| ObjectiveTo study the effects of Chuankezhi injection on IL-12/STAT4and IL-4/STAT6signal channels in chronic obstructive pulmonary disease (COPD) rats and the relations between the unbalance of Thl/Th2, COPD and Chuankezhi injection, chronic obstructive pulmonary disease rat model was prepared.60SD rats were randomized into normal group, COPD model group and Chuankezhi injection treatment groups (high, medium and low dosage group). During the experimental study, the variation of IFN-γ/IL-12/IL-12R/IL-4/IL-4R levels in serum and bronchoalveolar lavage fluid (BALF), and the STAT4/6proteins and STAT4/6mRNA levels in lung tissue were observed.Methods(1) Rats were induced by cigarette smoke inhalation and LPS tracheal instillation. The quality of COPD rat model and the effects of Chuankezhi injection on pathological changes and inflammatory cells of COPD rats were evaluated by general state observation, visual inspection and HE stained pathological observation of lung tissue, cell count of BALF;(2) Investigated the therapeutic effect of Chuankezhi injection on COPD rats by determined the levels of IFN-γ/IL-12/IL-12R/IL-4/IL-4R in serum and BALF and analyzed the effects of Chuankezhi injection on serum and cytokines in BALF;(3) Observed the effects of Chuankezhi injection on the levels of STAT4/6proteins in lung tissue by Western blot; and evaluated the effects of Chuankezhi injection on COPD rats from the perspective of the differentiation regulation of Th1/Th2; (4) Observed the effects of Chuankezhi injection on the levels of STAT4/6mRNA in lung tissue by real-time PCR; and tried to evaluate the effects of Chuankezhi injection on COPD rats from the perspective of immune imbalance of Thl/Th2.Results(1) According to general state observation, visual inspection, cell count of BALF and HE dyeing pathological changes in COPD rats which had been successfully replicated, the pathomorphology of the COPD rat model was similar humans’. Chuankezhi injection can improve the clinical symptoms in COPD rats for cough and respiratory secretions etc, making them better at coat shiny, action, spirit, food and water intakes than the rats in model group. Comparing with model group, it can also significantly improve the pathological lesions in lung tissue and the situations of lung volume, surface smoothness and elasticity, pulmonary bullae of COPD rats. From pathology observation, it can be seen that the amount of goblet cells and the infiltration of inflammatory cells significantly reduced. In addition, the bronchial wall did not incrassate obviously and alveolar was relatively complete.(2) Study found that the COPD model group had the highest total leukocyte count, three groups of Chuankezhi injection had no significant difference (P>0.05) with normal group, suggesting Chuankezhi injection had obvious inhibitory effect on inflammatory cells of COPD rats. In cell count, each group was given priority to mononuclear macrophages. Compared with normal group, mononuclear macrophage percentage in model group decreased significantly (P<0.01), whereas percentages of lymphocyte, neutrophils and eosinophils were significantly increased (P<0.01). Compared with model group, mononuclear macrophages in three groups of Chuankezhi injection increased significantly (P<0.01or P<0.05), the percentages of lymphocyte, neutrophils and eosinophils reduced significantly (P<0.01or P<0.05) which had no significant difference with normal group (P>0.05), indicating the rat model dripped with LPS by the methods of smoking plus trachea may induce mononuclear macrophages percentage reduction, and increase the percentages of lymphocyte, neutrophils and eosinophils. The Chuankezhi injection can obviously increase mononuclear macrophages percentage of COPD rats, reduce the percentages of lymphocyte, neutrophils and eosinophils and significantly inhibit the secretion of inflammatory cells.(3) In the model group, IL-4, IL-4R concentration significantly lower (P <0.01), while the levels of IFN-γ、IL-12、IL-12R and the ratio of IFN-γ to IL-4were significantly increased (P<0.01), suggesting that the rats dripped with LPS by the methods of smoking plus trachea induced the occurrence of unbalance of TH1/Th2differentiations in which Thl cells had an advantage. Compared with model group, Chuankezhi injection treatment groups can significantly reduce the levels of IFN-γ、IL-12、IL-12R(P<0.01) and the ratio of IFN-Y to IL-4, and increase the levels of IL-4and IL-4R(P<0.01) in serum and PALF of COPD rats, which suggested that Chuankezhi injection regulated the unbalance of Th1/Th2by lowering the levels of IFN-γ, IL-12and IL-12R, raising the levels of IL-4and IL-4R and reducing the ratio of IFN-γ to IL-4to inhibit the polarization of Thl cells. In addition.