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OBJECTIVE To investigate the improvement effect and mechanism of different extracts from Tylophora yunnanensis on non-alcoholic steatohepatitis （NASH）. METHODS Normal human liver LO2 cells were induced to steatosis by free fatty acid， then were divided into normal group， model group， silybin group （100 μmol/mL）， T. yunnanensis ethanol extracts （TYS） group （50 μg/mL）， T. yunnanensis ethyl acetate extracts （TYSA） group （50 μg/mL）， and T. yunnanensis n-butanol extracts （TYSB） group （50 μg/mL）. After 24 hours of drug intervention， the deposition of lipid droplets was observed in LO2 cells in each group. The contents of total cholesterol （TC）， triacylglycerol （TG）， malondialdehyde （MDA） and glutathione （GSH）， the activities of aspartate transaminase （AST）， alanine transaminase （ALT） and superoxide dismutase （SOD）， the mRNA expressions of Kelch-like ECH-associated protein 1（ Keap1）， nuclear factor E2-related factor 2（ Nrf2） and heme oxygenase 1（ HO- 1） were detected. NASH rat model was induced by a high-fat diet， and then divided into normal group， model group， silybin group （12.6 mg/kg）， TYS group （80 mg/kg）， TYSA group （80 mg/kg） and TYSB group （80 mg/kg）， with six rats in each group. The liver indexes of rats in each group were calculated after 6 weeks of drug intervention. The liver histopathological changes were observed， and the contents of TC， TG， HDL-C and LDL-C， AST and ALT activities in serum， the contents of MDA and GSH， SOD activities in liver tissue were detected. RESULTS Compared with model group， TYS， TYSA and TYSB could reduce lipid droplet deposition， intracellular TC， TG and MDA contents， AST and ALT activities， and increase SOD activity， GSH content， and Keap1， Nrf2， HO-1 mRNA expression levels in LO2 cells after steatosis to varying degrees， with some differences being statistically significant （P＜0.05）. They also significantly improved liver injury in NASH model rats， reduced their liver indexes， TC， TG， LDL-C and MDA contents， AST and ALT 1-042） activities， and increased HDL-C （except for TYS and TYSB）， GSH contents and SOD activity， with TYSA having the most significant effect （P＜0.05）. CONCLUSIONS TYS， TYSA and TYSB have a certain improvement effect on NASH， among which TYSA has the most obvious effect. Its mechanism of action may be related to upregulating the Keap1/Nrf2/HO-1 signaling pathway and inhibiting oxidative stress
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The forkhead protein (FoxO) family plays a crucial role in regulating oxidative stress, cell proliferation, and apoptosis.FoxO6, a member of the FoxO family, helps regulate oxidative stress in gastric cancer and hepatocellular carcinoma.However, it is unclear whether FoxO6 participates in the protective effect of isoflurane preconditioning in liver injurycaused by oxidative stress in ischemia. In this study, we explored the role and mechanism of FoxO6 in the protective effectof isoflurane preconditioning during hepatocyte injury caused by oxygen-glucose deprivation (OGD). Cells from the humanfetal hepatocyte (LO2) line were incubated with 0%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, or 5% isoflurane for 3 h and thenexposed to OGD. Data showed that 3% isoflurane preconditioning inhibited FoxO6 expression, caspase-3 activity, andreactive oxygen species production and promoted cell viability. FoxO6 overexpression abolished the effects of 3%isoflurane preconditioning on caspase-3 activity, reactive oxygen species production, and cell viability in these cells.Moreover, FoxO6 regulated nuclear factor erythroid 2-related factor (Nrf2) expression via c-Myc after 3% isofluranepreconditioning and OGD exposure. Thus, isoflurane preconditioning prevented OGD-induced injury in LO2 cells bymodulating FoxO6, c-Myc, and Nrf2 signaling
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The study was aimed to investigate the protective effect and pharmacodynamic difference of the ethanol extracts of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus on the drug-induced liver injury induced by acetaminophen.The cell activations of LO2 cells treated by Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extracts were tested by CCK-8 essay.The effects of ethanol extracts on cell survival rate,the activities of ALT and AST in culture medium were detected based on the injury model of LO2 cells induced by APAP.Further,in purpose to observe the protective effect of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extracts on a mouse model of liver injury induced by intraperitoneal injectionof acetaminophen was established.Mice were randomly divided into control group,model group,positive drug group and Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extracts administration groups.The activities of ALT and AST in the serum and the levels of MDA,SOD,GSH and GSH-PX in the liver homogenate of the mice were detected by commercial kits.The HEstaining was used to observe the histopathological changes of liver tissue in each group and the TUNEL staining was used to observe the hepatocyte apoptosis.The results showed that the ethanol extracts at less than 1 g·L~(-1)did not affect the activity of LO2 cell.Compared with the model group,the cell survival rates of the Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extract administration groups was significantly increased;the ALT and AST in the culture medium were distinct decreased(P<0.05 or P<0.01).The survival rate of Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extract from different batches were similar,while that of the Schisandrea Sphenatherae Fructus ethanol extract from different batches were quite different(P<0.05or P<0.01).Further,animal experiments showed that Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extract administration groups could markedly inhibit the increase of ALT and AST levels in serum(P<0.01),decrease MDA content significantly(P<0.01),and increase GSH,GSH-PX and SOD activity significantly(P<0.01).Among them,compared with other groups,Schisandrae Sphenantherae Fructus ethanol extract-2 group showed the best effect(P<0.05 or P<0.01)while Schisandrae Sphenantherae Fructus ethanol extract-1 showed a poor effect(P<0.05 or P<0.01).In conclusion,both Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extracts have protective effect on APAP-induced drug-induced liver injury and there was a certain difference in the efficacy between Schisandrae Sphenantherae Fructus and Schisandrae Chinensis Fructus ethanol extracts from different habitats.
Subject(s)
Animals , Mice , Acetaminophen , Chemical and Drug Induced Liver Injury , Drugs, Chinese Herbal , Fruit , LiverABSTRACT
The aim of this paper was to investigate the mechanism and effect of psoralen and isopsoralen in the treatment of lipid accumulation in LO2 cells. Human LO2 cells nonalcoholic fatty liver models were established by using palmitic acid( PA). Then psoralen and isopsoralen were administered for intervention. Intracellular triglyceride( TG) and total cholesterol( TC) content,the cell supernatant alanine aminotransferase( ALT) and aspartate aminotransferase( AST) levels were determined by enzyme method. Cell supernatant proinflammatory cytokines( IL-6,TNF-α) and chemokines( IL-8,MCP-1) were determined by ELISA method. Western blot method was conducted to detect the protein expression of intracellular nuclear factor( NF-κB) p65 phosphorylation( p-p65),nonphosphorylated protein( p65),and transforming factor TGF-β1. Result showed that as compared with the model group,intracellular TG and TC levels,the cell supernatant ALT and AST levels,proinflammatory cytokines and chemokines were decreased( P < 0. 01,P <0. 05); the p-p65/p65 ratio and TGF-β1 protein expression were also significantly decreased( P< 0. 01,P< 0. 05) in psoralen intervention group. As compared with the model cells,intracellular TG content had no significant changes,but all the other indexes were reduced( P<0. 01,P<0. 05) in the cells of isopsoralen intervention group. Psoralen exhibited better effect than isopsoralen( P< 0. 01,P<0. 05). It is concluded that psoralen could improve the adipogenesis of LO2 cells induced by PA; both psoralen and isopsoralen are effective in ameliorating LO2 cells injury induced by PA,reducing inflammation via inhibiting the activation of NF-κB and down-regulating the expression of TGF-β1.
Subject(s)
Humans , Cell Line , Ficusin , Pharmacology , Furocoumarins , Pharmacology , Lipid Metabolism , NF-kappa B , Metabolism , Non-alcoholic Fatty Liver DiseaseABSTRACT
To address the controversial issue of the toxicity of dental alloys and silver nanoparticles in medical applications, an in vivo-like LO2 3-D model was constructed within polyvinylidene fluoride hollow fiber materials to mimic the microenvironment of liver tissue. The use of microscopy methods and the measurement of liver-specific functions optimized the model for best cell performances and also proved the superiority of the 3-D LO2 model when compared with the traditional monolayer model. Toxicity tests were conducted using the newly constructed model, finding that four dental castings coated with silver nanoparticles were toxic to human hepatocytes after cell viability assays. In general, the toxicity of both the castings and the coated silver nanoparticles aggravated as time increased, yet the nanoparticles attenuated the general toxicity by preventing metal ion release, especially at high concentrations.
