Probiotics Regulating Notch Signaling Pathway In Acute Liver Failure

Background: Liver failure is a group of clinical syndromecharacterised by coagulopathy, jaundice, hepatic encephalopathy and ascites,with varous precipitating factors containing virus, drugs, alcohol, toxin, andmetabolic disorders. Acute liver failure (ALF) is a subtype known as promptonset, rapid aggravation which presenting hepatic encephalopaghy in nomore than two weeks, and high mortality. Intestinal endotoxemia has beenconsidered to play a key role in the complicating mechanisms of ALF.Lipopolysaccharide (LPS), served as a main component of endotoxin, hasbeen found to activate Notch signaling passway which is closely related tohepatic inflammation, whereas the effects is still controversal. Recent studieshave reported that Notch signaling passway could promote the secretion ofIL-10, while others have inverse results. It was reported, in macrophages,that Notch signaling pathway activated by LPS was associated with thesecretion of high mobility group protein B1(HMGB1), which is animportant mediator in the late phases of inflammation in endotoxemia and issupposed to play a pivotal role in the progression of ALF. Microecologicshave been used to treat liver disease for the therapeutic effects of maintainingthe balance of intestinal flora, decreasing the production and absorption ofthe intestinal endotoxin, and reducing the serum level of endotoxin. However,the definite mechanisms are uncertain. In addition, liver transplantation, themost effective therapy to liver failure, is still limited by the donor shortageand expensive cost. Therefore, the prevention of liver failure is seemedparticularly important. Currently, nonalcoholic fatty liver disease (NAFLD)is an important cause of liver disease second to viral hepatitis, the NAFLDpatients are more likely to progress to liver failure than healthy individuals.To some extent, early diagnosis and early treatment of NAFLD is needed to prevent liver failure. Therefore, nonalcoholic steatohepatitis (NASH), knownas the limited step of NAFLD, should be diagnosed as soon as possible toinhibit the progression of liver failure.Objection: In this study, the mice model of ALF was establishedthrough intraperitoneal injection with D-galactosamine, and probiotics wereadministrated to treat the model mice. To investigate changes andsignificance differences after probiotics, we detected the levels of serumalanine aminotransferase (ALT), aspartate aminotransferase (AST), IL-10,HMGB1, plasma LPS, liver tissue Jagged1, Notch1, Hes5, NICD, and CD68(a specific marker for macrophage activation). Liver tissue pathologicalforms were observed via HE staining. We cultured murine macrophageRAW264.7in vitro with LPS in the absence and presence of DAPT, which isa specific inhibitor of the Notch signaling pathway. To understand theregulatory role of the Notch signaling pathway induced by LPS inmacrophages, the changes in the expressions of Jagged1, Notch1, Hes5, andNICD in RAW264.7, as well as the levels of cells supernatant IL-10andHMGB1were observed. In addition, we cultured RAW264.7with the miceplasma of the normal control, ALF model and probiotic groups, respectively.To study the possible mechanism of ALF treatment with probiotic, theorganization, cellular and molecular levels, levels of LPS, IL-10, andHMGB1in the cell supernatant, and expressions of Jagged1, Notch1, Hes5,and NICD in cells were measured. The results provided a novel andexperimental basis for the prevention of ALF with probiotics. We alsodetected the serum indexes that were in close relationship with thepathogenesis of NAFLD, and then established the diagnostic model ofNASH using statistical methods. This study afforded new ideas for earlydiagnosis of NASH and prevention of liver failure.Methods:1The effects of probiotics on Notch signaling pathway in mice model ofALFThirty BALB/c male