Leukocyte immunoglobulin-like receptor B2 overexpression as a promising therapeutic target and noninvasive screening biomarker for colorectal cancer

BACKGROUND Colorectal cancer(CRC)has become the second most deadly malignancy in the world,and the exploration of screening markers and precise therapeutic targets is urgent.Our previous research identified leukocyte immunoglobulin-like receptor B2(LILRB2)protein as a characteristic protein of CRC,but the association between LILRB2 expression and clinicopathological features,the internal mechanism related to CRC progression,and screening diagnostic efficacy are not clear.Therefore,we hypothesized that LILRB2 is significantly highly expressed in CRC tissues,correlated with advanced stage and a poor prognosis,and could be used as a therapeutic target and potential screening biomarker for CRC.AIM To explore whether LILRB2 can be used as a potential therapeutic target and noninvasive screening biomarker for CRC.METHODS Patients who underwent radical surgery for CRC at China-Japan Friendship Hospital between February 2021 and October 2022 were included.Cancer and paracancerous tissues were collected to verify LILRB2 expression,and the association between LILRB2 expression and clinicopathological features was analysed.Serum was collected from CRC patients,adenoma patients and healthy controls during the same period to assess the diagnostic value of LILRB2 as a noninvasive screening biomarker,and its diagnostic value was further compared with that of the traditional markers carcinoembryonic antigen(CEA)and carbohydrate antigen 19-9(CA19-9).RESULTS A total of 58 CRC patients were included,and LILRB2 protein was significantly overexpressed in cancer tissues compared with paracancerous tissues(P<0.001).Angiopoietin-like protein 2(ANGPTL2)protein,as the ligand of LILRB2,was synergistically overexpressed in CRC tissues(P<0.001),and overexpression of LILRB2 and ANGPTL2 protein was significantly correlated with poor to moderate differentiation,vascular involvement,lymph node metastasis,distant metastasis,advanced tumor-node-metastasis stage and a poor prognosis(P<0.05),which suggested that LILRB2 and ANGPTL2 are closely associated with CRC progression.In addition,serum LILRB2 concentrations increased stepwise in healthy individuals,adenoma patients and CRC patients with statistically significant differences.The sensitivity of serum LILRB2 for the diagnosis of CRC was 89.74%,the specificity was 88.89%,the area under the curve was 0.95,and the diagnostic efficacy was better than that of conventional CEA and CA19-9.CONCLUSION LILRB2 protein can be used as a potential novel therapeutic target and noninvasive screening biomarker for CRC,which is beneficial for early screening and precise treatment.

microRNA-627-5p inhibits colorectal cancer cell proliferation,migration and invasion by targeting Wnt2

BACKGROUND microRNA-627-5p(miR-627-5p)dysregulation has been observed in several cancer types,such as hepatocellular carcinoma,oral squamous cell carcinoma,glioblastoma multiforme,and gastric cancer.The biological function of miR-627-5p in colorectal cancer(CRC)growth and metastasis is yet unclear.AIM To investigate the effects of miR-627-5p on the malignant biological properties of colorectal malignant tumour cells by targeting Wnt2.METHODS The levels of miR-627-5p in colorectal tumour tissues were assessed in Gene Expression Omnibus datasets.In order to identify Wnt2 transcript expression in CRC tissues,quantitative real-time polymerase chain reaction(qRT-PCR)analysis was used.Luciferase reporter tests were used to explore whether miR-627-5p might potentially target Wnt2.Wnt2 transcript and protein levels were detected in CRC cells with high miR-627-5p expression.To learn more about how miR-627-5p affects CRC development,migration,apoptosis,and invasion,functional experiments were conducted.Cotransfection with the overexpression vector of Wnt2 and miR-627-5p mimics was utilized to verify whether overexpression of Wnt2 could cancel the impact of miR-627-5p in CRC.Western blot and qRT-PCR were conducted to investigate the effects of miR-627-5p on the Wnt/β-catenin signalling pathway.RESULTS miR-627-5p was notably decreased in colorectal tumour tissues,while the gene level of Wnt2 was notably upregulated.A dual luciferase reporter assay revealed that miR-627-5p specifically targets the 3’-untranslated regions of Wnt2 and miR-627-5p upregulation markedly reduced the protein and gene expression of Wnt2 in CRC cells.In vitro gain-of-function assays displayed that miR-627-5p overexpression decreased CRC cells’capabilities to invade,move,and remain viable while increasing apoptosis.Wnt2 overexpression could reverse the suppressive functions of miR-627-5p.Moreover,upregulation of miR-627-5p suppressed the transcript and protein levels of the downstream target factors in the canonical Wnt/β-catenin signalling,such as c-myc,CD44,β-catenin,and cyclinD1.CONCLUSION miR-627-5p acts as a critical inhibitory factor in CRC,possibly by directly targeting Wnt2 and negatively modulating the Wnt/β-catenin signalling,revealing that miR-627-5p could be a possible treatment target for CRC.

Development and validation of machine learning models for nonalcoholic fatty liver disease

Background: Nonalcoholic fatty liver disease(NAFLD) had become the most prevalent liver disease worldwide. Early diagnosis could effectively reduce NAFLD-related morbidity and mortality. This study aimed to combine the risk factors to develop and validate a novel model for predicting NAFLD. Methods: We enrolled 578 participants completing abdominal ultrasound into the training set. The least absolute shrinkage and selection operator(LASSO) regression combined with random forest(RF) was conducted to screen significant predictors for NAFLD risk. Five machine learning models including logistic regression(LR), RF, extreme gradient boosting(XGBoost), gradient boosting machine(GBM), and support vector machine(SVM) were developed. To further improve model performance, we conducted hyperparameter tuning with train function in Python package ‘sklearn’. We included 131 participants completing magnetic resonance imaging into the testing set for external validation. Results: There were 329 participants with NAFLD and 249 without in the training set, while 96 with NAFLD and 35 without were in the testing set. Visceral adiposity index, abdominal circumference, body mass index, alanine aminotransferase(ALT), ALT/AST(aspartate aminotransferase), age, high-density lipoprotein cholesterol(HDL-C) and elevated triglyceride(TG) were important predictors for NAFLD risk. The area under curve(AUC) of LR, RF, XGBoost, GBM, SVM were 0.915 [95% confidence interval(CI): 0.886–0.937], 0.907(95% CI: 0.856–0.938), 0.928(95% CI: 0.873–0.944), 0.924(95% CI: 0.875–0.939), and 0.900(95% CI: 0.883–0.913), respectively. XGBoost model presented the best predictive performance, and its AUC was enhanced to 0.938(95% CI: 0.870–0.950) with further parameter tuning. Conclusions: This study developed and validated five novel machine learning models for NAFLD prediction, among which XGBoost presented the best performance and was considered a reliable reference for early identification of high-risk patients with NAFLD in clinical practice.

