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Objective:To study the effect of ZhuJing pill variant formula medicated serum on hydrogen peroxide (H 2O 2)-induced epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial (ARPE-19) cells and its mechanism. Methods:Thirty female SPF grade SD rats aged 2 months old were selected.The rats were randomized into blank control group and Zhujing pill variant formula group according to random number table method, with 15 in each group, which were intragastrically administered with normal saline and ZhuJing pill variant formula solution for 7 days accordingly to prepare blank control serum and medicated serum.ZhuJing pill variant formula medicated serum was prepared with SD rats.ARPE-19 cells were divided into normal control group, model control group, blank serum group as well as 2.5%, 5.0% and 10.0% medicated serum groups, SB216763 group and SB216763+ medicated serum group.Normal and blank control groups were cultured in normal culture medium, while the other six groups were cultured in blank rat serum medium, medicated serum medium of corresponding concentration, 10 μmol/L SB216763 medium and 10 μmol/L SB216763+ 10.0% medicated serum medium, respectively.Normal control group was routinely cultured, while the other groups were routinely cultured for 24 hours, and then added with H 2O 2 with the final concentration of 200 μmol/L for 24 hours.Cell viability was assessed by cell counting kit-8 (CCK-8) assay, and cell migration ability was detected by Transwell assay.Intracellular reactive oxygen species (ROS) level was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay, and MDA level was identified by sulfhydryl barbituric acid assay.The expression levels of Nrf2 pathway related proteins including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (NQO-1) and EMT-related proteins including transforming growth factor-β2 (TGF-β2), protein kinase B (AKT), glycogen synthase kinase-3β (GSK-3β), snail family zinc finger 1 (SNAIL1), α-smooth muscle actin (α-SMA), epithelial cadherin (E-cadherin) in cells were measured by western blot assay.The use and care of animals complied with Regulations for the Administration of Affairs Concerning Experimental Animals. Results:There was no significant difference in cell survival rate among blank serum group, 2.5%, 5.0% and 10.0% medicated serum groups ( F=0.163, P>0.05). The cell survival rates were (100.50±5.91)%, (60.87±4.30)%, (73.27±4.46)%, (80.73±5.67)% and (89.90±4.97)% in normal control group, model control group, 2.5%, 5.0% and 10.0% medicated serum groups, and the number of migrating cells was (84.67±8.33), (222.33±13.58), (215.67±10.02), (174.67±10.60), (143.67±8.02) and (107.67±6.66) pcs/visual field in normal control group, model control group, blank serum group, 2.5%, 5.0% and 10.0% medicated serum groups, respectively, with significant differences among the groups ( F=26.628, 99.289; both at P<0.01). The contents of ROS and MDA in model control group were significantly increased in comparison with normal control group (both at P<0.01). The contents of ROS and MDA of 2.5%, 5.0% and 10.0% medicated serum groups were significantly decreased in comparison with model control group (all at P<0.01). The relative expression levels of SNAIL1, α-SMA, TGF-β2, p-AKT and p-GSK-3β proteins were significantly higher and the relative expression level of E-cadherin protein was significantly lower in model control group compared with normal control group, 2.5%, 5.0% and 10.0% medicated serum groups (all at P<0.05). Compared with normal control group, the relative expression level of cytoplasmic Nrf2 in model control group was decreased, while the relative expression levels of nuclear Nrf2, HO-1 and NQO-1 were increased, and the differences were statistically significant (all at P<0.05). Compared with model control group, the relative expression levels of cytoplasmic Nrf2 in 2.5%, 5.0% and 10.0% medicated serum groups were reduced, and the relative expression levels of nuclear Nrf2, HO-1 and NQO-1 were enhanced, and the differences were statistically significant (all at P<0.01). Compared with model control group, the relative expression level of cytoplasmic Nrf2 in SB216763 group was decreased, and the relative expression level of nuclear Nrf2 was increased, and the differences were statistically significant (both at P<0.05). Compared with SB216763 group, the relative expression levels of cytoplasmic Nrf2, SNAIL1 and α-SMA in SB216763+ medicated serum group were decreased, and the relative expression levels of nuclear Nrf2 and E-cadherin protein were increased, and the differences were statistically significant (both at P<0.05). Conclusions:ZhuJing pill variant formula medicated serum can inhibit H 2O 2-induced EMT in ARPE-19 cells.The mechanism may be related to the inhibition of AKT/GSK-3β pathway and the activation of Nrf2 signaling pathway.
