Metformin exerts anti-liver fibrosis effect based on the regulation of gut microbiota homeostasis and multi-target synergy.

Liver fibrosis can progress to cirrhosis if left untreated. Therefore, identifying effective antifibrotic drugs is crucial. This study aimed to investigate the role and potential mechanism of metformin in treating hepatic fibrosis based on the synergistic effect of multiple targets and the "intestine-liver axis" theory. A CCl4-induced liver fibrosis mouse model was established. We measured liver function, liver fibrosis indicators, oxidative stress and inflammation indices. Hematoxylin and eosin and Masson's trichrome staining were used to detect collagen deposition. The expression of apoptotic proteins, TGF-ß/Smads and TIMP-1/MMPs was assessed. 16S rRNA and untargeted metabolomics (liquid chromatography-mass spectrometry) were used to assess mouse intestinal flora and metabolites, performing a comprehensive correlation analysis. Metformin improved the general status and liver function and decreased liver collagen deposition in CCl4-induced liver fibrotic mice. Compared with the control group, IL-6, TNF-α and COX-2 serum levels in the liver fibrosis group increased. Although not significantly different, the serum inflammatory marker levels in the metformin group were lower than those in the model group. Metformin decreased serum MDA and increased serum SOD activity, which increased and decreased, respectively, in the model group. Furthermore, metformin inhibited liver cell apoptosis, TGF-ß1 expression and TIMP-1, while promoting Smad7 expression, MMP-1 and MMP-2 in fibrotic mice. 16S rRNA analysis indicated that metformin significantly ameliorated the Bacteroides, Helicobacter, Parabacteroides and Parasutterella imbalance. We identified 385 differential metabolites between the metformin and model groups. Prevotella abundance significantly decreased in the metformin group and positively correlated with decreased taurocholic acid levels. Metformin potentially reverses liver fibrosis by inhibiting inflammation, mitigating oxidative stress damage and suppressing hepatocyte apoptosis via intestinal flora metabolite regulation. Metformin also regulates the TGF-ß/Smads and TIMP-1/MMPs signalling pathways. This study provides a theoretical basis for the clinical use of metformin in patients with liver fibrosis.

miRNA-486-5p Promotes COPD Progression by Targeting HAT1 to Regulate the TLR4-Triggered Inflammatory Response of Alveolar Macrophages.

Purpose: The aim of this study was to investigate the role of miRNA-486-5p in chronic obstructive pulmonary disease (COPD) progression and the underlying molecular mechanisms. Materials and Methods: Aberrant miRNA expression profiles between smokers and nonsmokers, and those between COPD patients and normal subjects were analyzed using microarray datasets and reverse-transcriptase quantitative polymerase chain reaction (qPCR). Enzyme-linked immunosorbent assay was used to determine the levels of inflammatory cytokines in cell supernatants. Expression levels of inflammatory cytokines, HAT1, TLR4, and miR-486-5p, were determined using qPCR or Western blotting. Luciferase reporter assays and fluorescence in situ hybridization were used to confirm the regulatory interaction between miR-486-6p and HAT1. Results: miR-486-5p was significantly upregulated in the COPD and smoker groups compared to the control group, as demonstrated using bioinformatics analysis and validated using qPCR assay of alveolar macrophages and peripheral monocytes. Moreover, miR-486-5p expression was significantly correlated with the expression of IL-6, IL-8, TNF-α, and IFN-γ. Luciferase reporter assays confirmed that miR-486-5p directly targeted HAT1, and cellular localization showed that miR-486-5p and HAT1 were highly expressed in the cytoplasm. miR-486-5p overexpression led to a significant upregulation of TLR4 and a significant downregulation of HAT1. Inversely, miR-486-5p inhibition led to a significant downregulation of TLR4 and a significant upregulation of HAT1. HAT1 knockdown using siRNA significantly upregulated the expression of TLR4, IL-6, IL-8, TNF-α, and IFN-γ. Conclusion: miR-486-5p was differentially expressed in the alveolar macrophages of COPD patients. miR-486-5p overexpression may enhance the TLR4-triggered inflammatory response in COPD patients by targeting HAT1.

FAPP2 Accelerates the Proliferation and Invasion of Hepatocellular Carcinoma Cells via Wnt/ß-Catenin Signaling.

Four-phosphate adaptor-2 (FAPP2) has been recently identified as a tumor-associated regulator that is closely related to tumorigenesis. However, the precise role of FAPP2 in hepatocellular carcinoma (HCC) is still largely unknown. This study was designed to determine the function and molecular mechanisms of FAPP2 in HCC. Elevated expression of FAPP2 commonly occurred in the tumor tissue of HCC compared with normal controls. High expression of FAPP2 was also detected in HCC cell lines, and its knockdown markedly decreased the proliferation, colony formation, and invasion of HCC cells. Upregulation of FAPP2 using a FAPP2 expression vector markedly promoted the proliferation colony formation and invasion of HCC cells. FAPP2 was found to promote the activation of Wnt/ß-catenin signaling. Importantly, inhibition of Wnt/ß-catenin signaling abrogated the FAPP2 overexpression-conferred oncogenic effect in HCC cells. In addition, xenograft tumor experiments revealed that knockdown of FAPP2 significantly decreased the tumorigenicity of HCC cells in vivo. Taken together, our data reveal a tumor-promotion function of FAPP2 in HCC and demonstrate that knockdown of FAPP2 is capable of suppressing HCC cell proliferation and invasion through downregulation of Wnt/ß-catenin signaling. FAPP2 may be an attractive candidate anticancer target for HCC.

