1,25(OH)2VD3 对大鼠肺纤维化中IL-9 及PU-1 表达水平的影响

目的:探讨肺纤维化发生发展中IL-9 及PU.1 的作用以及活性维生素D3 [1,25(OH)2 VD3 ]在纤维化过程中对两种因子表达水平的影响。方法:90 只SPF 级雄性SD 大鼠随机分成三个组:对照组、模型组和治疗组(n =30)。模型组和治疗组经气管注入博来霉素(5 mg/ kg)建立肺纤维化模型,对照组注入等体积生理盐水。治疗组于手术后第2 天腹腔注射活性维生素D3 ,模型组注射等量的活性维生素D3 溶剂(丙二醇),对照组注射等量的生理盐水。各种处理均为两天一次。分别于手术后第14、21 和28 天处死大鼠取材,各小组每个时间点10 只大鼠。HE 染色法观察各组实验大鼠肺部病理变化,Masson染色法观察胶原纤维的差异,碱水解法检测羟脯氨酸含量的变化。应用Real-time PCR 和免疫组化技术分别从mRNA 水平和蛋白质水平检测大鼠肺组织中IL-9 及PU.1 的表达,ELISA 法检测血清中IL-9 的表达水平。结果:博来霉素处理后,第14 天大鼠肺部已经出现纤维化,随着时间推移,纤维化进一步加重。在三个时间点,模型组和治疗组的羟脯氨酸含量明显高于对照组,而治疗组明显低于模型组。在三个时间点,治疗组和模型组中IL-9 及PU.1 的表达量均逐渐增高,且都显著高于对照组;第14、21 天治疗组两种因子的表达量均明显低于相应时间点的模型组,第28 天时治疗组与模型组比较差异无统计学意义(P>0.05)。第21 天模型组和治疗组IL鄄9 和PU郾1 的表达水平明显高于第14 天,第28 天与第21 天比较差异无统计学意义。结论:IL-9 和PU.1 在博来霉素引起的大鼠肺纤维化早期可能发挥促纤维化作用。活性维生素D3 可能通过降低PU.1 的表达水平,进而减少IL-9 的分泌,从而对大鼠肺纤维化的发生发展起一定的抑制作用。

Impact of 1,25 (OH)2 VD3 on expression levels of IL-9 and PU.1 in rats with pulmonary fibrosis

Objective:To explore the function of IL-9 and PU.1 on genesis and development of pulmonary fibrosis,and the effect of active vitamin D3 [1,25(OH)2 VD3 ] on the expression levels of this two factors during the pathogenesis of fibrosis.Methods: 90 male SD rats were randomly divided into model group,treatment group,control group (n =30).Bleomycin(5 mg/ kg) was injected into the trachea of rats to establish the model of pulmonary fibrosis in the model group and treatment group,while the control group was injected with isopyknic sterile saline.The treatment group,the model group and the control group were injected intraperitoneally with active vitamin D3 ,solvent of vitamin D3(propylene glycol) and sterile saline on the 2nd day after surgery respectively.All injections were carried out once every other day.10 rats were euthanized at 14th,21st and 28th day in each group in turn.After obtaining lung tissues from experimental rats,the pathological change of lung was compared by hematoxylin-eosin staining.The difference of collagen fiber and hydroxyproline content were compared by the Masson staining and basic-hydrolysis method respectively.The mRNA and protein expression of IL-9 and PU.1 in lung tissue were detected by Real-time PCR and immunohistochemical technology respectively.The expression of IL-9 in serum was detected by ELISA.Results: Fibrosis appeared in lungs of experimental rats treated with bleomycin after 14 days,and more and more aggravated with time.At three time points,the hydroxyproline content in model group and treatment group were significantly higher than that of control group,and the treatment group was significantly lower than the model group.At three time points,the expression of IL-9 and PU.1 in model group and treatment group were risen gradually,and obviously higher than that in control group.On the 14th and 21st day,the expression of two factors in treatment group was significantly lower than model group;on the 28th day,there was no statistically significant difference between treatment group and model group(P>0.05).In model group and treatment group,the expression of two factors on 21st day was significantly higher than that on 14th day;there was no statistically significant difference between the 28th day and the 21st day. Conclusion: IL-9 and PU.1 may play a profibrotic role at early stage of pulmonary fibrosis induced by bleomycin.The active vitamin D3 may lower the expression level of PU.1,and then reduce the secretion of IL-9,thus may play an inhibiting effect on genesis and development of pulmonary fibrosis in rats.