CD47 Deficiency In Mice Exacerbates Chronic Fatty Diet-induced Steatohepatitis Through Its Role In Regulating Hepatic Inflammation And Lipid Metabolism

NAFLD is a clinical syndrome with hepatic steatosis as its hallmark.Its spectrum includes diseases from simple hepatic steatosis,known as non-alcoholic fatty liver(NAFL),to non-alcoholic steatohepatitis(NASH).Compared to NAFL,NASH is more severe.In condition to steatosis,the features of NASH include hepatocyte injury,inflammation,fibrosis and angiogenesis.NAFLD is closely related to obesity,insulin resistance,and genetic susceptibility,and is considered to be a manifestation of the metabolic syndrome(MS)in the liver.In recent years,the prevalence of NAFLD has been increasing worldwide,and has become the leading cause of end-stage liver disease and liver transplantation in some countries.Patients with NAFLD also have an increased risk of cardiovascular disease and hepatocellular carcinoma.There are currently no specific drugs for NAFLD.Therefore,investigating the pathogenesis and pathophysiology of NAFLD and NASH is very important to develop new therapeutic strategies.It is now believed that the pathogenesis of NAFLD / NASH follows the "multiple hits" hypothesis,including abnormal lipid metabolism,lipotoxicity,oxidative stress,endoplasmic reticulum stress,mitochondrial dysfunction,intestinal dysbacteria,and genetic susceptibility.These “hits” interact and progress liver from NAFL to NASH or even end-stage liver disease.Hepatocyte steatosis is one of the important characteristics of NASH.Long-term high-fat diet increases the intake of triglycerides(TG)on the one hand,and on the other hand induces insulin resistance(IR),and increases the hydrolysis of adipocyte and de novo synthesis of lipid in liver,leading to an increase in liver free fatty acid(FFA)content.Excess FFA is esterified into TG and is present in liver cells as lipid droplets.When its content exceeds the storage capacity of lipid droplets,FFA will cause lipotoxicity and further induce oxidative stress and endoplasmic reticulum stress.The stress response further affacts lipid homeostasis in hepatocytes.In addition to lipid disorder,FFA and LPS are also involved in inflammation.They can activate transcription factors such as NF-?B,therefore upregulate the expression and secretion of pro-inflammatory cytokines and chemokines.The pro-inflammatory cytokines activate Kupffer cells(KCs),and the chemokines recruit circulating monocytes/ macrophages to the liver,further exacerbate the inflammatory response.KCs can activate hepatic stellate cells(HSCs)to hepatic myofibroblasts(HMFs)through the TGF-? pathway.HMFs are the main sources of collagen and promote liver fibrosis.The hypoxic environment induced by fibrosis promotes pathological angiogenesis in the liver.As mentioned above,the pathological elements such as steatosis,inflammation and fibrosis affect each other and promote the development of NASH.NF-?B is an important transcription factor that can regulate a variety of apoptosis and inflammation-related gene expression,thus plays an important role in the pathogenesis of NASH.Under the normal condition,NF-?B binds to the inhibitory factor I?B in the cytoplasm.When I?B is phosphorylated by I?B kinase(IKK)and then degraded,NF-?B is released from inhibition and activated,and its p65 subunit is phosphorylated and transferred into the nucleus,initiating the target gene transcription.PPAR? is also an important transcription factor,which mainly regulates FA transport and mitochondrial ?-oxidation,but it is also involved in liver inflammation and fibrosis.PPAR? can directly bind to the p65 subunit of NF-?B,preventing it from binding to the target genes,thereby down-regulating the genes expression.PPAR? can also inhibit the activity of NF-?B by promoting the expression of I?B.Since NF-?B can promote the expression of various pro-inflammatory factors,PPAR? may exert an anti-inflammatory effect through its negative regulation of NF-?B.SIRT1 is a NAD +-dependent deacetylase that increase fatty acid oxidation(FAO)and down-regulate the expression of adipogenic genes by increasing PPAR? activity.SIRT1 can deacetylate the Rel / p65 subunit of NF-?B and reduce the transcriptional activity of Rel / p65,thereby inhibiting the activation of NF-?B.Thus SIRT1 can regulate NF-?B in both direct and indirect ways and exerting an anti-inflammatory effect.In summary,PPAR? and SIRT1 may reduce the expression level of pro-inflammatory factors by inhibiting the NF-?B pathway,and at the same time improve the lipid metabolism of liver cells,thereby improving the progress of NASH.CD47,also known as integrin-related protein(IAP),is widely expressed on the surface of many cells.CD47,interacting with its endogenous ligand thrombospondin(TSP-1),inhibits the effects of NO on endothelial cells(ECs)and vascular smooth muscle cells(VSMCs),reduces blood flow and angiogenesis.Studies have shown that the TSP-1 / CD47 pathway is involved in the pathogenesis of ischemia-reperfusion injury and atherosclerosis.CD47 can also interact with signal-regulating proteins(SIRP-?)expressed on the surface of macrophages,inhibiting phagocytosis,transducing an “do not eat me” signal,thus playing an important role in transplantation immunity.Recently,studies have shown that in a HFD-induced obese mouse model,compared with wild type(WT)mice,CD47 KO mice can resist weight gain caused by a