IL-6RFP Can Protect Liver Function In The Model Of Mice Infected With Schistosoma Japonicum

Objective IL-6 is an important contributor to both inflammation and cancer. Critical roles of IL-6 in production of acute-phase proteins along with its role in expansion and activation of T cells and differentiation of B cells, makes it an important factor in inflammatory diseases. Moreover, IL-6 influences multiple signaling pathways that together promote tumor cell survival and proliferation, angiogenesis and vasculogenesis, metastasis and drug resistance. As a result, inhibitors of IL-6 and IL-6R mediated signaling are the subject of increasing research and clinical evaluations in the treatment of inflammatory diseases and cancer. IL-6 in the model of C57BL/6 mice infected with Schistosoma japonicum increased significantly, but the role of IL-6 signaling pathway in the pathogenesis of schistosomiasis was not clear. The objective of this article is to explore the role of m IL-6RFP for the protection of liver cells in the model of BALB/c mice infected with Schistosoma japonicum.Methods Expression of recombinant m IL-6RFP protein was taken by the using of baculovirus insect cell expression system, nickel ion column was used to obtain purified recombinant m IL-6RFP protein. Hep G2 cells were set with the normal group, IL-6 stimulation group, IL-6 blocked group and Protein eluent group, in vitro experiment. RT-PCR method was used to detect fibrinogen(FGG) and haptoglobin(HP) m RNA expression in each group. So as to verify the biological activity of m IL-6RFP. Six-to eight-week-old female BALB/c mice(Experimental Animal Center, Chinese Science Academy, Shanghai, China) were used in all the experiments. Schistosoma japonicum(Chinese mainland strain) infected Oncomelania hupensissnails were provided by Jiangsu Institute of Parasitic Diseases. After anesthetized by intraperitoneal injection ofketamine, mice were infected with 20±2 freshly shed cercaria percutaneously. Some BALB/c mice were administrated by tail vein injection with 100 μg of m IL-6RFP or equal volumes of protein solvent, at 24, 26, 28, 30, 32, 34, 36, 38 and 40 days after infection. Mice were sacrificed 48 h after the last antibody injection. All mice were housed under specific pathogen-free barrier conditions in Anhui Medical University animal facility. Liver samples from 6-week S. Japonicum infected mice were fixed in 10% buffered formalin and processed through the routine paraffin embedding procedures. Sections(4 μm) were stained with H&E, and examined for quantitative and qualitative changes. In the evaluation of hepatic granuloma size, only those containing clearly identifiable central ovum were selected. The granuloma size were measured by Image Tool 3.0 software. Total RNA from individual liver of schistosoma infected mice as well as uninfected mice was extracted using the Trizol Reagent. For each sample, 1 μg of total cellular RNA was reverse-transcribed to c DNA with random primers and MMLV reverse transcriptase in a total reaction volume of 20 μl. RT-PCR method were used to detect IL-6、IL-13、IL-1β、CXCL1 and TNF-α m RNA expression in each group. Real-time quantitative RT-PCR on 1μl of c DNA from each sample was performed in duplicates by SYBR Premix Ex Taq according to the manufacturer’s instructions. For analysis, the “housekeeping” gene encoding β-actin was used as a normalization control. Values are shown as mean ±SEM from six independent experiments. Dissociation curves were used to verify the specificity of the PCR products. Determination of ALT/AST levels, Serum levels of the liver-associated ALT and AST were measured as indicators of hepatocellular damage. The concentrations of the enzymes were determined using a commercial kit according to the manufacturer’s instructions.Results Hep G2 cell experiments showed that mIL-6RFP can reduce the effect of IL-6 on it, the m RNA expression of FGG and HP were significantly reduced(P<0.05). The recombinant protein m IL-6RFP has biological activity, it can block IL-6. In the BALB/cmice model infected with Schistosoma japonicum, blockade of IL-6 the serum levels of the liver-associated ALT and AST were decreased significantly(P<0.05) compared with the eluent group, while no significant difference on granuloma formation.Conclusion In the BALB/c mice infected with Schistosoma japonicum, m IL-6RFP can significantly improve the liver function but no obvious effect on granuloma formation.