Effect Of Butyrate On Alleviating High-Fat Diet-Induced NAFLD And Its Mechanisms In Rats

Non-alcoholic fatty liver disease(NAFLD),an invisible killer to animal and human health,has become the most common liver disease in the world.It is suggested that NAFLD occurred due to over intake of saturated fat,obesity and insulin resistance.Moreover,liver inflammation and oxidative stress play important roles in the complicated process of NAFLD progressing from simple steatosis to steatohepatitis(NASH),hepatofibrosis and hepatocarcinoma.Butyrate is a monocarboxylic acid composed of four C atoms,and is one of the most intensively studied short-chain fatty acids(SCFA).It is produced endogenously by the colonic bacterial anaerobic fermentation of fiber polysaccharides.The butyrate formed in gut is transported through the portal vein and eventually leads to a variety of biological effects on different tissues and organs.Butyric acid is not only the principle energy source of intestinal epithelial cell,but also a G protein coupled receptor activator and a deacetylase inhibitor regulating gene expression.Butyric acid can exert physiological functions such as anti-inflammatory,anti-tumor,anti-oxidative stress and inhibition of insulin resistance.It is reported that sodium butyrate(NaB)can alleviate NAFLD,however,the targeting genes and the molecular mechanism are still unclear.Besides,previous studies have focused more on the preventive effect of NaB on NAFLD and few on treatment efficacy.Therefore,to depict the alleviation of NaB on high-fat diet-induced and obesity and NAFLD,we establish high-fat diet-induced obese rat model and followed by gavage of NaB to the obese rats.To further understand the mechanism of the effects,AML12 cells were treated with oleic acid to mimic high-fat model in vitro followed by knockdown of a key gene in the signaling pathway.1 Sodium butyrate alleviates high-fat diet-induced obesity and insulin resistanceTwenty-four male specific pathogen-free(SPF)Sprague Dawley rats(6-week-old)were obtained from Beijing Vital River Laboratory Animal Technology Co.Ltd.and fed inAnimal Core Facility of Nanjing Medical University.The animals were housed in conventional conditions at 22±2? and 50-60%humidity with a lighting regime of 12 h light/12 h dark cycles.Within the first week,rats were allowed to access to food and water without restraint for assimilating into their environment.Then rats were randomly assigned to two groups,fed either a basal diet(CON,n=8)or a high-fat diet(HF,45%energy from fat,n=16)for 9 weeks to establish obesity.Afterwards,rats in the control group were continually kept on basal diet,while HF rats were then divided into two groups(n=8):HFB group was gavaged with 300 mg/kg NaB per rat every other day for 7 weeks;the other HF group as well as the CON group received same amount of saline.At the end of the 7th weeks,rats were fasted overnight and sacrificed with an anesthetic(1%barbital sodium,500 mg/kg).The blood and liver samples were collected and stored at-80?.The results have shown that the obese mouse model was established successfully by 9 weeks high-fat diet feeding with significantly(P<0.05)higher body weight compared to the rats fed control diet.Seven weeks of NaB treatment significantly(P<0.05)reduced the body weight,liver weight,epididymal fat weight and liver index in HFB rats.GTT test has shown that compared with the HF,HFB group had significantly lower glucose level in plasma after 30 min of intraperitoneal injection of glucose.High-fat diet decreased hepatic IRS1,PI3K and p-AKT protein levels,which was up-regulated by NaB treatment.Meanwhile,high-fat diet-induced up-regulation of AST and ALT as significantly reversed by NaB.These results indicate that oral administration of NaB was able to alleviate high-fat diet induced obesity,insulin resistance and liver damage in rats.2 Sodium butyrate alleviates high-fat diet-induced NAFLD by regulating the lipid metabolismHE and OR staining of liver sections showed obvious alleviation in hepatic lipid accumulation in HFB group.In liver,high-fat diet-induced increase of TG concentration was partially alleviated by NaB treatment,but no effect was observed on Tch concentration.There was no significant changes in Hormone sensitive lipase(HSL),adipose triglyceride lipase(ATGL)or p-HSL levels,but acetyl CoA carboxylase(ACC)and fatty acid synthase(FAS)protein level were significantly(P<0.05)decreased after NaB treatment.In the mean time,high-fat diet-induced down-regulation of hepatic PPAR? and CPT1 mRNA expression was significantly(P<0.05)reversed by NaB.Hepatic content of PPARa and CPT1 protein was not affected by high-fat diet,but it was