mice of clean grade, with6-8weeks old and 18-20g, were purchased from the Animal Experimental Center of HebeiMedical University. All mice were fed adaptively for1week, and thendivided into three groups randomly. All mice were continued to be fed withstandard diet. In addition, mice in the probiotics group (n=10) were perfusedwith probiotics (900mg/kg/d) for2weeks, whereas mice in normal control(n=10) and ALF model groups (n=10) were perfused with the same volumeof normal saline to meet the criteria of body mass for2weeks. At the end of2weeks, D–galactosamine (3.0g/kg) was administered throughintraperitoneal injection in ALF model and probiotics groups. The samevolume of normal saline was administered through intraperitoneal injectionin normal control group. At36h after the intraperitoneal injection, all micewere killed for the experiment. Liver tissue, serum, and plasma samples werecollected. Serum ALT and AST were measured by automatic biochemicalanalyzer, IL-10and HMGB1were examined by enzyme-linkedimmunosorbent assay (ELISA), and plasma LPS was checked by limuliodreagent inspection method. Some liver tissues were fixed in10%formaldehyde, embedded in paraffin, and then stained withHematoxylin-eosin (HE) The expressions of Jagged1, Notch1, NICD andHes5in the liver were detected via real-time quantitative polymerase chainreaction (real time-PCR) and western blot, respectively. The expression ofCD68in mice liver was measured via immunohistochemical stainingmethod.2The regulatory role of Notch signaling pathway in the macrophageswith LPSMurine macrophage RAW264.7were purchased from the Cell Centerof the Institute of Basic Medical Sciences, Chinese Academy of MedicalSciences. RAW264.7in logarithmic phase were inoculated in sixwell plates(5×105cells per well), and cultured in DMEM medium with high glucosecontaining10%fetal bovine serum. After they were grown to70%confluence, the cells were divided into three groups randomly. Normalcontrol group: RAW264.7cells were cultured in DMSO (the same volume of DAPT) for24h, then with1×PBS buffer solution (the same volume ofLPS) for30h. LPS group: RAW264.7cells were cultured in DMSO (thesame volume of DAPT) for24h, then with LPS (100ng/ml) for30h.LPS+DAPT group: RAW264.7cells were cultured in DAPT (10μM) for24h, then with LPS (100ng/ml) for30h. All culture cells and supernatantswere collected. The levels of cell supernatants IL-10and HMGB1wereexamined via ELISA. Moreover, the expressions of Notch1, Hes5and NICDwere measured by real time-PCR and western blot, respectively.3Molecular mechanism of cytokine secretion regulation bymacrophages with probiotics mice plasmaRAW264.7cells in logarithmic phase were inoculated in sixwell plates(5×105cells per well), and cultured in DMEM medium with high glucosecontaining10%fetal bovine serum. After they were grown to70%confluence, the cells were divided into three groups randomly. Normal miceplasma group: RAW264.7cells were cultured in20%normal mice plasmafor48h. ALF mice plasma group: RAW264.7cells were cultured in20%ALF mice plasma for48h. Probiotics mice plasma group: RAW264.7cellswere cultured in20%plasma of probiotics mice for48h. All cells andsupernatants were collected. The levels of cell supernatants IL-10andHMGB1were examined by ELISA, the levels of supernatant LPS waschecked by limuliod reagent inspection method. Moreover, the expressionsof Jagged1, Notch1, NICD and Hes5in RAW264.7cells were detected byreal time-PCR and western blot, respectively.4Cytokeratin18, alanine aminotransferase, platelets and triglyceridespredict the presence of nonalcoholic steatohepatitisLiver biopsy has been done in all enrolled patients, and they werediagnosed with NAFLD based on the guideline for diagnosis and treatmentof NAFLD (new revised edition2010) by the Chinese national workshop onfatty liver and alcoholic liver disease for the Chinese liver diseaseassociation. The