and nnate as well as

9

ABSTRACT

Steatosis is one of the most common liver diseases in humans. It is caused by an accumulation of free fatty acids, mainly triglycerides, which entails several damage that may ultimately lead to cellular necrosis and apoptosis. Different causes determine the development of this pathology such as obesity, diabetes, excessive consumption of alcohol or metabolic diseases. Fatty liver disease may be caused by an excessive alcohol consumption, called alcoholic fatty liver disease (NAFLD), or not, called non-alcoholic fatty liver disease. When NAFLD develops an inflammation, it progresses in non- alcoholic steatohepatitis (NASH), associated with a whole range of hepatocytes injury.

In NAFLD/NASH pathogenesis a clear role is played by gut microbiota because the liver is constantly exposed to gut-derived bacterial fractions or metabolites, and it is an important site for bacterial phagocytosis and clearance. Because of this association, probiotics should be considered as a potential terapeutic tool for NAFLD treatment. The word probiotic refers to viable microorganisms which, when administered in adequate amount, confer health benefits to the host by improving the properties of the indigenous microflora. Although not all probiotics give the same benefits to the host, the main mechanisms of action include a direct effect on the other microorganisms, commensally

and nnate as well as

the acquired immune system. Generally, the optimal concentration to detect a beneficial effect is between 10

and 10

colony forming units (CFU)/ml. Although the most widely used commercial probiotic species belong to the genera Lactobacillus and Bifidobacterium and are important components of the human gastrointestinal

microflora, is also known that some soil bacteria may play a probiotic role. Suitable

animal models of steatosis are an essential tool to study the mechanisms of development

of NAFLD and to test potential drugs for the treatment of this pathology. The most used

models are nutritional ones, (involving the use of a high fat diet HFD-, a diet lacking

in methionine and choline, a diet rich in cholesterol or with high doses of sucrose and

fructose) or models in which diabetic animals are used. A recent model, used in

literature to evaluate the protective effect of some compounds (drugs, natural

substances) on type 2 diabetes mellitus and hepatic steatosis, combines the feeding of

high-fat diet and low dose of streptozotocin treatment (35 mg/Kg). The peculiarity of

this model lies in the fact that it closely mimic the natural history of steatosis and type 2

10 diabetes mellitus, in contrast with only high-fat diet or streptozotocin treatment.

However, few examples of this model exist in literature. The available studies discuss many features of the model, but none of them consider the modulation of drug- metabolizing enzymes or perform a deep analysis of the antioxidant enzymes activity.

The first aim of this study was to feature the model in this way, providing new informations about it, and then to evaluate the potential benefic effect of a soil-bacterial mixture (Bacillus amyloliquefaciens and Pseudomonas fluorescens) on liver steatosis based upon certain characteristics of the two bacteria that suggested their potential probiotic role (such as absence of toxicity in animal models).

The first step of this study was the in vivo treatment (8 weeks) on male Wistar rats (n=15) randomly divided into three groups. The first group was fed with normal diet only, the second group was diabetic and fed a high fat diet (streptozotocin induced diabetic rats on an HFD), the third group was diabetic, fed a high fat diet and it was administered the bacterial-mixture (1 ml containing 10

CFU) per os daily in the last 4 weeks of treatment. At the end of experiment rats were anesthetized by ether inalation and blood was collected, rats were then sacrificed and their liver dissected for biochemical, molecular, immunological and histological assays. Several parameters (ALT, AST, cholesterol, LDL, HDL, glucose, triglycerides) were measured on blood samples. Lipids and glutathione content, lipid peroxidation, some cytochrome P450- dependent activities, heme oxygenase-1 activity and certain antioxidant activities (DT- diaphorase, catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase) were measured on liver samples. Histological analysis (hematoxylin-eosin stain and red-oil stain) were performed in liver samples. Expressions of genes involved -6) and of SREBP1c were analyzed in liver samples by real-time PCR. Some P450-isoforms were detected by Western blotting.

Hepatic lipid quantification and histological analysis show the presence of steatosis in an early stage, with no measurable oxydative stress or inflammation. The results obtained from this study demonstrate that the model utilized leads to a general modulation of the cytochrome P450 monooxygenase system

CYP2E1 and CYP3A and downregulating CYP2C11 activities. The model did not

affect the activity of the main antioxidant enzymes. The bacterial mix did not show a

protective effect against fatty liver disease.