significantly(P<0.05)increased in NaB-treated obese rats.Hepatic expression of mitochondrial DNA(mtDNA)-encoded genes was not significantly affected by either high-fat diet or NaB.However,the protein content of COX1 and COX4,as well as the enzyme activity of mitochondrial complex III and mitochondrial complex V was significantly decreased in HF group,which was completely(P<0.05)rectified after NaB treatment.These results suggest that NaB alleviates high-fat diet-induced NAFLD by down regulating lipid synthesis,improving hepatic mitochondrial function and stimulating fatty acid ?oxidation.3 Sodium butyrate alleviates high-fat diet-induced NAFLD by inhibiting liver inflammationHigh-fat diet increased hepatic IL-1?,TNFa and IL-18 mRNA expression as well as IL-1?and TNF? protein levels,which was alleviated after NaB treatment.High-fat diet significantly(P<0.05)increased IL-6 protein concentration,yet the mRNA expression was not altered.However,NaB treatment significantly(P<0.05)decreased IL-6 at both mRNA and protein levels.Moreover,the pro-inflammatory M1 macrophage marker F4/80 protein expression was up-regulated in the liver of high-fat diet-induced obese rats,which was completely rectified(P<0.05)by NaB.Meanwhile,the anti-inflammatory M2 macrophage marker CD206 protein tended to be lower(P=0.086)in HF group,and was significantly(P<0.05)increased in HFB group.Such alleviation effects were confirmed with immunofluorescence of another M1 macrophage marker CD68 and CD206.NaB significantly(P<0.05)attenuated high-fat diet-induced upregulation of p65 and TLR4 mRNA expression.IKKy decreased significantly(P<0.05)in HF group and restored in HFB group.In contrast,p-IKK?/? and p-p65 were significantly(P<0.05)increased in HF group and rectified by NaB.Furthermore,high-fat diet increased NLRP3 expression at both mRNA and protein levels,which was rectified by NaB.In addition,cleaved caspase-1 was significantly(P<0.05)up-regulated in HF group and suppressed obviously by NaB.Although apoptosis-associated speck-like protein(ASC)was not altered in HF group,NaB treatment significantly(P<0.05)decreased its mRNA expression level.Although GPR41 and GPR43 protein level in liver tissues remained unchanged,high-fat diet significantly increased(P<0.05)the protein level of HDAC1 in the liver,which was completely normalized(P<0.05)by NaB.Histone H3 acetyl K9(H3K9Ac)was detected on the promoter of TLR4,p65,IL-1?,NLRP3,ASC,CPT1,and PPARa genes with ChIP assays.H3K9Ac on PPARa promoter was significantly diminished in HF group,which was completely restored after NaB treatment(P<0.05).Moreover,protein-protein interaction between PPARa and p-p65 was detected by Co-IP assay.The ratio of immuoprecipitated PPARa to p-p65 was significantly decreased in HF group,and completely normalized in HFB.These results suggest that NaB gavage is effective in treating high-fat diet-induced adult-onset fatty liver disease through epigenetic regulation of PPARa to stimulate hepatic fatty acid ? oxidation and inhibit inflammation.4 Sodium butyrate alleviates high-fat diet-induced NAFLD by inhibiting oxidative stressHigh-fat diet-induced down-regulation of anti-oxidative stress markers GSH and SOD was significantly(P<0.05)reversed by NaB.The product of lipid peroxidation MDA was increased in HF group,which was completely(P<0.05)rectified after NaB treatment.Additionally,high-fat diet decreased hepatic expression Nrf2 at both mRNA and protein levels,which was up-regulated after NaB treatment.GCLC,GCLM,NQO-1 and HO-1,the enzymes associated with oxidative stress and be regulated by Nrf2,were decreased significantly(P<0.05)in HF group and restored in HFB group.Furthermore,siRNA was used to clarify the role of Nrf2 in alleviating NAFLD.Sodium butyrate can alleviate the increase of ROS caused by oleic acid,improve the enzyme activity of GSH and SOD(P<0.05)and up-regulate Nrf2,GCLC,HO-1 and NQO1 expression.In Nrf2 knockdown group,the effect of butyrate on the oxidative stress was diminished and PPARa protein expression was also decreased.To explore the mechanism of sodium butyrate in regulating the expression of Nrf2,level of H3K9Ac on the promoter of Nrf2 genes was detected with ChIP assays.H3K9Ac on Nrf2 promoter was significantly diminished in HF group,which was completely restored after NaB treatment(P<0.05).These results indicated that NaB gavage is effective in treating high-fat diet-induced adult-onset NAFLD through improving hepatic anti-oxidative stress by epigenetic regulation of Nrf2 that can.In conclusion,oral administration of NaB is able to alleviate NAFLD though regulating lipid metabolism,inhibiting inflammation and decreasing anti-oxidative stress by co-activating PPARa and Nrf2.