How does cigarette smoking aggravate hepatic steatosis to non-alcoholic steatohepatitis?
Creators: name: Fouda, Sherouk
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320507
subject: Metabolic medicine
classificationCode: 321401
subject: Basic pharmacology

description: Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disorders. It is closely associated with key aspects of metabolic disease such as obesity and hypertriglyceridemia. The benign stage of NAFLD is characterised by excessive lipid accumulation in over 5% of hepatocytes, which can further progress into non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HC). While a high-fat diet is recognised as a major contributor to NAFLD via delivering excess free fatty acids to the liver, there is very little study on the effect of cigarette smoking (CS) on the progression of NAFLD based on the general literature review in Chapter 1.
 
The overall aim of this project was to investigate the effects of two major lifestyle factors, overconsumption of a fat-rich diet and smoking (individually and in combination), on the development of NASH. We hypothesised that smoking is an important cofactor in the progression of NAFLD.
 
Initially, we conducted a systematic review of the literature (Chapter 2) to examine the clinical evidence for the hypothesised additive effects of diet-induced obesity and CS on NAFLD progression. Among the 1662 screened literature reports, only 8 met the inclusion criteria for evaluating CS on the progression of NAFLD in NAFLD patients. Results indicate that CS is an independent risk factor in the progression of NAFLD. Interestingly, there was apparent inadequacy of clinical studies reporting on the diagnostic criteria as recommended by the American Association for the Study of Liver Diseases (AASLD) for quantifiable examination of NAFLD progression. Furthermore, there was a clear indication that cigarette smoking is a risk factor implicated in NAFLD progression. Our results also show a strong correlation between smoking and advanced fibrosis in NAFLD patients. This led to the conclusion that cigarette smoking has adverse effects on the liver and is associated with NAFLD progression and advanced fibrosis.
 
Given the lack of clinical data on the effect of CS on the progression of NAFLD from a high-fat diet (HFD), we investigated this relationship using an animal model that largely mimics diet-induced obesity and chronic smoking behaviours in humans. We also performed several long-term animal studies to investigate disease progression by combining the two lifestyle factors (HFD and smoking) and comparing different intervention strategies to determine the best treatment options. The overall research design, approaches and methods used in this project are described in Chapter 3.

Chapter 4 investigated the progression of NAFLD in relation to chronic smoking and HFD feeding. The hypothesis was that concurrent HFD feeding and smoking worsens fatty liver disease through increased oxidative stress. Briefly, age matched male C57bl/6 mice were used for all experiments. Results show that mice fed an HFD and exposed to mainstream cigarette smoke-developed liver steatosis with evident lipotoxicity and displayed more significant levels of hepatic inflammation and fibrosis. This study suggests that CS aggravates HFD-induced steatosis towards NASH and that there is a close association of this progression with exacerbated oxidative stress in the liver.

The aim of Chapter 5 was to determine the effectiveness of caloric restriction on the progression of NAFLD in the presence of smoke exposure. This study was conducted in HFD fed mice. As a first intervention, mice were pair-fed with their control smoke-exposed mice. Interestingly, pair-fed mice developed less steatosis and inflammation compared to HFD-fed mice. Smoke exposed groups who were pair-fed also had lower inflammation and steatosis levels compared with their control counterparts. These results indicate that caloric restriction is effective in halting the progression of NAFLD and correcting all phenotypes of NASH. This study also indicates that lipid metabolites resulting from HFD and CS-induced factors contribute to NASH development by mechanisms independent of caloric intake.

Oxidative stress has been suggested as one of the multiple factors that aggravate simple hepatic steatosis to NASH. To follow up the observation of the additive effects of HFD and CS in increasing oxidative stress in the liver, Chapter 6 aimed to examine the role of oxidative stress in NAFLD progression by using a potent intracellular antioxidant, Tempol. Mice exposed to CS and HFD feeding were either treated with tempol or vehicle. This study showed that mice treated with tempol had less pronounced hepatic inflammation with no change in steatosis severity compared to CS/HFD-fed mice. As this effect of antioxidant occurred even when hepatic steatosis remained unchanged, this finding strongly indicates that the increased oxidative stress is a key mechanism for CS to precipitate hepatic steatosis towards NASH.
 
In summary (Chapter 7), the research in this thesis provides new conceptual insights into the effects of dietary fat and cigarette smoking on the pathogenesis of NAFLD and its progression to NASH. Findings in the mouse model suggest a crucial role of CS on the progression from simple hepatic steatosis towards a NASH-like phenotype with exacerbation of steatosis, dyslipidaemia, inflammation, and fibrosis. We discovered that the progression of NAFLD as a result of smoking led to a sustained increase in oxidative stress and depletion of key antioxidant enzymes, causing a worsening of NAFLD induced by dietary high fat consumption. This mechanistic approach provides a unique model to explore novel therapeutic approaches for the prevention and treatment of NAFLD and its progressive forms. Chapter 7 also discussed the limitations of this thesis and future studies that could address the remaining issues and their implications to humans.