Roles of microRNAs in immunopathogenesis of non-alcoholic fatty liver disease revealed by integrated analysis of microRNA and mRNA expression profiles.

BACKGROUND: The integrative analysis of microRNA and mRNA expression profiles can elucidate microRNA-targeted gene function. We used this technique to elucidate insights into the immunological pathology of non-alcoholic fatty liver disease (NAFLD). METHODS: We analyzed differentially expressed microRNA and mRNA expression profiles of CD4+ T lymphocytes from the liver and mesenteric lymph nodes (MLNs) of mice with NAFLD using microarrays and RNA sequencing. Normal mice were used as controls. The target genes of microRNAs were predicted by TargetScan. Integrative analysis showed that the mRNAs were overlapped with microRNAs. Furthermore, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the key genes and pathways. Then, 16 microRNAs and 10 mRNAs were validated by qRT-PCR. RESULTS: Microarray analysis suggested that 170 microRNAs were significantly de-regulated in CD4+ T lymphocytes from the liver between the two groups. Eighty mRNAs corresponded with microRNA targeted genes. KEGG analysis indicated that the MAPK pathway was consistently augmented in the liver of NAFLD mice. miR-23b, let-7e, miR-128 and miR-130b possibly played significant parts in the MAPK pathways. Furthermore, between the two groups, 237 microRNAs were significantly de-regulated in CD4+ T lymphocytes from MLNs. 38 mRNAs coincided with microRNA target genes. The metabolic pathway was consistently enriched in the MLNs of NAFLD mice. miR-206-3p, miR-181a-5p, miR-29c-3p and miR-30d-5p likely play important roles in the regulation of metabolic pathways. CONCLUSION: The results of this study presented a new perspective on the application of integrative analysis to identify complex regulation means involved in the immunological pathogenesis of NAFLD.

PANX1-mediated ATP release confers FAM3A's suppression effects on hepatic gluconeogenesis and lipogenesis.

BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet ß cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed. METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays. RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers. CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.

Polyamines are essential in embryo implantation: expression and function of polyamine-related genes in mouse uterus during peri-implantation period.

Polyamines are key regulators in cell growth and differentiation. It has been shown that ornithine decarboxylase (Odc) was essential for post-implantation embryo development, and overexpression of spermidine/spermine N1-acetyltransferase will lead to ovarian hypofunction and hypoplastic uteri. However, the expression and function of polyamine-related genes in mouse uterus during early pregnancy are still unknown. In this study we investigated the expression, regulation, and function of polyamine-related genes in mouse uterus during the peri-implantation period. Odc expression was strongly detected at implantation sites and stimulated by estrogen treatment. The expression of Odc antizyme 1 and spermidine/spermine N1-acetyltransferase was also highly shown at implantation sites and regulated by Odc or polyamine level in uterine cells. Embryo implantation was significantly inhibited by alpha-difluoromethylornithine, an Odc inhibitor. Moreover, the reduction of Odc activity caused by alpha-difluoromethylornithine treatment was compensated by the up-regulation of S-adenosylmethionine decarboxylase gene expression. Collectively, our results indicated that the coordinated expression of uterine polyamine-related genes may be important for embryo implantation.

Study of Clinical and Genetic Risk Factors for Aspirin-induced Gastric Mucosal Injury.

BACKGROUND: Current knowledge about clinical and genetic risk factors for aspirin-induced gastric mucosal injury is not sufficient to prevent these gastric mucosal lesions. METHODS: We recruited aspirin takers as the exposed group and healthy volunteers as the control group. The exposed group was categorized into two subgroups such as subgroup A as gastric mucosal injury diagnosed by gastroscopy, including erosion, ulcer or bleeding of the esophagus, stomach, or duodenum; subgroup B as no injury of the gastric mucosa was detected by gastroscopy. Clinical information was collected, and 53 single nucleotide polymorphisms were evaluated. RESULTS: Among 385 participants, 234 were in the aspirin-exposed group. According to gastroscopy, 82 belonged to subgroup A, 91 belonged to subgroup B, and gastroscopic results of 61 participants were not available. Using the Chi-square test and logistic regression, we found that peptic ulcer history (odds ratio [OR] = 5.924, 95% confidence intervals [CI]: 2.115-16.592), dual anti-platelet medication (OR = 3.443, 95% CI: 1.154-10.271), current Helicobacter pylori infection (OR = 2.242, 95% CI: 1.032-4.870), male gender (OR = 2.211, 95% CI: 1.027-4.760), GG genotype of rs2243086 (OR = 4.516, 95% CI: 1.180-17.278), and AA genotype of rs1330344 (OR = 2.178, 95% CI: 1.016-4.669) were more frequent in subgroup A than subgroup B. In aspirin users who suffered from upper gastrointestinal bleeding, the frequency of the TT genotype of rs2238631 and TT genotype of rs2243100 was higher than in those without upper gastrointestinal bleeding. CONCLUSIONS: Peptic ulcer history, dual anti-platelet medication, H. pylori current infection, and male gender were possible clinical risk factors for aspirin-induced gastric mucosal injury. GG genotype of rs2243086 and AA genotype of rs1330344 were possible genetic risk factors. TT genotype of rs2238631 and TT genotype of rs2243100 may be risk factors for upper gastrointestinal bleeding in aspirin users.

