Effect Of IL-10 On Expression Of CTGF And TIMP-1 In Hepatic Stellate Cells Mediated By TGFβ₁

ObjectiveHepatic fibrosis (HF) is a common pathologic change in chronic liver diseases. With further development, it can become liver cirrhosis, which seriously affect health and life of patients. Hepatic stellate cells (HSC) play a central role in the process of hepatic fibrosis. Transfoming growth factorβ₁ (TGFβ₁) is a powerful cytokine resulting in fibrosis, promoting proliferation and activation of HSC, as so as magnanimous synthesis of ECM, playing an important role in the process of fibrosis. Interleukin-10 is a inhibiting factor of inflammation, clinical and animal experiments indicate that,IL-10 may lessen inflammation of liver, inhibit development of hepatic fibrosis, so it may become an anti-hepatic fibrosis medicine. But its mechanism of action is not yet clear. In this study ,hepatic stellate cells (rHSC-99) cultured in vitro was exposed in various concentrations of IL-10 and/ or TGFβ₁,the levels of CTGF and TIMP-1 mRNA were measured by the quantitative reverse-transcription polymerase chain reaction (RT-PCR) method, the expression ofα-smooth muscle action (α-SMA) in the HSC was measured by immunocytochemical stain method, to investigate antifibrogenic mechanisms of IL-10.Materials and MethodsMaterials1.Y-HSC99 cellsactivated hepatic stellate cells of liver from rat.2. AgentTGFβ₁ and Recombinant Rat IL-10; Reverse Transcription Kit; Taq enzyme; Primer; DMEM nutrient medium;α-SMA; SP Kit; DAB coloration system.Cell Culture γ-HSC99 cells were inoculated in 25cm2 culture flask after reanimated. Addin 5 ml DMEM nutrient medium containing 10% fetal calf serum, put it in 37℃,5% CO₂ incubator,0.25% trypsin digestion and passage.Measurement of CTGF and TIMP-1 mRNA by RT-PCRAfter intervention With different concentrations of IL-10(10,20,40 ug/L) and/or TGFβ₁ and(8 ug/L) for 24,48, 72h,Extract total RNA of HSC with Trizol; Determine the content and purity of RNA by spectrophotometric method. Reverse Transcription polymerase chain reaction with RNA PCR Kit,the product of RT-PCR was determined by electrophoresis of agarose gel (100v, 30s ),the result was shoot by GDS gel imaging system, the staps were scaned and quantitated by computer analysis system.Measurement ofα-SMA by immunocytochemical stain methodAfter intervention With IL-10(20 ug/L) and/or TGFβ₁ and(8 ug/L) for 24h, immunocytochemical staining was completed to detect the expression ofα-SMA , Observe the dyeing condition ofα-SMA under light microscope, analyze the expression ofα-SMA qualitatively by image analyzer.Statistics analysisAll the datas were demonstrated in mean±SD,weather is there significant difference among groups or not was analyzed by One-way ANOVA analysis, LSD-t test. Weather is it significant or not was determined by P<0.05.Results1. RT-PCR resultsClear amplification sraps can be seen at 383bp and 335bp after electrophore- sis of agarose gel. Gray scale analysis results demostrate that, the expression of CTGF and TIMP-1 mRNA in the TGFβ₁ group had remarkably increased contrasting to control group,the difference had statistical significance(p<0.05);when HSC was exposed in various concentrations of IL-10(C,D,E group),the destinational gene expression contrasting to control group had no distinct statistical significance (p>0.05); when exposed in both TGFβ₁ and different concentrations of IL-10(F,G,H group), the expression of CTGF and TIMP-1 mRNA had remarkably decreased contrasting to TGF β₁ group,the difference had statistical significance (p<0.05),and with IL-10 concentration increased,there was a much more remakable inhibition. 2. Immunocytochemical stain resultsAfter DAB coloration, cells with brown pigmentosus particle were positive cells, the positive cell population in TGFβ₁ group had remarkably increased and the majority had much more deeper color, so the expression ofα-SMA increased, when HSC was only exposed in IL-10, there was no distinct statistical significance contrasting to control group; when exposed in both TGFβ₁ and IL-10, the positive cell population had remarkably decreased and had much light color contrasting to TGFβ₁ group, so the expression ofα-SMA decreased.Conclusions1. TGFβ₁ can induce further activation of HSC, increase expression ofα-SMA, and can promote the expression of CTGF and TIMP-1 mRNA on gene level.2. Intervention only by IL-10 has no much effect on expression ofα-SMA, as so as expression of CTGF and TIMP-1 mRNA.3. IL-10 can remarkably inhibit the expression of CTGF and TIMP-1 mRNA mediated by TGFβ₁ .