Uncovering The Mechanisms For The Beneficial Effects Of Probucol, A Lipid-lowering Drug, On Non-alcoholic Steatohepatitis

Part1: To establish a rat model of non-alcoholicsteatohepatitis and investigate the effects on serumparameters and liver histopathologyObjective: To establish a rat model that recapitulates the pathogenesisof non-alcoholic steatohepatitis (NASH) and use this model to determine theeffects of probucol on serum parameters and liver histopathology.Methods: Forty male Sprague-Dawley rats were randomly assigned toone of the4groups:(1) normal control group (NC group)(n=10) withstandard diet feeding,(2) high-fat diet group (HD group)(n=10) fed ahigh-fat diet composed of15%lard oil,2%pure cholesterol, and83%standard chow,(3) probucol control group (NP group)(n=10) fed high-fatdiet and received normal saline, and (4) probucol group fed high-fat diet andreceived probucol (500mg/kg body weight/day)(HP group)(n=10).Normal saline and probucol were given by gastric gavage for15weeks.After the feeding trial, rats were weighted prior to sacrifice. Heart blood samples were collected and subjected to biochemical analysis. Serumglucose and serum concentrations of alumina aminotransferase (ALT),aspartate aminotransferase (AST), triglyceride, and total cholesterol weredetermined using standard biochemical methods with commerciallyavailable kits. Serum insulin levels were measured using an insulinradioimmunoassay kit. Serum tumor necrosis factor-alpha (TNF-α) andadiponectin concentrations were quantified using enzyme-linkedimmunosorbent assay kits. Insulin resistance was assessed using thehomeostasis model of insulin resistance (HOMA-IR) index, which iscalculated as fasting serum glucose×fasting serum insulin/22.5. The liversof all experimental animals were removed and weighted, and the ratio ofliver to body weight was calculated. Liver samples were routinely fixed in10%buffered formalin, embedded in paraffin, and cut into5-μm-thicksections. The sections were subjected to haematoxylin and eosin (H&E)staining using standard procedures. Histological assessment was performedblindly according to the NAFLD Activity Score (NAS) system. The NAS isthe unweighted sum of the scores for steatosis (0-3), lobular inflammation(0-3), and hepatocellular ballooning (0-2).Results: Compared to the HD group, probucol-treated animals showeda significant reduction in liver weight (17.6±3.6g vs21.1±1.3g, p <0.05)and liver/body weight (0.039±0.007vs0.045±0.0043, p <0.05), but nosignificant difference in body weight (439.4±47.3g vs466.7±30.9g, p> 0.05). There were significantly lower levels of serum ALT (80.18±22.90U/Lvs126.40±52.56U/L, p <0.01), AST (179.45±41.14U/L vs250.40±30.45U/L, p <0.01), cholesterol (2.4±0.3mmol/L vs2.7±.0.2mmol/L, p<0.01),and free fatty acid (0.557±0.19mmol/L vs0.734±.0.11mmol/L, p<0.05) inthe probucol group than in the HD group. The levels of serum triglyceridedid not differ between the two groups (0.8±0.1vs0.9±0.3, p>0.05).Probucol-treated animals had significantly lower concentrations of seruminsulin (12.02±1.90mU/L vs15.75±2.14mU/L, p<0.01), as well asHOMA-IR index (3.72±0.70vs5.21±1.00, p<0.01), when compared to theHD group. Moreover, probucol intervention resulted in a significant increasein the concentration of serum adiponectin (15.31±2.41μg/ml vs11.98±2.28μg/ml/L, p<0.05) and a concomitant decrease in the concentration of serumTNF-α (162.8±28.9pg/ml vs311.0±40.3pg/ml/L, p<0.01) in comparisonwith the HD group. H&E staining analysis revealed that livers from the HDgroup exhibited severe macrovesicular steatosis, lobular inflammation, andhepatocellular ballooning. Probucol treatment led to a marked decrease inthe degree of steatosis (2.10±0.316vs3.00±0.00, p<0.01) and severity ofinflammation (1.00±0.471vs2.30±0.48, p<0.05) in livers of rats fed theHFD. However, the ballooning degeneration of hepatocytes remainedunchanged upon the probucol treatment (0.20±0.421vs0.30±0.48, p>0.05).Conclusions: Probucol treatment has beneficial effects on serumparameters, hepatic steatosis, and lobular inflammation in HFD-induced NASH, which may be associated with an improvement in insulin resistance.Part2: Effects of probucol treatment on adipocyte size,lipogenesis, and adipokine secretion in a rat model ofnon-alcoholic steatohepatitisObjective: To determine the effects of probucol treatment on adipocytesize, lipogenesis, and adipokine secretion in a rat model of non-alcoholicsteatohepatitis (NASH).Methods: Forty male Sprague-Dawley rats were randomly assigned toone of the4groups:(1) normal control group (NC group)(n=10) withstandard diet feeding,(2) high-fat diet group (HD group)(n=10) fed ahigh-fat diet composed of15%lard oil,2%pure cholesterol, and83%standard chow,(3) probucol control group (NP group)(n=10) fed high-fatdiet and received normal saline, and (4) probucol group fed high-fat diet andreceived probucol (500mg/kg body weight/day)(HP group)(n=10).Normal saline and probucol were given by gastric gavage for15weeks. Atthe