Silencing p53 inhibits interleukin 10-induced activated hepatic stellate cell senescence and fibrotic degradation in vivo

Abstract

Activated hepatic stellate cells are reported to play a significant role in liver fibrogenesis. Beside the phenotype reversion and apoptosis of activated hepatic stellate cells, the senescence of activated hepatic stellate cells limits liver fibrosis. Our previous researches have demonstrated that interleukin-10 could promote hepatic stellate cells senescence via p53 signaling pathway in vitro. However, the relationship between expression of p53 and senescence of activated hepatic stellate cells induced by interleukin-10 in fibrotic liver is unclear. The purpose of present study was to explore whether p53 plays a crucial role in the senescence of activated hepatic stellate cells and degradation of collagen mediated by interleukin-10. Hepatic fibrosis animal model was induced by carbon tetrachloride through intraperitoneal injection and transfection of interleukin-10 gene to liver was performed by hydrodynamic-based transfer system. Depletions of p53 in vivo and in vitro were carried out by adenovirus-based short hairpin RNA against p53. Regression of fibrosis was assessed by liver biopsy and collagen staining. Cellular senescence in the liver was observed by senescence-associated beta-galactosidase (SA-β-Gal) staining. Immunohistochemistry, immunofluorescence double staining, and Western blot analysis were used to evaluate the senescent cell and senescence-related protein expression. Our data showed that interleukin-10 gene treatment could lighten hepatic fibrosis induced by carbon tetrachloride and induce the aging of activated hepatic stellate cells accompanied by up-regulating the expression of aging-related proteins. We further demonstrated that depletion of p53 could abrogate up-regulation of interleukin-10 on the expression of senescence-related protein in vivo and vitro. Moreover, p53 knockout in fibrotic mice could block not only the senescence of activated hepatic stellate cells, but also the degradation of fibrosis induced by interleukin-10 gene intervention. Taken together, our results suggested that interleukin-10 gene treatment could attenuate carbon tetrachloride-induced hepatic fibrosis by inducing senescence of activated hepatic stellate cells in vivo, and this induction was closely related to p53 signaling pathway.

Keywords: Interleukin 10; gene therapy; hepatic stellate cells; liver fibrosis; p53; senescence.

Figure 1.

IL-10 gene intervention attenuated CCL₄-induced hepatic fibrosis in rats. (a) Schematic diagram of drug intervention in the process of hepatic fibrosis induced by CCL₄ in rats. Liver fibrosis in SD male rats was induced by intraperitoneal injection of 40% (v/v) CCL₄-olive oil mixed reagent, and pcDNA 3.1-rIL-10 recombinant plasmid and control reagent were injected through tail vein from the fourth week. (b) Content of IL-10 in rat plasma. After IL-10 gene intervention, the expression of IL-10 in rat plasma was significantly increased. (c) H&E (200× magnification) and Masson stain (100× magnification) were used to estimate the degree and grade of liver fibrosis; Sirius Red stain (100× magnification) was used to observe deposition of collagen I and III. (d) Semi-quantitative analysis of grading of inflammatory and staging of fibrosis according to modified HAI score. (e) Expression of collagen I and III. (f) Serum level of HA. (g) Serum levels of ALT and AST. n = 10, Data were displayed as average ± SD. ***P < 0.001, compared with group control; ^##P < 0.01, ^###P< 0.001, compared with group model; ^$P < 0.05, ^$$$P < 0.001, compared with group pcDNA3.1. (A color version of this figure is available in the online journal.)

Figure 2.

