The Effect And Mechanism Study Of Active Components (Group) From TianHuang Tablets In Treating Fatty Liver

The Chinese was facing an enormous health problem of rapid weight growth at present. With the growing prevalence of obesity, and type II diabetes, dyslipidemia,insulin resistance and related metabolic syndrome in 10 years lately, China is already in danger of fatty liver disease.Fatty liver occurs mainly in hepatic lobulethe, in which it is a clinical pathological syndrome of diffuse hepatic steatosis. The chronic fatty liver is main type of fatty liver that divided into simple fatty liver, fatty liver cirrhosis and steatohepatitis pathology. It is well known that alcohol, excessive intake of nutrients is an important cause of fatty liver. However, the current rapid growth in the incidence of fatty liver is closely associated with a high incidence of obesity and its associated metabolic syndrome. Fatty liver is the intake of body's excess energy or entopic lipid accumulation in the liver. For a better understanding,Liver can be imaged as the "chemical" of energy substances. When excess energy input exceeds the metabolic capacity, and energy metabolism get slow, the imbalance of liver energy will happen between production capacity and output capacity.The most effective strategies for treating fatty liver of nutrition type is to restricte diet (reducing production) and do exercise (to promote the output). Another important method to treat is to use a drug to activate the operating efficiency of energy metabolism in the liver cells workshop. The study found that liver lipid metabolism is a complex network control system,including a variety of blood-borne lipoproteins (chylomicrons, LDL, HDL), cytokines (adiponectin, leptin), the intracellular enzyme systems (AMPK, ACC, FAS, CPT-1), receptor (CD36, FAT)nuclear receptor (PPAR, PXR, LXR, SREBP), etc. Thankfully, a number of important transcription factors and metabolic regulation is a key regulator of the entire network node, containing PPAR-a, SREBP-lc, AMPK. The regulation of these key targets will accelerate the catabolism of the liver, thereby treating fatty liver.We think about that are AMPK (AMP-activated protein kinase) is in the upstream network of lipid metabolism, so the target of AMPK is paid closed attention. When the rate of AMP/ATP increased, the phosphorylation of AMPK be enhanced, leading to the cascade of accelerated lipid metabolism. The activation of AMPK pathways in hepatocytes both accelerate catabolism of fatty acids and inhibit the synthesis and metabolism of lipids. It is found that some drugs or active pharmaceutical ingredient can activate AMPK, such as (metformin, salicylic acid).The herbal medicine in treating metabolic diseases has been trend. Recently, we found that Coptidis and Sanqi can treat fatty liver in vivo and in vitro, the mechanism may be the activation of AMPK signaling pathway. It is very excited that Coptidis and Sanqi play a synergistic remarkable effect on the treatment of fatty liver.Such findings attract us to explore Coptidis coupled with Sanqi as a new drug discovery.From effects of the active ingredient to clear mechanism of action, but some problems was still unclear.(1) The material basis of coptidis on efficacy of lipid-lowering is unclear.Whether there are other alkaloids of lipid-lowering activity apart from berberine?(2) Whether the activity of the active ingredient in vitro may occur in animal model?(3) Whether the mechanism of coptisine is same with the report of berberine by activating AMPK signaling pathway?(4) The material basis of Panax on lipid-lowering efficacy contain unclear,which is better components in lipid-lowering activity of saponins?(5) Why the lipid-lowering effect of berberine combined saponins is remarkable in vivo, better than single effect, whether there is a chemical synergistic effect?