ABSTRACT
Objective:To investigate the effect of epigallocatechin gallate (EGCG) on the activation of human Tenon fibroblasts (HTFs) and its mechanism.Methods:Tenon capsule tissues from nine eyes of nine advanced primary open angle glaucoma patients during trabeculectomy were obtained for primary cell culture.HTFs harvested were identified by immunofluorescence staining for vimentin and keratin.Cells at passage 4-6 were used for experiment.Viability of HTFs treated with EGCG at 0, 10, 20, 30, 40, 50, 60, 70 and 80 μmol/L was detected by cell counting kit-8 (CCK-8) assay.The cells were divided into blank control group, transforming growth factor (TGF)-β1-induced group, and EGCG-treated group, which were cultured in normal medium, medium containing 10 ng/ml TGF-β, medium containing 10 ng/ml TGF-β+ 50 μmol/L EGCG, respectively.The proliferation rate of HTFs was detected by BrdU labeling assay.Cell migration was observed by scratch wound healing assay.The expression of α-smooth muscle actin (α-SMA) was measured by immunofluorescence staining.The protein relative expression levels of Smad2/3, phosphoinositide-3-kinase (PI3K), protein kinase B (Akt) as well as the phosphorylated Smad2/3 (p-Smad2/3) and phosphorylated Akt (p-Akt) were measured by western blot.This study was approved by the Ethics Committee of Guangdong Provincial People's Hospital (NO.GDREC2019331H[R1]).Results:The HTFs harvested had spindle shape, grew regularly and were vimentin-positive.CCK-8 assay showed that there was no significant difference in the variability of HTFs treated with EGCG at 10, 20, 30, 40 and 50 μmol/L in comparison with 0 μmol/L EGCG treatment (all at P<0.05). BrdU labeling assay showed that cell proliferation in the TGF-β1-induced group was (66.37±12.65)%, which was significantly higher than (14.75±12.33)% in EGCG-treated group ( P<0.05). Three days after scratch, the relative scratch area in the TGF-β1-induced group was (47.33±12.22)%, which was significantly lower than (92.67±4.04)% in the EGCG-treated group ( P<0.05). Immunofluorescence assay showed that α-SMA fluorescence was significantly enhanced in the TGF-β1-induced group in comparison with the blank control group, which was reduced to blank control group level in EGCG-treated group.Western blot analysis showed that there were significant differences in the relative expression levels of p-Smad2/3, PI3K and p-Akt protein among the various groups ( F=58.820, 121.153, 69.289; all at P<0.001). The relative expressions of p-Smad2/3, PI3K and p-Akt in the TGF-β1-induced group were significantly higher than those in the blank control group, 10 μmol/L and 50 μmol/L EGCG-treated groups (all at P<0.05). Conclusions:EGCG can suppress TGF-β1-induced HTFs activation through Smad and PI3K/Akt signaling pathways.
ABSTRACT
Objective To evaluate the clearance efficacy of resin hemoperfusion(HP) on the removal of organophosphorus in the rabbits poisoned by methamidophos(MAP) and its effects on organ injury. Method Six-teen healthy Japanese white rabbits were randomly divided into HP group and non-HP group. MAP was given through gastric tube in a dosage of 20 mg/kg to rabbits of both groups. Rabbits of liP group received resin hemop-ersion plus conventional treatment including early gastric lavage, atropine and pralidoxime. Rabbits of non-HP group received only conventional treatment. The plasma concentration of MAP was determined by using gas chro-matography before and after rabbits were poisoned at different intervals. Serum choline esterase (ChE),lactic de-hydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) of rabbits of both groups were assayed 6 hours after rabbits poisoned. Pathological changes in lung, liver, kidney and muscle were investigated simutaneously. SPSS 10.0 software was used for statistical analysis. The comparison between groups was carried out by using t -test. Results ① The typical symptoms of organophospborus poisoning were occurred in rabbits within 5 - 10 minutes after ingestion of MAP. In HP group, the plasma levels of rabbits before,and 30 min,60 min,90 min and 120 min after hemoperfu-sion were (11.43±1.56),(7.82±1.54),(4.97±1.58),(5.66±1.75) ,(5.49±1.68) μg/mL, respectively (P <0.01). After hemoperfusion, the plasma MAP levels of rabbits in HP group were lower than those in non-HP groups (P < 0.01). The improveme, nt of clinical presentation of rabbits was observed shortly after HP. ② The blood choline esterase activity of rabbits were depressed in hoth groups without significant difference. In contrast, the blood levels of ALT, AST,LDH,CK and CK-MB of rabbits in non-HP group elevated significantly than in HP group (P < 0.01). ③ The more severe injury of muscle, liver, kidnet and lung of rabbits can could be seen in non-HP group. Conclusions ① HP can effectively eliminate the plasma MAP and has the potential to improve the clinical presentation of intoxication in rabbits. ② Early intervention of Hp exerts a protection from organ dam-age of organophosphorus pesticide.