Author: Zhang, Huan
Title: Polygonum cuspidatum (Hu Zhang), a traditional Chinese medicine, ameliorates high-fat diet-induced fatty liver in rats and free fatty acid-induced hepatic steatosis in LO2 cells
Advisors: Wong, Wing-tak (ABCT)
Chan, Shun-wan (ABCT)
Degree: Ph.D.
Year: 2018
Subject: Hong Kong Polytechnic University -- Dissertations
Medicine, Chinese
Fatty liver -- Treatment
Liver -- Diseases -- Treatment
Department: Department of Applied Biology and Chemical Technology
Pages: xxviii, 156 pages : color illustrations
Language: English
Abstract: Non-alcoholic fatty liver disease (NAFLD) is a burgeoning health problem worldwide and includes a spectrum of disease from simple steatosis to inflammatory nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. One forth of the NAFLD patients progress from simple steatosis to NASH and have fibrosis in 3 years. Due to the changes in the modern diet and people's lifestyle, NAFLD has become one of the leading causes of chronic liver diseases. Moreover, NAFLD is an emerging risk factor for both cardiometabolic and hepatic mortality. Though the pathogenesis of NAFLD is not entirely understood, it is believed that lipid accumulation arises from an imbalance between lipid acquisition (i.e., fatty acid uptake and de novo lipogenesis) and removal (i.e., mitochondrial fatty acid oxidation and export as a component of very-low-density lipoprotein particles) in liver is one of the major causes. Currently, only limited treatments for NAFLD are available. Lifestyle interventions such as dietary change, weight loss and increase physical activity are strongly recommended for improving the outcome of patients with NAFLD. However, it is hard to maintain the lifestyle change. International pharmaceutical companies put huge resources to develop drugs targeting different stages of NAFLD. Although a few pharmaceutical agents are effective, long term usage of these agents might cause many adverse effects. For example, vitamin E might increase the risk of prostate cancer or stroke and pigolitazone might cause bladder cancer. As a result, alternative therapeutic strategies are therefore warranted. Traditional Chinese medicine (TCM) might be the answer for the alternative treatment for NAFLD based on two main reasons. First, NAFLD is caused by multi-factors, the multiple-targeted TCM could regulate the dysfunction of multiple targets concurrently. Second, using TCM to prevent and treat NAFLD has received increasing attention due to its effectiveness and acceptable prices. Therefore, the patients with NAFLD can tolerate the long term treatment. In the TCM's view, NAFLD is not recognized as a particular disease. Instead, the symptoms of NAFLD including obesity, right rib hypochondriac pain, discomfort, jaundice and fatigue have been classified as "Feiqi" and "liver swelling". According to the book "Chinese medicine diagnosis" published in 1997, such symptoms have been named as liver swelling. Phlegm and blood stasis is considered as the main pathogenesis. It was mainly divided into five types: phlegm and blood stasis, liver and spleen deficiency, intrinsic damp heat, qi stagnation and blood stasis, liver and kidney deficiency. Polygonum cuspidatum sieb. et Zucc. (HZ, Hu Zhang in Chinese) is a perennial herb with both medicinal and food dual value, native to eastern Asia such as China, Japan and Korea, whose young stems are edible as a spring vegetable with a flavor similar to lemon or rhubarb. The root of HZ has been used for conditions of the heart and blood vessels including atherosclerosis and hyperlipemia and for digestion problems including constipation, liver disease (hepatitis), and gallstones. Literature review showed that four traditional Chinese herbal formulae, Qushi Huayu Decoction, Danning Tablet, Huganning tablet, QuYuhuatan tong luo Decotion were studied as the treatment for NAFLD. Interestingly, all these four herbal formulae contained HZ, indicating that their effect on treating NAFLD may be due to the present of HZ. In addition, the channel tropism of HZ is liver, which is consistent with the main site of fatty liver. HZ can alleviate blood stasis and damp heat, which is consistent with pathogenesis and treatment of fatty liver. Based on previous findings, a potential role of HZ in the treatment of NAFLD is of considerable interest. Previous studies have revealed that there are three possible pathologies which might induce lipid accumulation in liver, they are oxidative stress, impaired lipophagy and bile acids synthesis. Based on the preliminary results, we further hypothesized that HZ might possess lipid reduction effects by regulating these factors concurrently.
