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This paper was to investigate the effect of Huanglian Jiedu Decoction(HLJD) on ulcerative colitis(UC) in mice, and determine the effective components in plasma, and virtually screen its therapeutic target, and predict its mechanism. Sixty Balb/c mice were randomly divided into blank group, model group, mesalazine treatment group(0.3 g·kg~(-1)), and HLJD treatment groups(24.66, 12.33, 6.17 g·kg~(-1)). Excepted for the blank group, all the mice in HLJD and mesalazine treatment groups were gavage administration. All mice freely drank 2.5% DSS solution for seven days to induce UC. The disease activity index(DAI) was detected each day. At the end of the experiment, HE staining was used to observe the pathological changes in colon. The content of IL-1β, IL-6 and TNF-α in colon were determined by ELISA. The effective components in plasma were determined by UPLC-Q-TOF-MS. The reverse docking in PharmMapper was used to screen the component targets. The disease targets of UC were collected by searching TTD, OMIM and GeneCards databases. The intersection of the component targets and disease targets was selected as the therapeutic targets. Then the therapeutic targets were imported into the STRING for GO and KEGG enrichment analysis. Discovery Studio was used to simulate the docking between the components and the targets. RESULTS:: showed that the DAI in the model group increased significantly(P<0.05), and the number of inflammatory cells and infiltration degree increased significantly compared with the blank group. The DAI in HLJD treatment group was significantly reduced(P<0.05), and the number and infiltration degree of inflammatory cells were reduced compared with the model group. The ELISA results showed that the levels of IL-1β, IL-6 and TNF-α were increased significantly in the model group(P<0.01) compared with the blank group, and significantly down regulated in the HLJD treatment group(P<0.05) compared with the model group. After UPLC-Q-TOF-MS analyse, ten components were identified. The network pharmacology analysis showed that the action targets were significantly enriched in 129 of biological processes, such as response to organic substance, chemical and oxygen-containing compound, etc., as well as 16 of signal pathways, such as IL-17, TNF and hepatitis B signal pathways, were enriched too. The results of molecular docking showed that limonin, palmatine and berberine could bind to CASP3 and MMP9 by hydrogen bond. In conclusion, HLJD could alleviate the colonic mucosal inflammatory infiltration and mucosal damage in UC mice. The mechanism may be related to the anti-inflammatory effect on UC mice by reducing the levels of IL-1β, IL-6 and TNF-α in colon through limonin, palmatine and berberine regulating IL-17 signal pathway and TNF signal pathway via CASP3 and MMP9 meditated.
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Animals , Mice , Anti-Inflammatory Agents/therapeutic use , Colitis, Ulcerative/drug therapy , Colon , Dextran Sulfate/therapeutic use , Drugs, Chinese Herbal , Molecular Docking Simulation , PlasmaABSTRACT
Objective To construct a dual-luciferase reporter gene vector and validate the targeting relation ship between miR-299 and the COL4A3 gene,laying a foundation for the study on the effect of miR-299 in the chondrogenic differentiation of stem cells by regulating the COL4A3 gene.Methods This study was made from March,2018 to December,2018.Firstly,the potential binding sites between miR-299 and COL4A3-3'UTR were pre dicted using bioinformatics.Then,the wild and mutant COL4A3-3'UTR sequences were amplified by PCR and cloned into psiCHECK-2 plasmid to construct corresponding recombinant vectors.The vectors were validated by enzyme digestion and gene sequencing.Finally,the cells were resuscitated,amplified,transfected and divided into 4 groups:COL4A3-WT+miR-299/NC group,COL4A3-WT+miR-299-inhibitor/NC-inhibitor group,COL4A3-MUT+miR-299/NC group and COL4A3-MUT+miR-299-inhibitor/NC-inhibitor group.Each group contains 3 holes,respectively.Luciferase activity in each group was determined using a dual-luciferase assay kit.The statistical analysis was conducted and differences between groups were compared by t test.Probabilities lower than 5％(P＜0.05) were considered statistically significant.Results Enzyme digestion and DNA sequencing showed that the dual-luciferase reporter gene vector of psiCHECK-2-COL4A3 was constructed successfully.Luciferase assay demonstrated that in wild COL4A3 gene,luciferase activity reduced in the miR-299 transfection group (The average R/F value was 59.38％) compared with the NC group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In wild COL4A3 gene treated with inhibitor,luciferase activity increased in the miR-299-inhibitor group (The average R/F value was 153.98％) compared with the NC-inhibitor group (The average R/F value was 100.00％),with a statistical significant difference (P＜0.05).In mutant COL4A3 gene treated with inhibitor,no obvious statistical differences in luciferase activity were found between miR-299 transfection group (The average R/F value was 102.09％),miR-299-inhibitor group (The average R/F value was 108.51％) and NC group (The average R/F value was 104.70％),NC-inhibitor group (The average R/F value was 105.13％) and/9＞0.05.Conclusion The dual-luciferase reporter gene vector of the 3'UTR of the COL4A3 gene is constructed successfully.In addition,dual-luciferase assay further verifies the authenticity of miR-299 directly targeting the 3'UTR of the COL4A3 gene.
