| ObjectiveTaking ApoE-/-mice as the research object,to explore the lipid-lowering efficacy and the effect of liver lipid synthesis and lipid oxidation of HuaTanJiangZhuo Decoctionon?HTJZD?in the high fat introduced hyperlipidemia ApoE-/-mice with phlegm turbidity syndrome through the blood lipid test and quantitative analysis of hepatic AMPK ?,SREBP-lc,PPARa and downstream related metabolic enzyme gene.Methods???The molding of hyperlipidemia ApoE-/-mice with phlegm turbidity syndrome and intervention of HTJZD6 weeks of male ApoE-/-mice were chose as experimental animals and randomly divided into 2 groups according to the weight,control group and experimental group after 2 weeks of adaptation.The mice in control group were fed with ordinary diet,while the experimental group was fed with high fat diet.The cycle of molding was 4 weeks.At the end of sixth week,blood was collected from mice and texted for plasma lipid to determine the establishment of hyperlipidemia.Experimental mice were divided into 5 groups randomly according to the level of serum TC level,namely model group,high dose group of HTJZD?H-HTJZD?,medium dose group of HTJZD?M-HTJZD?,low dose group of HTJZD?L-HTJZD?and positive control group.All mice were intragastric administration one time everyday according to body weight for 4 weeks.At the end of week ten,6 groups of mice were fasted for blood collection and sampled to test plamsa lipid level and the lipidosis of liver and aorta.???The efficiency of liver lipid metabolism of HTJZD on hyperlipidemia ApoE-/-mice with phlegm turbidity syndromeThe fresh liver samples of mice were colleted and stored after euthanasia.The efficiency of liver lipid metabolism of HTJZD on hyperlipidemia ApoE-/-mice with phlegm turbidity syndrome was explored through quantitative analysis of several liver samples which were randomly selected from 6 groups.1.Detect liver lipid synthesis-related factors such as SREBP-lc and its downstream lipase genes ACC,FAS,SCD-1 and lipid oxidation-related factors like PPARa and its downstream lipid peroxidation genes of CPT-1 via the Q-PCR technique;2.Detect the protein expression of lipid synthesis-related factors SREBP-lc and ACC,lipid oxidation-related factors PPARa,AMPKa,p-AMPK ? and downstream target p-ACC via Western Blots techniques.Results???The molding of hyperlipidemia ApoE-/-mice with phlegm turbidity syndrome and intervention of HTJZD1.The results of baseline analysis:?1?Baseline weight after the initial groupingAfter 2 weeks of adaptability,the weight of control group was?18.49±1.29?g while the experimental group was?17.95±1.42?g.The difference between two groups was no statistical significance?P>0.05?,which prompted that two groups were comparable.?2?Random grouping of the experimental group:Experimental mice were divided into 5 groups randomly according to the level of serum TC level at the end of fourth week.The difference of TC,TG and LDL-C and weight between 5 groups were not statistically significant?P>0.05?.Results indicated that the random grouping was ideal.2.Analysis of hyperlipidemia moldingThe blood lipid levels,food and water intake,weight of 2 groups were tested after the end of molding.?1?plasma lipid resultsthe plasma lipid of TC,TG,LDL-C of experimental group is 2 fold,3 folds,15 folds increased compared to control group,respectively.The differences between two groups was statistically significant?P<0.05?which prompted that hyperlipidemia model was successfully built.?2?Results of food intakeThe food intake was obviously decreased compared to control group,the difference was statistically significant?P<0.05?which prompted that obvious anorexia was seen in experimental mice.?3?Results of water intakeThe water intake was slightly decreased compared to control group,there was no statistically significant difference?P>0.05?.?4?Results of weightThe weight of experimental group was increased compared with control group,there was no statistically significant difference between two groups?P>0.05?.3.Evaluation of lipid-lowering efficacy of HTJZDThe general situations,palsma lipid levels,liver and aortic pathological sections of 6 groups were analyzed to evaluate lipid-lowering efficacy of HTJZD after the treatment.?1?General situation after the treatment?The water and food intake of 6 groupsThe food intake of mice which were fed with high fat diet was obviously decreased compared to the control group.The difference was statistically significant?P<0.05?.The water intake of 2 groups?H-HTJZD and M-HTJZD?was also declined,the difference was statistically significant?P<0.05?.The food intake of L-HTJZD and H-HTJZD was decreased compared to model group,there was no statistically significant difference?P>0.05?.The water intake of 3 groups of HTJZD and the positive control group was reduced,especially the former groups,the difference was statistically significant?P<0.05?.Results suggested that obvious anorexia can be seen in high-fat fed mice.?Results of terminal weightThe weight of model group was increased,while the 3 groups of HTJZD was decreased compared to the control group,but there was no statistically significant difference?P>0.05?.The terminal weight of L-HTJZD and H-HTJZD was decreased compared to model group and there was statistically significant difference?P<0.05?.Results suggest that high-fat fed mice owm the syptoms of weight gain and body fat.?Results of liver wet weightThe wet weight of liver of model group was increased compared with the control group,the difference was statistically significant?P<0.05?.The weight of 3 groups of HTJZD was decreased compared