Proteomics-based identification of proteins in tumor-derived exosomes as candidate biomarkers for colorectal cancer

BACKGROUND Colorectal cancer(CRC)is the second leading cause of cancer-related death,with high morbidity worldwide.There is an urgent need to find reliable diagnostic biomarkers of CRC and explore the underlying molecular mechanisms.Exosomes are involved in intercellular communication and participate in multiple pathological processes,serving as an important part of the tumor microenvironment.AIM To investigate the proteomic characteristics of CRC tumor-derived exosomes and to identify candidate exosomal protein markers for CRC.METHODS In this study,10 patients over 50 years old who were diagnosed with moderately differentiated adenocarcinoma were recruited.We paired CRC tissues and adjacent normal intestinal tissues(>5 cm)to form the experimental and control groups.Purified exosomes were extracted separately from each tissue sample.Data-independent acquisition mass spectrometry was implemented in 8 matched samples of exosomes to explore the proteomic expression profiles,and differentially expressed proteins(DEPs)were screened by bioinformatics analysis.Promising exosomal proteins were verified using parallel reaction monitoring(PRM)analysis in 10 matched exosome samples.RESULTS A total of 1393 proteins were identified in the CRC tissue group,1304 proteins were identified in the adjacent tissue group,and 283 proteins were significantly differentially expressed between them.Enrichment analysis revealed that DEPs were involved in multiple biological processes related to cytoskeleton construction,cell movement and migration,immune response,tumor growth and telomere metabolism,as well as ECM-receptor interaction,focal adhesion and mTOR signaling pathways.Six differentially expressed exosomal proteins(NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28)were validated by PRM analysis and evaluated by receiver operating characteristic curve(ROC)analysis.The area under the ROC curve was 0.93,0.96,0.97,0.78,0.75,and 0.88(P<0.05)for NHP2,OLFM4,TOP1,SAMP,TAGL,and TRIM28,respectively,indicating their good ability to distinguish CRC tissues from adjacent intestinal tissues.CONCLUSION In our study,comprehensive proteomic profiles were obtained for CRC tissue exosomes.Six exosomal proteins,NHP2,OLFM4,TOP1,SAMP,TAGL and TRIM28,may be promising diagnostic markers and effective therapeutic targets for CRC,but further experimental investigation is needed.

Increased expression of brain-derived neurotrophic factor is correlated with visceral hypersensitivity in patients with diarrheapredominant irritable bowel syndrome

BACKGROUND Visceral hypersensitivity is considered to play a vital role in the pathogenesis of irritable bowel syndrome(IBS). Neurotrophins have drawn much attention in IBS recently. Brain-derived neurotrophic factor(BDNF) was found to mediate visceral hypersensitivity via facilitating sensory nerve growth in pre-clinical studies. We hypothesized that BDNF might play a role in the pathogenesis of diarrhea-predominant IBS(IBS-D).AIM To investigate BDNF levels in IBS-D patients and its role in IBS-D pathophysiology.METHODS Thirty-one IBS-D patients meeting the Rome IV diagnostic criteria and 20 ageand sex-matched healthy controls were recruited. Clinical and psychological assessments were first conducted using standardized questionnaires. Visceral sensitivity to rectal distension was tested using a high-resolution manometry system. Colonoscopic examination was performed and four mucosal pinch biopsies were taken from the rectosigmoid junction. Mucosal BDNF expression and nerve fiber density were analyzed using immunohistochemistry. Mucosal BDNF mRNA levels were quantified by quantitative real-time polymerase chain reaction. Correlations between these parameters were examined.RESULTS The patients had a higher anxiety score [median(interquartile range), 6.0(2.0-10.0) vs 3.0(1.0-4.0), P = 0.003] and visceral sensitivity index score [54.0(44.0-61.0)vs 21.0(17.3-30.0), P < 0.001] than controls. The defecating sensation threshold[60.0(44.0-80.0) vs 80.0(61.0-100.0), P = 0.009], maximum tolerable threshold[103.0(90.0-128.0) vs 182.0(142.5-209.3), P < 0.001] and rectoanal inhibitory reflex threshold [30.0(20.0-30.0) vs 30.0(30.0-47.5), P = 0.032] were significantly lower in IBS-D patients. Intestinal mucosal BDNF protein [3.46 E-2(3.06 E-2-4.44 E-2) vs3.07 E-2(2.91 E-2-3.48 E-2), P = 0.031] and mRNA [1.57(1.31-2.61) vs 1.09(0.74-1.42), P = 0.001] expression and nerve fiber density [4.12 E-2(3.07 E-2-7.46 E-2) vs1.98 E-2(1.21 E-2-4.25 E-2), P = 0.002] were significantly elevated in the patients.Increased BDNF expression was positively correlated with abdominal pain and disease severity and negatively correlated with visceral sensitivity parameters.CONCLUSION Elevated mucosal BDNF may participate in the pathogenesis of IBS-D via facilitating mucosal nerve growth and increasing visceral sensitivity.

Importance of SALL4 in the development and prognosis of hepatocellular carcinoma

AIM: To detect the expression of sal-like protein 4(SALL4) and to explore its relationship with clinicopathological characteristics and prognosis of hepatocellular carcinoma(HCC).METHODS: One hundred and twenty-six samples of HCC tissue, 44 of adjacent noncancerous cirrhotic tissue and 10 of liver hemangioma tissue, were obtained from patients who underwent hepatectomy for HCC at the Fourth Hospital of Hebei Medical University. None of the patients had received any form of treatment before the operation. After resection, all the tissues were fixed in 10% neutral formaldehyde and embedded in paraffin. Expression of SALL4 was detected by immunohistochemistry. Patients were followed up for postoperative survival until February 2014. The relationships between SALL4 expression level and clinicopathological data and prognosis of HCC were analyzed. RESULTS: SALL4 expression was negative in the 10 samples of tissue from liver hemangioma, was weakly positive in the two samples from adjacent noncancerous cirrhotic tissue, and positive in 58 samples of HCC tissues. The differences were statistically significant(P < 0.05). Expression of SALL4 was higher in patients with higher α-fetoprotein(AFP) levels, portal vein tumor thrombus, and later clinical stage based on the Barcelona Clinic Liver Cancer classification(P < 0.05). Among patients with negative expression, weakly positive expression, positive expression, and strongly positive expression of SALL4, the median survival time was 39, 25, 23, and 9 mo, respectively(P < 0.001). When both AFP and SALL4 were detected, patients who were negative for both AFP and SALL4, SALL4-positive only, AFP-positive only, and positive for both AFP and SALL4, had a median survival time of 41, 38, 31, and 12 mo, respectively(P < 0.001).CONCLUSION: Expression of SALL4 is relevant to the prognosis of HCC patients. Patients with higher expression levels of SALL4 and AFP have worse prognosis.

