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. 2018 Dec 15;841:33-48.
doi: 10.1016/j.ejphar.2018.08.040. Epub 2018 Sep 5.

Sparstolonin B (SsnB) Attenuates Liver Fibrosis via a Parallel Conjugate Pathway Involving P53-P21 Axis, TGF-beta Signaling and Focal Adhesion That Is TLR4 Dependent

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Free PMC article

Sparstolonin B (SsnB) Attenuates Liver Fibrosis via a Parallel Conjugate Pathway Involving P53-P21 Axis, TGF-beta Signaling and Focal Adhesion That Is TLR4 Dependent

Diptadip Dattaroy et al. Eur J Pharmacol. .
Free PMC article

Abstract

SsnB previously showed a promising role to lessen liver inflammation observed in a mouse model of NAFLD. Since NAFLD can progress to fibrosis, studies were designed to unravel its role in attenuating NAFLD associated fibrosis. Using both in vivo and in vitro approaches, the study probed the possible mechanisms that underlined the role of SsnB in mitigating fibrosis. Mechanistically, SsnB, a TLR4 antagonist, decreased TLR4-PI3k akt signaling by upregulating PTEN protein expression. It also decreased MDM2 protein activation and increased p53 and p21 gene and protein expression. SsnB also downregulated pro-fibrogenic hedgehog signaling pathway, inhibited hepatic stellate cell proliferation and induced apoptosis in hepatic stellate cells, a mechanism that was LPS dependent. Further, SsnB decreased fibrosis by antagonizing TLR4 induced TGFβ signaling pathway. Alternatively, SsnB augmented BAMBI (a TGFβ pseudo-receptor) expression in mice liver by inhibiting TLR4 signaling pathway and thus reduced TGFβ signaling, resulting in decreased hepatic stellate cell activation and extracellular matrix deposition. In vitro experiments on human hepatic stellate cell line showed that SsnB increased gene and protein expression of BAMBI. It also decreased nuclear co-localization of phospho SMAD2/3 and SMAD4 protein and thus attenuated TGFβ signaling in vitro. We also observed a significant decrease in phosphorylation of SMAD2/3 protein, decreased STAT3 activation, alteration of focal adhesion protein and stress fiber disassembly upon SsnB administration in hepatic stellate cells which further confirmed the antagonistic effect of SsnB on TLR4-induced fibrogenesis.

Keywords: Apoptosis; Cyclin; E Cell cycle; Hedgehog signaling; NAFLD; NASH; TLR4; p21; p53.

Conflict of interest statement

Conflict of interest

The authors declare that there is no conflict of interest.