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Objective To identify the Y-chromosomal genetic types for the soldier's remains from Huaihai Campaign,and to offer a clue for search of their paternal relatives.Methods DNA of the remains were extracted by the ancient DNA extraction method.Yfiler kit was used for the multiplex amplification of 17 Y-STR loci.The haplogroups of the samples were speculated.Detailed genotyping of the selected Y-SNP was performed based on the latest Y-chromosome phylogenetic tree.Haplotype-sharing analysis was done based on the data of Y-SNP and Y-STR,the closest modern individual information to the genetic relationship of remains was gained.Results A total of 8 Y-STR haplotypes were observed on 17 Y-STR loci of 8 male individuals.Furthermore,6 Y-SNP haplogroups were identified,which were O2a1-M95+,O1a1-P203+,O3*-M122+/M234-,D1-M15+,C3*-ST and R1a1-M17+.Conclusion Identification of Y-chromosomal genetic types for the soldier's remains from Huaihai Campaign shows a reference value on inferring the geographical origins of old materials.
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BACKGROUND: Preliminary experimental study found that the human amniotic mesenchymal stem cells (hAMSCs)transplantation can improve nerve injury symptoms of rats with cerebral infarction.OBJECTIVE: To observe the survival, colonization and differentiation of hAMSCs in the infarct area of cerebralinfarction rats.METHODS: Sixty Sprague-Dawley rats were randomly assigned into hAMSCs transplantation, model or shamoperation groups (n=20/group). Animal models of middle cerebral artery occlusion were produced in the model andtransplantation groups by Zea-Longa method. One day after modeling, rats in the hAMSCs transplantation groupwere given in situ transplantation of 10 μL of hAMSCs (2×106) into the damaged striatum and cortex, while those inthe model and sham operation group were given the same volume of PBS. Within 1 week after transplantation, ratneurological defects were assessed and changes in their body mass were continuously monitored. Two weeks aftertransplantation, TTC staining was used to observe cerebral infarct size, hematoxylin-eosin staining was used forpathological observation of brain tissues, and immunofluorescent staining was used to detect expression ofneuron-specific nuclear protein.RESULTS AND CONCLUSION: With time, weight loss was increased while neurologic deficit scores were graduallyreduced in the hAMSCs and model groups. Compared with the model group, the weight loss and neurologic deficitscores were lower in the hAMSCs group,; however, there was a significant difference in the neurologic deficit scoresbut not in the weight loss between the two groups. Additionally, the hAMSCs significantly reduced infarct size,attenuated pathologic injury, and decreased the number of inflammatory cells. Immunofluorescence stainingshowed that the hAMSCs were observed at 1 week after transplantation under inverted luorescence microscope,and gradually differentiated into nerve cells at 2 weeks after transplantation. In conclusion, transplanted hAMSCsmay migrate to and survive in the cerebral infarct region, and differentiate into nerve cells in situ in rats with cerebralinfarction.