Altered profiles of fecal metabolites correlate with visceral hypersensitivity and may contribute to symptom severity of diarrhea-predominant irritable bowel syndrome

BACKGROUND Fecal metabolites are associated with gut visceral sensitivity,mucosal immune function and intestinal barrier function,all of which have critical roles in the pathogenesis of irritable bowel syndrome(IBS).However,the metabolic profile and pathophysiology of IBS are still unclear.We hypothesized that altered profiles of fecal metabolites might be involved in the pathogenesis of IBS with predominant diarrhea(IBS-D).AIM To investigate the fecal metabolite composition and the role of metabolites in IBSD pathophysiology.METHODS Thirty IBS-D patients and 15 age-and sex-matched healthy controls(HCs)underwent clinical and psychological assessments,including the IBS Symptom Severity System(IBS-SSS),an Italian modified version of the Bowel Disease Questionnaire,the Bristol Stool Form Scale(BSFS),the Hospital Anxiety and Depression Scale,and the Visceral Sensitivity Index.Visceral sensitivity to rectal distension was tested using high-resolution manometry system by the same investigator.Fecal metabolites,including amino acids and organic acids,were measured by targeted metabolomics approaches.Correlation analyses between these parameters were performed.RESULTS The patients presented with increased stool water content,more psychological symptoms and increased visceral hypersensitivity compared with the controls.In fecal metabolites,His[IBS-D:0.0642(0.0388,0.1484),HC:0.2636(0.0780,0.3966),P=0.012],Ala[IBS-D:0.5095(0.2826,0.9183),HC:1.0118(0.6135,1.4335),P=0.041],Tyr[IBS-D:0.1024(0.0173,0.4527),HC:0.5665(0.2436,1.3447),P=0.018],Phe[IBS-D:0.1511(0.0775,0.3248),HC:0.3967(0.1388,0.7550),P=0.028],and Trp[IBS-D:0.0323(0.0001,0.0826),HC:0.0834(0.0170,0.1759),P=0.046]were decreased in IBS-D patients,but isohexanoate[IBS-D:0.0127(0.0060,0.0246),HC:0.0070(0.0023,0.0106),P=0.028]was significantly increased.Only Tyr was mildly correlated with BSFS scores in all subjects(r=-0.347,P=0.019).A possible potential biomarker panel was identified to correlate with IBS-SSS score(R2 Adjusted=0.693,P<0.001).In this regression model,the levels of Tyr,Val,hexanoate,fumarate,and pyruvate were significantly associated with the symptom severity of IBS-D.Furthermore,visceral sensation,including abdominal pain and visceral hypersensitivity,was correlated with isovalerate,valerate and isohexanoate.CONCLUSION Altered profiles of fecal metabolites may be one of the origins or exacerbating factors of symptoms in IBS-D via increasing visceral sensitivity.

Increased intestinal mucosal leptin levels in patients with diarrhea-predominant irritable bowel syndrome

AIM To measure the leptin levels in patients with diarrheapredominant irritable bowel syndrome(IBS-D) and analyze the relationship of leptin with clinical features, visceral sensitivity, mast cells, and nerve fibers. METHODS Forty-two patients with IBS-D fulfilling the Rome Ⅲ criteria and 20 age-and sex-matched healthy controls underwent clinical and psychological evaluations using validated questionnaires(including IBS Symptom Severity Scale, IBS-specific Quality of Life, Hamilton Anxiety Scale, and Hamilton Depression Scale), along with colonoscopy, colonic mucosal biopsy, and visceral sensitivity testing. Serum leptin levels were assayed using enzyme-linked immunosorbent assay. Mucosal leptin expression and localization were evaluated using immunohistochemistry and immunofluorescence.Mucosal leptin m RNA levels were quantified using quantitative real-time reverse transcription polymerase chain reaction. Mast cell counts and activation rates were investigated by toluidine blue staining. Correlation analyses between these parameters were performed.RESULTS There were no statistically significant differences in age, gender, or body mass index between the IBS-D group and the control group. The median IBS Symptom Severity Scale score in the IBS-D group was 225.0(range, 100-475). IBS-D patients had significantly increased anxiety [IBS-D: median, 6.5; interquartile range(IQR), 3.3; control: median, 2.0; IQR, 2.0; P < 0.001] and depression(IBS-D: median, 7.0; IQR, 3.0; control: median, 3.0; IQR, 2.0; P < 0.001) scores. IBS-D patients had significantly lower first sensation threshold(IBS-D: median, 50.6; IQR, 25.9; control: median, 80.5; IQR, 18.6; P < 0.001), defecation sensation threshold(IBS-D: median, 91.5; IQR, 29.3; control: median, 155.0; IQR, 21.1; P < 0.001) and maximum tolerable threshold(IBS-D: median, 163.2; IQR, 71.2; control: median, 226.2; IQR, 39.3; P < 0.001). Mucosal leptin expression, as reflected by integrated optical density(IBS-D: median, 4424.71; IQR, 4533.63; control: median, 933.65; IQR, 888.10; P < 0.001), leptin mR NA expression(IBS-D: median, 1.1226; IQR, 1.6351; control: median, 0.8947; IQR, 0.4595; P = 0.009), and mast cell activation rate(IBS-D: median, 71.2%; IQR, 12.9%; control group: median, 59.4%; IQR, 18.88%; P < 0.001) were significantly increased in IBS-D patients. The colocalization of leptin and leptin receptors was observed on mast cells and PGP9.5-positive nerve fibers in the intestinal mucosa. Also, leptin expression was positively correlated with anxiety, depression, and the mast cell activation rate, but negatively correlated with the defecation sensation threshold and the maximum tolerance threshold during visceral sensitivity testing(adjusted P < 0.0038).CONCLUSION Increased levels of mucosal leptin may interact with mast cells and the nervous system to contribute to the pathogenesis of IBS-D.