high-fat diet and reduce hepatic steatosis.Circulating monocytes/macrophages infiltration and inflammatory response decreased,meanwhile glucose tolerance and insulin sensitivity were improved.These data reveal new functions of CD47 in obesity and related metabolic syndrome.Our previous work showed that the body weight and subcutaneous fat accumulation of HFD-fed CD47 KO mice were down-regulated compared with those of WT mice,but the hepatic steatosis increased,which was inconsistent with previous studies.Therefore,we designed the following experiments to further explore the role of CD47 in the pathogenesis of NASH.We used CD47 KO and C57 BL / 6(WT)mice,fed them with a high-fat diet(HFD)for 40 weeks to replicate a mouse NASH model.Mice body weight is measured monthly,and subtracted by baseline weight to get the monthly weight gain.At the designated time points,the mice were dissected,the tissues and serum were taken.First,we performed H & E and oil red O staining of liver sections,and measured the liver TG content to analyze the severity of liver lipid accumulation.At the same time,serum ALT was measured to assess the degree of liver damage.In order to investigate the mechanism of mouse hepatic steatosis,we measured the TC,HDL and LDL of serum,and measured the m RNA expression levels of liver apolipoproteins Apo A1,Apo B,Apo C2 and MTTP by q RT-PCR.Subsequently,we performed Sirius Red staining on liver sections to evaluate the liver fibrosis,and measured the m RNA and protein expression of liver profibrotic cytokine TGF-?,as well as the TGF-? activator TSP-1.Next,we used immunofluorescence staining to observe the quantity and distribution of CD31 + Tie + vascular endothelial cells,to evaluate the liver microvessel density.Together with the m RNA levels of CD31 and VEGFR,we are able to assess the pathological angiogenesis associated with fibrosis.Finally,we investigated the liver inflammation in this mouse NASH model.The expressions of IL-1?,TNF-?,IL-6,IL-10 and CCL2 were measured by q RT-PCR,and the protein expressions of IL-6,IL-10 and CCL2 were obtained by immunoblotting.In addition to pro-inflammatory factors and chemokines,we also observed the infiltration of CD68 + macrophages by immunofluorescence staining.As mentioned earlier,NF-?B is an important transcription factor that regulates the inflammatory response.Therefore,we also measured the phosphorylation and nuclear translocation of NF-?B by immunoblotting and immunofluorescence methods to analyze the NF-?B activation.In order to explore the mechanism of activation of inflammation in CD47 KO mice,we measured the gene expression of PPAR? and SIRT1,two transcription factors that regulate NF-?B activity in mouse liver.The results indicated that in the HFD-induced mouse liver NASH model,compared with WT mice,CD47 KO mice represented as:1.CD47 KO mice had decreased weight gain and subcutaneous fat accumulation,but hepatomegaly was more severe and liver weight / weight ratio was higher.Histopathological results showed that the size and number of lipid droplets in the liver increased,together with the increased liver TG content.It is suggested that HFD-induced liver steatosis is more serious in CD47 KO mice.2.Although the expression of apolipoprotein was higher in CD47 KO mice under the condition of low-fat diet(LFD),HFD significantly down-regulated the expression of apolipoprotein,and both CD47 KO and WT mice were down-regulated to the similar extent.This indicates that the significantly aggravated hepatic steatosis observed in CD47 KO mice cannot be explained by changes in the expression of apolipoproteins.3.The liver collagen deposition of CD47 KO mice increased,and the expression of pro-fibrotic cytokines TGF-? and TSP-1 was up-regulated,indicating that liver fibrosis was exacerbated in CD47 KO mice.Immunofluorescence staining showed that the number of CD31 + Tie + vascular endothelial cells increased,so were the m RNA expression of CD31 and VEGFR,suggesting that the absence of CD47 promoted angiogenesis associated with fibrosis.4.CD47 KO mice had more intense inflammatory reactions in their livers,with increased expression of pro-inflammatory factors such as IL-1?,TNF-?,and IL-6,and decreased expression of anti-inflammatory factors such as IL-10.In addition to cytokines,the infiltration of CD68 + macrophages increased,and the expression of chemokine CCL2 which recruited the macrophages were also up-regulated.As an important inflammatory regulator,NF-?B has increased phosphorylation and nuclear translocation of p65 subunits,indicating that the activation of NF-?B is enhanced in CD47 KO mice.5.PPAR? and SIRT1 are important transcription factors regulating inflammatory response and lipid metabolism,and they can exert anti-inflammatory effects through negative regulation of NF-?B.Under LFD conditions,the expression of these two molecules was higher in CD47 KO mice than in WT mice.But HFD significantly inhibited the expression of them in CD47 KO mice.It is suggested that CD47 deletion may indirectly promote the activity of NF-?B by inhibiting the activities of PPAR? and SIRT1,thereby up-regulating the expression of a lot of inflammatory and fibrotic factors and promoting liver inflammation.Taken together,our results indicate that CD47 plays a significant role in the pathogenesis of HFD-induced hepatosteatosis and fibrosis through its role in regulation of inflammation and lipid metabolism.