Subject(s)
Humans , Cells, Cultured , Dental Casting Technique , Hepatocytes/drug effects , Metal Nanoparticles/toxicity , Silver/toxicity , Toxicity TestsABSTRACT
Objective:To explore the protective effect of the water-soluble total flavonoids from Isodon lophanthoides var.gerardia-nus (Benth.) H.Hara on LO2 cells damage.Methods:The cytotoxicity was evaluated by MTT cell viability determination to confirm the concentration range .Hepatocyte damage model was established by H 2 O2 treatment.After the oxidative stress hepatocyte was coin-cubated with WSTF at different concentrations for various times , the protective effect of WSTF on H 2 O2-induced hepatocyte damage was evaluated by MTT cell viability determination and the content determination of ALT , AST and MDA in cell supernatant .The inhibition of WSTF against H 2 O2-induced LO2 cells apoptosis was evaluated by the quantitative determination of Rhodamine 123 fluorescence and intracellular ROS.Results:The LO2 cells injured by 0.3 mmol· L-1 H2 O2 treatment for 4 h were used as the hepatocyte damage model.The concentration range of WSTF was 0.0312-0.125 mg· ml-1.WSTF could inhibit H2O2-induced injury in LO2 cells and obviously reduce ALT, AST and MDA.Moreover, WSTF could reverse mitochondrial membrane potential depolarization and decrease the amount of intracellular ROS .Conclusion:WSTF exhibits notable protective and curative effects on hepatocyte damage in vitro.
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To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions.
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Objective: To clarify the structure features of polysaccharides from Chrysanthemum morifolium (PCM) and to study their activities against tumor cells and NF-κB. Methods: Six homogeneous neutral polysaccharides were obtained from three kinds of C. morifolium (Hangju, Huaiju, and Boju) flowers by successive hot water extraction, followed by ethanol precipitation, ion-exchange chromatography, and gel permeation chromatography. Their primary structures were characterized by HPGPC, IR, GC, and GC-MS analyses. Their bioactivities were examined by MTT assay using PANC-1 and LO2 cells. In addition, NF-κB signaling activation in PANC-1 and LO2 cells treated by polysaccharides were also measured. Results: The weight-average molecular mass of the six PCM, CMTA0S1, CMTA0S3, CMJA0S1, CMJA0S2, CMBA0S1, and CMBA0S3 was 7.523 × 104, 7.80 × 103, 7.80 × 104, 1.04 × 104, 5.79 × 104, and 1.35 × 104, respectively. CMTA0S1, CMJA0S1, and CMBA0S1 mainly contained galactose (Gal), arabinose (Ara) and glucose (Glc) residues in molar ratio of 1.23:1.00:0.20, 2.18:1.00:0.53, and 3.30:1.00:0.75, while CMTA0S3, CMJA0S2, and CMBA0S3 mainly contained Gal, Ara, Glc, and mannose (Man) residues in molar ratio of 0.73:1.00:0.40:0.21, 1.39:1.00:0.84: 0.55, and 1.19:1.00:0.48:0.19. Methylation analysis indicated that six PCM primarily consisted of T-arabinofuranosyl, 1, 5-arabinofuranosyl, 1, 4-galactopyranosyl, 1, 3, 6-galactopyranosyl, and 1, 4-glucopyranosyl residues. The biological activity study suggested that all the PCM could inhibit the growth of PANC-1 cells. Among them the inhibitory rates of CMTA0S3 and CMJA0S2 were at most to 70% with concentration-effect relationship. The NF-κB inhibition test indicated that only the crude polysaccharide CMBA had strong immunosuppressive activity, and homogeneous polysaccharides CMTA0S1 and CMJA0S1 showed potential immunostimulation. Conclusion: The six homogeneous polysaccharides share similar structures and inhibition on PANC-1 cells growth. Meanwhile they also may regulate the NF-κB activation.
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Objective To study the influence of serum containing Ziqi Ruangan Pill on inhibitory effect of alcohol LO2 and to investigate the regulation on apoptosis related genes and the protective mechanism for LO2. Methods Rabbit sera saline containing Ziqi Ruangan Pill was prepared. The apoptotic LO2 model was established by being cultured with alcohol. The effect of serum on the model was detected by MTT method. Identification of apoptosis was carried out using flow cytometry and Hoechst3325 fluorescent staining. The expression of apoptotic inducing gene,casepase3 and NF-?B was detected by Real-time RT PCR and Western-Blot. Results The serum containing Ziqi Ruangan Pill could regulate the expression of the casepase3,NF-?B and reduce the apoptosis. Conclusion Ziqi Ruangan Pill can protect the LO2 by regulating the expression of apoptosis stimulative gene casepase3 and NF-?B.