Serotonin transporter and cholecystokinin in diarrhea-predominant irritable bowel syndrome: Associations with abdominal pain, visceral hypersensitivity and psychological performance

BACKGROUND Visceral hypersensitivity and psychological performance are the main pathophysiological mechanisms of irritable bowel syndrome(IBS).Previous studies have found that cholecystokinin(CCK)can enhance colon movement and that serotonin transporter(SERT)is a transmembrane transport protein with high affinity for 5-hydroxytryptamine,which can rapidly reuptake 5-hydroxytryptamine and then regulate its action time and intensity.We speculate that SERT and CCK might play a role in the pathogenesis of diarrheapredominant IBS(IBS-D)by affecting visceral sensitivity and the brain-gut axis.AIM To determine SERT and CCK levels in IBS-D patients diagnosed using Rome IV criteria and to analyze their associations with abdominal pain,visceral hypersensitivity and psychological performance.METHODS This study collected data from 40 patients with IBS-D at the China-Japan Friendship Hospital from September 2017 to April 2018 and 18 healthy controls.The severity of abdominal pain,visceral sensitivity and psychological performance were evaluated in IBS-D patients and healthy controls,the levels of SERT and CCK in plasma and colonic mucosa were evaluated,and the correlations between them were analyzed.RESULTS There were significant differences in the initial sensation threshold(31.00±8.41 mL vs 52.22±8.09 mL,P<0.001),defecating sensation threshold(51.75±13.57 mL vs 89.44±8.73 mL,P<0.001)and maximum tolerable threshold(97.25±23.64 mL vs 171.11±20.83 mL,P<0.001)between the two groups.IBS-D patients had more severe anxiety(7.78±2.62 vs 2.89±1.02,P<0.001)and depressive(6.38±2.43 vs 2.06±0.73,P<0.001)symptoms than healthy controls.Significant differences were also found in mucosal CCK(2.29±0.30 vs 1.66±0.17,P<0.001)and SERT(1.90±0.51 vs 3.03±0.23,P<0.001)between the two groups.There was a significant positive correlation between pain scores and mucosal CCK(r=0.96,0.93,0.94,P<0.001).Significant negative correlations between anxiety(r=-0.98;P<0.001),depression(r=-0.99;P<0.001),pain evaluation(r=-0.96,-0.93,-0.95,P<0.001)and mucosal SERT were observed.CONCLUSION IBS-D patients had psychosomatic disorders and visceral hypersensitivity.SERT and CCK might be involved in the pathogenesis of IBS-D by regulating the braingut axis and affecting visceral sensitivity.This provides a new potential method for identifying a more specific and effective therapeutic target.

Increased international normalized ratio level in hepatocellular carcinoma patients with diabetes mellitus

AIM: To determine the association of diabetes mellitus (DM) and international normalized ratio (INR) level in hepatocellular carcinoma (HCC) patients. METHODS: Our present study included 375 HCC patients who were treated at the China-Japan Friendship Hospital, Ministry of Health (Beijing, China), in the period from January 2003 to April 2012, and with a hospital discharge diagnosis of HCC. The demographic, clinical, laboratory, metabolic and instrumental features were analyzed. χ2 test, Student's t test and Mann-Whitney U test were used to compare the differences between HCC patients with and without DM. Unconditional multivariable logistic regression analysis was used to determine the association of DM and INR level in HCC patients. A sub-group analysis was performed to assess the effect of liver cirrhosis or hepatitis B virus (HBV) infection on the results. The Pearson correlation test was used to determine the relationship between INR level and fasting glucose. In addition, association between diabetes duration, and diabetes treatment and INR level was determined considering the potentially different effects. RESULTS: Of the total, 63 (16.8%) patients were diabetic (diabetic group) and 312 (83.2%) patients were diagnosed without diabetes (non-diabetic group). Their mean age was 56.4 ± 11.0 years and 312 (83.2%) patients were male. Compared with patients without DM, the HCC patients with diabetes were older (59.5 ± 10.3 vs 55.8 ± 11.1, P=0.015), had a lower incidence of HBV infection (79.4% vs 89.1%, P=0.033), had increased levels of systolic blood pressure (SBP) (133 ± 17 vs 129 ± 16 mmHg, P=0.048) and INR (1.31 ± 0.44 vs 1.18 ± 0.21, P=0.001), had lower values of hemoglobin (124.4 ± 23.9 vs 134.2 ± 23.4, P=0.003) and had a platelet count (median/interquartile-range: 113/64-157 vs 139/89-192, P=0.020). There was no statistically significant difference in the percentages of males, overweight or obesity, drinking, smoking, cirrhosis and Child classification. After controlling for the confounding effects of age, systolic blood pressure, hemoglobin, platelet count and HBV infection by logistic analyses, INR was shown as an independent variable [odds ratio (OR)=3.650; 95%CI: 1.372-9.714, P=0.010]. Considering the effect of liver cirrhosis on results, a sub-group analysis was performed and the study population was restricted to those patients with cirrhosis. Univariate analysis showed that diabetic patients had a higher INR than non-diabetic patients (1.43 ± 0.51 vs 1.25 ± 0.23, P=0.041). After controlling for confounding effect of age, SBP, hemoglobin, platelet count and HBV infection by logistic analyses, INR level remained as the sole independent variable (OR=5.161; 95%CI: 1.618-16.455, P=0.006). No significant difference in the relationship between INR level and fasting glucose was shown by Pearson test (r=0.070, P=0.184). Among the 63 diabetic patients, 35 (55.6%) patients had been diagnosed with DM for more than 5 years, 23 (36.5%) received oral anti-diabetic regimens, 11 (17.5%) received insulin, and 30 (47.6%) reported relying on diet alone to control serum glucose levels. No significant differences were found for the association between DM duration/treatment and INR level, except for the age at diabetes diagnosis. CONCLUSION: The INR level was increased in HCC patients with DM and these patients should be monitored for the coagulation function in clinical practice.