end of the feeding experiment, rats were sacrificed and adipose tissuesamples were collected and subjected to histological analysis. Tissuesamples were routinely fixed in10%buffered formalin, embedded inparaffin, and cut into5-μm-thick sections. The sections were subjected tohaematoxylin and eosin (H&E) staining using standard procedures. The diameter and cross-sectional area of adipocytes were determined using theNIS-Elements BR3.2software. Total RNA and protein were extracted fromadipose tissues, and the adiponectin mRNA level and the phosphorylatedAMP-activated protein kinase (p-AMPK) and acetyl-CoA carboxylase1(ACC1) protein amounts were assessed.Results: Animals in the HD group had a significant elevation in thediameter and cross-sectional area of adipocytes compared to the normalgroup (p <0.05). Probucol intervention significantly decreased the diameterand cross-sectional area of adipocytes in animals fed the HFD(p <0.05relative to the HFD group). The adiponectin mRNA level was significantlyelevated by probucol treatment (p <0.05relative to the HD group).Moreover, compared to the HD group, probucol-treated animals hadsignificantly increased levels of p-AMPK and decreased concentrations ofACC1(p <0.05).Conclusions: Probucol treatment diminishes the adipocyte size inanimals fed the HFD and enhances the adiponectin production fromadipocytes. Moreover, probucol treatment interferes with fatty acid synthesisin adipocytes through the AMPK pathway. Part3: Beneficial effects of probucol on lipid metabolism,oxidative stress, and inflammation response in a rat model ofnon-alcoholic steatohepatitisObjective: To uncover the mechanisms for the beneficial effects ofprobucol on lipid metabolism in non-alcoholic steatohepatitis (NASH) anddetermine the influence of probucol on NASH-associated oxidative stressand inflammation response.Methods: Forty male Sprague-Dawley rats were randomly assigned toone of the4groups:(1) normal control group (NC group)(n=10) withstandard diet feeding,(2) high-fat diet group (HD group)(n=10) fed ahigh-fat diet composed of15%lard oil,2%pure cholesterol, and83%standard chow,(3) probucol control group (NP group)(n=10) fed high-fatdiet and received normal saline, and (4) probucol group fed high-fat diet andreceived probucol (500mg/kg body weight/day)(HP group)(n=10).Normal saline and probucol were given by gastric gavage for15weeks.After the feeding trial, rats were euthanized, and their livers were removedand snap-frozen in liquid nitrogen until use. The mRNA abundances of liverX receptor alpha (LXR-α), acetyl-CoA carboxylase1(ACC1), sterolregulatory element binding protein1C (SREBP-1C), and fatty acid synthase(FAS) were analyzed using a semi-quantitative reversetranscription-polymerase chain reaction (RT-PCR) technique. The proteinlevels of phosphorylated AMP-activated protein kinase (p-AMPK),3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase, LXR-α,SREBP-1C, and ACC1in the liver were determined using Western blotanalysis.10%(w/vol) liver homogenate was prepared in physiological saline. The homogenate was centrifuged at2,500×g for10min at4°C. Thesupernatant was used for assays. Malondialdehyde (MDA) levels and theactivities of total superoxide dismutase (SOD), catalase (CAT), andglutathione peroxidase (GSH-Px) were measured using commerciallyavailable kits. The enzyme activities were expressed as units per milligramof protein (U/mg protein). Protein concentration was measured with thebicinchoninic acid method using a commercially available kit. The nuclearfactor kappa B (NF-κB) protein level in the liver was assessed byimmunohistochemistry.Results: Compared to the normal group, high-fat-diet feeding resultedin a significantly increase in the mRNA and protein levels of LXR-α,SREBP-1c, FAS, and ACC1and in the protein levels of HMG-CoAreductase (p <0.05). The p-AMPK protein level was significantly decreasedin the HD group relative to the normal group (p <0.05). Notably, these geneexpression changes were reversed by probucol intervention (p <0.05between the HD and probucol groups). Immunohistochemistry revealed asignificant reduction in the NF-κB level in the probucol-treated animalscompared to the HD group (p <0.05). There was a significant elevation inthe MDA concentration in the HD group relative to the normal group,whereas the activities of SOD, CAT, and GSH-Px were significantly lower inthe HD group than in the normal group (p <0.05). Interestingly, probucoltreatment significantly reduced the MDA level and concomitantly increased the activities of SOD, CAT, and GSH-Px in animals with the HFD (p <0.05relative to the HD group).Conclusions: Probucol treatment reduces hepatic de novo fatty acidsynthesis in the rat fed the HFD through alteration of the expression of a setof genes involved in lipid metabolism. Moreover, probucol intervention canattenuate oxidative stress and inflammation responses, thereby contributingto an improvement of hepatic steatosis and lobular inflammation inHFD-induced NASH.