E maintained per cage in an animal area maintained at a continual temperature of 22 1 C, using a relative humidity involving 50 and 60 , and light/dark cycle of 12/12 h, and had been fed having a common diet plan and water ad Adrenergic Receptor drug libitum. Each of the experimental procedures and methodsAntioxidants 2021, 10,four ofhave been authorized by the Animal Management Committee of China Health-related University (IACUC approval quantity: CMUIACUC-2016-376). 2.five. Experimental Protocol Right after about 1 week of adaptation, the male ICR mice were randomly assigned to six groups (n = six per group): the handle group, paracetamol group, paracetamol + NAC (600 mg/kg) group, paracetamol + SS (125 mg/kg) group, paracetamol + SS (250 mg/kg) group, and paracetamol + SS (500 mg/kg) group. SS was suspended in 0.5 carboxymethyl cellulose (CMC) option and orally administered to mice in 3 treatment groups at doses of 125, 250, and 500 mg/kg, respectively, for 6 days, together with the final dose offered 1 h prior to paracetamol administration. The handle and paracetamol mice had been only treated with 0.five CMC option, within the similar way. A single hour soon after the final SS dose, mice were administered with paracetamol (400 mg/kg) with standard saline by single intraperitoneal injections in each of the groups (except the handle group). The mice in the NAC group were orally pretreated with NAC (600 mg/kg) 1 h prior to the paracetamol challenge. Twelve hours just after paracetamol administration, the mice had been ROCK1 supplier euthanized with CO2 , after which, blood and liver tissues had been collected for additional evaluation [24]. The blood was collected by cardiac puncture just after euthanasia and centrifuged at 1700g for 30 min at 4 C for plasma collection. To evaluate the function of an AMPK inhibitor (Compound C) in regulating paracetamolinduced hepatotoxicity, mice had been randomly divided into 5 groups (n = six per group): a control group, paracetamol (400 mg/kg) group, paracetamol (400 mg/kg) + compound C (25 mg/kg) group, paracetamol (400 mg/kg) + SS (500 mg/kg) group, and paracetamol (400 mg/kg) + SS (500 mg/kg) + compound C (25 mg/kg) group. SS was administered to mice for six days at doses of 500 mg/kg, respectively. The handle and paracetamol mice were only treated with 0.five CMC remedy, within the similar way. Compound C (25 mg/kg) was given intraperitoneally to the animals of the intervention groups 1 h prior to paracetamol administration. Just after fasting for 12 h, mice have been intraperitoneally injected with paracetamol option. Twelve hours soon after paracetamol administration, the mice have been anesthetized for harvesting blood for additional analysis. 2.6. Analysis of Biochemical Markers The blood was centrifuged (five min at 12,000g at four C) to separate the serum. The serum levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBil (total bilirubin), TC (total cholesterol), and TG (triglyceride) have been measured making use of commercial detection kits (HUMAN Diagnostics Worldwide, Ahrensburg, Germany). 2.7. Histopathological Examination The liver samples had been fixed in 10 formalin for at least 24 h prior to paraffin embedding. The slides had been stained with hematoxylin and eosin (H E), and examined applying a Nikon Compound Microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan), to evaluate the cellular and morphological structure. The severity of liver disease was graded from 0 to five: 0 points signifies normal (normal–no hepatocyte necrosis); 1 point signifies minimal ild (much less 1 ) (focal and limited to centrilobular area; fewer than 1/4 from the impacted lobules are necrotic); two.