Effects Of IL-13 And Alcohol On Collagen Expression Of Human Lung Fibrolasts

Objectives:Interleukine-13(IL-13) is considered to be one of the most potent profibrotic type2 cytokines. IL-13 can enhance fibrosis when it binds with its receptor interleukine-13 receptor al(IL-13Ral), interleukine-4 receptor a(IL-4Ra), but interleukine-13 receptor a2(IL-13Ra2) which acts as a decoy receptor is the other receptor of IL-13.IL-13Ra2 alone binds IL-13 with high affinity compared with IL-13Ral.However,it has a short cytoplasmic tail that is devoid of signaling motifs.So when IL-13 binds with IL-13Ra2,it can inhibit the signal transduction and function of IL-13.In this study, we will detect the effects on HFL-1 cells proliferation and the gene expression of IL-13Rα1,IL-4Rαand IL-13Rα2 mRNA when alcohol stimulate HFL-1 cells, and investigate the effects of alcohol combination with IL-13 on COL1A1,COL3A1 mRNA expression and on the collagen I synthesis of cultured HFL-1 cells in vitro. To investigate the relationship between alcohol and IL-13 signaling pathway in pulmonary fibrosis is for exploring its mechanism from protein and molecular levels.Methods:1.Activity of HFL-1 cells was detected by trypan blue staining.2. Modality of HFL-1 cells was observed by HE staining.3. MTT assay was used to determine the proliferation of HFL-1 cells which were stimulated with alcohol in different concentrations.4.The expression of IL-13Rα1,IL-4Rα,IL-13Rα2,COL1A1 and COL3AlmRNA was evaluated by Real time RT-PCR after HFL-1 cells were stimulated with alcohol or/and IL-13. HFL-1 cells were cultured and divited to four groups:control group, IL-13 group, alcohol group and IL-13+alcohol group.5. The expression of COL1A1 protein was evaluated by ELISA after HFL-1 cells were stimulated with alcohol and/or IL-13.Results: 1. The activity of HFL-1 cells was well, and low level of alcohol was no toxicity to HFL-1 cells.2. The modality of HFL-1 cells was clostridid, it/s cytoplasm was caught red and it/s nucleus was ellipse form and caught blue.3. Alcohol single(<200 mM) can't stimulate the proliferation of HFL-1 cells, IL-13 has great effect on the proliferation of HFL-1 cells in dose-dependent manner.Combination of IL-13 and alcohol could greatly up-regulate the proliferation of HFL-1 cells.4. The resulte of Real time RT-PCR demonstrated that alcohol (<200 mM) has no effect on the mRNA expression of COL1A1 and COL3A1 of HFL-1 cells,but has great effect on the mRNA expression of IL-13Rα1,IL-13Rα2 and IL-4Rα. Combination of alcohol and IL-13 significantly up-regulated the collagen production and expression of COL1A1 and COL3A1 of HFL-1 cells, compared with IL-13 alone.5. The resulte of ELISA demonstrated that the expression of COL1A1 protein is consistent with the resulte of Real time RT-PCR.Conclusion:1. Low level of alcohol can't stimulate the proliferation of HFL-1 cells, Combination of IL-13 and alcohol could greatly up-regulate the proliferation of HFL-1 cells.2. Low level of alcohol could enhance the expression of IL-13Rαl and IL-4RαmRNA in HFL-1 cells, but ownregulate the expression the expression of IL-13Ra2 mRNA.3. Low level of alcohol have no effect on the expression of COL1A1 and COL3A1 mRNA,but the expression of COL1A1 and COL3A1 mRNA in a dose-dependent manner was induced when HFL-1 cells were stimulated with IL-13.4. The mRNA and protein expression of COL1A1 and COL3A1 was enhanced when HFL-1 cells were stimulated with IL-13 and alcohol.