miR-761-hepcidin/Gpx4 pathway contribute to unexplained liver dysfunction in polycystic ovary syndrome by regulating liver iron overload and ferroptosis.

Aims: To investigate the underling mechanisms of liver dysfunction in patients with polycystic ovary syndrome (PCOS).Materials and methods: PCOS patients were enrolled according to the Amsterdam criteria while PCOS animal model was established by dihydrotestosterone (DHEA) sustained release tablet implantation on its neck. Further liver damage and iron overload were detected by HE and Prussian blue staining. The liver related enzymes, mRNA and protein levels of hepcidin and GPX4 were tested by ELISA, qRT-PCR and Western blot. RNA interference and miR-761 transfection were routinely performed while the regulation of miR-761 on hepcidin and GPX4 was confirmed by luciferase reporter gene analysis.Results: We found that a part of PCOS patients and animal model had unexplained liver damage, which is independent of nonalcoholic fatty liver disease (NAFLD) and accompanied by increased ferrum (Fe) deposition. Besides, the expression of hepcidin and GPX4 that is important effector proteins for ferroptosis was down regulated in liver, showing the importance of iron metabolism in this unexplained liver damage. Based on the miR-761-hepcidin/GPX4 axis, we systematically studied the effects of miR-761 on ferroptosis and Fe deposition, which further influence the phenotype and liver function of PCOS model. From both in vivo and in vitro levels, changes in PCOS disease phenotype and ferroptosis were observed through hierarchical antagonism or overexpression of miR-761, hepcidin and GPX4.Conclusions: our results provide a novel explanation for unexplained liver damage in PCOS and a potential therapeutic target.

Association between Serum Free Fatty Acids to HDL-Cholesterol Ratio and Nonalcoholic Fatty Liver Disease: a Cross-Sectional Study.

BACKGROUND: This cross-sectional study aimed to investigate the association between serum free fatty acids and high-density lipoprotein-cholesterol ratio (FHR) and nonalcoholic fatty liver disease (NAFLD) in a Chinese population. METHODS: A total of 760 NAFLD subjects and 379 healthy controls who took their annual health checkups were enrolled during 2019. Fasting blood samples were obtained from the population. NAFLD was diagnosed based on hepatic ultrasound examination. RESULTS: Serum FHR (*100) in NAFLD subjects was significantly higher than that in controls. We found that the serum FHR in NAFLD participants was positively correlated with BMI, DBP, WBC, HGB, ALT, AST, GGT, TG, FPG, UA, and hsCRP. Univariate and multivariate logistic regression analysis showed that FHR was independently associated with the presence of NAFLD. The area under curve (AUC) of the receiver operating characteristic (ROC) curve of FHR for NAFLD was 0.781 with the 95% confidence interval from 0.753 to 0.810. The optimal cutoff point of FHR for predicting NAFLD was 41.14 with 78.8% sensitivity and 77.3%, respectively. CONCLUSIONS: FHR was significantly associated with NAFLD and may serve as an effective indicator in NAFLD patients.

Antagonizing circRNA_002581-miR-122-CPEB1 axis alleviates NASH through restoring PTEN-AMPK-mTOR pathway regulated autophagy.

Circular RNAs (circRNAs) have been shown to play critical roles in cancer biology, but their functions in nonalcoholic steatohepatitis (NASH) remain unexplored. Full length of circRNA_002581 was amplified and sequenced, followed by RNA immunoprecipitation, RNA-Fluorescence in Situ Hybridization and dual luciferase reporter gene analysis to confirm the existence of the circRNA_002581-miR-122-CPEB1 regulatory axis in vitro. CircRNA_002581 knockdown was used to study its roles in high concentration of free fatty acids-induced NASH-like cell model and a methionine and choline deficiency (MCD) diet-induced NASH mice model. Autophagy flux and related potential PTEN-AMPK-mTOR pathway were tested by western blot. CircRNA_002581 overexpression significantly relieved the inhibitory role of miR-122 on its target CPEB1 by sponging miR-122. CircRNA_002581 knockdown markedly attenuated lipid droplet accumulation, reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), pro-inflammatory cytokines, apoptosis, H2O2, and increased ATP level in both mice and cellular models of NASH. Mechanistically, circRNA_002581 interference significantly rescue the defective autophagy evidenced by increased autophagosome number, upregulated LC3-II/I level, and decreased p62 level. Further chloroquine-mediated total autophagy inhibition antagonizes the protective effect of circRNA_002581 knockdown. Finally, CPEB1-PTEN-AMPK-mTOR pathway is shown to link the autophagy and circRNA_002581 knockdown-mediated NASH alleviation. CircRNA_002581-miR-122-CPEB1 axis actively participates in the pathogenesis of NASH through PTEN-AMPK-mTOR pathway-related autophagy suppression. Targeting circRNA_002581 is a potential therapeutic strategy for NASH through partial autophagy restoration.