Figures

Fig. 1.
Fig. 1.. SsnB treatment ameliorates liver fibrosis in NASH mice.
A: Representative images of picrosirius red stain of Control, NASH and NASH+SsnB mice. Images were taken at 20× magnification. (*P < 0.05). B: Morphometric analysis of picrosirius red immunohistochemistry in liver slices from Control, NASH and NASH+SsnB mice groups. (*P < 0.05). C: The degree of fibrosis by METAVIR scoring system. (*p < 0.05).
Fig. 2.
Fig. 2.. SsnB treatment decreased microRNA21(miR21) expression and upregulated PTEN protein expression in NASH liver.
A: qRT-PCR analysis of miR21 expression of Control, NASH, and NASH+SsnB mouse liver samples normalized against Control (*P < 0.05). B: Western blot analysis of β-actin and PTEN protein levels of Control, NASH, and NASH+SsnB liver homogenates. C. Bar diagram representing levels of PTEN protein normalized against β-actin of respective samples. Y-axis represent arbitrary unit of PTEN band of mice groups from Control, NASH and NSH+SsnB liver homogenate. (*) P < 0.05 is considered statistically significant when compared with control. (#) P < 0.05 is considered statistically significant when compared with NASH.
Fig. 3.
Fig. 3.. SsnB treatment induced PTEN expression increases p53, p21 upregulation and decreases hedgehog signaling in liver.
A and B: Representative images of MDM2 (A) and Gli1 (B) immunoreactivity as shown by immunofluorescence microscopy on liver slices of Control, NASH and NASH+SsnB mice, images taken at 20× magnification using immunofluorescence microscopy. C and D: Representative images of p21 (C) and p53 (D) immunoreactivity as shown by immunohistochemistry on liver slices of Control, NASH and NASH+SsnB mice, taken at 20× magnification.
Fig. 4.
Fig. 4.. Morphometric analysis of Fig. 3.
Morphometric analysis of immunoreactivities of (A)MDM2, (B)Gli1, (C)p21 and (D)p53. Between group comparisons were compared with a Students t-test and analyses with a *p < 0.05 were considered significant.
Fig. 5.
Fig. 5.. SsnB treatment increased p53, p21 expression in vitro.
A: qRT-PCR analysis of mRNA expression of p53 and p21 from control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate cells, normalized against control (*P < 0.05). B. Immunofluorescence dual labeling of Control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate cells depicting α-SMA (green)-p53 (red) co-localization (yellow), taken at 20× magnification. C. Immunoreactivity of p21 (red) in control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate as shown by immunofluorescence microscopy at 20× magnification. D. Western blot analysis of p53, p21 and β-actin protein levels in Control, LPS and LPS+SsnB10 (10 μM) primary Rat hepatic stellate cell homogenates. E and F. Bar diagram representing the levels of p53 protein (E) and p21 (F) normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant.
Fig. 5.
Fig. 5.. SsnB treatment increased p53, p21 expression in vitro.
A: qRT-PCR analysis of mRNA expression of p53 and p21 from control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate cells, normalized against control (*P < 0.05). B. Immunofluorescence dual labeling of Control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate cells depicting α-SMA (green)-p53 (red) co-localization (yellow), taken at 20× magnification. C. Immunoreactivity of p21 (red) in control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate as shown by immunofluorescence microscopy at 20× magnification. D. Western blot analysis of p53, p21 and β-actin protein levels in Control, LPS and LPS+SsnB10 (10 μM) primary Rat hepatic stellate cell homogenates. E and F. Bar diagram representing the levels of p53 protein (E) and p21 (F) normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant.
Fig. 6.
Fig. 6.. SsnB treatment decreased gene expression of hedgehog signaling markers and reduces Cyclin E protein expression in vitro.
A: qRT-PCR analysis of mRNA expression of Gli1, Gli2, IHH, Ptc from Control (untreated), LPS-treated, LPS+SsnB10(10 μM) and LPS+SsnB100 (100 μM) treated human immortalized hepatic stellate cells (LX2), normalized against control (*P < 0.05). B: Western blot analysis of Cyclin E and β-actin protein levels of Control (untreated), LPS-treated, LPS+SsnB10(10 μM) and LPS+SsnB100(100 μM) treated human immortalized hepatic stellate cells (LX2). C. Morphometric analysis of western blot: Bar diagram represents the level of cyclin E normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant.
Fig. 7.
Fig. 7.. SsnB treatment decreases proliferation and induces apoptosis in hepatic stellate cells.
A. Cell cycle analysis of untreated cells (control), cells treated with LPS, LPS+SsnB10(10 μM), and LPS+SsnB100(100 μM). Quantitation of the PI staining data is presented as the cell cycle distribution percentages. B. Apoptosis is indicated by TUNEL based ApopTag® technology (EMD Millipore, MO) which labels 3’-OH ends of DNA fragments by fluorescent antibody as detected by immunofluorescence microscopy in Control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated LX2 cells. C. Western blot analysis of p53, cleaved caspase3 (Casp3), total caspase3 (Casp3), cleaved PARP1 and β-actin protein levels of Control (untreated), LPS-treated, LPS+SsnB10 (10 μM), and LPS +SsnB100(100 μM) treated LX2 cells. D. Morphometric analysis of western blot where the bar diagram represents the level of p53, cleaved caspase3, total caspase3, cleaved PARP1 normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant. E. TUNEL assay-based apoptosis analysis of Control, NASH, and NASH+SsnB treated mice liver samples. F. Morphometric analysis of apoptotic events/3 microscopic field. (*) P < 0.05 is considered statistically significant.