exclusion criteria are listed as follows:(a) alcoholic fattyliver disease (alcohol consumption≥40g/d for male or≥20g/d for female during past five years) or excessive alcohol consumption (≥140g/wk formale or≥70g/wk for female);(b) viral hepatitis;(c) autoimmune liverdiseases;(d) drug-or toxin-induced liver steatosis (with no drugs or toxinswhich can induce hepatotoxicity);(e) genetic or metabolic liver diseases,such as Wilson’s disease, hemochromatosis, and alfa-1antitrypsin deficiency;(f) biliary obstruction (by ultrasonography). The patients were divided intotwo groups, namely, non-nonalcoholic steatohepatitis (non-NASH) andNASH (by liver histology). Body mass index (BMI) and waist-on-hip ratio(WHR) were calculated in all patients, and the history of diabetes mellitus(DM), hypertension, dyslipidemia and smoking habits were obtained byregular doctor visit. The laboratory evaluation in all patients included: ALT,AST, total bilirubin (TB), albumin (ALB), alkaline phosphatase (ALP),γ-glutamyl transpeptidase (γ-GT), international normalized ratio (INR),platelets, white blood cell (WBC), creatinine, Uric acid (UA), fasting glucose(FG), triglycerides (TG), total cholesterol (TC), hemoglobin andhs-C-reactive protein (hs-CRP). The level of serum CK-18-Asp396wasdetected by ELISA.The results were presented as means±standard deviation. Normalityand homogeneity of variance were tested before single-factor analysis ofvariance. All data were compared between the two groups throughLeast-Significant-Difference method. Two-tailed P <0.05was consideredstatistically significant, whereas P <0.01was considered to exhibit verysignificant difference. Linear correlation analysis tested the relationshipsamong the variables. SPSS13.0version was used to compute the collecteddata.Results:1The effects of probiotics on Notch signaling pathway in mice model ofALF1.1General condition of mice:Normal mice in good spirits and with good appetite, as well as normalresponse to external stimuli and pain were used. ALF mice in low spirits and with poor appetite, bradykinesia and diminished response to external stimuli.The mental condition, appetite and response to external stimuli in probioticsmice had been ranged between normal mice and model mice.1.2Changes in levels of serum ALT, AST, HMGB1, IL-10, and plasma LPSin mice:The levels of serum ALT (848.404±94.828U/L), AST (911.490±67.652U/L), HMGB1(101.909±12.428μg/L), IL-10(4627.884±842.453pg/ml)and plasma LPS (11.801±0.887EU/ml) in the ALF model group were higherthan those in normal control group (38.994±9.628U/L,55.279±7.499U/L,20.733±5.369μg/L,1064.924±238.455pg/ml,0.578±0.119EU/ml)(all P <0.01). Compared with the ALF model group, the levels of ALT(689.891±84.649U/L), AST (776.026±61.892U/L), HMGB1(82.556±9.719μg/L), IL-10(3182.596±769.235pg/ml), and LPS (7.396±0.919EU/ml) inthe probiotics group were significantly lower (all P <0.01).1.3Changes in liver histopathology:Under a light microscope, normal hepatocytes were observed to bearranged radially from the central veins based on HE staining in normalcontrol group. Histopathological examinations of the liver biopsies in theALF model showed that the structure of liver tissues was destroyed, thehepatic cord in the hepar was disarranged, and large necrosis area andsignificant inflammatory cellular infiltration were present. In addition, theremaining hepatocytes exhibited swelling and ballooning. The degrees ofnecrosis, degeneration, and infiltration of inflammation cells in the liver werelesser in the probiotics group than those in the ALF group.1.4Expressions of Jagged1, Notch1, Hes5and NICD in mice hepatic tissues:Compared with the normal control group, the expressions of Jagged1,Notch1, Hes5and NICD were increased in the liver of ALF model mice (allP <0.01). The expressions of the above indexes were decreased significantlyin the probiotics group compared with those in the ALF model group (P <0.05or P <0.01). 