Altered metabolism of bile acids correlates with clinical parameters and the gut microbiota in patients with diarrhea-predominant irritable bowel syndrome

BACKGROUND Bile acids(BAs)have attracted attention in the research of irritable bowel syndrome with predominant diarrhea(IBS-D)due to their ability to modulate bowel function and their tight connection with the gut microbiota.The composition of the fecal BA pool in IBS-D patients is reportedly different from that in healthy populations.We hypothesized that BAs may participate in the pathogenesis of IBS-D and the altered BA profile may be correlated with the gut microbiome.AIM To investigate the role of BAs in the pathogenesis of IBS-D and the correlation between fecal BAs and gut microbiota.METHODS Fifty-five IBS-D patients diagnosed according to the Rome Ⅳ criteria and twentyeight age-,sex-,and body mass index-matched healthy controls(HCs)were enrolled in this study at the gastroenterology department of China-Japan Friendship Hospital.First,clinical manifestations were assessed with standardized questionnaires,and visceral sensitivity was evaluated via the rectal distension test using a high-resolution manometry system.Fecal primary BAs including cholic acid(CA)and chenodeoxycholic acid(CDCA),secondary BAs including deoxycholic acid(DCA),lithocholic acid(LCA),and ursodeoxycholic acid(UDCA)as well as the corresponding tauro-and glyco-BAs were examined by ultraperformance liquid chromatography coupled to tandem mass spectrometry.The gut microbiota was analyzed using 16S rRNA gene sequencing.Correlations between fecal BAs with clinical features and gut microbiota were explored.RESULTS Fecal CA(IBS-D:3037.66[282.82,6917.47]nmol/g,HC:20.19[5.03,1304.28]nmol/g;P<0.001)and CDCA(IBS-D:1721.86[352.80,2613.83]nmol/g,HC:57.16[13.76,1639.92]nmol/g;P<0.001)were significantly increased,while LCA(IBSD:1621.65[58.99,2396.49]nmol/g,HC:2339.24[1737.09,2782.40];P=0.002)and UDCA(IBS-D:8.92[2.33,23.93]nmol/g,HC:17.21[8.76,33.48]nmol/g;P=0.025)were significantly decreased in IBS-D patients compared to HCs.Defecation frequency was positively associated with CA(r=0.294,P=0.030)and CDCA(r=0.290,P=0.032)and negatively associated with DCA(r=−0.332,P=0.013)and LCA(r=−0.326,P=0.015)in IBS-D patients.In total,23 of 55 IBS-D patients and 15 of 28 HCs participated in the visceral sensitivity test.The first sensation threshold was negatively correlated with CDCA(r=−0.459,P=0.028)in IBS-D patients.Furthermore,the relative abundance of the family Ruminococcaceae was significantly decreased in IBS-D patients(P<0.001),and 12 genera were significantly lower in IBS-D patients than in HCs(P<0.05),with 6 belonging to Ruminococcaceae.Eleven of these genera were negatively correlated with primary BAs and positively correlated with secondary BAs in all subjects.CONCLUSION The altered metabolism of BAs in the gut of IBS-D patients was associated with diarrhea and visceral hypersensitivity and might be ascribed to dysbiosis,especially the reduction of genera in Ruminococcaceae.

Untargeted metabolomics characteristics of nonobese nonalcoholic fatty liver disease induced by high-temperature-processed feed in Sprague-Dawley rats

BACKGROUND Nonalcoholic fatty liver disease(NAFLD)has become one of the most common chronic liver diseases in the world.In our early clinical data and questionnaire analysis of NAFLD,it was found that the body mass index of some patients did not meet the diagnostic criteria for overweight or obesity.The consumption of high-temperature-processed foods such as fried food,hot pot and barbecue is closely related to the occurrence of nonobese NAFLD.Reducing the intake of this kind of food can reduce disease severity and improve prognosis.AIM To explore the untargeted metabolomics characteristics of nonobese nonalcoholic fatty liver disease in Sprague-Dawley rats induced by high-temperatureprocessed feed.METHODS Fifty-four male Sprague-Dawley rats were divided into three groups:The control group received a standard diet;the nonfried soybeans(NDFS)group received 60%NDFS and 40%basic feed and the dry-fried soybeans(DFS)group received 60%DFS and 40%basic feed.Six rats were sacrificed at week 4,8,and 12 in each group.The food intake,body weight,Lee’s index,liver index,serological index and hepatic histopathology were assessed.Untargeted metabolomics characteristics were used to analyze the changes in liver metabolites of rats at week 12.Correlations between metabolites and pathology scores between the DFS and control groups and between the DFS and NDFS groups were analyzed.We selected some of the metabolites,both within the pathway and outside of the pathway,to explain preliminarily the difference in liver pathology in the three groups of rats.RESULTS There were no statistically significant differences in the food intake,body weight,Lee's index or serological index between the DFS group and the control group(P>0.05).At week 8 and week 12,the steatosis scores in the DFS group were significantly higher than those in the other two groups(P<0.05).At week 12,the liver index of the DFS group was the lowest(NDFS group vs DFS group,P<0.05).The fibrosis score in the DFS group was significantly higher than those in the other two groups(P<0.05).The correlation analysis of the liver pathology score and differential metabolites in the DFS and NDFS groups showed that there were 10 strongly correlated substances:Five positively correlated substances and five negatively correlated substances.The positively correlated substances included taurochenodeoxycholate-3-sulfate,acetylcarnitine,20a,22bdihydroxycholesterol,13E-tetranor-16-carboxy-LTE4 and taurocholic acid.The negatively correlated substances included choline,cholesterane-3,7,12,25-tetrol-3-glucuronide,nicotinamide adenine dinucleotide phosphate,lysoPC[16:1(9Z)]and glycerol 3-phosphate.The correlation analysis of the liver pathology score and differential metabolites in the DFS and control groups showed that there were 13 strongly correlated substances:Four positively correlated substances and 9 negatively correlated substances.The positively correlated substances included 4-hydroxy-6-eicosanone,3-phosphoglyceric acid,13-hydroxy-9-methoxy-10-oxo-11-octadecenoic acid and taurochenodeoxycholate-3-sulfate.The negatively correlated substances included lysoPC[16:1(9Z)],S-(9-hydroxy-PGA1)-glutathione,lysoPC[20:5(5Z,8Z,11Z,14Z,17Z)],SM(d18:1/14:0),nicotinamide adenine dinucleotide phosphate,5,10-methylene-THF,folinic acid,N-lactoylglycine and 6-hydroxy-5-methoxyindole glucuronide.CONCLUSION We successfully induced liver damage in rats by using a specially prepared hightemperature-processed feed and explored the untargeted metabolomics characteristics.