Fig. 7.
Fig. 7.. SsnB treatment decreases proliferation and induces apoptosis in hepatic stellate cells.
A. Cell cycle analysis of untreated cells (control), cells treated with LPS, LPS+SsnB10(10 μM), and LPS+SsnB100(100 μM). Quantitation of the PI staining data is presented as the cell cycle distribution percentages. B. Apoptosis is indicated by TUNEL based ApopTag® technology (EMD Millipore, MO) which labels 3’-OH ends of DNA fragments by fluorescent antibody as detected by immunofluorescence microscopy in Control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated LX2 cells. C. Western blot analysis of p53, cleaved caspase3 (Casp3), total caspase3 (Casp3), cleaved PARP1 and β-actin protein levels of Control (untreated), LPS-treated, LPS+SsnB10 (10 μM), and LPS +SsnB100(100 μM) treated LX2 cells. D. Morphometric analysis of western blot where the bar diagram represents the level of p53, cleaved caspase3, total caspase3, cleaved PARP1 normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant. E. TUNEL assay-based apoptosis analysis of Control, NASH, and NASH+SsnB treated mice liver samples. F. Morphometric analysis of apoptotic events/3 microscopic field. (*) P < 0.05 is considered statistically significant.
Fig. 8.
Fig. 8.. SsnB treatment decreases hepatic stellate cell activation in murine NASH.
A. Representative images of α-SMA immunoreactivity as shown by immunofluorescence microscopy on liver slices of Control, NASH and NASH+SsnB mice, taken at 20× magnification using immunofluorescence microscopy. B. Morphometric analysis of α-SMA immunoreactivity in A. (*P < 0.05).
Fig. 9.
Fig. 9.. SsnB treatment in NASH mice upregulates BAMBI in liver.
A. Representative images of BAMBI immunoreactivity as shown by immunohistochemistry on liver slices of Control, NASH and NASH+SsnB mice, taken at 20× magnification. B. Morphometric analysis of BAMBI immunoreactivity in A. (*P < 0.05).
Fig. 10.
Fig. 10.. SsnB treatment decreases fibronectin deposition in NASH liver.
A. Representative images of fibronectin immunoreactivity as shown by immunohistochemistry on liver slices of Control, NASH and NASH+SsnB mice, taken at 20× magnification. B. Morphometric analysis of fibronectin immunoreactivity in A. (*P < 0.05).
Fig. 11.
Fig. 11.. SsnB upregulates BAMBI and decreases TGFβ signaling in vitro.
A. qRT-PCR analysis of mRNA expression of BAMBI from control (untreated), LPS-treated, LPS+SsnB10 (10 μM), and LPS+SsnB100 (100 μM) treated Rat primary hepatic stellate cells, normalized against control (*P < 0.05). B. Western blot analysis of β-actin and BAMBI protein levels of Control (untreated), LPS-treated, LPS+SsnB10 (10 μM), and LPS+SsnB100 (100 μM) treated rat primary hepatic stellate cells, C. Morphometric analysis of western blot where the bar diagram represents the level of BAMBI normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant. D. Immunofluorescence dual labeling depicting SMAD2/3 (red)-SMAD4 (green) co-localization (yellow) on Control (untreated), LPS-treated, and LPS+SsnB100 (100 μM) treated human immortalized hepatic stellate cells (LX2) taken at 40× magnification. E. Western blot analysis of phosphor SMAD2/3, total SMAD2/3 and β-actin protein levels of Control (untreated), LPS-treated, LPS+SsnB10 (10 μM), and LPS+SsnB100 (100 μM) treated human immortalized hepatic stellate cells (LX2). F. Morphometric analysis of western blot where the bar diagram represents the level of pSMAD2/3 normalized against total SMAD2/3 of respective samples. (*) P < 0.05 is considered statistically significant.
Fig. 12.
Fig. 12.. SsnB downregulates STAT3 phosphorylation, decreases stellate cell activation and connective tissue growth factor in vitro.
A. Western blot analysis of phospho STAT3, α-SMA, CTGF and β-actin protein levels of Control (untreated), LPS-treated, LPS+SsnB10 (10 μM), and LPS+SsnB100 (100 μM) treated human immortalized hepatic stellate cells (LX2). B. Morphometric analysis of western blot where the bar diagram represents the level of pSTAT3, α-SMA, and CTGF normalized against β-actin of respective samples. (*) P < 0.05 is considered statistically significant.
Fig. 13.
Fig. 13.. SsnB treatment decreases focal adhesion protein expression and inhibits stress fiber formation in vitro.
A. Immunofluorescence microscopy depicting paxillin (green) immunoreactivity in Control (untreated), LPS-treated, LPS+SsnB10(10 μM), and LPS+SsnB100(100 μM) treated human immortalized hepatic stellate cells (LX2) taken at 20× magnification B and D. Immunofluorescence microscopy depicting paxillin (green) immunoreactivity at 40× magnification in Control (untreated), LPS-treated, and LPS+SsnB100(100 μM) treated human immortalized hepatic stellate cells (LX2) (B) and Control (untreated), LPS-treated, LPS +SsnB10(10 μM), and LPS+SsnB100(100 μM) treated Rat primary hepatic stellate cells (D). Paxillin immunoreactivity (on the edges of the cells) is pointed by white arrows. C and E. Immunofluorescence dual labeling depicting α-SMA(red)-Vinculin(green) on Control (untreated), LPS-treated, and LPS+SsnB100(100 μM) treated human immortalized hepatic stellate cells (LX2) (C) and Control (untreated), LPS-treated, LPS+SsnB10(10 μM) and LPS+SsnB100(100 μM) treated in rat primary hepatic stellate cells (E) taken at 40× magnification. α-SMA immunoreactivity (as stress fibers) is pointed by white arrows. Nucleus stained with DAPI (Blue).

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