Role of intrahepatic B cells in non-alcoholic fatty liver disease by secreting pro-inflammatory cytokines and regulating intrahepatic T cells.

OBJECTIVE: To investigate the alterations and functions of intrahepatic B (IHB) cells in non-alcoholic fatty liver disease (NAFLD) model induced by high-fat diet (HFD). METHODS: C57BL/6 J mice were fed with HFD for 16 weeks to induce NAFLD. Flow cytometry was used to analyze lymphocytes from liver, spleen and peripheral blood mononuclear cells (PBMC). Real-time polymerase chain reaction and immunofluorescence stain were applied to investigate cytokine expression in the intrahepatic lymphocytes and IHB cells. CD4(+) intrahepatic T (IHT) cells and IHB cells were enriched by a magnetic-activated cell sorting method and cultured in vitro. The cytokines and immunoglobulin (Ig) levels in the plasma, cultural supernatants and liver homogenates were monitored with cytometric bead arrays or multiplex immunoassays. RESULTS: The percentage of IHB cells in CD45(+) cells was significantly higher in the NAFLD group than in the control group (P < 0.05). IHB cells expressed higher levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the NAFLD group, and produced higher levels of IL-6 and TNF-α under the stimulation of lipopolysaccharide (LPS) than the control group. IgG2a levels were higher in the plasma, liver homogenates and the culture supernatants of IHB cells after stimulated by LPS and anti-CD40/IgM in the NAFLD group than in the control group. Moreover, IHB cells enhanced the activation of CD4(+) IHT cells and promoted the differentiation into T helper (Th) 1 cells in the NAFLD group. CONCLUSION: IHB cells might be involved in NAFLD both by inducing the secretion of IL-6, TNF-α and IgG2a and by enhancing the activation of CD4(+) IHT cells and their differentiation into Th1 cells.

Cyclic adenosine monophosphate-induced argininosuccinate synthase 1 expression is essential during mouse decidualization.

L-Arginine (L-Arg), a conditional essential amino acid in adults, has been shown to enhance pregnancy outcome. Argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl) are the key enzyme for L-Arginine (L-Arg) biosynthesis. Based our microarray analysis, Ass1 expression is upregulated significantly at implantation site on day 5 of pregnancy compared to that at inter-implantation site. However, the expression, regulation and function of Ass1 during early pregnancy remain unknown. Here we found that Ass1 is highly expressed in mouse decidua and uterine stromal cells undergoing decidualization, and Asl is weakly expressed in mouse decidua and uterine stromal cells undergoing decidualization. α-Methyl-DL-aspartic acid (MDLA), a specific inhibitor for Ass1, can significantly increase the rate of embryonic reabsorption. Under in vitro induced decidualization, MDLA clearly inhibits the expression of decidual/trophoblast prolactin-related protein (Dtprp), a marker for decidualization in mice. Only Ass1 expression is induced by cAMP through PKA/p-Creb signaling pathway. Results from our cell culture models further indicates that the high level of L-Arg enhances stromal proliferation, while enzymatic activity or Ass1 expression level is essential to determine the magnitude of both mouse and human decidualization. Interestingly, L-Arg at high concentration down-regulates Ass1 and Asl expression by negative feedback to maintain L-Arg homeostasis. These findings highlight that cAMP-induced Ass1 expression is important in controlling the magnitude of decidualization through regulating L-Arg level.