الفهرس 
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Abstract
the present work aimed to study some dysfunction criteria associated with hepatic steatosis and to compare the effects of the SGLT2 inhibitor canagliflozin and the biguanide metformin on oxidative stress and inflammatory conditions associated with hepatic steatosis and their correlation with the brain functions and their complications.
The animals used for this study are fifty adults male Wistar rats, each with average weight 170-180g.
The animals were divided into two groups at random; the first served as a control group, with the animals fed a conventional chow diet. While the second group acted as the HFD group, in which the animals were fed a 70% fructose diet, the experiment continued for five weeks.
Five weeks later, the HFD group of animals was divided into four sub-groups, while the control group was divided into three sub-groups, with each group consisting of eight rats, and were treated with the medicines for another four weeks in addition to HFD; the total groups were as follows.
group 1 “Standard diet control”: the animals were fed on standard chow diet throughout the experiment (9 weeks) and received the respective vehicle in comparable volume to the other administered drugs.
group 2 “HFD Model”: the animals were fed on high fructose diet throughout the experiment (9 weeks) without any treatment.
group 3 “HFD/Canagliflozin 10 mg”: the animals were fed on high fructose diet throughout the experiment (9 weeks) and received canagliflozin on daily basis at a dose level of 10 mg/kg. (The last 4 weeks).
group 4 “HFD/Canagliflozin 20 mg”: the animals were fed on high fructose diet throughout the experiment (9 weeks) and received canagliflozin on daily basis at a dose level of 20 mg/kg. (The last 4 weeks).
group 5 “HFD/Metformin 300 mg”: the animals were fed on high fructose diet throughout the experiment (9 weeks) and received metformin on daily basis at a dose level of 300 mg/kg. (The last 4 weeks).
group 6 “Canagliflozin 20 mg”: the animals were fed on standard chow diet throughout the experiment (9 weeks) and received canagliflozin on daily basis at a dose level of 20 mg/kg. (The last 4 weeks).
group 7 “Metformin 300 mg”: the animals were fed on standard chow diet throughout the experiment (9 weeks) and received metformin on daily basis at a dose level of 300 mg/kg. (The last 4 weeks).
The rats had free access to food and water during the trial, and all treatments were provided orally once in the morning. The rats were fasted overnight and weighed at the end of the trial, and blood samples were obtained for analysis of the following parameters:
1. Fasting blood glucose levels.
2. Fasting serum insulin levels.
3. Liver functions “AST and ALT” activities.
4. Lipid profile “Total cholesterol, Triglycerides, HDL-cholesterol and LDL-cholesterol” levels.
5. Inflammatory markers “TNF-α and NF-κB.
Following the collection of blood samples, the animals were decapitated, and their livers and brains were exposed and excised, washed, and homogenized before being utilized in the following assays.
1. Assessment of oxidative stress markers “MDA levels, catalase activity and reduced glutathione levels” in brain and liver.
2. Detection of phosphorylated and non- phosphorylated AMPK-1 protein expression in brain and liver.
Other halves of the tissues were prepared for histopathological investigation. They were fixed in 10% formalin, processed routinely, and embedded in paraffin; the paraffin-embedded tissues were sectioned at 5 µm thickness and subjected to histopathological investigation.
The results of the present study revealed that:
- HFD-fed male Wistar rats had higher fasting blood glucose and fasting serum insulin levels. Furthermore, as compared to the conventional diet control group, they clearly demonstrated the standard symptoms of steatohepatitis, as evidenced by liver dysfunction and severe degradation of the liver structure.
- Dyslipidemia was detected in HFD-fed rats, which was characterized by a significant increase in serum levels of total cholesterol, triglycerides, and LDL-cholesterol, with a decrease in HDL-cholesterol levels when compared to the conventional diet control group.
- Functional impairment of the liver as demonstrated by a significant increase in AST and ALT enzyme activity in the serum of HFD-fed rats compared to the conventional diet control group.
- When compared to the conventional diet control group, oxidative stress was significantly elevated in both liver and brain tissues, as evidenced by increased MDA content, decreased catalase activity, and lower glutathione levels in HFD-fed rats.
- Fatty liver, characterized by liver inflammation and fibrosis, was found after nine weeks of HFD feeding, as demonstrated by enhanced hepatic production of TNF-α and NF-ҡB.
- When compared to the conventional diet control group, HFD-fed rats had higher area percent GFAP expression, lower intact neuron and intact neuron dentate counts, and higher intact neuron dentate counts.
- In HFD-fed rats, oral treatment of canagliflozin 10, 20 mg/kg and metformin 300 mg/kg significantly reduced high fasting blood levels, fasting glucose levels, serum insulin, serum AST and ALT activity. They also improved their lipid profile, as seen by lower serum total cholesterol, triglycerides, LDL cholesterol, and an increase in HDL cholesterol.
- When compared to the untreated HFD-model group, the treatment of HFD-fed rats with canagliflozin 10, 20 mg/kg and metformin 300 mg/kg inhibited the state of oxidative stress in the hepatic and brain tissue.
However, oral administration of canagliflozin 10 and 20 mg/kg resulted in a considerable restoration of the normal ratio of activated to non-activated (phosphorylated/non phosphorylated) AMPK-α in both liver and brain tissues, whereas metformin 300 mg/kg resulted in an inconsequential ratio.
- In addition, the oral administration of canagliflozin 10, 20 mg/kg and metformin 300 mg/kg improved liver inflammation and progression of fibrosis through down-regulation of the hepatic expression of TNF- and NF-B with consequent improvement of the brain functions as manifested by reduced area of percent GFAP expression, increased intact neuron dentate and intact neuron dentate counts which may be through down-regulation of the brain expression of TNF- and NF-B when compared to untreated HFD-model group.
In conclusion, canagliflozin 10 and 20 mg/kg therapy of HFD fed rats reduced the progression of NAFLD, which can be related to its antihyperglycemic impact with subsequent amelioration of oxidative stress via attenuating mitochondrial dysfunction., improving insulin resistance, inflammation, and apoptosis in dose dependent manner. Fortunately, these effects were reflected as improvements in brain functions manifested through biochemical and histopathological investigations.
Thus, it can be concluded that canagliflozin provides protection against brain dysfunction through alleviating NAFLD aggravating factors. In conclusion, the present study suggests canagliflozin as dual therapeutic agent of significant effects concerning protection from steatosis and its associated neurological disorders in dose dependent manner.
Despite metformin exerts comparable results concerning the liver axis, yet concerning the brain dysfunction, it gave less beneficial results compared to canagliflozin.