to model group,but there was no statistically significant difference?P>0.05?.Results suggested that the wet weight of liver in high-fat fed mice increased significantly and HTJZD can improve the symptoms.?2?Lipid levels after drug deliveryThe plasma level of TC?TG?LDL-C in mice which were fed with high fat diet was obviously increased compared to the control group.The difference was statistically significant?P<0.05?.Results reflected the stability of hyperlipemic animal model.The plasma level of TC in HTJZD groups was elevated compared with model group,but there was no statistically significant difference?P>0.05?.The plasma level of TG in HTJZD groups was elevated also,only the difference between L-HTJZD and model groud was statistically significant?P<0.05?.The plasma level of LDL-C in H-HTJZD and M-HTJZD was elevated compared with model group,the difference was statistically significant?P<0.05?.Results indicated that the lipid-lowering efficacy of HTJZD is not obvious.?3?Results of liver HE stainingThe structure of hepatic lobule was not clear in all mice,fat vacuolesliver could be seen all over the cell cytoplasm.Nucleus were in the middle of cell and hepatic sinusoids expanded filled with erythrocyte and lymphocyte.The symptom in control group was lighter while worse in the model group accompanied with massive necrosis,flaky and infiltrated with large number of lymphocyte.The structure of hepatic lobule,necrotic area and steatosis were improved in HTJZD groups.Partial necrosis filled with large number of lymphocyte and expansive hepatic sinusoids was seen in positive control group.Results suggested that HTJZD can lighten the degree of hepatic steatosis and improve liver injury and necrosis.???The efficiency of liver lipid metabolism of HTJZD on hyperlipidemia ApoE-/-mice with phlegm turbidity syndrome1.The efficiency of liver lipid synthesis of HTJZD on hyperlipidemia ApoE-/-miceGene and protein expression of liver SREBP-lc and its downstream lipase ACC,FAS,SCD-1 were Quantitative detected.Results showed that the gene expression of liver SREBP-lc was increased while protein expression was decreased compared with control group,the gene expression of ACC,FAS,SCD-1 were decreased too,but the difference was not statistically significant?P>0.05?.Results suggested that liver lipid synthesis process in high-fat fed ApoE-/-mice is not increased.Gene expression of liver SREBP-lc in L-HTJZD was obviously increased compared with model group,the difference was statistically significant?P<0.05?,but the elevatory of SREBP-lc protein expression was not obvious and there was no statistically significant difference?P>0.05?.The expression of ACC,FAS gene was also appeared to rise while SCD-1 gene expression appeared slight decline,and there were no statistically significant difference?P>0.05?.Gene expression of liver SREBP-lc,ACC,FAS,SCD-1 in H-HTJZD and M-HTJZD decreased while SREBP-1c,ACC protein expression was slightly higher compared with model group,but there was no statistically significant difference?P>0.05??Results suggested a trend that HTJZD can reduce the process of lipid synthesis in high fat feed ApoE-/-mice.2.The efficiency of liver lipid oxidation of HTJZD on hyperlipidemia ApoE-/-miceGene and protein expression of liver PPAR? and its downstream lipid peroxidase gene CPT-1 were Quantitative detected.Results showed that:the gene expression of liver PPARa and CPT-1 was increased while protein expression was no change compared with control group,and there was no statistically significant difference?P>0.05?.Results suggested that liver lipid oxidation process in high-fat fed ApoE-/-mice is increased slightly.Gene expression of liver PPARa and CPT-1 in L-HTJZD was increased compared with model group,so was the protein expression of PPARa,the difference was not statistically significant?P<0.05?.Gene expression of liver PPARa and CPT-1 and protein expression of PPARa was gradually reduced with dosage increase compared with model group,and the difference between H-HTJZD and model group was statistically significant?P<0.05?.Results indicated that HTJZD can restrain the lipid oxidation process in high fat fed ApoE-/-mice.3.The efficiency of phosphorylation of hepatic AMPK ? of HTJZD on hyperlipidemia ApoE-/-miceprotein expression of liver AMPK ?,p-AMPK ? and p-ACC were Quantitative detected to detect the effect of HTJZD.Results showed that:the protein expression of liver AMPK?,p-AMPK? and p-ACC were slightly increased compared with control group,and there was no statistically significant difference?P>0.05?.The protein expression of liver AMPK?,p-AMPK? and the ratio of p-AMPK ?/AMPK? in 3 groups of HTJZD were increased while the p-ACC protein expression decreased compared with model group,but there were no statistically significant difference?P>0.05?.Results suggested a trend that HTJZD can promote the phosphorylation of AMPK ?.Conclusion???Hyperlipidemia can be molded by high fat diet for 4 weeks in ApoE-/-mice,and the type is mixed hyperlipidemia.Mice with hyperlipidemia show obvious symptoms of obesity,while HTJZD can relieve these symptoms.???HTJZD can lighten the degree of hepatic steatosis and improve liver injury and necrosis.???HTJZD may have the ability of reducing the process of lipid synthesis and promoting the phosphorylation of AMPK? in high fat feed ApoE-/-mice to improve the state of hyperlipidemia.???The lipid-lowering efficacy of HTJZD is not obvious and whether HTJZD can promote the lipid oxidation process mediated by PPARa is remain further studied. |
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