| Objective:In this study,a mouse model of nonalcoholic fatty liver disease(NAFLD)was established based on a high fat and high fructose diet.The effect and mechanism of lycopene intervention on NAFLD mice by regulating hepatic NOD-like receptor proteins 3(NLRP3)inflammasome signaling pathway and intestinal microbiota were investigated.These results provide theoretical basis for the development and utilization of phytochemicals such as lycopene in the treatment of NAFLD.Method:1.Study design:Fifty 8-week-old male C57 mice were randomly divided into 5groups(10/group)after adaptive feeding for one week:the control group(LFD)was fed with control diet(10%fat)+drinking purified water;the high-fat and high fructose module(HFFD)was fed with high-fat feed(45%fat)+10%fructose water;the low-dose lycopene group(LLY)was fed with high-fat diet+10%fructose water+lycopene 20 mg/(kg·d)by gavage;the high-dose lycopene group(HLY)was fed with high-fat diet+10%fructose water+lycopene 60 mg/(kg·d)by gavage;resveratrol control group(RSV)was fed with high-fat diet+10%fructose water+resveratrol 50 mg/(kg·d)by gavage.During 8 weeks of intervention,the mice were free to feed and drink,weighed every week to adjust the dose of intragastric administration,and given equal volume of soybean oil intragastric administration in LFD group and HFFD group.At the end of the intervention,mice were fasted for 12 hours,and their serum,liver and cecum contents were collected for subsequent index detection.2.Laboratory analysis:Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)and fasting blood glucose(FBG)were detected by automatic biochemical analyzer.Histopathological changes of mouse liver were observed by HE staining.Serum insulin(INS)was detected by radioimmunoassay,and the homeostasis model insulin resistance index(HOMA-IR)was calculated.The activity of superoxide dismutase(SOD)and the level of malondialdehyde(MDA)in liver were detected by chemical kits.Liver tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA).Real-time PCR was used to detect cathepsin-B,thioredoxin interacting protein(TXNIP),toll-like receptor-4(TLR-4),nuclear factor-κB(NF-κB),NLRP3,interleukin-1β(IL-1β)and cysteine aspartase-1(Caspase-1)gene expression levels.Western blotting was used to detect the protein expression levels of Cathepsin-B,TXNIP,TLR-4,NF-κB,NLRP3,Caspase-1 and its precursor(Pro-Caspase-1),IL-1βand its precursor(Pro-IL-1β)in liver of mice.Microbial composition of cecal contents of mice was detected based on 16S r DNA intestinal flora sequencing.Results:1.After 8 weeks of high fat and high fructose feeding,weight gain,energy intake,fat weight and fat index of HFFD group were significantly higher than those of LFD group(P<0.05).Compared with HFFD group,weight gain and fat weight of mice in LLY group,HLY group and RSV group declined significantly(P<0.05).2.Compared with LFD group,the level of serum ALT in HFFD group was significantly higher(P<0.05).Compared with HFFD group,the level of serum ALT in LLY group,HLY group and RSV group were decreased significantly(P<0.05).The results of liver histological observation showed that the structure of liver lobules in LFD group was clear,hepatocytes were arranged neatly,and there were almost no fat vacuoles.In HFFD group,more than 1/3 hepatocytes in unit area had steatosis,disorder of hepatic cord arrangement and inflammatory cell infiltration.Compared with HFFD group,after administration of 20,60 mg/(kg·d)lycopene and 50 mg/(kg·d)resveratrol,the steatosis and inflammatory cells of mouse liver cells decreased in varying degrees.3.Serum TG,TC,HDL-C,LDL-C,FBG,INS,HOMA-IR,MDA,TNF-αand IL-6levels in HFFD group were significantly higher than those in LFD group(P<0.05).Compared with HFFD group,serum TG,TC,HDL-C,LDL-C and INS levels in HLY group were significantly decreased(P<0.05).Serum FBG level in LLY group and RSV group declined significantly(P<0.05).In addition,the levels of MDA,TNF-αand IL-6 in liver of mice in HLY group and RSV group were significantly lower than those in HFFD group(P<0.05).4.Compared with LFD group,the expression of Caspase-1 gene,NLRP3,Pro-Caspase-1 and Caspase-1 protein in HFFD group were significantly up-regulated(P<0.05).Compared with HFFD group,20,60 mg/(kg·d)lycopene and 50 mg/(kg·d)resveratrol significantly decreased the expression of Caspase-1 gene,NLRP3,Pro-Caspase-1 and Caspase-1 protein(P<0.05).The results showed that compared with LFD group,the expression levels of TXNIP protein,TLR-4 and NF-κB gene and protein in HFFD group were significantly up-regulated(P<0.05).Compared with HFFD group,the expression level of TXNIP protein in liver of HLY group decreased significantly(P<0.05).The expression levels of TLR-4,NF-κB gene and protein in liver of mice in HLY group and RSV group decreased significantly(P<0.05).5.16S rDNA sequencing results showed thatβ-diversity of intestinal flora was significantly isolated in the five groups(R~2=0.36183,P<0.001).At the phylum-level,the relative abundance of Frimicutes in 20,60 mg/(kg·d)lycopene treated groups were decreased significantly compared with the other three groups(P<0.05).At the genus-level,the relative abundance of Lachnospiraceae＿NK4A136＿group,Desulfovibrio and Alistipes in HFFD group were increased significantly compared with LFD group(P<0.05);the relative abundance of Alloprevotella and Blautia were decreased significantly(P<0.05).Compared with the HFFD group,the relative abundance of Lachnospiraceae＿NK4A136＿group,Desulfovibrio and Alistipes in LLY,HLY and RSV groups were reduced significantly(P<0.05).The relative abundance of Alloprevotella in LLY group and HLY group was significantly higher than that in HFFD group(P<0.05).Correlation analysis showed that there was a significant negative correlation between Alloprevotella and the expression of FBG,INS,HOMA-IR and NLRP3 related genes in serum of mice(P<0.05).Lachnospiraceae＿NK4A136＿group was significantly positively correlated with serum lipid level and liver IL-6 and TLR-4 gene expression levels of mice(P<0.05).Desulfovibrio has a significant positive correlation with the expression levels of IL-6 and NLRP3 related genes in mice liver(P<0.05).There was a significant positive correlation between Alistipes and TLR-4 gene expression level in liver of mice(P<0.05).Conclusion:Lycopene supplementation reduced the levels of hepatic lipid deposition,oxidative stress and inflammatory response in NAFLD mice.The potential mechanisms were related to the improved composition of intestinal microbiota and the regulating of NLRP3inflammasome signaling pathway in the liver by lycopene. |
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