(6) Whether berberine combined with ginsenoside can co-activate AMPK target that played a pharmacological synergy?In order to answer these questions, this study develop screening method for antihepatic steatosis active components within Coptidis Rhizoma and Sanqi using liver cell extraction with HPLC analysis and a free fatty acid-induced hepatic steatosis HepG2 cell assay. We aim to AMPK target to explore the molecular mechanisms of lipid-lowering of coptsine, berberine coupled with ginsenoside Rbl.Then animal models of SD rats were used to observe the treatment of fatty liver effects administrated with coptisine, berberine, ginsenoside Rbl, and berberine combined with Ginsenoside Rbl. Finally, the pharmacokinetics was used to look for the evidence of berberine enhanceing ginsenosides Rbl bioavailability.Methods1. A method is developed for screening bioactive components in treating hepatic steatosis in vitro. This method used liver cell extraction with HPLC analysis and a free fatty acid-induced hepatic steatosis HepG2 cell assay. The application of this method focus on Coptidis Rhizoma alkaloids extract and Panax notoginseng to screen the possible active components in treating hepatic steatosis.2. HepG2 cell line is used as research tool. We will select coptisine, berberine,coupled with ginsenoside Rbl as research object. Western Blot method will be carried out to observe whether AMPK phosphorylation are activated by coptisine,berberine,coupled with ginsenoside Rbl. we will compare the effect relationship of alone and in combination and dose-response relationship.3. HepG2 cell line will be used as research tool. We will examine changes in AMPK signaling pathway downstream of the lipid gene. RT-PCR will be used to observe the rate-limiting enzyme of fatty acid ? oxidation of CPT-1 and cholesterol synthesis rate-limiting enzyme HMG-CoA gene expression by coptisine, berberine coupled with ginsenoside Rbl.4. We will develop a method to determine the content of ATP, ADP and AMP in liver cell using HepG2 cell line. The intracellular energy substances will be observed by coptisine, berberine coupled with ginsenoside Rbl. The increase in intracellular AMP is the direct cause of the activation of AMPK.5. Using liquid chromatography-mass spectrometry (LC-MS) technique to compare the pharmacokinetics of combined with berberine and notoginsenoside Rbl in single-use or in combined ues. SD rats will be orally administered therapeutic,drug monitoring multi-point and drug curve will be analyzed. We will look for the scientific evidence of berberine promotion of bioavailability of ginsenosides Rbl.6. We will use high-fat diet-induced fatty liver SD animal models and oral administration of drug, modeling 6 weeks, treating 5 weeks. We will observe the treat effects of high-dose group (50mg/kg/d) coptisine, low-dose group (10mg/kg/d)coptisine for hepatic steatosis. We will compare with the treat effects of berberine group (50mg/kg/d), notoginsenoside Rbl group (100mg/kg/d), berberine combined with Rbl group notoginsenoside. A series of blood parameters were measured (TG,TC, Glu, HDL, LDL, AST, ALT), hepatic lipid indicators (TG) and liver histopathology analysis (HE staining) were analyzed to evaluate lipid-lowering effect of active component.Results1. Two alkaloids within CAE, berberine and coptisine, were identified by HepG2 cell extraction with HPLC analysis as high affinity components for HepG2.These alkaloids were also determined to be active and potent compounds capable of lowering triglyceride (TG) accumulation in the FFA-induced hepatic steatosis HepG2 cell assay. Other alkaloids were found to have a lower affinity for cellular components from HepG2 cells and a lower inhibition of TG accumulation. Two notoginseng winthin Panax notoginseng, notoginseng Rbl and notoginseng Rgl were identified by HepG2 cell extraction with HPLC analysis as high affinity components for HepG2. Berberine, coptisine, notoginseng Rbl and notoginseng Rgl were also determined to be active and potent compounds capable of lowering triglyceride (TG) accumulation in the FFA-induced hepatic steatosis HepG2 cell assay. This remarkable inhibition of TG accumulation by coptisine and notoginseng Rbl occurred at concentrations of 0.2?g/mL and 100.0?g /mL, respectively.2. Coptisine, berberine and ginsenoside Rbl can all induce phosphorylation of AMPK in HepG2 cells. The result of Western Blot showed that compared with normal cells, coptisine high concentration (25.0 ?g / mL), low concentration (5.0 ?g/ mL) can promote certain degrees of phosphorylation of AMPK, high concentrations group of coptisine is better low concentrations group in induced effect of phosphorylation of AMPK. The results of Western Blot showed that compared with normal cells group, Berberine (25.0?g/ mL), ginsenoside Rbl (100?g/mL) alone can significantly promote AMPK phosphorylation. The gropu of berberine combined with ginsenosides Rbl was significantly better than single group.3. RT-PCR technique can observed the effect on AMPK signaling pathway downstream of protein