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Background Choroidal neovascularization (CNV) leads to blindness in many fundus diseases.Study showed that naringenin suppresses CNV,but it presents with poor bioavailability because of its poor solubility in water.β-cyclodextrin (β-CD) can increase the water-solubility of drugs, however, whether the inhibitory effect of naringenin on CNV can be improved after clathrated with β-CD remains unclear.Objective This study was to compare the inhibitory effects of naringenin with naringenin/β-CD compounds on CNV in rats.Methods Naringenin/β-CD clathrate compounds were prepared with saturated solution,the solubility of naringenin in water was calculated based on standard curve.Thirty-two male Brown Norway rats were randomized into normal control group, model control group, naringenin group and naringenin/β-CD group.Laser-induced CNV models were created in the right eyes of rats from the model control group, naringenin group and naringenin/β-CD group.Naringenin and naringenin/β-CD clathrate compounds were intraperitoneally injected at a dose of 20 mg/kg in the rats of naringenin group and naringenin/β-CD group since the day after modeling, respectively, once per day for 4 weeks, and equal volume of DMSO was injected in the same way in the model control group.Fluorescein isothiocyanate dextran (FITC-D) was injected via rat hypoglossal vein for the preparation of flatmounts of choroid in the fourth week,and the areas of CNV were measured and compared among the groups.The retinal pigment epithelium (RPE)-choroid-sclera tissues were isolated from the rats, and the relative expression levels of vascular endothelial growth factor (VEGF) , cyclooxygenase-2 (COX-2),phosphatidylinositol-3-kinase (PI3K),p38mitogen-activated protein kinase (p38MAPK), matrix metalloproteinase (MMP)-2, MMP-9 mRNA and their proteins in RPE-choroid-sclera tissue were detected using real-time PCR and Western blot.Results The solubility of naringenin in water increased by 11.8 folds after encapsulated with β-CD.The CNV areas in the model control group, naringenin group and naringenin/β-CD group were (34.56± 1.67), (20.90± 1.47) and (13.20± 1.38) × 103 μm2 , respectively, showing significant reduces in the naringenin group and naringenin/β-CD group compared with the model control group (t =3.973 ,P＜O.05;t =5.532, P＜0.01) ,and the CNV area in the naringenin/β-CD group was significantly smaller than that in the naringenin group (t =3.605,P＜0.05).The relative expression levels of VEGF, COX-2, PI3K, p38MAPK, MMP-2, MMP-9 mRNA and their proteins were significantly declined in the normal control group,naringenin group and naringenin/β-CD group in comparison with the model control group (all at P＜0.05).In addition, the expression levels of VEGF mRNA and COX-2 mRNA and their proteins were significantly lower in the naringenin/β-CD group than those in the naringenin group (all at P＜0.05).Conclusions The naringenin/β-CD clathrate compounds can improve the water solubility of naringenin and enhance their inhibitory effect on rats CNV.The inhibitory effect of naringenin on rats CNV probably is associated with anti-inflammatory pathway.
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Objective To study and compare the protective effects of pmtease inhibitor and corticosteroid on endotoxin-indueed acute lung injury in order to guide the choice of appropriate drugs. Method Thirty-two New Zealand rabbits were randomly divided(random number) into four groups with 8 rabbits in each, namely normal controls(C) ; lipopolysaecharide(LPS) group(L) ; ulinastatin(UTI) group(U) and dexamcthasone(DEX) group (D) .Except group C, all rabbits were injected with a dose of LPS 600 μg/kg iv. Meanwhile the rabbits in group U,group D received UTI(100 000 μ/kg), DEX(5 mg/kg), respectively. The specimens were collected 4 hours later for detecting the levels of TNF-α and NO in serum, and blood gas analysis, histological manifestations, the lung wet/dry weight ratio, lung tissue MPO and SOD activity, lung tissue MDA. Data were analyzed by ANOVA (SNK- q test), and P < 0.05 was considered as significantly different. Results Compared with group C, the lungs of the rabbits in group L had inflammatory granulocyte infdtration, diffused alveolar septum thickening and hemorrhagic spots were observed in pathological examinations. The histological changes of group U and group D were much lessened than those in group L. As groups U and D were compared with group L, there were significant differences inmany biomarkers including lung wet/dry weight ratio[(5.02±0.11),(4.93±0.13) vs.(5.37 ±0.29)],lung tissue MPO activity[(0.51 ± 0.05),(0.54±0.07) vs.(0.82 ± 0.09)] and MDA[(0.82 ±0.05),(0.81 ±0.04) vs.(0.96±0.05)], NO[(296.2± 11.7),(291.7 ± 15.8) vs.(351.8±19.6)] and TNF-α[group D(2.021 ± 0.122) vs. group L(4.999 ± 0.139)],lung tissue SOD activity[(120.3 ± 6.1),(122.6±3.5) vs.(105.1 ± 8.5)] and blood gas analysis[pH(7.30±0.23),(7.30±0.17) vs.(7.22±0.45) and PaO_2( 101.9 ± 6.8).( 102.5 ± 4.7) vs.(80.3 ± 3.3)] ; but there were no differences of above mentioned biomarkers between group U and D( P > 0.05). And there were no significant differences in PaCO_2 betweeu group U and D and group L[(37.0 ± 3.3),(37.6 ± 3.0) vs.(34.8 ± 2.3)]( P > 0.05). Conclusions The protective effects of ulinastatin on endotoxin-induced acute lung injury is comparable to those of dexamethasone, thus the former may be a clinical substitute for the latter with less side effects.