There are three major objectives of my project: (1) to investigate the lipid reduction effects of HZ and characterize the molecular mechanisms of lipid reduction of HZ in free fatty acid (FFA)-induced steatotic cells using human liver cell line (LO2) cells in vitro; (2) to evaluate the lipid reduction effects of HZ in HFD-induced steatosis in rats in vivo; (3) to investigate the possible mechanistic pathways of HZ on lipid reduction by using molecular and metabolomics approaches. Recent studies have revealed that impaired autophagy, indeed, may be a fundamental mechanism of some lipid metabolism disorders such as NAFLD, obesity and other metabolic syndrome. Lipophagy may therefore represent a potential therapeutic target for the treatment of NAFLD. HZ could decrease the lipid content in FFA-induced steatosis model in LO2 cells. The lipid-lowering effect maybe related to the restoration of the down-regulated phosphorylation of adenosine 5'-monophosphate activated protein kinase (p-AMPK), peroxisome proliferator activated receptor α (PPARα), low density lipoprotein receptor (LDLR), and phosphorylation of acetyl-CoA carboxylase (p-ACC) protein expressions. HZ induced autophagy to promote lipid clearance. The current study reported (for the first time) that treatment of HZ could be able to upregulate LDLR and p-ACC expressions in the absent of autophagy, suggesting that HZ reduced lipid accumulation partially by regulating autophagy induction. However, treatment of HZ was found to be not able to upregulate PPARα expression in the absent of autophagy, suggesting that HZ upregulated PPARα expression by regulating autophagy induction. Further investigation was performed on the anti-NAFLD effects of HZ in vivo. Our in vivo study demonstrated that in response to HZ supplementation, both hepatic triglyceride (TG) and total cholesterol (TC) accumulation, lipid content were significantly reduced. HZ attenuated oxidative stress by elevating antioxidant enzyme such as catalase (CAT) and inhibiting malondialdehyde (MDA) levels in the liver. The restoration of the down-regulated p-AMPK, PPARα, LDLR, and p-ACC protein expressions induced by HFD were achieved in animals treated with either simvastatin or HZ. A cytosolic form of LC3 (LC3-I) is conjugated to form LC3-PE conjugate (LC3-II), is regarded as the most reliable marker protein for autophagy. HZ induced LC3-II expression, which indicated HZ may promote lipid clearance in the fatty liver by enhancing lipophagy. It is well established that bile acids' synthesis has profound effect on facilitating cholesterol removal from the body. In the present work, combination of ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to examine the metabolite changes in the serum samples from rats to investigate the therapeutic effect of HZ on HFD-induced steatosis rats. Using untargeted mass spectrometry-based metabolomic platforms, multivariate statistics revealed that HFD significantly perturbed bile acids. HZ supplement to HFD rats resulted in significant increases in three primary bile acids [chenodeoxycholate (CDCA), glycochenodeoxycholate (GCDCA) and glycocholate (GCA)] and two secondary bile acids [deoxycholate (DCA) and glycodeoxycholate (GDCA)]. Decreased utilization of cholesterol [bile acid synthesis due to cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1)] could contribute to the accumulation of free cholesterol in NAFLD. Regarding the mechanism of bile acid disturbance, up-regulated CYP7A1 may affect the bile acid synthetic pathway and lead to lower cholesterol content in the body. HZ significantly promoted CYP7A1 activity, indicating that HZ could accelerate the transformation of cholesterol into bile acids in the liver. Another factor that might contribute to the cholesterol lowering effect of HZ is through inhibiting dietary cholesterol absorption in the small intestine with the aids of binding effects of HZ and conjugated bile acids. Our result showed that HZ increased three conjugated bile acids (GCA, GDCA and GCDCA). These conjugated bile acids lowered the solubility of cholesterol, resulting in lowering the reabsorption of cholesterol from intestine to the hepatocytes. In conclusion, our studies have shown that HZ reduced the lipid accumulation in vitro and in vivo mainly by regulating the imbalance between lipid acquisition and removal, especially through lipophagy and bile acids synthesis to remove lipid from the body. In addition, we demonstrated that the lipid reduction effects of HZ were achieved by regulating multiple targets, including the suppression of oxidative stress, activation of lipophagy and enhancement of bile acid synthesis. Taken all the results together, this project indicated that HZ might be a novel drug target for the prevention and treatment of NAFLD.
Rights: All rights reserved
Access: open access

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