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Objective To investigate the impact of transplantation of human amniotic mesenchymal stem cells(hAMSCs)on the histopathological change in paraquat-induced pulmonary fibrosis in rats. Methods Forty-six female SD rats were randomly divided into the sham surgery group and the hAMSCs transplant group. Pulmonary fibrosis model was induced by 2％ of paraquat intragastric administration(100 mg/kg/rat). hAMSCs were injected through caudal vein(2 × 106 cells/mL/rat). The histopathological changes were observed through microscopy after HE and the immunohistochemical staining. Results General conditions in rats received hAMSCs transplantation were better than those of the model rats. More large area and white fibrosis nidus were observed in bilateral lung of model rats,with less dispersal spot or nidus. The construction of lung tissue was disordered in the model rats. The thickness of alveolar wall was found increased. There were large area interstitial hyperplasia and a large number of inflammatory cells infiltrations. The construction of lung tissue was apparently improved. A majority of alveolar wall was monolayer cell. There were only less and small area with interstitial hyperplasia. Inflammatory cell infiltration was significantly decreased. The anti-human nucleus specific antibody positive hAMSCs were observed planted and survived in lung interstitial tissue. And few hAMSCs were observed planted in alveolar wall. Conclusion The transplanted hAMSCs can be planted and survived in lung tissue ,and may play a therapeutic role in araquat-induced pulmonary fibrosis.
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Objective To investigate the impact of transplantation of human amniotic mesenchymal stem cells(hAMSCs)on the histopathological change in paraquat-induced pulmonary fibrosis in rats. Methods Forty-six female SD rats were randomly divided into the sham surgery group and the hAMSCs transplant group. Pulmonary fibrosis model was induced by 2％ of paraquat intragastric administration(100 mg/kg/rat). hAMSCs were injected through caudal vein(2 × 106 cells/mL/rat). The histopathological changes were observed through microscopy after HE and the immunohistochemical staining. Results General conditions in rats received hAMSCs transplantation were better than those of the model rats. More large area and white fibrosis nidus were observed in bilateral lung of model rats,with less dispersal spot or nidus. The construction of lung tissue was disordered in the model rats. The thickness of alveolar wall was found increased. There were large area interstitial hyperplasia and a large number of inflammatory cells infiltrations. The construction of lung tissue was apparently improved. A majority of alveolar wall was monolayer cell. There were only less and small area with interstitial hyperplasia. Inflammatory cell infiltration was significantly decreased. The anti-human nucleus specific antibody positive hAMSCs were observed planted and survived in lung interstitial tissue. And few hAMSCs were observed planted in alveolar wall. Conclusion The transplanted hAMSCs can be planted and survived in lung tissue ,and may play a therapeutic role in araquat-induced pulmonary fibrosis.
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An electrochemical sensor has been developed for the selective determination of chlortetracycline ( CTC) using the molecularly imprinted technique. A molecular imprinted polymer ( MIP) on the surface of a glassy carbon electrode ( GCE ) was prepared by electropolymerization of o-aminophenol ( OAP ) in the presence of CTC in the sodium perchlorate ( NaClO4 ) solution using cyclic voltammetry ( CV ) . The electrochemical performance of the sensor was studied by using differential pulse voltammetry ( DPV ) . A linear relationship between the peak current difference and the CTC concentration was found in the range of 2. 0×10-8-6. 1×10-7 mol/L with the detection limit of 1. 5×10-8 mol/L (3σ). After regeneration by washing with the mixture of methanol and sulfuric acid, the sensor showed excellent reproducibility and good stability. The MIP electrode exhibited almost no response to chloramphenicol and penicillin, and very weak responses to tetracycline and oxytetracycline, proving a good selectivity. Recoveries of standard addition measured in the actual samples of milk and chicken meat were between 86 . 4% -96 . 9%. Compared with the reported methods, this sensor showed a low detection limit, simple operation without derivatization, rapid response and low cost.