Altered profiles of fecal bile acids correlate with gut microbiota and inflammatory responses in patients with ulcerative colitis

BACKGROUND Gut microbiota and its metabolites may be involved in the pathogenesis of inflammatory bowel disease.Several clinical studies have recently shown that patients with ulcerative colitis(UC)have altered profiles of fecal bile acids(BAs).It was observed that BA receptors Takeda G-protein-coupled receptor 5(TGR5)and vitamin D receptor(VDR)participate in intestinal inflammatory responses by regulating NF-ĸB signaling.We hypothesized that altered profiles of fecal BAs might be correlated with gut microbiota and inflammatory responses in patients with UC.AIM To investigate the changes in fecal BAs and analyze the relationship of BAs with gut microbiota and inflammation in patients with UC.METHODS The present study used 16S rDNA sequencing technology to detect the differences in the intestinal flora between UC patients and healthy controls(HCs).Fecal BAs were measured by targeted metabolomics approaches.Mucosal TGR5 and VDR expression was analyzed using immunohistochemistry,and serum inflammatory cytokine levels were detected by ELISA.RESULTS Thirty-two UC patients and twenty-three HCs were enrolled in this study.It was found that the diversity of gut microbiota in UC patients was reduced compared with that in HCs.Firmicutes,Clostridium IV,Butyricicoccus,Clostridium XlVa,Faecalibacterium,and Roseburia were significantly decreased in patients with UC(P=3.75E-05,P=8.28E-07,P=0.0002,P=0.003,P=0.0003,and P=0.0004,respectively).Proteobacteria,Escherichia,Enterococcus,Klebsiella,and Streptococcus were significantly enriched in the UC group(P=2.99E-09,P=3.63E-05,P=8.59E-05,P=0.003,and P=0.016,respectively).The concentrations of fecal secondary BAs,such as lithocholic acid,deoxycholic acid,glycodeoxycholic acid,glycolithocholic acid,and taurolithocholate,in UC patients were significantly lower than those in HCs(P=8.1E-08,P=1.2E-07,P=3.5E-04,P=1.9E-03,and P=1.8E-02,respectively)and were positively correlated with Butyricicoccus,Roseburia,Clostridium IV,Faecalibacterium,and Clostridium XlVb(P<0.01).The concentrations of primary BAs,such as taurocholic acid,cholic acid,taurochenodeoxycholate,and glycochenodeoxycholate,in UC patients were significantly higher than those in HCs(P=5.3E-03,P=4E-02,P=0.042,and P=0.045,respectively)and were positively related to Enterococcus,Klebsiella,Streptococcus,Lactobacillus,and pro-inflammatory cytokines(P<0.01).The expression of TGR5 was significantly elevated in UC patients(0.019±0.013 vs 0.006±0.003,P=0.0003).VDR expression in colonic mucosal specimens was significantly decreased in UC patients(0.011±0.007 vs 0.016±0.004,P=0.033).CONCLUSION Fecal BA profiles are closely related to the gut microbiota and serum inflammatory cytokines.Dysregulation of the gut microbiota and altered constitution of fecal BAs may participate in regulating inflammatory responses via the BA receptors TGR5 and VDR.

Potential role of diabetes mellitus in the progression of cirrhosis to hepatocellular carcinoma:a cross-sectional case-control study from Chinese patients with HBV infection

BACKGROUND: Diabetes mellitus (DM) is regarded as a new risk factor for hepatocellular carcinoma (HCC), but few studies have focused on the potential role of DM in the progression of cirrhosis to HCC as well as in patients with simple HBV infection. METHODS: A cohort of 1028 patients, treated at our hospital and with a hospital discharge diagnosis of HCC and/or cirrhosis, was screened. Among them, 558 were diagnosed with chronic HBV infection and 370 were analyzed statistically according to the diagnostic, inclusion and exclusion criteria. The demographic, clinical, metabolic, virological, biochemical, radiological and pathological features were analyzed and the multivariate logistic regression model was used to determine the potential role of DM. RESULTS: In 248 cirrhotic patients, 76 were diabetic and their mean duration of DM was 4.6 years. In 122 HCC patients with cirrhosis, 25 were diabetic and their mean duration of DM was 4.4 years. Univariate analysis showed that compared with cirrhotic patients, the HCC patients had a higher percentage in males (P=0.001), a lower percentage in DM patients (P=0.039), a higher percentage in cigarette smokers (P=0.005), a higher percentage in patients with AFP】400 ng/mL (P【0.001), higher values of white blood cells (P【0.001), hemoglobin (P【0.001) and platelet (P【0.001), increased levels of ALT (P【0.001) and GGT (P【0.001), higher total bilirubin (P=0.018) and albumin levels (P【0.001), and a lower international normalized ratio (P【0.001). Multivariate logistic regression analysis showed that DM was anindependent associated factor for HCC [odds ratio (OR)=0.376; 95% CI, 0.175-0.807; P=0.012]. Even after the HCC patients were restricted to those with decompensated cirrhosis and compared with decompensated cirrhotic patients, the similar result was observed (OR=0.192; 95% CI, 0.054-0.679; P=0.010). CONCLUSIONS: DM is an independent factor in the progression of cirrhosis to HCC, but the role may be contrary to our current viewpoint. To clarify the causal relationship of DM and HCC, prospective and experimental studies are required.

Identification of the circRNA-miRNA-mRNA regulatory network and its prognostic effect in colorectal cancer

BACKGROUND The high morbidity and mortality of colorectal cancer(CRC)have posed great threats to human health.Circular RNA(CircRNA)and microRNA(miRNA),acting as competing endogenous RNAs(ceRNAs),have been found to play vital roles in carcinogenesis.However,the biological function of ceRNAs in CRC pathogenesis and prognosis remains largely unexplored.AIM To identify the CRC-specific circRNA-miRNA-mRNA regulatory network and uncover the subnetwork associated with its prognosis.METHODS CircRNAs,miRNAs and mRNAs differentially expressed(DE)in CRC tissues were selected by expression file analysis in the Gene Expression Omnibus(GEO)database,and the downstream target molecules of circRNAs and miRNAs were predicted.Then,the intersection of differentially expressed RNA molecules with the predicted targets was determined to obtain a ceRNA network.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were conducted to elucidate the possible mechanism of pathogenesis.A survival analysis using the gene profiles and clinical information in The Cancer Genome Atlas(TCGA)database was performed to identify the mRNAs associated with the clinical outcome of CRC patients and construct a prognostic subnetwork.RESULTS We downloaded three datasets(GSE126095,GSE41655 and GSE41657)of largescale CRC samples from the GEO database.There were 55 DEcircRNAs,114 DEmiRNAs and 267 DEmRNAs in CRC tissues compared with normal tissues.After intersecting these molecules with predicted targets,19 circRNAs,13 miRNAs and 28 mRNAs were chosen to develop a circRNA-miRNA-mRNA network.GO and KEGG functional enrichment analyses indicated that the retinol metabolic process,leukocyte chemotaxis,extracellular matrix remodeling,endoplasmic reticulum stress,alcohol dehydrogenase activity,gastric acid secretion,nitrogen metabolism and NOD-like receptor signaling pathway might participate in the tumorigenesis of CRC.After verifying the identified mRNA effect in the TCGA database,we finally recognized 3 mRNAs(CA2,ITLN1 and LRRC19)that were significantly associated with the overall survival of CRC patients and constructed a ceRNA subnetwork including 5 circRNAs(hsa_circ_0080210,hsa_circ_0007158,hsa_circ_0000375,hsa_circ_0018909 and hsa_circ_0011536)and 3 miRNAs(hsa-miR-601,hsa-miR-671-5p and hsa-miR-765),which could contain innovative and noninvasive indicators for the early screening and prognostic prediction of CRC.CONCLUSION We proposed a circRNA-miRNA-mRNA regulatory network closely associated with the progression and clinical outcome of CRC that might include promising biomarkers for carcinogenesis and therapeutic targets.