MiR-218-XOR-ROS Pathway Regulates the Progression of Nonalcoholic Steatohepatitis.

BACKGROUND: To investigate the role of the miR-218-xanthine oxidoreductase (XOR) pathway in the pathogenesis of nonalcoholic steatohepatitis (NASH) and to explore the potential downstream mechanisms involving oxidative stress and energy metabolism. METHODS: The NASH animal model was established by feeding BALB/c mice with an MCD diet, while BRL-3A cells were cultured with a mixture of oleate and palmitate for 72 hours to mimic the steatosis and inflammation of NASH in vitro. The steatosis and inflammation levels were assessed by H-E/oil-red staining and serum/supernatant TG, ALT, and AST levels. The apoptosis degree was tested by the TUNEL/flow cytometry method both in animals and cultured cells. The XOR and miR-218 levels were detected by western blotting and qRT-PCR. RESULTS: Decreased miR-218 and increased XOR levels were identified in the NASH animal and cell models, while the regulation of miR-218 on XOR was also confirmed. NASH alleviation was achieved after miR-218 over-expression in vivo and in vitro, according to the declination of steatosis and inflammation-related markers. Although H2O2 and ATP levels were increased and decreased in NASH models, respectively, antagonizing miR-218 could significantly alleviate those changes. CONCLUSIONS: The miR-218-XOR pathway may provide a novel mechanism and treatment option for NASH.

Association between Serum Ferritin Level and Nonalcoholic Fatty Liver Disease in a Non-Obese Chinese Population: a Cross-Sectional Study.

BACKGROUND: The aim of the study is to evaluate the cross-sectional association between serum ferritin level and nonalcoholic fatty liver disease (NAFLD) in a non-obese Chinese population. METHODS: A cross-sectional study was performed among 1,020 non-obese subjects (body mass index < 25 kg/m2) who took their annual health examination at the First Affiliated Hospital, College of Medicine, Zhejiang University. Serum ferritin level and other clinical and laboratory parameters were measured in the population. Liver ultrasound examinations were performed to diagnose NAFLD. RESULTS: Of the 1,020 enrolled participants, 148 (14.51%) fulfilled the diagnostic criteria for NAFLD. Subjects with NAFLD had a higher level of serum ferritin than individuals without NAFLD in non-obese subjects. Serum ferritin level was significantly and positively correlated with parameters of MS (BMI, SBP, TG and FPG) in NAFLD group. Stepwise logistic regression analysis showed that serum ferritin level was significantly associated with the risk factor for NAFLD. After adjusting for confounders, serum ferritin level was an independent factor predicting advanced fibrosis (FIB-4 ≥ 1.3) in NAFLD participants. CONCLUSIONS: Increased serum ferritin level is significantly associated with NAFLD, and elevated serum ferritin level is an independent factor predicting advanced fibrosis for NAFLD in a non-obese Chinese population.

miRNA-133a-UCP2 pathway regulates inflammatory bowel disease progress by influencing inflammation, oxidative stress and energy metabolism.

AIM: To investigate the role of the miR-133a-UCP2 pathway in the pathogenesis of inflammatory bowel disease (IBD) and to explore the potential downstream mechanisms with respect to inflammation, oxidative stress and energy metabolism. METHODS: C57BL/6 mice were fed dextran sulfate sodium (DSS) liquid for 7 consecutive days, followed by the administration of saline to the DSS group, UCP2 siRNA to the UCP2 group and a miR-133a mimic to the miR-133a group on days 8 and 11. Body weight, stool consistency and rectal bleeding were recorded daily, and these composed the disease activity index (DAI) score for the assessment of disease severity. After cervical dislocation was performed on day 14, the length of the colon in each mouse was measured, and colonic tissue was collected for further study, which included the following: haematoxylin and eosin staining, UCP2 and miR-133a detection by immunohistochemical staining, western blot and quantitative real-time PCR, measurement of apoptosis by TUNEL assay, and the assessment of inflammation (TNF-α, IL-1ß, IL-6 and MCP1), oxidative stress (H2O2 and MDA) and metabolic parameters (ATP) by ELISA and colorimetric methods. RESULTS: An animal model of IBD was successfully established, as shown by an increased DAI score, shortened colon length and specific pathologic changes, along with significantly increased UCP2 and decreased miR-133a levels. Compared with the DSS group, the severity of IBD was alleviated in the UCP2 and the miR-133a groups after successful UCP2 knockdown and miR-133a overexpression. The extent of apoptosis, as well as the levels of TNF-α, IL-1ß, MDA and ATP, were significantly increased in both the UCP2 and miR-133a groups compared with the DSS group. CONCLUSION: The miR-133a-UCP2 pathway participates in IBD by altering downstream inflammation, oxidative stress and markers of energy metabolism, which provides novel clues and potential therapeutic targets for IBD.