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Objective To investigate the etiological and epidemiological characteristics of fungal keratitis in Jilin province of China and to establish a rapid and specific method for molecular identification of the prevalent fungal pathogens.Methods Corneal scrapings were collected from 225 patients with suspected fungal keratitis.Fungal strains were isolated and identified based on their morphology and physiological characteristics.The epidemiological characteristics of all isolated strains causing fungal keratitis were statistically analyzed.Species-specific primers of Fusarium solani (F.solani) were designed and used together with the universal fungal primers to establish a multiplex PCR assay for identification of F.solani in corneal scrapings.Results 156 out of 225 patients (69.3％) were diagnosed as fungal keratitis by fungal culture followed by the examination of morphological and physiological characteristics.A total of 168 pathogenic fungi strains were isolated,most of which were Fasarium spp.(49.4％),followed by Aspergillus spp.(17.9％) and Candida spp.(14.3％).F.solani was the predominant pathogen accounting for 34.5％ in all patients.Most of the patients (87.5％) were farmer and male patients (57.1％) accounted for the majority of 156 patients as well.Corneal trauma (38.5％) was considered as the main predisposing factor.The established multiplex PCR could specifically amplify a 300 bp nucleotide fragment of F.solani.It could be used for a rapid identification of F.solani in corneal scrapings.Conclusion Fusarium genus,particularly the species of F.solani,was the predominant pathogen for fungal keratitis in Jilin province of China.Corneal trauma was the most important predisposing factor.The established multiplex PCR could identify fungal infection from corneal scrapings rapidly and specifically.These findings are very important for the early diagnosis and treatment of fungal keratitis.
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<p><b>OBJECTIVE</b>To observe the effect of intestinal trefoil factor (ITF) combined with mucin on immune function of intestinal epithelial cells (IEC) after being treated with burn rat serum.</p><p><b>METHODS</b>The rat IEC-6 cell lines were divided into control group (C, cultured in DEME medium containing 10% calf serum), burn control group (BC, cultured in DEME medium containing 10% burn rat serum), burn serum + ITF group (B + I, cultured in DEME medium containing 10% burn rat serum and 25 microg/mL ITF), burn serum + mucin group (B + M, cultured in DEME medium containing 10% burn rat serum and 250 microg/mL mucin), and burn serum + ITF + mucin group (B + I + M, cultured in DEME medium containing 10% burn rat serum, 25 microg/mL ITF, and 250 microg/mL mucin) according to the random number table. Meanwhile, 200 microL suspension of E. coli with density of 1 x 10(8) CFU/mL was added to each culture. At post culture minute (PCM) 15, 30 and post culture hour (PCH) 1, 2, 3, the number of bacteria adherent to IEC-6 was counted after Wright-Giemsa staining, and cell survival rate was calculated after trypan blue staining, with 20 samples in each group at each time point. (2) Other samples of IEC-6 cells without addition of E. coli were divided into BC, B + I, B + M, and B + I + M groups with the same treatment as above. The supernatant contents of IL-6, IL-8, and TNF-alpha were determined by radioimmunoassay at PCH 3, 6, 12, 24, 48, with 6 samples in each group at each time point. Data were processed with t test.</p><p><b>RESULTS</b>(1) Compared with that in C group, count of adherent bacteria to IEC-6 in BC group at each time point was significantly increased (with t values from 2.947 to 8.149, P values all below 0.01). Compared with those in BC group, the counts in B + I, B + M, B + I + M groups at the major time points were significantly decreased (with t values from -4.733 to -2.180, P < 0.05 or P < 0.01). (2) Compared with that in C group, cell survival rate in BC group at each time point was obviously lowered (with t values from -4.126 to -2.363, P values all below 0.05). Cell survival rates in B + I and B + M groups at some time points were significantly elevated as compared with those in BC group (with t values from 2.120 to 3.423, P < 0.05 or P < 0.01). Cell survival rate in B + I + M group at PCM 15 and PCH 3 was respectively (96.7 +/- 2.4)% and (84.0 +/- 6.7)%, which was respectively higher than that in B + I and B + M groups [(94.5 +/- 3.1)%, t = 2.507, P < 0.05; (77.1 +/- 8.2)%, t = 2.934, P < 0.01]. (3) The contents of TNF-alpha in supernatant of B + I + M group at PCH 6, 12, 24, 48 were significantly lower than those in the other 3 groups (with t values from -6. 