Effects And Mechanisms Of Improvement Of Emodin On Simple Hepatic Steatosis Of Non-alcoholic Fatty Liver

Non-alcoholic fatty liver (NAFLD) as a chronic liver disease is prevalent in many countries. the Mothidity of of Non-alcoholic fatty liver in Western countries is about15%-40%and9%-40%in Asia, and with rapid growth in the past15years. NAFLD consists of as pectrum of liver disease, ranging from simple steatosis to non-alcoholic steato hepatitis(NASH), fibrosis, cirrhosis and Hepatocellular carcinoma finally. The presence of non-alcoholic fatty liver disease is in connection with pericardial fat, carotid artery stenosis and increase in cardiovascular mortality, which makes the prognosis more serious. The disease has become one of the major diseases of serious impact on physical and mental health of our people, and the national economy, social development and stability in our country.Pathogenesis of NAFLD is no tvery clear until now,"two hits"theory is approved by most medical scientists."firsthit", is insulin resistance(IR). NAFLD almost exist insulin resistance. Insulin resistance lead liver cells to fatty degeneration through increasing lipolysis, in the mean time, sensitivity of damage factor inside and outside is rose."seeond hit", is lipid Peroxidation caused by exceed lipid peroxide(LPO) and some cytokine. The "second hit" leads liver cells to inflammation, necrosis, fibrosis.Recent research suggests that cytokines secreted by fat cells regulate lipid metabolism in liver cells. Signaling Pathways of adiponectin closely linked to two hits. Its regulation mechanism beeome the hot spot being researched by most medical scientists. Adiponectin receptor2existing in liver cells is suggested to relate to the pathogenesis of NAFLD. Adiponectin binding to its receptor in liver cells regulates the expression of nuclear transcription factor PPAR-alpha related to liver lipid oxidation, and increase expression of rate-limiting enzyme in lipid oxidation, thereby increases Liver lipid oxidation and reduces liver lipid deposition.There is no NAFLD in chinese tradional medicine, NAFLD is equivalent to Chinese "obesity""hypochondriac pain""jaundicet"..According toTCM theory, intake excessive energy caused dampness and heat stagnating in spleen, dampness-heat in middle jiao, then qiji is blocked by dampness-heat, with the passing of time blood stasis is indueed. To sum up, dampness, heat and blood stasis are the key for NAFLD. Emodin is one of the main active ingredient of Polygonum multiflorum and rhubarb. Polygonum multiflorum have the function of tonifing the liver and kidney, loosening bowel to relieve constipation. Rhubarb have the function of removing stagnated heat. Emodin has some effect in terms of lipid-lowering, protecting the liver cells, the role of the key pathological factor in NAFLD..The experiment aims to observe the systematic effect of Emodin on NAFLD, and make further reseach the mechanism. The conclusion of this experiment provid for exploratory research to develop the treatment of NAFLD drugs from Chinese herbal medicine.Objectives1. Observation of influence of emodin on nonalcoholic fatty liver early in vivo and in vitro models, including body weight, liver weight, liver steatosis, liver function, blood lipid metabolism and TG and liver enzymes in liver cell lines.2. Study on the possible mechanism of emodin in treating nonalcoholic fatty liver, including the improvement of lipid deposition, and protection on liver cells, reducing oxidative stress, and protein and gene expression of adiponectin receptor2, PPAR-alpha, CPT1, ACOX1.Methods62C57BL6mice were randomly divided into blank control group, model group and ginsenoside group and emodin group, the rosiglitazone group. The blank control group had been fed with normal diet, and other groups were given a high fat diet(88%ordinal forage+10%lard+2%cholesterin)for8w. After8W,two rats were randomly choosed from each group, liver tissue sections that stained with HE method is observed by light microscope to confirm NAFLD model mice replicated successfully. Then, the model group were randomly divided into4groups, namely blank model group, emodin group, ginsenoside group, rosiglitazone group. After eight weeks, the blank control group was given DMSO (5%DMSO20mg/kg)by intragastric administration, Ginsenoside group was given ginsenoside(20mg/kg),Emodin group was given emodin(20mg/kg), The rosiglitazone group was given given rosiglitazone(1Omg/kg). After treating for4w, all mice were fasted for12h,then were killed after being narcotized with chloraldurat to get serum and hepatic tissue rapidly according to routine, which is affixed by formalin, Packed with Paraffin, sectioned, stained