Concurrent measurement of dynamic changes in viral load, serum enzymes, T cell subsets, and cytokines in patients with severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infection caused by a novel Bunyavirus. Analysis on the dynamic changes of clinical, laboratory, and immunological abnormalities associated with SFTS in a concurrent study is lacking. Thirty-three SFTS patients were admitted to Jiangsu People's Hospital, Nanjing, China, and diagnosis was made based on the clinical symptoms and positive viral RNA detected by RT-PCR. Four patients deceased and twenty-nine survived. Blood samples were collected every other day between Day 5 and Day 15 from the onset of fever. Samples from healthy volunteers were used as normal controls. Peak viral RNA load, serum enzymes, IL-6, and IL-10 were significantly higher in deceased patients compared to survivors. Viral load, serum enzymes, and cytokines declined in survivors within 2 weeks from onset of fever. CD69+ T cells were elevated early after infection while HLA-DR+ and CTLA4+ T cells were elevated during the recovery phase of those who survived. High level SFTSV viral load was concurrently observed with reduced PLT, elevated serum enzymes, elevated pro-inflammatory and anti-inflammatory cytokines, and activation of CD69+ T cells. The degree and pattern of changes in these parameters may indicate the clinical outcome in SFTSV-infected patients.

A case of brucellosis displaying Parkinsonian-like tremor.

We report a rare case of brucellosis with Parkinsonian-like tremor and simple partial motor seizure. This patient worked as a sheep butcher and the sheep were imported from brucellosis-endemic areas. He presented with classical manifestations of brucellosis; infection was confirmed using the Rose Bengal Plate and Standard Tube Agglutination tests. The patient also suffered from headache, partial seizures, changes of personality and static tremor of both upper limbs. After anti-infection therapy, but without the use of anti-Parkinson drugs, the patient fully recovered and remained free of Parkinsonian-like tremor. Brucellosis can present with atypical symptoms, clinicians should widen their diagnostic view of brucella infection.

[Hepatitis B e antigen from chronic hepatitis B patients induces Th1/Th2 cytokine imbalance in vitro].

OBJECTIVE: To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs). METHODS: PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis. RESULTS: Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01). CONCLUSION: HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.

[Role of T lymphocyte activation in the development of severe fever accompanied by thrombocytopenia syndrome].

OBJECTIVE: To explore the effect of peripheral blood T lymphocyte activation on the blood cells, tissue injury and the development of disease in patients with severe fever with thrombocytopenia syndrome (SFTS). METHODS: The expressions of CD69, HLA-DR, CD28 and CTLA-4 on peripheral blood T lymphocytes were determined dynamically by flow cytometry and the relationships between the above immune molecules and ALT, AST, leukocytes, platelets were analyzed respectively. RESULTS: The expressions of CD69 and HLA-DR on peripheral blood T lymphocytes in patients with SFTS were elevated significantly during the whole course of disease (P<0.05). CD28 expression on CD4(+); lymphocyte subset decreased in the early stage and gradually increased to the normal range. Meanwhile, CTLA-4 expression on T lymphocytes went up in the late stage of viral infection. The levels of serum ALT, AST, LDH and CK were significantly higher than the upper limit of the normal and the counts of WBC and PLT dropped to the lowest at the outset. But all of them returned back to the normal range gradually with the down-regulation of CD69 and HLA-DR and the up-regulation of CTLA-4 on T lymphocyte. CONCLUSION: The overactivation of T lymphocytes may contribute to tissue injury and the high expression of CTLA-4 may be a negative feedback regulation to the overactivation of T lymphocytes, which plays an important role in immunoregulation of SFTS patients.

[The influence of hepatitis B e antigen on the expression of toll-like receptor 2 on peripheral monocytes].

OBJECTIVES: In order to investigate the relationship among the toll-like receptor 2 (TLR2), hepatitis B e antigen and HBV DNA, the expression levels of TLR2 on peripheral blood monocytes of chronic hepatitis B (CHB) patients as well as on their monocytes stimulated by ligand of TLR2 (Pam3CSK4) and HBeAg were analyzed. METHODS: Sixty-eight adults with CHB were enrolled, including 37 HBeAg-positive patients, 17 HBeAg-negative and HBV DNA negative patients, and 14 HBeAg-negative and HBV DNA positive patients. Sixteen healthy volunteers were also studied as controls. TLR2 expression levels on their peripheral blood monocytes stimulated with Pam3CSK4 or not stimulated were analyzed by FACS Caliber. The relationship of the expression levels of TLR2, HBeAg and HBV DNA were also analyzed. The level of TLR2 on peripheral blood monocytes of healthy volunteers and HBeAg-negative CHB patients stimulated by HBeAg was examined for six hours. RESULTS: The TLR2 expression levels on CD14+ cells were significantly reduced in HBeAg-positive patients (47.57%+/-21.40 %) compared to both healthy volunteers (76.51%+/-7.46%) and HBeAg-negative patients (HBV DNA positive group 73.2%+/-14.2%, HBV DNA negative group 75.2%+/-11.3%); but there was no difference between those of the HBeAg-negative patients and the healthy volunteers. Expression levels of TLR2 on monocytes stimulated by TLR2 ligand in HBeAg-positive patients were obviously increased, and reached the basic levels of the healthy volunteers and the HBeAg-negative patients. The expression levels of TLR2 on monocytes stimulated by HBeAg of the healthy volunteers and the HBeAg-negative patients were markedly reduced. CONCLUSIONS: In the presence of HBeAg, HBV down-regulates the expressions of TLR2 on CD14+ cells from peripheral blood, and there is no correlation between HBV-DNA and TLR2. Pam3CSK4 can boost the TLR2 expression in HBeAg-positive patients. The proposed interaction between HBV and TLR2 may provide an important clue to explain the reasons of the establishment of persistent HBV infection.