914 to -2.889, P < 0.05 or P < 0.01). The contents of IL-6 in supernatant of B + I + M group at some time points were significantly lower than those in the other 3 groups (with t values from -7. 657 to -2.580, P < 0.05 or P < 0.01). The contents of IL-8 in supernatant of B + I + M group at PCH 6, 12, 24, 48 were significantly lower than those in BC and B + M groups (with t values from - 8.802 to - 3.640, P values all below 0.01), and those in B + I + M group at PCH 12, 24 were lower than those in B + I group (with t value respectively -2.786, -2.740, P value all below 0.05).</p><p><b>CONCLUSIONS</b>ITF can maintain immune function and homeostasis of IEC, prevent bacterial adherence, decrease cell death rate, and reduce release of inflammatory mediators. The effect can be strengthened with addition of mucin.</p>
Subject(s)
Animals , Rats , Bacterial Adhesion , Burns , Blood , Cell Line , Epithelial Cells , Allergy and Immunology , Metabolism , Interleukin-6 , Metabolism , Interleukin-8 , Metabolism , Intestines , Cell Biology , Allergy and Immunology , Metabolism , Mucins , Pharmacology , Peptides , Pharmacology , Serum , Allergy and Immunology , Trefoil Factor-2 , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the effect of intestinal trefoil factor (ITF) combined with mucin on the ability of proliferation and migration of intestinal epithelial cells (IEC) after being treated by burn rat serum.</p><p><b>METHODS</b>The rat IEC-6 cell lines were subcultured and divided into control group (C, cultured with DMEM medium containing 10% calf serum), burn serum group (BS, cultured with DMEM medium containing 10% burn rat serum), burn serum + ITF group (B + I, cultured with DMEM medium containing 10% burn rat serum and 25 microg/mL ITF), burn serum + mucin group (B + M, cultured with DMEM medium containing 10% burn rat serum and 250 microg/mL mucin), and burn serum + ITF + mucin group (B + I + M, cultured with DMEM medium containing 10% burn rat serum, 25 microg/mL ITF, and 250 microg/mL mucin) according to the random number table. Cells were counted on post culture day (PCD) 0, 1, 2, 3, 4, reflecting cell proliferation ability. Cell migration distance was measured at post scratch hour (PSH) 12, 24, 36, 48, 72. Then, cells of each group were placed in upper compartment of Transwell chamber while the corresponding medium was respectively added into lower compartment of Transwell chamber. Cells in lower compartment of Transwell chamber were counted at post culture hour (PCH) 4, 6, 8, 10, 12, reflecting cytomorphosis ability. Data were processed with t test.</p><p><b>RESULTS</b>(1) Cell proliferation ability. The cell numbers in BS group on PCD 0, 1, 2, 3, 4 were significantly less than those in C group (with t values from -16.569 to -2.613, P < 0.05 or P < 0.01). The cell number showed no statistical difference between B + I and BS groups, and between B + M and BS groups at each time point (with t values respectively from 0.037 to 0.740 and 0.116 to 0.429, P values all above 0.05). The cell number in B + I + M group on PCD 2 was respectively larger than that in BS group (t = 6.484, P < 0.01) and B + I group ( t = 3.838, P < 0.01). (2) Cell migration distance in BS group at PSH 12, 24, 36, 48, 72 was significantly shorter than that in C group (with t values from -37.594 to -6.727, P values all below 0.01). There was no obvious difference in cell migration distance between BS and B + M groups at each time point (with t values from 0.055 to 0.589, P values all above 0.05). Cell migration distance in B + I group at PSH 12, 24, 36 was respectively (47 +/- 6), (126 +/- 13), (170 +/- 11) microm, all longer than those in BS group [(42 +/- 7), (98 +/- 14), (154 +/- 22) microm, with t values from 2.230 to 4.817, P < 0.05 or P < 0.01]. Cell migration distance in BS group at PSH 12, 24, 36, 48, 72 and B + I group at PSH 12, 24, 36, 48 was respectively shorter than