High alcohol-producing Klebsiella pneumoniae causes fatty liver disease through 2,3-butanediol fermentation pathway in vivo.

High alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the gut microbiota had been demonstrated to be the causative agent of fatty liver disease (FLD). However, the catabolic pathways for alcohol production in vivo remain unclear. Here, we characterized the genome of HiAlc and medium alcohol-producing (MedAlc) Kpn and constructed an adh (an essential gene encoding alcohol dehydrogenase) knock-out HiAlc Kpn W14 strain (W14Δadh) using CRISPR-Cas9 system. Subsequently, we established the mouse model via gavage administration of HiAlc Kpn W14 and W14 Δadh strains, respectively. Proteome and metabolome analysis showed that 10 proteins and six major metabolites involved in the 2,3-butanediol fermentation pathway exhibited at least a three-fold change or greater during intestinal growth. Compared with HiAlc Kpn W14-fed mice, W14Δadh-fed mice with weak alcohol-producing ability did not show apparent pathological changes at 4 weeks, although some steatotic hepatocytes were observed at 12 weeks. Our data demonstrated that carbohydrate substances are catabolized to produce alcohol and 2,3-butanediol via the 2,3-butanediol fermentation pathway in HiAlc Kpn, which could be a promising clinical diagnostic marker. The production of high amounts of endogenous alcohol is responsible for the observed steatosis effects in hepatocytes in vivo.

Astragaloside IV attenuates high glucose-induced EMT by inhibiting the TGF-ß/Smad pathway in renal proximal tubular epithelial cells.

In the present study, we examined the molecular mechanism of astragaloside IV (AS-IV) in high glucose (HG)-induced epithelial-to-mesenchymal transition (EMT) in renal proximal tubular epithelial cells (PTCs). NRK-52E cell viability and apoptosis were determined by the cell counting kit-8 (CCK-8) assay and flow cytometric analysis, respectively. Expressions of E-cadherin, N-cadherin, vimentin, and occludin were measured by Western blot, and those of E-cadherin and N-cadherin were additionally measured by immunofluorescence analysis. Transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The expressions of Smad2, Smad3, phosphorylated-Smad2 (p-Smad2), and p-Smad3 were measured using Western blot. We found that AS-IV could recover NRK-52E cell viability and inhibit HG-induced cell apoptosis. TGF-ß1, α-SMA, Smad2, Smad3, p-Smad2, and p-Smad3 expressions were decreased in the AS-IV-treated groups compared with the HG group. Moreover, the expressions of E-cadherin and occludin were remarkably up-regulated and those of N-cadherin and vimentin were down-regulated in the AS-IV-treated groups compared with the HG group. Interestingly, the TGF-ß1 activator SRI-011381 hydrochloride had an antagonistic effect to AS-IV on HG-induced EMT behavior. In conclusion, AS-IV attenuates HG-induced EMT by inhibiting the TGF-ß/Smad pathway in renal PTCs.

Regulation and related mechanism of GSN mRNA level by hnRNPK in lung adenocarcinoma cells.

Gelsolin (GSN) is an actin filament-capping protein that plays a key role in cell migration. Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNPK) regulates GSN expression level by binding to the 3'-untranslated region (3'UTR) of GSN mRNA in non-small cell lung cancers (NSCLC) H1299 cells which are highly metastatic and express high level of GSN. We found that hnRNPK overexpression increased the mRNA and protein level of GSN, whereas hnRNPK knockdown by siRNA decreased the mRNA and protein level of GSN in both H1299 and A549 cells, indicating a positive role of hnRNPK in the regulation of GSN expression. Furthermore, hnRNPK knockdown affected the migration ability of H1299 and A549 cells which could be rescued by ectopic expression of GSN in those cells. Conversely, GSN knockdown in hnRNPK-overexpressing cells could abort the stimulatory effect of hnRNPK on the cell migration. These results suggest that hnRNPK function in the regulation of cell migration is GSN-dependent. Taken together, these data unveiled a new mechanism of regulation of the GSN expression by hnRNPK and provides new clues for the discovery of new anti-metastatic therapy.

PPAR­Î³ agonist rosiglitazone protects rat peritoneal mesothelial cells against peritoneal dialysis solution­induced damage.

Long-term peritoneal dialysis (PD) leads to ultrafiltration failure (UFF). Peritoneal mesothelial cells, which form the innermost monolayer of the peritoneal cavity, have been shown to regulate various responses, including inflammation, in UFF. The present study was designed to investigate the effect of the peroxisome proliferator­activated receptor­Î³ (PPAR­Î³) agonist, rosiglitazone, on peritoneal dialysis solution (PDS)­induced injuries in rat peritoneal mesothelial cells (RPMCs). RPMCs were cultured for different durations and with different concentrations of PDS. The gene expression levels of aquaporin­1 (AQP­1) and zonula occluden­1 (ZO­1) were determined using reverse transcription­quantitative polymerase chain reaction analysis. The protein levels of AQP­1, ZO­1 and PPAR­Î³ were measured using western blot analysis. Interleukin (IL)­6 and IL­8 were detected using ELISA. The RPMCs were damaged by stimulation with 4.25% PDS for 72 h. The expression levels of AQP­1 and ZO­1 were increased, and the secretion of IL­6 and IL­8 were decreased by rosiglitazone. The use of the PPAR­Î³ inhibitor, GW­9662, completely prevented the effects of rosiglitazone. These results indicated that PDS exposure stimulated an inflammatory response in the RPMCs. The PPAR­Î³ activator, rosiglitazone, appeared to relieve the injury by inhibiting inflammation, and regulating the expression of AQP­1 and ZO­1, however further investigations are required to elucidate the potential underlying mechanism.

Protein Phosphatase 2C of Toxoplasma Gondii Interacts with Human SSRP1 and Negatively Regulates Cell Apoptosis.

OBJECTIVE: The protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability. METHODS: The yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry. RESULTS: We identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1. CONCLUSION: TgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.