Alteration of fecal tryptophan metabolism correlates with shifted microbiota and may be involved in pathogenesis of colorectal cancer

BACKGROUND Gut tryptophan(Trp)metabolites are produced by microbiota and/or host metabolism.Some of them have been proven to promote or inhibit colorectal cancer(CRC)in vitro and animal models.We hypothesized that there is an alteration of gut Trp metabolism mediated by microbiota and that it might be involved in the pathogenesis of cancer in patients with CRC.AIM To investigate the features of Trp metabolism in CRC and the correlation between fecal Trp metabolites and gut microbiota.METHODS Seventy-nine patients with colorectal neoplastic lesions(33 with colon adenoma and 46 with sporadic CRC)and 38 healthy controls(HCs)meeting the inclusion and exclusion criteria were included in the study.Their demographic and clinical features were collected.Fecal Trp,kynurenine(KYN),and indoles(metabolites of Trp metabolized by gut microbiota)were examined by ultraperformance liquid chromatography coupled to tandem mass spectrometry.Gut barrier marker and indoleamine 2,3-dioxygenase 1(IDO1)mRNA were analyzed by quantitative realtime polymerase chain reaction.Zonula occludens-1(ZO-1)protein expression was analyzed by immunohistochemistry.The gut microbiota was detected by 16S ribosomal RNA gene sequencing.Correlations between fecal metabolites and other parameters were examined in all patients.RESULTS The absolute concentration of KYN[1.51(0.70,3.46)nmol/g vs 0.81(0.64,1.57)nmol/g,P=0.036]and the ratio of KYN to Trp[7.39(4.12,11.72)×10^-3 vs 5.23(1.86,7.99)×10^-3,P=0.032]were increased in the feces of patients with CRC compared to HCs,while the indoles to Trp ratio was decreased[1.34(0.70,2.63)vs 2.46(1.25,4.10),P=0.029].The relative ZO-1 mRNA levels in patients with CRC(0.27±0.24)were significantly lower than those in HCs(1.00±0.31)(P<0.001),and the relative IDO1 mRNA levels in patients with CRC[1.65(0.47-2.46)]were increased(P=0.035).IDO1 mRNA levels were positively associated with the KYN/Trp ratio(r=0.327,P=0.003).ZO-1 mRNA and protein levels were positively correlated with the indoles/Trp ratio(P=0.035 and P=0.009,respectively).In addition,the genera Asaccharobacter(Actinobacteria)and Parabacteroides(Bacteroidetes),and members of the phylum Firmicutes(Clostridium XlVb,Fusicatenibacter,Anaerofilum,and Anaerostipes)decreased in CRC and exhibited a positive correlation with indoles in all subjects.CONCLUSION Alteration of fecal Trp metabolism mediated by microbiota is associated with intestinal barrier function and tissue Trp metabolism,and may be involved in the pathogenesis of CRC.

Comprehensive proteomic signature and identification of CDKN2A as a promising prognostic biomarker and therapeutic target of colorectal cancer

BACKGROUND The carcinogenesis of colorectal cancer(CRC)involves many different molecules and multiple pathways,and the specific mechanism has not been elucidated until now.Existing studies on the proteomic signature profiles of CRC are relatively limited.Therefore,we herein aimed to provide a more comprehensive proteomic signature profile and discover new prognostic markers and therapeutic targets by performing proteomic analysis of CRC and paired normal tissues.AIM To investigate the proteomic signature and identify novel protein prognostic biomarkers of CRC.METHODS Cancer tissues and paired normal tissues were collected from 48 patients who underwent surgical removal at the China-Japan Friendship Hospital from January 2020 to June 2021.Data independent acquisition(DIA)quantitative proteomic analysis was performed using high-performance liquid chromatography–mass spectrometry/mass spectrometry(nano-UHPLC–MS/MS)to identify differen tially expressed proteins,among which those with a P adj value(t test,BH correction)<0.05 and an absolute fold change(|log2FC|)>2 were identified as potential markers.Differentially expressed proteins were selected by bioinformatics analysis and validated by immunohistochemical tissue microarrays,and their association with prognosis was further analyzed with the Gene Expression Profiling Interactive Analysis database to identify prognostic protein biomarkers of CRC.RESULTS Significantly differential protein expression was observed between cancer tissues and normal tissues.Compared with normal tissues,1115 proteins were upregulated and 705 proteins were downregulated in CRC based on P adj<0.05 and|log2FC|>2,and bioinformatics analysis revealed that the differentially expressed proteins were involved in multiple biological processes associated with tumorigenesis,including ribosome biogenesis in eukaryotes,focal adhesion,extracellular matrix-receptor interactions and other tumor metabolism processes.Moreover,cyclin-dependent kinase inhibitor 2A(CDKN2A)expression was markedly upregulated in CRC,as validated by immunohistochemistry(0.228 vs 0.364,P=0.0044),and was significantly enriched in tumor proliferation and signal transduction pathways such as the cell cycle and p53 signaling pathways.High CDKN2A expression was significantly correlated with poor prognosis(P=0.021).These results demonstrated that CDKN2A functions as a driver of CRC.CONCLUSION Our study provides a comprehensive proteomic signature of CRC and highlights CDKN2A as a potential powerful prognostic marker and precision therapeutic target.

