The Role Of Sema4D In Metabolic-Associated Fatty Liver Disease

Background:As an important lipid metabolic organ in the body,the liver plays an important coordinating role in fatty acid synthesis,lipid transport,and lipid oxidative decomposition.Abnormal liver lipid metabolism can cause fatty liver.The long-term accumulation of lipids will produce lipotoxicity,leading to stress,injury,and death of hepatocytes.Metabolism-associated fatty liver disease(MAFLD),characterized by the infiltration and accumulation of lipid droplets in liver tissue,affects an increasing number of people and has become an important cause of primary liver cancer and liver transplantation,and currently,there are no approved clinical drugs for MAFLD.Although overweight/obesity,type 2 diabetes and metabolic dysfunction are considered risk factors for MAFLD,the pathogenesis still needs further study.To explore the pathogenesis of MAFLD,we queried the GWAS database and found that the SNPs of SEMA4D,a member of the axon guidance molecular family,are related to body mass index and other relevant metabolic parameters in the population,suggesting that Sema4D may be involved in the pathogenesis of metabolic diseases.This study used a high-fat diet to induce a mouse MAFLD model and found that knocking out Sema4D in mice exacerbated liver lipid infiltration induced by a high-fat diet,and knocking out Sema4D in mice was also accompanied by the worsening of obesity.Objective:This study aimed to explore whether Sema4D plays a role in MAFLD.Methods:1.Analysis of single nucleotide polymorphism(SNP)of SEMA4D corrected with metabolic disordersGWAS CENTRAL(https://www.gwascentral.org/)of genome-wide association analysis(GWAS)was used to search for the correlation between SEMA4D and metabolic disorders(P<0.05).2.Expression of Sema4D in the liverUsing the Human Protein Atlas and Tabula Mauris database,we investigated the expression of Sema4D in different cell types of liver tissue.The liver and peripheral blood leukocytes of mice were collected,and the mRNA expression level of Sema4D was detected by Q-PCR.3.Effect of Sema4D knockout on HFD-induced MAFLD in miceC57BL/6J wild-type(WT)mice and Sema4D knockout(Sema4D-/-)mice of the same background were fed a Western diet(WD).The weight changes of the mice were monitored during feeding,and the weights of the mice were recorded during sampling,as well as the weights of the fat,liver,and spleen.Liver lipid droplet content and morphological changes were observed by tissue section staining.Partial liver tissue was split for triglyceride content detection.Simultaneously,WT and Sema4D-/-mice were fed a high-fat diet(60%HFD),and the above experiments were conducted.4.Effect of Sema4D-specific knockout in myeloid on WD-induced MAFLD in miceBy using the method of hybridization between Sema4Dfloxp/floxp mice and LysM-Cre mice,a mouse model of Sema4D-specific knockout in myeloid(Sema4Dfl/fl;LysM-Cre)was constructed.Mice were fed a WD to establish MAFLD.The weights of the body,adipose tissue,liver and spleen were recorded,and the liver tissue was sectioned and stained to observe the accumulation of lipid droplets in the liver and the morphological changes.5.Effect of recombinant Sema4D(rSema4D)on hepatocyte lipid metabolismSix-to eight-week-old WT mice were perfused,the liver was digested,and liver tissue was removed.Under aseptic conditions,primary hepatocytes were prepared and cultured on seed plates.After 30 minutes of stimulation with rSema4D,free fatty acids(FFAs)were added,and after 24 hours,lipid droplets accumulated in oil red O-stained cells.Another HepG2 cell line was used and subjected to the same treatment method for lipid absorption experiments.HepG2 cells were collected,and Q-PCR was used to detect the expression changes in lipid absorption-related genes.Results:1.The SNP of SEMA4D is correlated with metabolic disordersThrough the collation and analysis of the GWAS database,we found that many SNP sites located on SEMA4D have a significant correlation with metabolic disorders(P<0.05).2.Through single-cell data analysis and Q-PCR detection,the results showed that Sema4D was expressed in immune cells in liver tissue,such as T cells,monocytes/macrophages,NK cells,and B cells.Hewever,Sema4D was not detected in liver cells.The Sema4D receptors PlexinB1 and PlexinB2 can be detected in liver cells.3.Sema4D knockout promotes MAFLD induced by HFD(1)After HFD or WD,the weight of subcutaneous adipose tissue(SWAT)and spleen of Sema4D-/-mice increased significantly compared with WT mice(SWAT P<0.05,Spleen P<0.05),and the body weight,liver weight,and the ratio of liver weight to body weight also increased significantly(liver weight P<0.05,body weight P<0.01,liver weight to body weight P<0.05).(2)HE and Oil Red O staining also showed that more lipid droplets appeared in the livers of Sema4D-/-mice.But the contents of triglycerides(TG)and total cholesterol(TC)in the liver were no significant difference.(3)In the SWAT of Sema4D-/-mice,the fat hydrolysis-related gene Pnpla2(patatin-like phospholipase domain containing 2)was significantly downregulated compared to that in WT mice(P<0.001).4.Sema4D-specific knockout in myeloid promotes MAFLD induced by WDCompared with Sema4Dfl/fl mice,after WD,Sema4Dfl/fl LysM-Cre mice showed a significant increase in their Liver/body weight(P<0.05).Moreover,HE staining and oil red O staining suggested that the livers of Sema4Dfl/fl LysM-Cre mice exhibited more significant lipid droplet accumulation than those of Sema4Dfl/fl mice.This result indicates that Sema4D from myeloid cells is involved in MAFLD.5.Recombinant Sema4D can inhibit lipid droplet accumulation by hepatocytes in vitroWhen further exploring the mechanism of Sema4D in lipid metabolism,we used the prepared rSema4D to test lipid droplet accumulation in hepatocytes in vitro.The results showed that rSema4D inhibited lipid droplet accumulation in mouse primary hepatocytes and HepG2 cell lines(primary hepatocytes,P<0.005;HepG2 cells,P<0.05).In HepG2 cell lines,the addition of rSema4D significantly downregulated the expression of CD36,PPARy,and FAS(Fas cell surface death receptor)(CD36 P<0.01,PPARγ P<0.01,FAS P<0.05).Conclusions:This article discussed the role of the axon guidance molecule Sema4D in metabolic fatty liver disease.1.The SEMA4D gene contains many SNP sites related to metabolic indicators.2.In Sema4D knockout mice,HFD or WD aggravated lipid infiltration and inflammation in the liver.3.In myeloid-specific Sema4D knockout mice,the fatty liver induced by WD was aggravated.4.Recombinant Sema4D inhibits lipid droplet accumulation by hepatocytes.