that in B + I + M group (with t values respectively from 2.982 to 7.390 and 2.707 to 2.918, P < 0.05 or P < 0.01). (3) Cytomorphosis ability. Compared with those of C group, cell counts in lower compartment of BS group at PCH 4, 6, 8, 10, 12 were significantly decreased (with t values from -23.965 to -6.436, P values all below 0.01). Cell count in lower compartment of BS group at PCH 4, 6, 8, 10, 12 was respectively less than that of B + I group (with t values from 3.650 to 10.028, P values all below 0.01) and similar to that of B + M group (with t values from 0.199 to 0.797, P values all above 0.05). Cell counts in lower compartment of B + I + M group at PCH 4, 6, 8, 10, 12 were significantly larger than those of BS group (with t values from 4.313 to 15.100, P values all below 0.01). Cell count in lower compartment of B + I + M group at PCH 10 (328 +/- 47) and PCH 12 (465 +/- 37) was respectively larger than that in B + I group (277 +/- 25, 353 +/- 34, with t value respectively 3.051, 6.945, P values all below 0.01).</p><p><b>CONCLUSIONS</b>ITF can improve cytomorphosis ability for promoting cell migration with limited effect on cell proliferation, which can be enhanced with addition of mucin. The main mechanism of ITF in maintaining intestinal mucosal barrier may be attributed to acceleration of cell migration.</p>
Subject(s)
Animals , Rats , Burns , Blood , Cell Line , Cell Movement , Cell Proliferation , Epithelial Cells , Cell Biology , Metabolism , Intestinal Mucosa , Intestines , Cell Biology , Metabolism , Mucins , Pharmacology , Peptides , Pharmacology , Serum , Allergy and Immunology , Trefoil Factor-2ABSTRACT
Objective To find out the potential risk factor of plague in Wanzhou section of the Three Gorges Reservoir area, and to provide scientific basis for prevention and control of plague. Methods Rodents were captured by rat traps/cages at night and identified into species in Wanzhou section of the Three Gorges Reservoir area from 2001 to 2009. Flea was counted and serum antibodies against plague F1 of rats, cats and dogs were detected by indirect hemagglutination (IHA). Plague surveillances were performed in human beings and rats. Results The rodents captured belonged to 9 species, 2 families, 2 orders and 1 classes. The average indoor rodent density was 1.16% (961/82 558), and was 1.12% (1345/119 671) outdoors. Rattus norvegicus was the dominant species,accounting for 50.37%. The proportion of R. Flavipectus was 3.80% in 2004, 4.50% in 2008 and 10.12% in 2009,showing an increasing trend year by year. There were three kinds of mice infected fleas in Wanzhou, which including Xenopsylla cheopis, Leptopsylla segnis and Ctenocephalides felis. The average rate of flea infected mice was 1.18%(82/6959) and the total flea index was 0.036. No F1 antibody against plague was detected in 6959 dogs and 160 cats serum samples. Conclusions No plague is found in Wanzhou section of the Three Gorges Reservoir area. But R.Flavipectus, Xenopsylla cheopis and Leptopsylla segnis are dominant species in Wanzhou section, and the proportion of which shows an increasing trends year by year. There is a potential risk of plague outbreaks in Wanzhou section of the Three Gorges Reservoir area.
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<p><b>OBJECTIVE</b>To investigate the correlation between nonalcoholic fatty liver disease (NAFLD) and three anthropometric indices, namely waist to hip ratio (WHR), body mass index (BMI) and waist to height ratio (WHtR).</p><p><b>METHODS</b>This retrospective case-control study involved 77 NAFLD patients and 50 patients without such disease, and their data of the 3 anthropometric indices were collected. Risk correlation analysis and Mantel-Haenszel chi-square test were used for correlation analysis.</p><p><b>RESULTS</b>NAFLD was significantly correlated to WHR (chi(2)(MH)=59.609, P<0.001; odds ratio=30.522, 95% CI 12.815-72.695), WHtR (chi(2)(MH)=45.316, P<0.001; odds ratio=21.037, 95% CI 8.665-51.072) and showed a dose-response relationship with BMI (chi(2)=25.017, P<0.001).</p><p><b>CONCLUSION</b>These results support a close correlation between NAFLD and the 3 anthropometric indices, indicating that BMI, WHR and WHtR can be significant predictors of NAFLD and have potential value for evaluating and predicting NAFLD.</p>