Mining The Cancer Genome Atlas database for tumor mutation burden and its clinical implications in gastric cancer

BACKGROUND Tumor mutational burden(TMB)is an important independent biomarker for the response to immunotherapy in multiple cancers.However,the clinical implications of TMB in gastric cancer(GC)have not been fully elucidated.AIM To explore the landscape of mutation profiles and determine the correlation between TMB and microRNA(miRNA)expression in GC.METHODS Genomic,transcriptomic,and clinical data from The Cancer Genome Atlas were used to obtain mutational profiles and investigate the statistical correlation between mutational burden and the overall survival of GC patients.The difference in immune infiltration between high-and low-TMB subgroups was evaluated by Wilcoxon rank-sum test.Furthermore,miRNAs differentially expressed between the high-and low-TMB subgroups were identified and the least absolute shrinkage and selection operator method was employed to construct a miRNA-based signature for TMB prediction.The biological functions of the predictive miRNAs were identified with DIANA-miRPath v3.0.RESULTS C>T single nucleotide mutations exhibited the highest mutation incidence,and the top three mutated genes were TTN,TP53,and MUC16 in GC.High TMB values(top 20%)were markedly correlated with better survival outcome,and multivariable regression analysis indicated that TMB remained prognostic independent of TNM stage,histological grade,age,and gender.Different TMB levels exhibited different immune infiltration patterns.Significant differences between the high-and low-TMB subgroups were observed in the infiltration of CD8+T cells,M1 macrophages,regulatory T cells,and CD4+T cells.In addition,we developed a miRNA-based signature using 23 differentially expressed miRNAs to predict TMB values of GC patients.The predictive performance of the signature was confirmed in the testing and the whole set.Receiver operating characteristic curve analysis demonstrated the optimal performance of the signature.Finally,enrichment analysis demonstrated that the set of miRNAs was significantly enriched in many key cancer and immune-related pathways.

Identification of circ_0000375 and circ_0011536 as novel diagnostic biomarkers of colorectal cancer

BACKGROUND Colorectal cancer(CRC)imposes a tremendous burden on human health,with high morbidity and mortality.Circular ribonucleic acids(circRNAs),a new type of noncoding RNA,are considered to participate in cancer pathogenesis as microRNA(miRNA)sponges.However,the dysregulation and biological functions of circRNAs in CRC remain to be explored.AIM To identify potential circRNA biomarkers of CRC and explore their functions in CRC carcinogenesis.METHODS CircRNAs and miRNAs differentially expressed in CRC tissues were identified by analyzing expression profiles from the Gene Expression Omnibus(GEO)database.Circ_0000375 and circ_0011536 were selected as CRC biomarker candidates.Quantitative real-time polymerase chain reaction was utilized to evaluate the expression of these 2 circRNAs in CRC tissues,serums and cell lines.Receiver operating characteristic curves were generated to assess the diagnostic performances of these 2 circRNAs.Then,functional experiments,including cell counting kit-8,wound healing and Transwell invasion assays,were performed after the overexpression of circ_0000375 and circ_0011536 in CRC cell lines.Furthermore,candidate target miRNAs of circ_0000375 and circ_0011536 were predicted via bioinformatics analysis.The expression levels of these miRNAs were explored in CRC cell lines and tissues from GEO datasets.A luciferase reporter assay was developed to examine the interactions between circRNAs and miRNAs.Based on the target miRNAs and downstream genes,functional enrichment analyses were applied to reveal the critical signaling pathways involved in CRC carcinogenesis.RESULTS Downregulated circ_0000375 and circ_0011536 expression was observed in CRC tissues in GSE126095,clinical CRC tissue and serum samples and CRC cell lines.The areas under the curve for circ_0000375 and circ_0011536 were 0.911 and 0.885 in CRC tissue and 0.976 and 0.982 in CRC serum,respectively.Moreover,the serum levels of these 2 circRNAs were higher in patients at 30 d postsurgery than in patients before surgery,suggesting that the serum expression of circ_0000375 and circ_0011536 is related to CRC tumorigenesis.Circ_0000375 and circ_0011536 overexpression inhibited the proliferation,migration and invasion of CRC cells.Furthermore,miR-1182 and miR-1246,which were overexpressed in CRC tissues in GSE41655,GSE49246 and GSE115513,were verified as target miRNAs of circ_0000375 and circ_0011536,respectively,by luciferase reporter assays.The downstream genes of miR-1182 and miR-1246 were enriched in some CRC-associated pathways,such as the Wnt signaling pathway.CONCLUSION Circ_0000375 and circ_0011536 may function as tumor suppressors in CRC progression,serving as novel biomarkers for CRC diagnosis and as promising candidates for therapeutic exploration.

Atherogenic index of plasma combined with waist circumference and body mass index to predict metabolic-associated fatty liver disease

BACKGROUND Early identification of metabolic-associated fatty liver disease(MAFLD)is urgent.Atherogenic index of plasma(AIP)is a reference predictor of obesity-related diseases,but its predictive value for MAFLD remains unclear.No studies have reported whether its combination with waist circumference(WC)and body mass index(BMI)can improve the predictive performance for MAFLD.AIM To systematically explore the relationship between AIP and MAFLD and evaluate its predictive value for MAFLD and to pioneer a novel noninvasive predictive model combining AIP,WC,and BMI while validating its predictive performance for MAFLD.METHODS This cross-sectional study consecutively enrolled 864 participants.Multivariate logistic regression analysis and receiver operating characteristic curve were used to evaluate the relationship between AIP and MAFLD and its predictive power for MAFLD.The novel prediction model A-W-B combining AIP,WC,and BMI to predict MAFLD was established,and internal verification was completed by magnetic resonance imaging diagnosis.RESULTS Subjects with higher AIP exhibited a significantly increased risk of MAFLD,with an odds ratio of 12.420(6.008-25.675)for AIP after adjusting for various confounding factors.The area under receiver operating characteristic curve of the A-W-B model was 0.833(0.807-0.858),which was significantly higher than that of AIP,WC,and BMI(all P<0.05).Subgroup analysis illustrated that the A-W-B model had significantly higher area under receiver operating characteristic curves in female,young and nonobese subgroups(all P<0.05).The best cutoff values for the A-W-B model to predict MAFLD in males and females were 0.5932 and 0.4105,respectively.Additionally,in the validation set,the area under receiver operating characteristic curve of the A-W-B model to predict MAFLD was 0.862(0.791-0.916).The A-W-B level was strongly and positively associated with the liver proton density fat fraction(r=0.630,P<0.001)and significantly increased with the severity of MAFLD(P<0.05).CONCLUSION AIP was strongly and positively associated with the risk of MAFLD and can be a reference predictor for MAFLD.The novel prediction model A-W-B combining AIP,WC,and BMI can significantly improve the predictive ability of MAFLD and provide better services for clinical prediction and screening of MAFLD.