Liver fibrosis is a multifactorial disease with microbial and non-microbial causes. In recent years, Helicobacter pylori infection has been thought to play a critical role in some extra-gastrointestinal manifestations especially liver disorders. Outer membrane vesicles (OMVs) are one of the most important discussed H. pylori virulence factors. In the current study, four different clinical strains of H. pylori were collected and their OMVs were purified using ultra-centrifugation. To investigate their effects on liver cell exosomes, co-incubation with hepatocytes was applied. After a while, hepatocyte-derived exosomes were extracted and incubated with hepatic stellate cells (HSCs) to investigate the HSC activation and fibrosis marker induction. The expression of α-SMA, TIMP-1, β-catenin, vimentin, and e-cadherin messenger RNAs (mRNA) was assessed using real-time RT-PCR, and the protein expression of α-SMA, TIMP-1, β-catenin, vimentin, and e-cadherin was evaluated by Western blotting. Our results showed that infected hepatocyte-derived exosomes induced the expression of α-SMA, TIMP-1, β-catenin, and vimentin in HSCs and e-cadherin gene and protein expression was downregulated. In the current study, we found that H. pylori-derived OMVs may aid the exosome alternation and modified exosomes may have a possible role in HSC activation and liver fibrosis progression.
Introduction. Helicobacter pylori is a prevalent pathogenic bacterium that resides in the human stomach. Outer membrane vesicles (OMVs) are known as nanosized cargos released by H. pylori, which have been proposed to have a key role in disease progression, pathogenesis, and modulation of the immune system. There are multiple evidences for the role of H. pylori in extragastroduodenal illnesses especially liver-related disorders. However, the precise mechanism of H. pylori extragastroduodenal pathogenesis still remains unclear. In the current study, we aimed to determine the impact of H. pylori-isolated OMVs on hepatic stellate cell (HSC) activation and expression of liver fibrosis markers. Materials and Methods. Five H. pylori clinical strains with different genotype profiles were used. Helicobacter pylori OMVs were isolated using ultracentrifugation and were analyzed by scanning electron microscopy (SEM) and dynamic light scattering (DLS). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis was applied to determine protein components of H. pylori-derived OMVs. Cell viability of LX-2 human hepatic stellate cell line exposed to OMVs was measured by MTT assay. LX-2 cells were treated with OMVs for 24 h. The gene expression of α-SMA, E-cadherin, vimentin, snail, and β-catenin was analyzed using quantitative real-time PCR. The protein expression of α-SMA, as a well-studied profibrotic marker, was evaluated with immunocytochemistry. Results. Our results showed that H. pylori strains released round shape nanovesicles ranging from 50 to 500 nm. Totally, 112 various proteins were identified in OMVs by proteomic analysis. The isolated OMVs were negative for both CagA and VacA virulence factors. Treatment of HSCs with H. pylori-derived OMVs significantly increased the expression of fibrosis markers. Conclusions. In conclusion, the present study demonstrated that H. pylori-derived OMVs could promote HSC activation and induce the expression of hepatic fibrosis markers. Further research is required to elucidate the definite role of H. pylori-derived OMVs in liver fibrosis and liver-associated disorders.
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