and enzyme gene level. Coptisine high concentration (25.0 jig/ mL), low concentrations (5.0?g /mL) can regulate the expression of CPT-1 mRNA and reduced the expression of HMG-CoA mRNA. Ginsenoside Rbl (100?g/ mL)alone or in combination with berberine both raised CPT-1 mRNA expression, and reduced the expression of HMG-CoA mRNA. But berberine combined with ginsenoside Rbl group is larger than the other drug groups in the two target enzyme induction.4. Coptisine and berberine can affect intracellular ATP, ADP, AMP ratio of energy substances. The result of EC showed that the EC of berberine and coptisine administered group was significantly lower than normal group'. The ginsenosides Rbl (100?g /mL) concentrations did not change significantly in the cell EC. HPLC results showed that the effect of berberine group on EC is better than coptisine groups in the same concentration.5. The results of drug curves showed that when berberine combined with ginsenoside Rbl, the plasma concentration of ginsenosides Rblwas significantly increased, in which Cmax had advanced and Tmax is than larger 2.5 times than single ginsenosides group. Compared with drug curves of berberine in alone or combined group, there is not significant difference.6. SD rats were administrated with high-fat diet for 6 weeks,each group of therapeutic drugs treated for 30 days. (1) Compared with normal group, model group in liver weight, liver index, serum TG, LDL, Glu levels, liver tissue TQ levels were significantly elevated, liver function of ALT, AST was increased, histopathology were showed to be severe steatosis. (2) Compared with the model group, coptisine can decrease liver index, serum TC, TG, LDL, Glu levels, liver tissue TG, blood levels of liver function in varying degrees, the effect of high dose group gifted are better than the efficacy of low-dose group. (3) Compared with the model group,berberine combined with ginsenoside Rbl group can significantly reduce liver weight, improve remarkable liver index, decrease serum TC, TQ LDL levels and TG in liver tissue, reduce fat vacuoles in liver tissue, all indexes in combined group were better than in berberine alone group and ginsenoside Rbl alone group, some indicators was more preference than positive drug fenofibrate. (4) Compared with the model group: the effect of hypoglycemic and lowering lipid of berberine were better than ginsenosides Rbl alone group, but ginsenoside Rbl group improved liver function better than berberine group.ConclusionsA method is developed for screening bioactive components in treating hepatic steatosis in vitro. This method was compsed of liver cell extraction with HPLC analysis and a free fatty acid-induced hepatic steatosis HepG2 cell assay. Two alkaloids within CAE, berberine and coptisine and two notoginseng winthin Panax notoginseng, notoginseng Rbl and notoginseng Rgl were identified by HepG2 cell extraction with HPLC analysis as high affinity components and active components of lowering triglyceride (TG) accumulation for HepG2. The molecular mechanisms of these components lowing lipid is the activity of phosphorylation of AMPK in HepG2 cells. This activity effect had possible close relationship that the amount of energy substances AMP, ADP, ATP in hepatocytes were changed.Using high fat diet fatty liver models, it were proved that coptisine, berberine, ginsenoside Rbl all have the effect of treatment of fatty liver, in which the effect of high-dose treatment group coptisine better than low-dose group. The treatment effect of berberine combined ginsenoside Rbl was significantly better than single berberine or ginsenosides Rbl in vivo model of fatty liver. This enhancement may be a synergistic effect of berberine promoting ginsenosides Rbl bioavailability. The object of this research is about active components in lowing lipid within Coptidis Rhizoma alkaloids extract and Panax notoginseng. Our self-developed screening method is the study of the fuse,AMPK signaling pathway is the main line of research. Using in vivo and in vitro models, the lipid-lowering effect of coptsine, berberine combined with ginsenosides Rblwere observed. The molecular mechanism of lowing lipid was explored in the gene and protein level. Pharmacokinetic analysis was discussed for the possible causes associated with the synergies. This study can provide reliable and in-depth scientific evidence that the active component from herbal medicine were used to treat of fatty liver disease.