Repurposing matrine for the treatment of non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome in the liver. It is the most common chronic liver disease with fast-growing prevalence worldwide that parallels the obesity epidemic and type 2 diabetes (T2D). NAFLD ranges from simple hepatosteatosis to non-alcoholic steatohepatitis (NASH) and may finally leads to liver cirrhosis and failure. NASH is the critical stage in the progression from reversible and asymptomatic hepatosteatosis towards irreversible liver disease with significantly worsen prognosis. However, there is no effective drug for treatment of NASH. Matrine (Mtr) is a small molecule (MW: 248) originally isolated from plants and it has been used as a hepatoprotective drug in humans with few reported adverse effects. Several lines of evidence suggest that Mtr may be repurposed for the treatment of NASH, including a previous study of this laboratory showing its ability to reduce hepatosteatosis in high fat diet (HFD)-fed mice. However, a single HFD rodent model only replicates simple hepatosteatosis due to increased exogenous lipid overload. Therefore, the overall aim of this thesis was to characterise the therapeutic properties of Mtr for NASH treatment and investigate the underlying mechanism/s. The hypothesis addressed in this study was that Mtr has therapeutic properties for the treatment of NASH by ameliorating hepatic steatosis and inflammation as well as associated metabolic risk factors. These therapeutic effects were examined in three mouse models mimicking different characteristics of NASH. The first aim was to examine the therapeutic effects of Mtr on hepatosteatosis and glucose intolerance induced by an over consumption of carbohydrates. This was assessed in high fructose (HFru) fed mice, a well-characterised model of hepatosteatosis causing an increased in hepatic de novo lipogenesis (DNL) pathway. This study (Chapter 3) showed that treatment with Mtr markedly ameliorates hepatosteatosis (reduced triglyceride content) and glucose intolerance in HFru-fed mice. Further studies revealed that the reduced hepatosteatosis by Mtr is due to its inhibition of hepatic DNL involving the blocking of the endoplasmic reticulum (ER) stress pathway. These effects are associated with an upregulation of heat shock protein 72 (HSP72), the chaperone protein which is known to be protective against metabolic diseases and inflammation. A separate aim was to evaluate the potential efficacy of Mtr in the treatment for hyperglycaemia as hepatosteatosis is an important contributor to hepatic insulin resistance leading to hyperglycaemia. In this study, the antisteatotic effects of Mtr on hyperglycaemia were investigated in a mouse model where type 2 diabetes is induced by HFD in combination with low doses of streptozotocin (HFD-STZ). The results showed that oral administration of Mtr to HFD-STZ mice reverses hyperglycaemia and hepatosteatosis. These findings support the use of Mtr to treat hepatosteatosis and associated disorders in glucose homeostasis. The second aim of this thesis (Chapter 4) was to examine the therapeutic efficacy of Mtr for hepatic inflammation and fibrosis. This was investigated in mice fed a methionine choline-deficient (MCD) diet, a well-established mouse model which shares pathologic features of severe NASH in humans, in particular hepatic inflammation and fibrosis. The study showed that Mtr treatment suppresses the increases in TNFα, CD68, MCP-1, NLRP3, and hepatic fibrosis markers (TGFβ and Smad3) induced by MCD diet. Along with these effects, Mtr inhibits mTOR activation and upregulates HSP72 expression, suggesting a likely role of mTOR-HSP72 pathway in coordinating the therapeutic effects of Mtr for NASH. The final aim further investigated cellular mechanisms underlying the effects of Mtr on the inflammatory pathway. This was conducted using cultured J774A cells, a macrophage cell line rused here as a model for Kupffer cells believed to be a major source of inflammatory cytokines in the liver. Incubation of J774A cells with LPS stimulated the production of TNFα and increased NLRP3, CD68 and TGFβ expression. Treatment with Mtr also upregulated HSP72, inhibiting inflammation and fibrosis induced by LPS (Chapter 5). Additionally, Mtr suppressed the level of mTOR. These results provide further evidence that Mtr may exert its effect against NASH through the mTOR-HSP72 pathway. In summary, this thesis employed three different animal models to characterise a wide range of therapeutic effects of Mtr on NASH. Findings from these studies led to the following conclusions. Firstly, Mtr is a promising therapy for hepatosteatosis and associated glucose disorders. Secondly, Mtr can attenuate NASH-associated inflammation and fibrosis. Finally, mTOR-HSP72 may play an important role in mediating the therapeutic effects of Mtr on NASH. Overall, these findings provide strong pre-clinical evidence to support the repurposing of Mtr as a promising new drug for the treatment of NAFLD, particularly on the progression towards NASH