| ObjectiveIn order to confirm the mechanism of treating non-alcoholic fatty liver disease(NAFLD)with decoction of ZhiGanHeJi,an experienced formula of tradition Chinese medicine,we integrated in vivo and in vitro NAFLD models.In this study,the animal experiment of ZhiGanHeJi formula include giving ZhiGanHeJi decoction to C57Bl/6 mouse NAFLD model induced by high-fat diet by gavage and detecting bio-marker with the tissues and serum.In vitro study of ZhiGanHeJi integrated giving medicated serum and lyophilized powder to cellular steatosis in HepG2 cells induced by free fatty acids(FFA)respectively.MethodsPart One The Mechanism study of ZhiGanHeJi formula ameliorating hepatocyte steatosis in mice with non-alcoholic fatty liver disease induced by high fat diet1.Preparation of ZhiGanHeJi decoctionZhiGanHeJi,the Chinese medicine formula decoction came from two extractions and filtration,with a final concentration of 5g/ml.The decoction was put into centrifuge tube of 50 ml,-20 degrees preservation spare.50 Male C57BL/6 mice were randomly divided into 10 control groups and 40 in a high-fat diet group.After high-fat diet for 12 weeks,mice were randomly divided into model group,low-dose group of ZhiGanHeJi(1.25g/ml),high-dose group of ZhiGanHeJi(5g/ml),positive drug group of probucol(500mg/kg)were administrated by gavage respectively,twice / day,continuous administration of 8 weeks.Mice were sacrificed after injection of the sodium pentobarbital with an concentration of 0.2% anesthesia,and the whole liver tissue of each mice were divided for several parts for different indicators detection for pathological slices,Western blot,qRT-PCR and other tests.Part TwoThe Mechanism Study of lyophilized powder of Zhiganheji ameliorating hepatocyte steatosis induced by free fatty acidPreparation of ZhiGanHeJi lyophilized powderThe decoction of ZhiGanHeJi was placed in a disposable sterile petri dish.After 24 hours in the freeze-vacuum dryer,we got the lyophilized powder of ZhiGanHeJi formula.We built the HepG2 cells model of hepatocyte steatosis with 1m M free fatty acid(PA:OA=1:2).HepG2 cells were cultured in 6-well plates at a density of 300,000 per hole,with 2 ml of DMEM high-sugar complete culture medium containing 10% FBS and 1% Penicillin and streptomycin mixture in each hole.The experiment was designed for 5 groups: normal control group,FFA group,FFA and low dose ZhiGanHeJi group,FFA and AMPK activator group,FFA and AMPK inhibitor group.After 24 hours cell walled,each group was added to DMEM high sugar medium dilution with a concentration of 1m M FFA except the normal group and cultured for 24 hours to continue the following experiment.Indicator detection:1.Collect each group of cells,according to the kit instructions,determine the triglyceride(TG)content of each group of cells.2.Oil red O staining observed the coloration of lipids in each group of cells.3.Western-blot detected the expression of AMPK,P-AMPK,SREBP-1C,FAS,ACC,CPT-1,PGC-1 alpha in each group of cells.4.Immunofluorescence detection of the expression of SREBP-1C in each group of cells.5.qRT-PCR detects mRNA expression AMPK,SREBP-1C,FAS,ACC,CPT-1,PGC-1alpha in each group.Part Three The Mechanism Study of medicated serum of ZhiGanHeJi ameliorating hepatocyte steatosis induced by free fatty acidPreparation of medicated serum of ZhiGanHeJi20 male rats aged 10 weeks and weight were about 330-360 g,randomly divided into normal groups and drug groups.Rats were given Chinese medicine or normal saline twice/day for a week by gavage respectively.After the last time gastric infusion,anaesthetized with 2% of barbiturate,took the whole blood from abdominal aorta blood,centrifugal separated medicated serum and control serum for 3500 rpm,15mins.Serum was heated in the water at 56 degree Celsius for30 minutes,filtrated with 0.22?m filter and then transferred to separate EP pipes(1.5ml),stored in the refrigerator of-80 degrees Celsius.HepG2 cells were cultured in 6-well plates at a density of 300,000 per hole,with 2 ml of DMEM high-sugar complete culture medium containing 10% FBS and 1% Penicillin and streptomycin mixture in each hole.The experiment was designed for 6 groups: control group,FFA group,FFA and 5% MS group,FFA and10% MS group,FFA and 15 %MS group,FFA and 20% MS group,After 24 hours,cells walled,each group was added to DMEM high sugar medium dilution with a concentration of 1m M FFA except the normal