Effects of Exosomes Derived From Helicobacter pylori Outer Membrane Vesicle-Infected Hepatocytes on Hepatic Stellate Cell Activation and Liver Fibrosis Induction

Abstract

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.

Keywords: Helicobacter pylori; exosome; liver fibrosis; outer membrane vesicle; α-SMA.

Figure 1

Morphological characterization of H. pylori-derived OMVs. (A) Scanning electron microscopic (SEM) image of OMVs showed the spherical and double-layered vesicles in different sizes. (B) Physicochemical characteristics of H. pylori-derived OMVs based on DLS. Size distribution is determined based on the intensity of OMVs in the ultracentrifugation technique. DLS confirmed nano-sized OMVs in a range of about 130–300 nm, peaked at 200 nm.

Figure 2

Characterization of hepatocyte-derived exosomes using Western blotting. Hepatocyte cell-derived exosomes were analyzed by Western blotting using antibodies against exosomal markers (CD9 and CD63).

Figure 3

Gene expression of liver fibrosis markers in LX-2 cells upon treatment with 10 µg/ml of H. pylori OMVs. Relative gene expression of fibrosis markers (vimentin, β-catenin, TIMP1, and α-SMA) was markedly increased after treatment of LX-2 cells with 10 µg/ml of H. pylori OMVs. mRNA level of E-cadherin was decreased in LX-2 cells treated with 10 µg/ml of H. pylori OMVs. Data presented as means ± standard error (SEM) for three independent experiments. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and by post-hoc one-way ANOVA statistical analysis.

Figure 4

Immunoblotting result of vimentin, β-catenin, TIMP1, α-SMA and E-cadherin protein expressions. As it shown vimentin, β-catenin, TIMP1, α-SMA protein expression of HSCs increased after treatment with infected hepatocytes-derived exosomes. Data presented as means ± standard error (SEM) for three independent experiments. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by post hoc one-way ANOVA statistical analysis. Scale bar: 20 µm, magnification 200X.