(4) It was found that there were significant differences about STAT4protein and STAT6protein expressions in the normal group, COPD model group and treatment groups (P<0.01). Compared with normal group, STAT4protein expression in the COPD model group increased significantly (P<0.01), while the expression of STAT6protein dropped significantly (P<0.01), which suggested that the rats dripped with LPS by the methods of smoking plus trachea may cause the occurrence of unbalance of TH1/Th2differentiations in which Thl cells had an advantage and showed a negative correlation between STAT4protein and STAT6protein expressions. Compared with model group, the Stripe mark colors of STAT4protein in the three dosages groups of Chuankezhi injection were lighter and the expression intensity significantly decreased (P<0.01), while STAT6stripe mark colors deepened and the expression intensity significantly enhanced (P<0.01or P<0.05), suggesting Chuankezhi injection can inhibit STAT4protein expression and promote STAT6protein expression. The three dosages groups had similar Stripe mark colors of STAT6proteins and had no obvious difference in expression intensity (P>0.05), shows that Chuankezhi injection affected the expressions of STAT6proteins without any obvious dose-effect relationship. The correlation analysis suggested that the more the STAT6protein was expressed, the less the STAT4was expressed after using Chuankezhi injection.(5) Compared with normal group, model group rats’expression level of STAT4mRNA in lung tissue significantly increased (P<0.01), while STAT6m RNA expression level was significantly reduced (P<0.01), which suggested that LPS dripped to the rats by the methods of smoking plus trachea caused STAT4mRNA expression hyperthyroidism and STAT6m RNA expression inhibition in lung tissue of COPD rats. Compared with model group, each treatment group’s STAT4mRNA expression in lung tissue was significantly lower (P<0.01or P <0.05), while STAT6mRNA expression significantly increased (P<0.01). With the dosage increase of Chuankezhi injection, STAT4and STAT6mRNA expressions in lung tissue moved towards the normal group, but there was no statistical significance between those differences (P>0.05), which suggested that Chuankezhi injection affected the expressions of STAT4and STAT6mRNA without any obvious dose-effect relationship.Conclusion(1) Unbalance of Thl/Th2differentiations was a characteristic feature of COPD model rats, in which Thl cells had an advantage. What’s more, IFN-γ, IL-12and IL-12R in serum and PALF expressed excessively, while the levels of IL-4and IL-4R were low and ratio of IFN-γ to IL-4increased.(2) Chuankezhi injection significantly inhibited the secretion of inflammatory cells and improved the pathological lesions of lung tissue in COPD rats. It also significantly reduced the percentages of lymphocyte, neutrophile granulocyte and eosnophils, and increased monocyte percentage.(3) Chuankezhi injection significantly reduced the levels of IFN-γ, IL-12, IL-12R and the ratio of IFN-γ to IL-4, and increased the levels of IL-4and IL-4R in serum and BALF of COPD rats, which suggested that Chuankezhi injection regulated the unbalance of Thl/Th2differentiations and reduced inflammation reaction by inhibited Thl cells polarization and increased Th2cell differentiation.(4) Chuankezhi injection significantly reduced STAT4protein and STAT4mRNA and increased STAT6protein and STAT6mRNA of lung tissue in COPD rats, which suggested that the effect of controlling the growth of Thl cells and promoting the expression of Th2cells, and regulating the unbalance of Thl/Th2may be one of the preventive mechanisms of Chuankezhi injection against COPD.(5) The three dosages groups of Chuankezhi injection changed the inflammatory cells, the cytokines and the expressions of STAT4proteins and STAT4which have obvious dose-effect relationship.In conclusion, the Thl/Th2differentiations are unbalanced in COPD rats; Chuankezhi injection can change the unbalance state of Thl/Th2of COPD rats by controlling the overexpressions of IFN-γ, IL-12, IL-12R and STAT4protein and STAT4mRNA which regulated the hyperthyroidism process of Thl cell differentiation and the cell differentiation of Th2; from the experimental study, it was speculated that COPD was closely related to the unbalance of Th1/Th2differentiations; the functions of Chuankezhi injection included controlling the secretion of inflammatory cells, improving the pathological lesions of lung tissue in COPD and preventing the action mechanisms of COPD by regulated the differentiations of Th1/Th2with methods of reducing the expression of STAT4mRNA and raising the expression of STAT6mRNA. |
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