group and cultured for 24 hours to continue the following experiment.Indicator detection:1.Collect each group of cells,according to the kit instructions,determine the triglyceride(TG)content of each group of cells.2.Oil red staining observed the coloration of lipids in each group of cells.3.Western-blot detected the expression of AMPK,P-AMPK,SREBP-1C,FAS,ACC,CPT-1,PGC-1 alpha in each group of cells.4.Immunofluorescence detection of the expression of SREBP-1C in each group of cells.5.qRT-PCR detected mRNA expression of AMPK,SREBP-1C,FAS,ACC,CPT-1,PGC-1 alpha in each group.Results Part OneAfter 8 weeks of ZhiGanHeJi treatment,although ZhiGanHeJi formula could not reduce weight gain in NAFLD mice during treatment for 8 weeks,it improved the liver index.Three groups of treatment comparison showed that the differences were statistically significant compared between probucol group,low-dose group of ZhiGanHeJi,high-dose group of ZhiGanHeJi and HFD model group.It was suggested that each dose group of ZhiGanHeJi and probucol could improve the liver index of mice in the NAFLD model.ZhiGanHeJi could improve serum lipid and liver fat deposition in NAFLD mice.After 8 weeks of ZhiGanHeJi or probucol treatment,serum TC,TG,LDL-C in each intervention group decreased significantly,and the difference was statistically significant.liver pathology examination with hematoxylin-eosin staining suggested that after 8 weeks of ZhiGanHeJi or probucol treatment,liver pathology of intervention group improved obviously with lipids deposition decreased apparently and high-dose group of ZhiGanHeJi was better than low-dose group of ZhiGanHeJi and group of probucol.liver pathology examination with Oil-Red O staining suggested that after8 weeks of ZhiGanHeJi or probucol treatment,liver pathology of intervention group improved obviously with lipids of Oil-Red O staining decreased apparently and high-dose group of ZhiGanHeJi was better than low-dose group of ZhiGanHeJi and group of probucol.ZhiGanHeJi could improve serum lipid and liver fat deposition in NAFLD mice.After 8 weeks of ZhiGanHeJi or probucol treatment,serum TC,TG,LDL-C in each intervention group decreased significantly,and the difference was statistically significant.liver pathology examination with hematoxylin-eosin staining suggested that after 8 weeks of ZhiGanHeJi or probucol treatment,liver pathology of intervention group improved obviously with lipids deposition decreased apparently and high-dose group of ZhiGanHeJi was better than low-dose group of ZhiGanHeJi and group of probucol.liver pathology examination with Oil-Red O staining suggested that after8 weeks of ZhiGanHeJi or probucol treatment,liver pathology of intervention group improved obviously with lipids of Oil-Red O staining decreased apparently and high-dose group of ZhiGanHeJi was better than low-dose group of ZhiGanHeJi and group of probucol.The results of Western blot suggested phosphorylation level increased after 8 weeks of ZhiGanHeJi or probucol treatment,and the related protein expression of lipid production decreased with down-regulated the expression of SREPBP-1C,FAS and ACC,promoting fatty acid beta-oxidation in mitochondria with up-regulated the expression of CPT-1,PGC-1alpha.The results of immunofluorescent corresponded to the results of western blot with up regulating the expression of p-AMPK alpha and CPT-1 and down-regulating the SREBP-1C.Statistical analysis of qRT-PCR showed that the AMPK gene and the relative gene expression of fatty acid beta-oxidation in mitochondria with CPT-1,PGC-1alpha gene mRNA expression in the liver of intervention group increased significantly(P<0.01),the relative gene expression of lipid production with SREBP-1C,FAS,ACC mRNA decreased significantly(P<0.01).Part TwoCompared with the normal group,the TG content in hepG2 cells in FFA group and AMPK inhibitor(DOX)group increased significantly(P<0.01),and compared with the FFA model group,the TG content decreased significantly in the AMPK activator(AICAR)group and the HepG2 cells in groups of lyophilized powder of ZhiGanHeJi.The fat droplets in hepG2 cells in the FFA model group and the AMPK inhibitor(DOX)group were obvious,and the TG and lipid droplet content in the AMPK activator(AICAR)group and HepG2 cells in the groups of lyophilized powder of ZhiGanHeJi decreased significantly compared with the FFA group.The results of Western blot suggested phosphorylation level