| Comprehensive learning of non-alcoholic steatohepatitis (NASH) in both modern medicine and traditional Chinese medicine (TCM) is demonstrated through literature review and experimental study in this paper. Efficacy and mechanism of GeGenQinLian Decoction and its componets are explored in in vivo and in vitro study. The research provided a basic reveal of the mechanism of GeGenQinLian Decoction and its componets, which may give a great contribution to clinical therapeutic levels and new grug discovery.1Literature reviewThrough literature analysis in recent10years both at home and abroad, the etiology, pathogenesis, and treatment of modern medicine, also etiology and pathogenesis, syndrome differentiation, physical sciencetreatment progress of TCM have been review.2Effects and Mechinsm of GeGenQinLian Decoction in NASH Rats induced by High-Fat DietObjectiveTo investigate the efficacy and mechinsm of GeGenQinLian Decoction on rat with non-alcoholic steatohepatitis (NASH) induced by high-fat-diet.MethodThe SD rats were fed with high-fat-diet for8weeks to make NASH rat model. At the beginning of modeling, the treatment groups were intervened by GeGenQinLian Decoction in high and low doses. The change of serum transaminase, serum lipid, serum inflammation factors, liver inflammation factors and the liver pathology had been tested to observe the efficacy. Meanwhile, PPAR γ protein and mRNA in liver were also detected.Outcome(1) General condition and pathomorphology:The hepatic steatosis and inflammation were increased in model group, which were improved in the treatment groups, together with significantly reduced NAS score. And there was no significant difference between treatment groups.(2) Serum transaminase:Compared with blank control group, the serum ALT, AST in model group were obviously increased. The serum ALT and AST decreased both in high and low dose of GeGenQinLian Decoction group.(3) Lipid:Serum HDL-C was reduced, serum LDL-C, TG and TC were increased in the model group compared with blank control group. The high dose groups of GeGenQinLian Decoction increased the serum HDL-C compared with the model group, while the low dose of GeGenQinLian Decoction decreased the serum LDL-C significantly. Both the high and low dose of GeGenQinLian Decoction decreased serum TC.(4) Serum and liver IL-6, IL-10and TNF-α:Compared with blank control group, the serum and liver IL-6and TNF-α in model group were obviously increased, while the serum and liver IL-10decrease. GeGenQinLian Decoction can significantly increase the leverl of serum and liver IL-10and decrease the serum and liver IL-6and TNF-α.(5) liver PPAR γ protein and mRNA expression:Compared with blank control group, the liver PPAR γ protein and mRNA expressions were both declined, which were increased in GeGenQinLian Decoction groups.ConclusionGeGenQinLian Decoction can significantly improve liver pathology of the high-fat diet-induced NASH rats; improving steatosis and infiltration of inflammatory cytokines; reducing serum transaminase levels;improving dyslipidemia; and achieving intervention on NASH rats by PPAR γ regulation.3A Cell Model of Nonalcoholic Steatohepatitis Induced by FFA:Establishment and Dynamic MonitoringObjectiveTo establish a cell model of nonalcoholic steatohepatitis (NASH) by free fatty acid (FFA) and to dynamically monitor the pathological features of steatosis, oxidative damage and inflammation in these cells.MethodsLiver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. For the treatment, palmitic and oleic acid0.1M stock solutions were prepare by dissolving FFA in DMSO (with a final concentration less than0.1%). The cells were exposed to increasing concentrations of a fresh mixture of exogenous FFA (250μmol/L,500μmol/L,1000μmol/L) in molar ratio1:2palmitic:oleic at different time points (12h,18,24h) respectively. Oil red0staining and the IL-8, TNF-α, SOD, MDA, GLUT4 in supernatant were evaluated.OutcomeSteatosis could be seen in FFA groups to different extents. IL-8, TNF-α, SOD, MDA and GLUT4gradually differed according to the change of stimulation time by different FFA concentrations, especially in the groups with0.5mM and1mM FFA concentrations for24h, of which groups there could be seen a typical steatosis.ConclusionA cell model of NASH has been created successfully by FFA.4Research of Gegenqinlian Decoction on NASH in vitroObjectiveFree fatty acid induced NASH model was used to evaluate the effects of Gegenqinlian Decoction.MethodLiver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μmol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of Gegenqinlian Decoction were meanwhile added. MTT, Oil red0staining and the IL-8, TNF-α, GLUT4in supernatant were evaluated.Outcome(1) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01), while the level of GLUT4was significantly lower than the control group (P<0.01).(2) Gegenqinlian Decoction can significantly improve cell degeneration and steatosis, reducing TNF-a, IL-8secretion and improving GLUT4content.ConclusionGegenqinlian Decoction can improve lipid metabolism and inflammation through influencing TNF-a, IL-8and GLUT4.5Experimental Studies of the Combination Effects of Berberine and Puerarin on NASH in vitroObjectiveTo discuss the combination effects of puerarin and berberine on NASH in vi tro. MethodLiver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μ g/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μ mol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of puerarin and berberine combination with different propotions were meanwhile added.MTT, Oil red0staining and the IL-8, TNF-a, GLUT4in supernatant were evaluated.Outcome(1) Compared with NASH model group, different concentrations of puerarin and berberine alone and combination (p500, p1000, p2000, b25, b50, b25p1000, b50p500, μmol/L) in the intervention group can decrease lipid droplets sidnificantly.