Study Of Intestinal Perfusion Of Unacylated Ghrelin In The Treatment Of Metabolic Associated Fatty Liver Disease Via Gut-Brain-Liver Axis

Objective: Metabolic associated fatty liver disease(MAFLD)has a prevalence of about 25% globally and 42% in Southeast Asia.However,there is no effective drug to treat MAFLD.Ghrelin is a polypeptide containing 28 amino acids,which is mainly synthesized and secreted by gastrointestinal endocrine cells and has a variety of biological activities.There are mainly two forms of circulating ghrelin,acylated ghrelin(AG)and unacylated ghrelin(UAG),and UAG accounts for about 80-90% of circulating ghrelin.However,there are few studies on UAG at present which mainly focuse on its role in energy metabolism.In the present study,the intervention effect of intestinal infusion of UAG on MAFLD,and its mechanism were preliminarily explored.Methods: MAFLD pathological model rats were constructed by a high fat diet and the changes of feeding and body weight were recorded.Serum levels of glucose,insulin,total cholesterol(TC),triglyceride(TG),malondialdehyde,malondialdehyde(MDA),total superoxide dismutase(SOD),alanine aminotransferase(ALT),aspartate aminotransferase(AST),interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor(TNF-α),hapetic TG,fasting insulin(FINS),and fasting plasma glucose(FPG)were measured by microplate kit method,and homestasis model assessment for insulin resistance(HOMA-IR)was calculated.Fatty acid synthase(FAS),acetyl-Co A carboxylase(ACC),carnitine palmitoyltransferase-1A(CPT-1A),stearoyl-Co A desaturase-1(SCD-1)and insulin signaling pathway related proteins including mammalian target of rapamycin(m TOR),phosphorylated m TOR(p-m TOR),phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B,(Akt)and phosphorylated Akt(p-Akt),and inflammatory factor protein expression in rat liver were measured by Western Blot(WB).Hematoxylin-eosin staining(HE)was used to observe the pathological changes of rat liver.The expression of GLP-1R and GLP-1 in rat brain PVN and NTS was observed by immunocytochemistry(IHC).GLP-1 neural pathway from NTS to PVN was observed by retrograde neuronal tracking combined with immunofluorescence staining.The co-localization of GLP-1and c-fos in NTS was observed by immunofluorescence double staining method.The effect of exendin(9-39),a GLP-1R antagonist,and hepatic vagotomy on the treatment of To intestinal perfusion of UAG for MAFLD were observed.Results: Compared with the control group,the levels of body weight,blood lipid,transaminase,HOMA-IR and serum inflammatory factors in the MAFLD model group were significantly increased(P<0.01),and the level of liver TG was significantly increased(P<0.01),the liver tissue structure was disorganized with obvious steatosis and inflammatory cell infiltration,indicating that the MAFLD pathological model was successfully constructed.Intestinal infusion of UAG significantly reduced the levels of blood lipid,transaminase,HOMA-IR,liver TG,serum inflammatory factor and MDA(P<0.01),increased the level of serum SOD,and upregulated the protein expressions of p-PI3 K,p-Akt and p-m TOR in liver(P<0.01).Meanwhile,the protein expressions of fat synthetase including ACC,FAS and SCD-1 were decreased(P<0.01),the protein expressions of fat decomposition enzyme CPT1 A were increased(P<0.05),and the protein expressions of liver inflammatory factors TNF-α,NF-κB and IL-6 were decreased(P<0.01),and the steatosis of liver tissue was improved with decreased inflammatory cell infiltration.Intestinal perfusion of UAG has therapeutic effect on MAFLD.Moreover,intestinal perfusion of UAG activated GLP-1 immunopositive neurons in NTS,increased the number of GLP-1R immunopositive neurons in PVN,and increased the projection of GLP-1 neurons from NTS to PVN.However,the therapeutic effect of UAG on MAFLD rats was weakened by GLP-1R receptor antagonist exendin(9-39)was injected into PVN or hepatic vagotomy prior to intestinal perfusion of UAG.Conclusions: Intestinal infusion of UAG has a therapeutic effect on MAFLD.This effect is related to the activation of GLP-1 pathway from NTS to PVN by intestinal infusion of UAG.This pathway receives UAG signals from the gut and transmits them to the liver via the vagus nerve to regulate inflammatory response,lipid metabolism and insulin signaling pathways in the liver,thus playing a therapeutic role in MAFLD.The results have important significance for providing new strategies for the treatment of MAFLD and clarifying the biological activity of UAG.The mechanism based on the gut-brain-liver axis may be one of the mechanisms of intestinal perfusion of UAG,and a deeper and more comprehensive mechanism needs to be further explored.