Low Serum Amylase is Associated with Gestational Diabetes Mellitus in Chinese Pregnant Women.

BACKGROUND: The association between low serum amylase levels and type 2 diabetes mellitus and metabolic syndrome has been clearly disclosed. However, the relationship between serum amylase levels and gestational diabetes mellitus (GDM) has not been extensively studied. This study aimed to assess the association of serum amylase with GDM. METHODS: A cross-sectional study was performed among 878 Chinese pregnant women who underwent detailed prenatal visits in Hangzhou, China. RESULTS: A total of 108 (12.30%) subjects fulfilled the diagnostic criteria of GDM. Patients with GDM had significantly lower levels of serum amylase than those without GDM. The prevalence rate of GDM decreased across serum amylase increasing tertiles (p for trend < 0.001). Correlation analysis showed that serum amylase level was negatively correlated with fasting plasma glucose, 1hPG, 2hPG, HOMA-IR, triglyceride, free fatty acid, and thyroid stimulating hormone (all with p < 0.05). Multiple logistic regression showed that low serum amylase level predicted increased risk of GDM. CONCLUSIONS: Our findings suggest that low serum amylase level is significantly associated with increased risk of GDM.

Meta-analysis of the association of AhR Arg554Lys, AhRR Pro185Ala, and ARNT Val189Val polymorphisms and endometriosis risk in Asians.

PURPOSE: Several studies have suggested an association between the polymorphisms AhR Arg554Lys, AhRR Pro185Ala, and ARNT Val189Val and endometriosis, but results have been inconclusive. The aim of the present study was to assess these associations by meta-analysis. METHODS: Eligible literatures were retrieved from PubMed, ISI Web of Science, Elsevier Science Direct, and several Chinese databases. The pooled odds ratios (ORs) and the corresponding 95 % confidence intervals (CIs) were calculated with a random or fixed-effect model. RESULTS: A total of six eligible studies were included. Regarding the AhR Arg554Lys and ARNT Val189Val polymorphisms, no obvious associations were found in either overall analysis or subgroup analysis based on the country, source of control, sample size, and genotyping method. For the AhRR Pro185Ala polymorphism, overall results suggested a marginal association with endometriosis susceptibility under the dominant model (OR = 1.65, 95 % CI = 1.00-2.72). Furthermore, a significantly increased risk for endometriosis was found in the subgroups which used the TaqMan method for genotype analysis or had a sample size ≥200. CONCLUSIONS: This meta-analysis suggested that the polymorphisms of AhR Arg554Lys and ARNT Val189Val are not associated with endometriosis, while the AhRR Pro185Ala polymorphism may be associated with endometriosis risk. However, further case-control studies with larger sample sizes are needed to confirm our results.

Circulating microRNAs in patients with polycystic ovary syndrome.

AIM: To explore the pattern of expression of circulating miRNAs in patients with polycystic ovary syndrome (PCOS). MATERIALS AND METHODS: Microarray and qRT-PCR were used to investigate circulating miRNAs in PCOS during clinical diagnosis. The targets of dys-regulated miRNAs were predicted using bioinformatics, followed by function and pathway analysis using the databases of Gene Ontology and the KEGG pathway. RESULTS: BMI, triglyceride, HOMA-IR, Testosterone and CRP levels were significantly higher, while estradiol was significantly lower in PCOS than in control groups. After SAM analysis, 5 circulating miRNAs were significantly up-regulated (let-7i-3pm, miR-5706, miR-4463, miR-3665, miR-638) and 4 (miR-124-3p, miR-128, miR-29a-3p, let-7c) were down-regulated in PCOS patients. Hierarchical clustering showed a general distinction between PCOS and control samples in a heat map. After joint prediction by different statistical methods, 34 and 41 genes targeted were up-and down-regulated miRNAs, in PCOS and controls, respectively. Further, GO and KEGG analyses revealed the involvement of the immune system, ATP binding, MAPK signaling, apoptosis, angiogenesis, response to reactive oxygen species and p53 signaling pathways in PCOS. CONCLUSIONS: We report a novel non-invasive miRNA profile which distinguishes PCOS patients from healthy controls. The miRNA-target database may provide a novel understanding of PCOS and potential therapeutic targets.

[Effect of modified cangfu daotan decoction in improving endometrial receptivity in infertility patients with polycystic ovarian syndrome].