(2) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01), while the level of GLUT4was significantly lower than the control group(P<0.01). Different concentrations of puerarin and berberine combination can significantly improve cell degeneration and steatosis, reducing TNF-a, IL-8secretion and improving GLUT4content(P<0.01).ConclusionPuerarin and berberine combination can improve cellular pathology, influencing the levels of TNF-a, IL-8and GLUT4, which showed a synergistic effect and better than a single drug effect.6Experimental Studies of the Combination Effects of Berberine, Puerarin and baicalin on NASH in vitroObjectiveTo discuss the combination effects of puerarin, berberine and baicalin on NASH in vitro.MethodLiver cells HepG2were cultured in DMEM medium supplemented with10%fetal bovine serum (FBS), penicillin (100units/ml) and streptomycin (100μg/ml) at37°C under5%CO2in a95%humidified atmosphere. The cells were exposed to a concentration of a fresh mixture of exogenous FFA (500μmol/L) in molar ratio1:2palmitic:oleic for24h. Different concentrations of puerarin, berberine and baicalin combination with different propotions were meanwhile added.MTT, Oil red0staining and the IL-8and TNF-a in supernatant were evaluated. Outcome(1) Compared with NASH model group, different concentrations of puerarin, berberine and baicalin alone and combination (bai25, bai50, bai100, bai200, b25p1000bai25, b25p1000bai50, b25p1000bai100, b50p500bai25, b50p500bai50, b50p500bai100, μmol/L) in the intervention group can decrease lipid droplets sidnificantly.(2) TNF-a and IL-8were significantly higher in model group than the control group (P<0.01). Different concentrations of puerarin, berberine and baicalin combination can significantly improve cell degeneration and steatosis and reduce TNF-a, IL-8secretion (P<0.01).ConclusionPuerarin, berberine, and baicalin combination can improve cellular pathology, influencing the levels of TNF-a and IL-8, which showed a synergistic effect. The better proportion of berberine, puerarin, baicalin combination was1:10:1-1:10:2in vitro.7The Related Studies between PPAR γ and Inflammatory Factors on NASH in vitroObjectiveTo clarify the relationship between PPAR γ and inflammatory factors.MethodNASH model in vitro was established using500μmol/L FFA for-24h. The agonists of PPAR γ Rosiglitazone and antaonist of PPAR γ GW9662were meanwhile added.Oil red0staining and the IL-8and TNF-a in supernatant were evaluated.Outcome(1) Compared with NASH model group, the GW966210μmol/L group can increase IL-8and TNF-a level; while IL-8and TNF-a were significantly reduced after adding rosiglitazone2μmol/L (P<0.05). In the groups of GW966210μmol/L plus with each monomer, IL-8and TNF-a levels were lowerd compared with the model group and GW966210μmol/L group (P<0.05).In order to avoid an increase caused by model, GW9662was added to the "normal" cells without FFA. The results showed that different concentrations of GW9662increased IL-8and TNF-a levels compared with the group without GW9662(P<0.05).(2) The intracellular lipid droplets content was analysized by reading OD values, lipid droplets levels were increased significantly in groups with different concentrations GW9662compared with the model group (P<0.05).ConclusionPPAR γ antagonist GW9662can promote inflammation by blocking PPAR γ; puerarin, berberine and baicalin hold the potential effects as PPAR γ agonist; PPAR γ is closely related to lipid metabolism.8Effects of Components in GeGenQinLian Decoction in NASH Rats Induced by High-Fat DietObjectiveTo investigate the efficacy of components in GeGenQinLian Decoction (puerarin, berberine, baicalin) on rat with non-alcoholic steatohepatitis (NASH) induced by high-fat-diet.MethodThe SD rats were fed with high-fat-diet for8weeks to make NASH rat model. At the beginning of modeling, the treatment groups were intervened by the componts of GeGenQinLian Decoction (number of groups identified by orthogonal table). The change of serum transaminase, serum lipid and the liver pathology had been tested to observe the efficacy.Outcome(1) General condition and pathomorphology:The hepatic steatosis and inflammation were increased in model group, which were improved in the treatment groups. And there was no significant difference between the all treatment groups.(2) Serum transaminase:Compared with blank control group, the serum ALT, AST in model group were obviously increased (P<0.01). The serum ALT and AST decreased both in all treatment groups, in which F, G, H groups were better than HH gtoup in inproving ALT and AST, and D, E group were better than HH group in inproving AST (P<0.01).(3) Lipid:Serum HDL-C was reduced, serum LDL-C, TG and TC were increased in the model group compared with blank control group (P<0.01). The A, B, D, E, I, HH groups increased the serum HDL-C compared with the model group, in which HH group was better than B, E groups (P<0.01). Compared with the model group, each treatment groups decreased serum LDL levels significantly (P<0.01), in which the HH group was better than E, F, H, I groups (P<0.01). For the serum triglycerides, the level in A, C, D, E, F, G, HH groups decreased compared with model group (P<0.01). For the serum total cholesterol, the level in A, B, C, D, E, F, G, HH group decreased compared with model group (P<0.01). ConclusionGeGenQinLian Decoction can significantly improve of the high-fat diet-induced NASH rats and achieve intervention on NASH rats by PPAR γ regulation.Different proportions of monomer composition gave positive effects to the liver pathology, serum transaminase levels and dyslipidemia. With some certain propotion, it showd an equal effect to the high dose of GeGenQinLian Decoction, suggesting a clear monomer combination could achieve the effect at some aspects with the original formula in traditional Chinese medicine. |
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