with HE, evaluated the degree of hpatic steatosis and inflammation under the optical microscope. Then calculate liver index, to detect the content of serurn ALT, AST, TG,CHO, LDL, HDL. Hepatic tissue was leave over to make10%liver homogenate at4℃for detecting content of TG, FFA, SOD, MDA, detected the protein expression of AdipoR2, PPAR-alpha, CPT1a, ACOX1by wersten blotting.The mRNA expression levels of AdipoR2, PPAR-alpha, CPT1a, ACOX1by real time quantitative RT-PCR.HepG2cells were cultured in1640medium supplemented with7%newborn calf serum.50Upenicillin/mL and50μg streptomycin/mL. For subculturing purposes, cells were detached by treatment with0.25%trypsin/0.02%EDTA at37℃. Cultures were used at75%confluency. Cultures were divided into5groups. To induce fat-overloading of cells, HepG2cells at75%confluency were exposed to a long-chain mixture of FFAs(oleate and palmitate) for24h. Emodin, ginsenoside and rosiglitazone were added to each group for24h,48h,72h. Cells were stained with Oil-Red-O to examine the amount of fat accumulation in the cells. TG,ALT, AST, SOD, MDA in homogenates from cells were measured using a commercial kit. The mRNA and protein expression levels of AdipoR2, PPAR-alpha, CPT1a, ACOX1were detected by real time quantitative RT-PCR and wersten blotting respectively. Data are expressed as mean±S.D. The Student's t-test was used to determine the statistical significance of the experimental data.ResultsExperiment1Emodin treatment of nonalcoholic fatty liver in vivo experiments1. According to the hepatic tissue pathology and the state of hepatic function of mice, we succeeded in replicating the models of rats with NAFLD.2. Compared with model group, the body weight and liver index of emodin group decrease, but has no considered significant (P>0.05). the liver weight of emodin group decrease significantly (P<0.05). Emodin can significantly improve liver fat deposition according to liver pathology (P<0.01). Emodin can improve Liver cell morph and arrangement significantly. Emodin can significantly reduce the serum ALT, AST, TG, FFA and CHO (P<0.05), reduce serum LDL and increase HDL, but has no considered significant(P>0.05). Emodin can reduce content of FFA and increase SOD in hepatic tissue significantly (P<0.05),can reduce content of TG and MDA in hepatic tissue, but has no considered significant (P>0.05)3. Compared with model group, protein and gene expression of adiponectin receptor2, PPAR-alpha, CPT1, ACOX1in emodin group increase significantly (P<0.01) Experiment2Emodin treatment of nonalcoholic fatty liver in vivo experiments1. After induced by FFAs for24h, Intracellular lipid vacuoles in HepG2cell line were seen by phase-contrast microscopy and confirmed by Oil Red O staining with fine cell viability and morph, indicating that the formation of the NAFLD lipid deposition in vitro model.2. Emodin can reduce intracellular lipid deposition reduce the TG, ALT and AST content significantly (P<0.05), and increase SOD content significantly (P<0.05)3. Emodin can increase protein and gene expression of adiponectin receptor2, PPAR-alpha, CPT1, ACOX1of the HepG2cell line after induced by FFAs (P<0.01)Conclusions1. Emodin can decrease significantly the body weight, improve hepatomegaly, reduce liver wet weight and the liver index. It is suggested that to reduce the abdomen fat may be one of the mechanism of emodin in the treatment of NAFLD.2. Emodin can decrease liver enzymes in serum and released from liver cell, improve damage on liver cells caused by lipid deposition in the simple fatty liver stage of NAFLD.3. Emodin can decrease the serum of FFA, TG, TC and LDL-C and increase HDL-C to correct the dyslipidemia. The possible mechanism for emodin to reverse liver fat deposition, improve liver function and protect the liver cells is reducing the entrance of free fatty acids into the liver cells, reducing triglyceride synthesis and reducing the toxic effects of excess FFA.4. Emodin can enhance the anti-oxidization ability, reduce lipid peroxidation. Emodin reduce liver cell free fatty acids flowing into the liver cells, reduce oxidative load of mitoehondrial, thereby improve (3-oxidation, reduce Ros and free radicals.5. Emodin can increase protein and gene expression of adiponectin receptor2and PPAR-alpha in vitro and in vivo, and raise protein and gene expression of CPTla and ACOX1, the rate-limiting enzyme in the fatty acid oxidation process regulated by PPAR-alpha in liver cells, indicating that the emodin can alleviate the NAFLD lipid deposition through this way. It is concluded that emodin can be an important target for the treatment of NAFLD.6. Ginsenoside Rbl has the same effect and mechanism on NAFLD as emodin.