巴瑞替尼调控巨噬细胞极化减轻硬皮病小鼠肺纤维化

Baricitinib attenuates pulmonary fibrosis in scleroderma mice by regulating macrophage polarization

目的 明确巴瑞替尼能否通过抑制酪氨酸蛋白激酶2(Janus kinase, JAK2)/信号传导与转录激活因子3(signal transducer and activator of transcription, STAT3)调控巨噬细胞极化,从而减轻硬皮病小鼠肺纤维化程度。方法 30只8周雌性C57BL/6小鼠按随机数字表法分为3组:对照组(Ctl组,n=6)、硬皮病组(SSc组,n=12)和硬皮病+巴瑞替尼组(SSc+Ba组,n=12)。SSc和SSc+Ba组小鼠背部皮下注射博来霉素(7.5 mg/kg),SSc+Ba组小鼠胃内灌注巴瑞替尼(5 mg/kg)。采用ELISA检测血浆中炎症因子白介素(interleukin, IL)-1β、IL-4、IL-6、IL-10浓度,HE、Masson染色评估皮肤真皮层厚度和肺组织病变及胶原含量,RT-PCR分析肺组织中炎症因子(IL-1β、IL-4、IL-6、IL-10)mRNA水平,Western blot分析肺组织中巨噬细胞标志物CD86、CD163及JAK2/STAT3的蛋白表达水平,流式细胞术检测F4/80、CD86和CD163以明确小鼠脾脏巨噬细胞极化情况,免疫荧光检测肺组织中CD86和CD163以明确肺组织中巨噬细胞极化情况。结果 与Ctl组相比,SSc组小鼠注射部位皮肤真皮层增厚(P<0.001),改良肺纤维化评分升高(P<0.001),皮肤和肺组织胶原增多(P<0.001)。SSc+Ba组小鼠上述表现均有不同程度的减轻(P<0.01)。与Ctl组相比,SSc组小鼠血浆中促炎因子IL-1β(P<0.05)和IL-6(P<0.001)浓度以及肺组织中上述两种因子的mRNA(P<0.05)水平明显上调。巴瑞替尼降低上述因子浓度(P<0.01)和肺组织中mRNA(P<0.01)水平。SSc组小鼠肺中JAK2/STAT3表达升高(P<0.01),巴瑞替尼降低上述蛋白的表达(P<0.05)。Western blot和免疫荧光检测结果均表明:与Ctl组相比,SSc组肺中CD86蛋白(M1巨噬细胞标志物)表达升高(P<0.01),CD163蛋白(M2巨噬细胞标志物)表达降低(P<0.05)。SSc+Ba组肺中CD86蛋白表达降低(P<0.05),CD163蛋白表达升高(P<0.05)。流式细胞术检测结果表明,SSc组脾脏巨噬细胞中F4/80+CD86+CD163-的M1型巨噬细胞比例升高(P<0.001),F4/80+CD86-CD163+的M2型巨噬细胞比例降低(P<0.01)。SSc+Ba组中,M1细胞比例降低(P<0.01),M2细胞比例升高(P<0.001)。结论 巴瑞替尼通过靶向JAK2/STAT3信号通路抑制M1巨噬细胞极化,来减轻SSc小鼠肺纤维化程度。

Objective To determine whether baricitinib regulates macrophage polarization through Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway to reduce the severity of pulmonary fibrosis in mice with scleroderma.Methods Thirty 8-week-old female C57BL/6 mice were randomly divided into control group(Ctl,n=6),scleroderma group(SSc,n=12)and scleroderma+baricitinib group(SSc+Ba,n=12).The mice of the SSc and SSc+Ba group were inflicted with a subcutaneous injection of 7.5 mg/kg bleomycin in the back,and an intragastric gavage of 5 mg/kg baricitinib.ELISA was used to detect the plasma levels of inflammatory factors,IL-1β,IL-4,IL-6 and IL-10.HE staining and Masson staining were employed to determine the thickness of skin dermis and pathological changes and collagen content in lung tissue.The mRNA expression of IL-1β,IL-4,IL-6 and IL-10 in lung tissue was measured by RT-PCR,and the protein levels of macrophage markers CD86,CD163 and JAK2/STAT3 in lung tissue were detected by Western blotting.Afterwards,flow cytometry was applied for the expression of F4/80,CD86 and CD163 to determine the polarization of macrophages.Immunofluorescence assay was used to detect CD86 and CD163 to clarify the polarization of macrophages in lung tissue.Results Compared with the Ctl group,the dermis at the injection site in the back was significantly thickened(P<0.001),the score of pulmonary fibrosis was obviously increased(P<0.001),and the collagen content in skin and lung tissue was notably elevated in the SSc group(P<0.001).The above changes were alleviated to varying degrees in the SSc+Ba group(P<0.01).The SSc group had significantly higher plasma levels of IL-1β(P<0.05)and IL-6(P<0.001)as well as elevated their mRNA levels in lung tissue(P<0.05)than the Ctl group.Baritinib treatment decreased the plasma concentrations of the above factors(P<0.01)and their mRNA levels in lung tissue(P<0.01).JAK2/STAT3 was highly expressed in the SSc group(P<0.01),and baricitinib reduced the activation of JAK2/STAT3 pathway(P<0.05).Western blotting and immunofluorescence assay indicated that CD86 protein(marker of M1 macrophages)was highly expressed(P<0.01)and CD163 protein(marker of M2 macrophages)was in low expression(P<0.05)in lung tissue in the SSc group than the Ctl group.In the SSc+Ba group,CD86 protein was reduced(P<0.05)and CD163 protein was increased(P<0.05)in lung tissue.Flow cytometry showed that the proportion of F4/80+CD86+CD163-M1 macrophages in the spleen was increased(P<0.001),and the proportion of F4/80+CD86-CD163+M2 macrophages was decreased in the SSc group when compared with Ctl group(P<0.01).In the SSc+Ba group,the proportion of M1 was decreased(P<0.01),and that of M2 was increased(P<0.001).Conclusion Baricitinib inhibits M1 macrophage polarization through JAK2/STAT3 signaling pathway,thus reducing the fibrosis of lung tissue in SSc mice.