis for clinical therapies and more chance for developing targeted therapeutic drugs.Understanding the biological function of the signal pathway in DR is helpful for physicians to selectively control its biological activities,make medical decisions and optimize new approaches to the treatment of DR.How to closely combine the study of signal pathway and targeted therapy in practice is a key link of improving the therapeutic effect of DR.Therefore,we should pay attention to translational medicine and reinforce targeted therapy in DR.