Hepatoprotective Effect of Mixture of Dipropyl Polysulfides in Concanavalin A-Induced Hepatitis

Abstract

The main biologically active components of plants belonging to the genus Allium, responsible for their biological activities, including anti-inflammatory, antioxidant and immunomodulatory, are organosulfur compounds. The aim of this study was to synthetize the mixture of dipropyl polysulfides (DPPS) and to test their biological activity in acute hepatitis. C57BL/6 mice were administered orally with DPPS 6 h before intravenous injection of Concanavalin A (ConA). Liver inflammation, necrosis and hepatocytes apoptosis were determined by histological analyses. Cytokines in liver tissue were determined by ELISA, expression of adhesive molecules and enzymes by RT PCR, while liver mononuclear cells were analyzed by flow cytometry. DPPS pretreatment significantly attenuated liver inflammation and injury, as evidenced by biochemical and histopathological observations. In DPPS-pretreated mice, messenger RNA levels of adhesion molecules and NADPH oxidase complex were significantly reduced, while the expression of SOD enzymes was enhanced. DPPS pretreatment decreased protein level of inflammatory cytokines and increased percentage of T regulatory cells in the livers of ConA mice. DPPS showed hepatoprotective effects in ConA-induced hepatitis, characterized by attenuation of inflammation and affection of Th17/Treg balance in favor of T regulatory cells and implicating potential therapeutic usage of DPPS mixture in inflammatory liver diseases.

Keywords: ConA hepatitis; anti-inflammatory activity; dipropyl polysulfides; hepatoprotective effects.

Figure 1

HPLC chromatogram of different dipropyl polysulfides in synthesized mixture.

Figure 2

Pretreatment with dipropyl polysulfides (DPPS) increases percentage of activated and regulatory macrophages and DCs in the liver. (a) Percentage of activated, CD69-positive, CD8+ and CD4+ cells, (b) F4/80+ macrophages and (c) CD11+ dendritic cells, expressing marker of classical (CD86) and alternative activation (CD206) and anti-inflammatory cytokine IL-10, determined by flow cytometry of mononuclear cells isolated from the livers of untreated mice and 6 h after oral treatment with DPPS, and gating strategy for detection of double-positive cells (d). Data are presented as mean + SD (* p < 0.05; two-tailed, unpaired Student’s t-test).

Figure 3

Pretreatment with DPPS attenuates markers of liver damage in ConA-induced acute hepatitis. DPPS pretreatment shows hepatoprotective effects in ConA-induced hepatitis. C57BL/6 mice were intravenously injected with 12.5 mg/kg of Concanavalin A. DPPS was administered orally (20 µL of 50% mixture solution), six hours before ConA injection. Livers were analyzed 12 and 24 h after ConA injection. (a) Representative H&E staining of paraffin-embedded liver sections, magnification 100×. (b) ALT levels determined in the serum 0, 8, 10, 18, and 30 h after oral administration of DPPS. (c) Serum levels of ALT determined 2, 4, 12, and 24 h after ConA injection (n = 7). (d) TUNEL staining of liver sections 6 h after ConA injection, magnification 400×. (e) Quantitative analysis of cell death rate: TUNEL-positive nuclei (brown) were counted in five random fields, and the data were summarized as the mean number of positive cells+SD (*** p < 0.001; ** p < 0.005; * p < 0.05; two-tailed, unpaired Student’s t-test).

Figure 4

Pretreatment with DPPS shows hepatoprotective effects in ConA-induced hepatitis. mRNA levels of (a) SOD1, SOD2, SOD3 and (b) p22^phox, p47^phox, p67^phox, iNOS in livers determined using real-time qRT-PCR with GPDH as an internal control, 8 h after infection (n = 5). Data are presented as mean + SE (*** p < 0.001; ** p < 0.005; * p < 0.05).

Figure 5

DPPS attenuates liver inflammation induced by ConA. (a) ICAM-1, VCAM-1, PECAM-1, and P-selectine mRNA expression in livers determined using real-time qRT-PCR with GPADH mRNA as an internal control, 12 h after infection (n = 5) presented as mean + SE. (b) Concentration of IL-1β, IL-6, IL-12, IL-17, TNF-α, and IL-10 in the liver tissue homogenate 6 and 12 h after ConA injection determined by ELISA presented as mean + SD (*** p < 0.001; ** p < 0.005; * p < 0.05).

Figure 6

Effects of DPPS on phenotype of lymphocytes in the livers of ConA-treated mice. Percentages of activated CD25+CD69+ (a), regulatory CD25+FoxP3+ (b), IL-17+ (c) and IFN-γ+ (d) CD4+ and CD8+ cells determined by flow cytometry of mononuclear cells isolated from the livers 12 h after ConA injection (n = 6). Data are presented as mean + SD (*** p < 0.001; ** p < 0.005; * p < 0.05).