The Preventive Effects And Mechanism Of Sodium Alginate On Nonalcoholic Fatty Liver Disease In Rats

Objective:In this study,a rat model of nonalcoholic fatty liver disease(NAFLD)induced by high-fat diet was established to explore the ameliorative effect and potential molecular mechanism of sodium alginate(SA)supplementation on NAFLD.The findings would provide a theoretical basis for the rational use of marine algae dietary fiber to prevent NAFLD.Method:1.Study design:Thirty-two 6-week-old male Sprague-Dawley rats were randomly divided into the following 4 groups(8 rats/group)after 1 week of acclimatization:normal control group(NC)fed with a control diet(10%fat)and gavage with purified water;model group(HFD)fed with a high-fat diet(45%fat)and gavage with purified water;low-dose sodium alginate group(LSA)fed with a high-fat diet and gavage with 50 mg/kg/d sodium alginate;high-dose sodium alginate group(HSA)fed with a high-fat diet and gavage with150 mg/kg/d sodium alginate.Each rat was provided free access to water and diet.After 16weeks,rats were fasted for 12 hours,and blood was collected from the abdominal aorta to separate serum.Serum,liver,fat,cecum and other tissues were collected for further use.2.Laboratory analysis:Serum triglycerides(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by automatic biochemical analyzer.The contents of liver triglycerides(liver TG)and liver total cholesterol(liver TC)were determined by the kit.The pathological changes were observed by hematoxylin-eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)and lipopolysaccharide(LPS)levels in serum;Quantitative real-time PCR was used to detect Toll-like receptor-4(TLR-4),nuclear factor-κB(NF-κB),NOD like receptor protein3(NLPR3),Cysteine aspartase-1(Caspase-1)and Interleukin-1β(IL-1β)gene relative expression levels in liver;the protein relative expression levels of TLR-4,NF-κB,NLRP3,Caspase-1 and IL-1βin liver were detected by Western Blotting(WB).Intestinal flora sequencing based on 16S r DNA was used to analyze the microbial composition of cecum contents in rats.The contents of short chain fatty acids(SCFAs)in rat cecum were detected by gas chromatography-mass spectrometry.Results:1.After 16 weeks of intervention,the body weight,epididymal fat weight,perirenal fat weight,serum TG,ALT,AST,TNF-αlevels,liver TG and TC levels of rats in HFD group were significantly higher than those in NC group,serum HDL-C levels was significantly lower than that in NC group(P<0.05).Compared with HFD group,the body weight,serum TG,ALT,TNF-α,liver TG and TC levels of rats in the SA intervention groups were significantly decreased,while serum HDL-C level was significantly increased(P<0.05).The HE staining of liver showed that HFD group exhibited a large number of fat vacuoles and accompanied inflammatory cell infiltration.Compared with HFD group,these symptoms were significantly improved after SA intervention.Meanwhile,NAS score was higher in the HFD group than that in the NC group(P<0.05).The NAS score was dramatically decreased after SA treatment in comparison with the HFD group(P<0.05).2.Gut microbiota analyze results showed thatβ-diversity was significantly isolated in the four groups(R~2=0.32354,P<0.001).At the genus level,HFD group displayed a higher abundance of Desulfovibrio and Ruminococcus＿torques＿group,and lower abundance of Oscillospiraceae＿UCG＿005,Lactobacillus,Ruminococcus and Turicibacter in comparison with the NC group(P<0.05).The gut community of LSA rats showed a significantly higher abundance of Oscillospiraceae＿UCG＿005,Butyricicoccaceae＿UCG＿009 and Colidextribacter compared with the HFD-associated gut community(P<0.05).The gut community of HSA rats showed a significantly higher abundance of Colidextribacter,unclassified＿Lachnospiraceae,Oscillibacter and lower levels of Allobaculum compared with the HFD-associated gut community(P<0.05).In addition,the serum LPS level of rats in the HFD group was significantly higher than that in the NC group,while the contents of acetic acid,propionic acid and isovaleric acid in cecum were significantly lower than those in the NC group(P<0.05).Compared with HFD group,the serum LPS content of rats in SA group was significantly decreased,and the acetic acid and propionic acid levels in cecum contents were significantly increased(P<0.05).3.Compared with NC group,TLR-4,NLRP3,Caspase-1 and IL-1βgene relative expression levels in the HFD group were significantly up-regulated(P<0.05).Compared with HFD group,50 mg/kg/d and 150 mg/kg/d SA intervention significantly down-regulated NLRP3 gene relative expression(P<0.05);In addition,the protein relative expression levels of TLR-4,NF-κB,NLRP3,Caspase-1 and IL-1βin HFD group were significantly higher than those in NC group(P<0.05).The protein relative expression levels of TLR-4 and NLRP3 in LSA group were significantly lower than those in HFD group(P<0.05).The protein relative expression levels of TLR-4,NF-κB,NLRP3,Caspase-1 and IL-1βin HSA group were significantly lower than those in HFD group(P<0.05).4.Correlation analysis between different bacteria and body weight,blood lipid,liver lipid,liver function,LPS,TNF-α,SCFAs and gene relative expression level of inflammatory pathway index showed that,the abundance of Oscillospiraceae＿UCG＿005and Butyricicoccaceae＿UCG＿009 showed negative correlations with body weight,serum ALT,LPS and other indexes,and showed positive correlations with acetic acid,propionic acid,butyric acid and other indicators(P<0.05).Colidextribacter and Oscillibacter displayed negative correlations with serum TNF-α(P<0.05).Ruminococcus,Lactobacillus and Turicibacter were inversely related to serum TG,liver TC,relative expression level of TLR-4 gene and other indicators(P<0.05).The relative abundance of Desulfovibrio,Allobaculum and Ruminococcus＿torques＿group exhibited a strong positive correlation with liver lipids,serum ALT and other indexes(P<0.05).Allobaculum and Ruminococcus＿torques＿group were also negatively correlated with acetic acid and other indexes(P<0.05).Conclusion:Sodium alginate supplementation improved the liver of lipid deposition and lipid metabolism disorder and decreased levels of serum inflammation in NAFLD rats.The mechanism may be related to the regulation of intestinal flora and its metabolites,further regulation of liver TLR-4/NF-κB/NLRP3 inflammatory signaling pathway.