Fig 1: Increased liver tissue stiffness promotes osteopontin which suppresses Interferon-Stimulated Gene (ISG) expression in the livers of CCl4-administered HBVTg mice: Liver fibrosis was induced by injecting CCl4 for 6 weeks in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice. RT-PCR analysis was done for mRNA expression of OPN, IFN-induced genes, and USP18. (A) Osteopontin (OPN); (C) 2’-5’ oligoadenylate synthetase-like 1 (OASL1); (D) Interferon-stimulated gene 15 (ISG15); (E) Apolipoprotein B Editing Complex (APOBEC3); (F) USP18. (B) OPN protein levels were measured by Elisa in serum. Data are expressed as Mean ± SEM (n =4). The data bars marked with the same alphabetical letter are considered not significantly different, while those marked with differing alphabetical letters are considered significantly different from each other (p ≤ 0.05).
Fig 2: The proposed model for increased liver stiffness inducing hepatitis B progression and end-stage liver diseases: In vitro and In vivo data show that an increased matrix stiffness upregulates the release of OPN, which limits the IFNα-induced innate immune response by suppressing the STAT-1 and ISGs activation, thereby upregulating the expression of HBV markers and inflammation and promoting the development of end-stage liver diseases. This provides an optional explanation for continued and fast HBV infection progression when the disease comes to the liver fibrosis stage.
Fig 3: Recombinant OPN treatment increases the expression of HBV marker by suppressing IFNα-inducible anti-viral genes activation in soft gel (2 KPa) attached HBV+ transfected HepG2.2.15 cells: To mimic overexpression, we treated soft (2 kPa) gel-attached HBV transfected HepG2.2.15 cells with rOPN for 24hrs. RT-PCR analysis was done for mRNA expression of IFN-induced genes and HBV RNA. (A) 2′–5′ oligoadenylate synthetase 1 (OAS1); (B) Interferon-stimulated gene 15 (ISG15); (C) Apolipoprotein B Editing Complex (APOBEC3); (D) HBV RNA. GAPDH acted as an internal control. (E) STAT-1 phosphorylation (pSTAT-1) was measured by immunoblotting. Total STAT-1 was used to normalize the data. (F) Quantification of immunoblotting data. Data are from three independent experiments presented as Mean ± SEM. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤0.05).
Fig 4: Silencing of OPN decreases expression of HBV marker by increasing IFNα-inducible anti-viral genes activation in soft gel (2 KPa)-attached HBV+ transfected HepG2.2.15 cells, and USP18 is partially responsible for the regulation of HBV infection markers by OPN in fibrotic stiffness gel-attached HBV+ transfected HepG2.2.15 cells: We silenced OPN by specific siRNA transfection, in soft (2 kPa) and stiff (25 kPa) gel-attached HBV transfected HepG2.2.15 cells. RT-PCR analysis was done for mRNA expression of IFN-induced genes and HBV RNA. (A) Transfection efficiency of OPN silencing and OPN mRNA; (B) 2′–5′ oligoadenylate synthetase 1 (OAS1); (C) Interferon-stimulated gene 15 (ISG15); (D) Apolipoprotein B Editing Complex (APOBEC3); (E) HBV RNA. GAPDH acted as an internal control. We silenced OPN by specific siRNA transfection in soft (2 kPa) and stiff (25 kPa) gel-attached HBV-transfected HepG2.2.15 cells, and to mimic overexpression, we treated only soft (2 kPa) gel-attached HBV transfected HepG2.2.15 cells with rOPN for 24hrs. RT-PCR analysis was done for mRNA expression of USP18 (F, G). GAPDH acted as an internal control for RT-PCR analysis. (H) USP18 protein was measured by immunoblotting and β-actin was also used as an internal control. Data are from three independent experiments presented as Mean ± SEM. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (P ≤0.05).
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