Study On The Activities Of Three Traditional Chinese Medicines And Their Effective Constituents In Treating NAFLD Of Rats

With the improvement of living standards, there is a rapid increase in incidence of non-alcohol fatty liver disease (NAFLD) and it may be the primary liver disease in future of China. However, effective drugs for NAFLD are still in shortage worldwidely. The study is based on understanding of "damp heat" and "blood stasis" in TCM (traditional Chinese medicines) theory, evaluated effective components of Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf.(traditional Chinese medicines which are used for clearing heat and eliminating dampness, activating blood and resolving stasis) in treatment of NAFLD, and to explore the possible effect mechanism primarily, hoping to provide reference for screening and finding effective herbs and ingredients in treatment of NAFLD.Research methods are as follows:(1) Construct NAFLD models in rats with high-fat diet, and investigate the effect of Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. in improving serum biochemical indicators, liver steatosis and inflammation in rats. And use real-time PCR, Western blotting and immunohistochemical staining to investigate the herbs’impact on expression of lipid regulation-associated protein-peroxisome proliferator-activated receptor-y (PPARy), sterol regulatory element binding protein-1c (SREBP-1c) and stearoyl-coenzyme A desaturase (SCD-1).(2) Use human liver L-02cells steatosis model to evaluate protective activity of berberine, emodin and curcumin (the effective components of Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf.) to hepatocyte steatosis and their impact on expression of SREBP-1c and caveolin-1protein.(3) Use NAFLD models in rats to investigate therapeutic effect of berberine, curcumin and their combinations in treatment of NAFLD, and impact on lipid regulation, expression of SREBP-1c, caveolin1, tumor necrosis factor-α(TNF-α), nuclear factor-κB p65(NF-κB p65), and phosphorylated c-jun N-terminal kinase (p-JNK).Research results are as follows:(1) Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. at a dose of1g crude drug·kg-1·d-1for6weeks showed significant improvement in hepatic steatosis of NAFLD rats, and improvement of Coptis chinensis Franch., Curcuma longa L.were better than lovastatin (10mg·kg-1·d-1). The three herbs could also reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in NAFLD rats (P<0.05or0.01), and improvement of ALP was better than lovastatin, which means Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. could treat NAFLD alone and have better effects than lovastatin. Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. could all significantly reduce serum levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) in NAFLD rats (P<0.05or0.01), which means the three herbs have an effect on regulation of blood lipids. All three herbs could significantly increase both PPARy gene and protein expression in liver of NAFLD rats, and Curcuma longa L. was the most effective (P<0.05or0.01). They could also reduce SREBP-lc, SCD-1gene and SREBP-lc p-JNK protein expression in liver of NAFLD rats. For SREBP-1, liver immunohistochemical staining results were consistent with Western blotting results.(2)The study have shown that in cellular level, both curcumin and emodin can reduce lipid droplets content, TG levels, and expression of SREBP-1c and caveolin-1protein in human hepatic cell L-02, while berberine has little effect on lipid droplets content and TG levels, no obvious effect on expression of SREBP-lc and caveolin-1protein in human hepatic cell L-02. This means that curcumin and emodin have a direct impact on hepatocellular lipid regulation, while berberine does not have direct effect on metabolism of liver cells. Indirect mechanism of berberine in treatment of NAFLD will be studied in later experiments.(3) Foregoing results showed that Coptis chinensis Franch. and Curcuma longa L. could be effective in treating NAFLD in animal level, but their effective components-berberine and curcumin had distinct roles in cellular level. It may be speculated that berberine and curcumin have different pathways and targets in treating NAFLD, and may have synergies between them. So, further experiment was carried out of8weeks observation of the effects of berberine and curcumin in treatment of NAFLD rats. Results showed that compared to NAFLD model, NAFLD rats that accepted normal diets for8weeks showed no significant recovery in pathological changes of liver tissue; compared to NAFLD model, berberine (100mg·kg-1·d-1) and curcumin (100mg·kg-1·d-1) administrated alone could significantly improve liver steatosis degree, reduce visceral adipose content in NAFLD rats, and combination therapy of both herbs (each50mg-kg-1·d-1) had a significantly better improvement in liver pathology changes. Combination therapy could reduce serum levels of TG, TC, LDL-C, ALT, AST, ALP, and increase high-density lipoprotein cholesterol (HDL-C) levels (P<0.05or0.01) to generally normal level as in normal groups. The difference was statistically significant compared to berberine and curcumin alone (P<0.05). Combination therapy could reduce serum levels of endotoxin, fasting blood glucose (FBG), insulin (INS) and MDA, and increase GSH-Px activity (P<0.01) to generally normal level as in normal groups. The difference was statistically significant compared to berberine and curcumin alone (P<0.05). PPARy gene expression was increased in liver tissue of rats in combination therapy, while gene expression of SREBP-lc, SCD-1, NF-κB and protein expression of SREBP-lc, TNF-a, NF-κB p65, p-JNK were down-regulated, caveolin-1protein expression was up-regulated to various levels. The difference was statistically significant compared to berberine and curcumin alone (P <0.05). Overall, it could be concluded that combined administration of berberine and curcumin (each50mg·kg-1·d-1) has a better effect than berberine or curcumin alone (100mg-kg-1·d-1), and a synergistic effect was shown.The results show that:(1) Coptis chinensis Franch., Curcuma longa L. and R. tanguticum Maxim. ex Balf. administrated alone all had a favorable effect on treating NAFLD rats, in which Coptis chinensis Franch. and, Curcuma longa L. had better effect.(2)10541Berberine, the effective component of Coptis chinensis Franch. and curcumin, the effective component of Curcuma longa L. all had a favorable therapeutic effect in NAFLD rats, and combination of the2components had synergistic effect. Curcumin can directly interfere with lipid metabolism in liver cells, with SREBP-lc and caveolin-1as its possible important targets; while berberine could not interfere with lipid metabolism directly, it may accomplish curative or synergistic effect by reducing endotoxin into the blood from gut and improving insulin resistance.