OBJECTIVE: To study the effect and potential mechanism of Modified Cangfu Daotan Decoction (MCDD) on endometrial receptivity in infertility patients with polycystic ovarian syndrome (PCOS). METHODS: Totally 298 women having normal ovulation who underwent artificial insemination were recruited as the control group, and they received no drug therapy. Another 355 infertility patients with PCOS who received ovarian stimulation therapy were recruited as the treatment group. Then they were further assigned to the treatment group I (195 cases) and the treatment group II (160 cases) according to random digit table. Patients in the treatment group I received clomiphene (CC) + human menopause gonadotropin (HMG) +human chorionic gonadotropin (HCG), while those in the treatment group II received CC + HMG + HCG and additionally took modified MCDD. The therapeutic course for all was three menstrual cycles. The pregnancy ratio, the endometrial thickness, and spiral artery pulsatility index (PI), resistance index (RI), and homeostasis model assessment-insulin resistance (HOMA-IR) were measured. Furthermore, the uncoupling protein 2 (UCP2) level was tested by Western blot. RESULTS: Compared with the control group, the endometrial thickness decreased and PI and RI increased in the treatment group I (all P < 0.05). Compared with the treatment group I , the endometrial thickness increased and PI and RI decreased in the treatment group II (all P < 0.05). Compared with before treatment, HOMA-IR levels were significantly decreased in the treatment group II after treatment (P < 0.05). Compared with the control group before treatment, the HOMA-IR level increased in the treatment group I and the treatment group II before treatment (P < 0.05). Compared with the control group after treatment, the HOMA-IR level increased in the treatment group I (P < 0.05). But there was no statistical difference in the post-treatment HOMA-IR level between the control group and the treatment group II (P < 0.05). Compared with the control group, the post-treatment UCP2 level was increased in the treatment group II (P < 0.05). After one year follow-up, the pregnancy rate was 16.1% (48/298) in the control group, 23.1% (37/160) in the treatment group I, and 33.8% (66/195) in the treatment group II. Compared with the control group, the pregnancy rate was significantly increased in the treatment group II (P < 0.05). CONCLUSION: MCDD was found to be capable of increasing the pregnancy rate of infertility patients with PCOS, which might be associated with improving endometrial blood flow and insulin resistance, increasing the UCP2 expression, and finally improving the endometrial receptivity.

[Prevalence of nonalcoholic fatty liver disease in patients with polycystic ovary syndrome: a case-control study].

OBJECTIVE: To explore the prevalence of nonalcoholic fatty liver disease (NAFLD) in patients with polycystic ovary syndrome (PCOS). METHODS: A case-control study employing 60 nonpregnant patients with PCOS and 60 non-pregnant patients without PCOS as control was conducted to compare the prevalence of NAFLD. RESULTS: The aminotransferase (ALT), fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR) levels were (29 +/- 15) U/L, (19 +/- 12) mU/L and 0.47 +/- 0.29 in PCOS group, which were significantly higher (P < 0.05) than corresponding parameters in control group [(15 +/- 13) U/L, (11 +/- 8) mU/L and 0.31 +/- 0.21)]. The occurrence of insulin resistance and NAFLD was63% (38/60) and 42% (25/60), higher than those in control group [35% (21/60) and 20% (12/60), P < 0.05]. The increment of ALT was 40% (24/60) in PCOS group, higher than that of 3% (2/60) in control group (P < 0.01). Compared with patients without NAFLD, patients with NAFLD had significantly increased body mass index (P < 0.01), waist-hip ratio, ALT, C-reaction protein, fasting insulin, insulin and HOMA-IR levels 2 hours after oral glucose tolerance test (P < 0. 05). CONCLUSION: The increased prevalence of NAFLD in PCOS patients suggests an association between these two conditions and the necessity of hepatic screening among PCOS patients for potential NAFLD.

Green tea consumption and liver disease: a systematic review.

OBJECTIVES: To present the effect of green tea consumption against liver disease. DATA SOURCES: Interventional and observational studies both in Western countries and in China and published between the years 1989 and December 2007. REVIEW METHODS: The articles were retrieved from Medline, Embase database, Chinese biomedicine web database and Chinese scientific journal's database using proper MESH headings and assessed by two independent investigators according to established inclusion criteria. The characteristics and outcomes of the chosen articles were displayed for further analysis and the quality of each study was also evaluated according to the widely acknowledged criteria. P<0.05 was defined as statistically significant in all enrolled trials. RESULTS: Ten qualified studies (eight from China, one from Japan and the other from the USA) with various outcomes such as liver cancer, cirrhosis and fatty liver disease were finally chosen. Among them, study designs differed in that there were four randomized-controlled clinical trials, two cohort, one case-control and three cross-sectional studies. The heterogeneity in the study design, outcomes, cofounders and amount of tea consumption precluded further meta-analysis. Nevertheless, eight studies showed a significant protective role of green tea against various liver diseases as determined by relative risk/odds ratio or P-value and among them, four studies showed a positive correlation between green tea intake and attenuation of liver disease. Moreover, the other two studies also presented the protective tendency of green tea against liver disease. CONCLUSIONS: An increased consumption of green tea may reduce the risk of liver disease.