1.5Expression of CD68protein in mice hepatic tissues:The expression of CD68protein in ALF model mice (0.631±0.067μm2)was higher than that in normal control mice (0.339±0.071μm2)(P <0.01).Compared with the ALF model group, the expression of CD68protein in thehepatic tissues of the probiotics group (0.460±0.094μm2) was decreased, thedifference was significant statistically (P <0.01).1.6Correlation analysis:The level of plasma LPS in mice was positively correlated with thelevels of ALT (r=0.936), AST (r=0.946), Jagged1mRNA (r=0.947), Notch1mRNA (r=0.945), Hes5mRNA (r=0.948), Jagged1protein (r=0.894),Notch1protein (r=0.829), NICD protein (r=0.926), Hes5protein (r=0.907),HMGB1(r=0.942), IL-10(r=0.900) and CD68protein (r=0.973), thedifference were significant statistically (all P <0.01).2The regulatory role of Notch signaling pathway in the macrophageswith LPS2.1Changes in levels of HMGB1and IL-10in RAW264.7cell supernatant:Low levels of HMGB1(0.213±0.046μg/L) and IL-10(59.192±23.304pg/ml) were observed in the culture supernatant of normal RAW264.7cells.The levels of HMGB1(7.441±0.634μg/L) and IL-10(315.188±79.133pg/ml) in the supernatant of RAW264.7cells with LPS increased, thedifference were significant statistically (all P <0.01). Compared with theLPS group, the levels of HMGB1(6.218±0.711μg/L), IL-10(252.060±57.633pg/ml) in the LPS+DAPT group were significantlydecreased (P <0.05or P <0.01).2.2Effects of DAPT on the expression of Notch1mRNA, NICD protein,Hes5mRNA and protein in RAW264.7cells through LPS stimulation:Compared with the normal RAW264.7cells group, the expressions ofNotch1mRNA (6.493±0.727), NICD protein (0.646±0.100), Hes5mRNA(7.301±0.851) and protein (0.957±0.097) in the LPS group were increased,the difference were significant statistically (all P <0.01). The expressions ofNotch1mRNA (3.203±0.682), NICD protein (0.424±0.045), Hes5mRNA(4.722±0.665) and protein (0.838±0.087) in the LPS+DAPT group were higher compared with those in the LPS group (all P <0.01).2.3Correlation analysis:The level of HMGB1in culture supernatant was positively correlatedwith Notch1mRNA (r=0.849), NICD protein (r=0.933), Hes5mRNA(r=0.833) and protein (r=0.866) in RAW264.7cells, the difference weresignificant statistically (all P <0.01). The level of IL-10in cells supernatantwas also positively correlated with the levels of cell Notch1mRNA(r=0.798), NICD protein (r=0.810), Hes5mRNA (r=0.821) and protein(r=0.834), the difference were significant statistically (all P <0.01).3Molecular mechanism of cytokine secretion regulation bymacrophages with probiotics mice plasma3.1Changes in levels of HMGB1, IL-10, and LPS in supernatant of RAW264.7cells with mice plasma:Low levels of HMGB1(3.433±0.674μg/L), IL-10(286.524±33.507pg/ml), and LPS (0.090±0.014EU/ml) were observed in the supernatant ofRAW264.7cells with normal mice plasma. The levels of HMGB1(34.691±9.058μg/L)， IL-10(1812.736±185.574pg/ml) and LPS(2.102±0.121EU/ml) in the supernatant of RAW264.7cells with ALF miceplasma were increased, the difference were significant statistically (all P <0.01). Compared with the ALF mice plasma group, the levels of HMGB1(20.058±2.278μg/L), IL-10(1338.322±151.339pg/ml), and LPS(1.220±0.069EU/ml) in the probiotics mice plasma group were significantlydecreased (all P <0.01).3.2Changes in expressions of Jagged1, Notch1, Hes5and NICD in RAW264.7cells with mice plasma:Compared with the normal mice plasma group, the expressions ofJagged1mRNA (6.671±0.790), Notch1mRNA (5.491±0.657), Hes5mRNA(6.301±0.930), Jagged1protein (0.631±0.067), Notch1protein(0.533±0.091), NICD protein (0.600±0.086), and Hes5protein (0.803±0.087)in the ALF mice plasma group were increased, the difference weresignificant statistically (all P <0.01). The expressions of Jagged1mRNA (4.120±0.623), Notch1mRNA (2.375±0.739), Hes5mRNA (3.657±0.755),Jagged1protein (0.460±0.094), Notch1protein (0.326±0.069), NICD protein(0.332±0.064), and Hes5protein (0.605±0.097) were decreased in theprobiotics mice plasma group compared with those in the ALF mice plasmagroup (all P <0.01).3.3Correlation analysis:The level of LPS in supernatant of RAW264.7cells with mice plasmawas positively correlated with the levels of Jagged1mRNA (r=0.969),Notch1mRNA (r=0.911), Hes5mRNA (r=0.955), Jagged1protein (r=0.931),Notch1protein (r=0.889), NICD protein (r=0.937), Hes5protein (r=0.940),HMGB1(r=0.913) and IL-10(r=0.965), the difference were significantstatistically (all P <0.01).4Cytokeratin18, alanine aminotransferase, platelets and triglyceridespredict the presence of nonalcoholic steatohepatitis4.1Characteristics of the study indexes:Compared with the non-NASH group, the levels of BMI, WHR, AST,ALT, ALP, γ-GT, platelets, UA, hs-CRP, and TG were significantly higherin the NASH group (all P <0.05), whereas ALB was lower (P <0.05).Numerous indexes showed no significant differences between two groups.These indexes include the age of patient, gender, smoking habits, systolicblood pressure (SBP), diastolic blood pressure (DBP), serum bilirubin, WBC,hemoglobin, creatinine, INR, Ferritin, FG, TC, and the medical history ofDM, hypertension, and dyslipidemia.4.2Changes of levels of CK-18fragments M30and the correlation analysisbetween levels of CK-18fragments M30and liver histology:CK-18fragments levels were significantly higher in patients withNASH (372.9U/L (319.6,431.4)) than that of the non-NASH group (248.1U/L (237.5,266.6))(P <0.001). The CK-18fragment levels showed asignificant positive correlation with steatosis severity (r=0.492), ballooning(r=0.211), lobular inflammation (r=0.346), and fibrosis stage (r=0.407)(P <0.05or P <0.01). 4.3Multivariate analysis:Multivariate analysis included BMI, WHR, serum AST, ALT, ALB,ALP, γ-GT, platelets, UA, hs-CRP, TG, and CK-18fragments. ALT,platelets, CK-18fragments, and TG were the predictive factors for NASH.The AUROC curves were0.811(95%CI:0.722-0.899),0.631(95%CI:0.515-0.746),0.892(95%CI:0.824-0.960) and0.714(95%CI:0.611-0.818)for the ALT, platelets, CK-18fragments, and TG, respectively (P <0.05or P<0.01). CK-18fragment levels showed a significant positive correlationwith ALT (r=0.639) and TG (r=0.390)(all P <0.01), while no correlationwith platelets (P>0.05).4.4The model to predict NASH in NAFLD patients:A new model that combines ALT, platelets, CK-18fragments, and TGwas established through logistic regression in the NAFLD patients. Theequation of this model was:-12.764+0.075×ALT (U/L)+0.013×platelets(×109/L)+0.012×CK-18fragment levels (U/L)+0.006×TG (mg/dL). TheAUROC curve for the prediction of NASH was0.920(95%CI:0.866-0.974).A cutoff value of0.361, with a sensitivity of89%and a specificity of86%,has a positive predictive value of89%and a negative predictive value of89%.Conclusions:1Mice models of ALF were established through intraperitonealinjection of D-galactosamine, the high level of plasma LPS has an importantfunction in the development of ALF.2Elevated levels of plasma LPS could activate the Notch signalingpathway in macrophages to promote the secretion of HMGB1, a potentiallate inflammatory mediator, and IL-10, an anti-inflammatory cytokine,especially for increasing HMGB1, which is involved in liver injury causedby inflammation and accelerating progression of ALF.3Probiotics protected the liver by reducing the levels of LPS,decreasing the activation of macrophages in the liver tissue, inhibiting theNotch signaling pathway, and lowering the secretion of HMGB1and IL-10, thereby presenting a new idea and direction for clinical prevention of ALF.4The novel scoring system, which combines ALT, platelets, CK-18fragments, and TG, can accurately predict the occurrence of NASH. It mayplay a certain role in improving the progression of NAFLD, the prognosis ofNAFLD patients and the prevention of ALF.