increased after intervention of lyophilized powder of ZhiGanHeJi and the related protein expression of lipid production decreased with down-regulated the expression of SREPBP-1C,FAS and ACC(P<0.01),promoting fatty acid beta-oxidation in mitochondria with up-regulated the expression of CPT-1,PGC-1alpha(P<0.01).The above results suggest that: inhibiting AMPK expression,could reduce the fatty acid beta oxidation in HepG2 cells and increase the lipid production effect,activating AMPK,could increase the fatty acid beta oxidation in HepG2 cells and reduce lipid production,and lyophilized powder of ZhiGanHeJi had a similar effect to AICAR.The results of immunofluorescent detection showed that expression of SREBP-1C in the normal group was in a low level,FFA group expression increased significantly,compared with the FFA group,the intensity of SREBP-1C fluorescence expression decreased significantly,and after inhibiting AMPK,SREBP-1C expression remained high expression level.Statistical results show that: compared with normal groups,the FFA group and the AMPK inhibitor group SREBP-1C,FAS,ACC gene mRNA expression increased significantly and AMPK,CPT-1,PGC-1 alpha gene mRNA expression decreased,there were significant differences(P<0.05),compared with the FFA group,compared to the FFA group,SREBP-1C,FAS,ACC gene mRNA expression decreased significantly(P<0.01)and AMPK,CPT-1,PGC-1 alpha gene mRNA expression increased(P<0.01).Part ThreeThe TG content in cells was detected by GPO-PAP enzyme method,and the fat droplet content in cytones was detected by oily O staining.Compared with the normal group,the TG content in HepG2 cells in FFA model group increased significantly(P<0.01)and the coloring of oily O fat drops was more obvious;Increased and decreased,the TG content of each intervention group in order:FFA group >5% MS>10% MS>15% MS>20% MS> normal group,this trend is consistent with the oily O staining.The results of Western blot suggested phosphorylation level increased after intervention of medicated serum of ZhiGanHeJi and the related protein expression of lipid production decreased with down-regulated the expression of SREPBP-1C,FAS and ACC,promoting fatty acid beta-oxidation in mitochondria with up-regulated the expression of CPT-1,PGC-1alpha.The results of immunofluorescent detection showed that expression of SREBP-1C in the normal group was in a low level,FFA group expression increased significantly,compared with the FFA group,the intensity of SREBP-1C fluorescence expression decreased significantly,and after inhibiting AMPK,SREBP-1C expression remained high expression level.Statistical results show that: compared with normal groups,the FFA group and the AMPK inhibitor group SREBP-1C,FAS,ACC gene mRNA expression increased significantly and AMPK,CPT-1,PGC-1 alpha gene mRNA expression decreased,there were significant differences(P<0.05),compared with the FFA group,compared to the FFA group,SREBP-1C,FAS,ACC gene mRNA expression decreased significantly and AMPK,CPT-1,PGC-1 alpha gene mRNA expression increased.Conclusion1)Although ZhiGanHeJi did not significantly reduce the weight gain of NAFLD model mice during the treatment period for 8 weeks,it decreased the liver index of mice and significantly reduced the serum lipids of NAFLD model animals including total cholesterol(TC),triglycerides(TG),low density ester protein(LDL-C)levels,improved hyperleptinemia and adipose pathological structure in NAFLD mice and effective reduction of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels to alleviate hepatocyte damage.The above efficacy is consistent with the expected clinical efficacy of ZhiGanHeJi.2)Not only the lyophilized powder but also the medicated serum of ZhiGanHeJi could correct the abnormal metabolism of fatty acids and reducing the lipid deposition of HepG2 cells,which is an important guiding significance for our research team on the study of ZhiGanHeJi formula.3)Both in vivo and in vitro experiments of Chinese medicine compound ZhiGanHeJi suggest that compound lipid liver compounding agent has reduced liver cell lipid deposition and effectively treating non-alcoholic fatty liver,which provides a preliminary theoretical basis for the research team to apply ZhiGanHeJi to the clinical treatment of non-alcoholic fatty liver disease and to further explore the pharmacological effects of ZhiGanHeJi. |
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