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Objective To study the effects and the mechanism of exogenous carbon monoxide on apoptosis of rat intestinal cells during endotoxemia. Methods The experimental rats were divided into 6 groups:control group, LPS(lipopolysaccharide: 5 mg/kg) group, CO inhalation(250?10~ -6) group, CO intraperitoneal injection(2 ml/kg) group, LPS(LPS 5 mg/kg) with CO inhalation(250?10~ -6) group and LPS(LPS 5 mg/kg) with CO intraperitoneal injection(2 ml/kg) group. The PaO_2, PaCO_2, SO_2 and COHb were monitored by blood gas analysis. The rat intestine malondialdehyde(MDA) was determined by thiobarbitric acid method and superoxide dismutase(SOD) was determined by hydroxylamine method after the rats were treated for 1, 3 and 6 hours. We also checked the apoptosis ratio of intestinal cells with flow cytometry(FCM). We also monitored the pathological changes with HE staining. Results Low concentration CO(250?10~ -6) inhalation and CO intraperitoneal injection(2 ml/kg) did not cause hypoxia. Comparing to control group and endotoxemia group,the intestineal MDA of the endotoxemic rats decreased after exposure to exogenous CO and the SOD activation increased. The apoptosis ratio of intestinal cells decreased after exposure to exogenous CO. On the apoptosis of endotoxemia rat intestinal cells, the effect of CO intraperitoneal injection was earlier than that of CO inhalation, but the effect of CO inhalation last longer. Conclusion Low concentration CO(250?10~ -6) inhalation and low dose CO(2 ml/kg) intraperitoneal injection were safe to rat. Exposure to exogenous CO could protect rat intestine against endotoxemia by inhibiting the apoptosis of intestinal cells. The effect of intraperitoneal CO injection was earlier than that of CO inhalation, but the effect of CO inhalation could last for longer than intraperitoneal CO injection.
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Carbon monoxide(CO) has been considered as a fatal gas.Recent researches show that tissues generate carbon monoxide,and carbon monoxide plays a key role in the tissue physiological and pathological processes.The endogenous CO is generated from the decomposition of heme which is catalyzed by heme oxygenase enzyme(HO).Studies show that CO involves in the process of anti-inflammation,anti-apoptosis,regulation of vascular tone,modulation of leukocyte adhesion and platelet aggregation.CO also functions as neurotransmitters.
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AIM: To observe the effects of norepinephrine (NE) on the cardiopulmonary and lung injury in goats with acute respiratory distress syndrome (ARDS) induced by endotoxin. METHODS: A model of septic ARDS was induced by intravenous infusion of low dose endotoxin in six goats. Then 40?10~ -6 NO inhalation was applied to the animals. After 30 min, combined intravenous infusion of NE at concentration of 0.5 mg?kg~ -1 ?min~ -1 was conducted. The dynamic changes in gas exchange and hemodynamics were measured with the aid of Swan-Ganz catheter. Arterial blood gas analysis before and after the onset of ARDS, 30 min after NO inhalation and combined NE was detected. Histology of the lung was also observed. RESULTS: Inhalation of NO rapidly reduced mean pulmonary arterial pressure (MPAP), increased PaO_2, decreased P_ (A-a) O_2 and Qs/Qt in septic ARDS goats. These decrease and increase were more significant than those in NO inhaled alone when animals received NE. The combination of NO inhalation and NE injection resulted in increase in mean arterial pressure. NO inhalation did not ameliorate lung injury and combined NE intravenous injection ameliorated lung injury. CONCLUSION: Injection of low dose norepinephrine improves the beneficial effects of inhaled nitric oxide on lung gas exchange and ameliorates lung injury in goats with acute respiratory distress syndrome induced by endotoxin.