IL-10-induced senescence of activated HSCs was associated with increased expression of p53 in vivo. (a) Immunohistochemistry staining of α-SMA and SA-β-Gal staining was used to detect the numbers of activated HSCs and senescent HSCs in rats, respectively (100× magnification). (b) Relative area of positive expression of α-SMA or SA-β-Gal (%) in liver tissue. (c) Immunofluorescence double staining to observe the expression of HMGA1 or p53 in fibrotic liver tissue. HSCs were specifically stained with antibody against α-SMA, and nuclei were stained with DAPI (100× magnification). (d) Bar graphs showed the percentage quantitation of fluorescent images of HMGA1 or P53 positive cells in all activated HSCs (α-SMA as marker). (e, f, g) Western blot analyzed the expression levels of α-SMA, P53, P21, P27, HMGA1 in liver tissue. n = 10, Data were displayed as average ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, compared with group control; ^#P < 0.05, ^##P < 0.01, ^###P< 0.001, compared with group model; ^$P < 0.05, ^$P < 0.001, compared with group pcDNA3.1. (A color version of this figure is available in the online journal.)

Figure 3.

Inhibition of p53 antagonized IL-10-induced expression of senescence-related proteins in HSC-T25 cells in vitro. (a) Vector structure of recombinant adenovirus. (b) Performance of GFP in HSC-T25 cells under fluorescence microscopy after adenovirus transfection for three days (200× magnification). The infection rate in all groups was above 80%. (c) Real-time PCR analyzed the mRNA expression of p53 after silencing p53. The knockdown efficiency at the RNA level was greater than 70% in all three groups. (d, e) Western blot analyzed the expression levels of p53 and its downstream target proteins P21, P27, and HMGA-1 after silencing p53. Data were displayed as average ± SD. *P < 0.05, ***P < 0.001, compared with group control; ^###P < 0.001, compared with group IL-10 intervention. (A color version of this figure is available in the online journal.)

Figure 4.

Depletion of p53 abrogated the anti-fibrotic effect of IL-10 on hepatic fibrosis in mice. (a) Schematic diagram of drug intervention in the process of hepatic fibrosis induced by CCL₄ in mice. Liver fibrosis in ICR male mice was induced by 10% (v/v) CCL₄-olive oil mixed reagent. From the third week, pcDNA3.1-mIL-10 recombinant plasmid and empty plasmid were injected into mice by tail vein, and adenovirus inhibiting p53 and control virus were injected through tail vein the next day. (b) H&E and Masson stain were used to assess the degree and grade of liver fibrosis, Sirius Red stain was used to detect accumulation of collagen I and III (100× magnification). (c) Semi-quantitative analysis of grading of inflammatory and staging of fibrosis according to modified HAI score. (d) Expression of collagen I and III. (e) Serum levels of ALT and AST. (f) Serum levels of HA. n = 6, Data were displayed as average ± SD. ***P < 0.001, compared with group control; ^#P < 0.05, ^##P < 0.01, ^###P< 0.001, compared with group CCL₄+Ad.Fc; ^$P < 0.05, ^$$$P < 0.001, compared with group CCL₄+Ad.Fc+IL-10. (A color version of this figure is available in the online journal.)

Figure 5.

Depletion of p53 attenuated IL-10-induced activated HSCs senescence in vivo. (a) Immunohistochemistry staining of α-SMA and SA-β-Gal staining was used to detect the numbers of activated HSCs and senescent HSCs in mice, respectively (100× magnification). (b) Relative expression of α-SMA or SA-β-Gal (%) in mice liver tissue. (c) Immunofluorescence double staining to observe the expression of p53 (200× magnification) or HMGA1 (100× magnification) in mice liver tissue. HSCs were specifically stained with antibody against α-SMA, and nuclei were stained with DAPI. (d) Bar graphs showed the percentage quantitation of fluorescent images of P53 or HMGA1 positive cells in all activated HSCs (α-SMA as marker). (e, f) Western blot analyzed the expression levels of α-SMA, P21, P27, and P16 in mice fibrotic liver. n = 6, Data were displayed as average ± SD. ***P < 0.001, compared with group control; ^#P < 0.05, ^###P < 0.001, compared with group CCL₄+Ad.Fc; ^$$$P < 0.001, compared with group CCL₄+Ad.Fc+IL-10. (A color version of this figure is available in the online journal.)