LIGHT在低氧性肺动脉高压形成中的作用及机制

Role of LIGHT in development of hypoxic pulmonary hypertension in mice

目的初步探讨LIGHT在低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)形成中的作用及其机制。方法将20只8周龄雌性C57BL/6J小鼠[体质量(17.90±0.91)g]和20只8周龄雌性LIGHT-/-C57BL/6J小鼠[体质量(17.55±0.93)g]分为4组(n=10):(1)野生小鼠常氧组(WT-C组)、(2)野生小鼠低氧组(WT-H组)、(3)LIGHT KO小鼠常氧组(LIGHT KO-C组)、(4)LIGHT KO小鼠低氧组(LIGHT KO-H组)。WT-H组和LIGHT KO-H组小鼠置于模拟6 000 m低压舱内连续低氧饲养30 d,WT-C组和LIGHT KO-C组小鼠舱外(海拔308 m)常规饲养。检测右心室收缩压(right ventricular systolic pressure,RVSP)和右心肥厚指数(right ventricular hypertrophy index,RVHI);HE染色观察肺小动脉结构;免疫组化检测LIGHT及其受体HVEM、LTβR表达;荧光定量PCR和Western blot检测肺组织LIGHT、HVEM和LTβR、IL-6的mRNA和蛋白水平。流式细胞术检测肺组织中各类炎症细胞的比例。结果与WT-C组相比,WT-H组肺组织中LIGHT的mRNA和蛋白水平均显著增高(P<0.05),WT-H组RVSP和RVHI明显升高(P<0.05),肺小动脉明显增厚;与LIGHT KO-C组相比,LIGHT KO-H组小鼠的RVSP、RVHI和肺小动脉厚度显著增加(P<0.05),但与WT-H组相比,LIGHT KO-H组的RVSP、RVHI和肺小动脉增厚程度明显降低(P<0.05)。与WT-C组相比,WT-H组LIGHT受体HVEM表达增加,LTβR表达降低,差异具有统计学意义(P<0.05)。LIGHT KO-H组与WT-H组相比,肺组织IL-6 mRNA和蛋白水平显著降低(P<0.05)。流式细胞检测发现,与WT-C组相比,WT-H组小鼠肺组织中单核细胞比例降低[(3.88±0.87)%vs(11.03±1.71)%,P<0.05],间质巨噬细胞比例升高[(15.56±2.69)%vs(8.57±2.17)%,P<0.05];与WT-H组相比,LIGHT KO-H组的肺组织单核细胞增加[(6.55±1.01)%vs(3.88±0.87)%,P<0.05],而间质巨噬细胞的比例降低[(10.87±1.68)%vs.(15.56±2.69)%,P<0.05]。结论慢性低氧诱导肺组织中LIGHT表达增加与HPH发病机制密切相关。LIGHT可能通过HVEM信号途径促进细胞增殖、上调肺组织IL-6表达、促进肺间质巨噬细胞产生,参与HPH的形成。

Objective To investigate the role of tumor necrosis factor superfamily member 14（ LIGHT/TNFSF14） in the development of hypoxic pulmonary hypertension in mice and explore the mechanism. Methods Twenty 8-week-old wild-type（ WT） female C57 BL/6 J mice and 20 age-matched LIGHT-deficient（ LIGHT-/-） transgenic female C57 BL/6 J mice were both randomized equally into normoxia groups and hypoxia groups. In the 2 hypoxia groups,the mice were housed in a hypobaric chamber simulating the condition of 6 000 m altitude for 30 d,and mice in normoxic groups were housed in routine conditions.The changes in right ventricular systolic pressure（ RVSP） were recorded and right ventricular hypertrophy index（ RVHI） was evaluated. HE staining was used to observe the changes in pulmonary vascular structures.Immunohistochemical staining was performed to observe the distribution of LIGHT,HVEM and LTβR in the lung of the mice. The expressions of LIGHT,HVEM,LTβR and IL-6 at both mRNA and protein levels in the lungs were tested using RT-PCR and Western blotting. The proportions of inflammatory cells in the lungs were determined using flow cytometry. Results Compared with the WT normoxic mice,the WT mice with chronic hypoxia showed significantly increased mRNA and protein levels of LIGHT in the lungs（ P〈0. 05） with also significantly increased RVSP and RVHI and obviously thickened pulmonary arterioles（ P〈0. 05）. These hypoxia-induced changes were markedly in attenuated LIGHT-/-mice. In WT mice, hypoxia induced significantly increased protein expression of LIGHT receptor HVEM（ P〈0. 05） and decreased expression of LTβR（ P〈0. 05）. LIGHT-/-mice with hypoxia showed significantly lower mRNA and protein levels of IL-6 in the lungs than WT hypoxic mice（ P〈0. 05）. Compared with WT normoxic mice,WT hypoxic mice showed significantly decreased proportions of monocytes [（ 3. 88 ± 0. 87） % vs（ 11. 03 ± 1. 71） %,P〈0. 05 ] and increased interstitial macrophages [（ 15. 56 ± 2. 69） % vs（ 8. 57 ± 2. 17） %,P〈0. 05 ] in the lungs. In LIGHT-/-mice with hypoxia,the proportion of monocytes was significantly increased to（ 6. 55 ± 1. 01） %（ P〈0. 05） and that of interstitial macrophage was significantly decreased to（ 10. 87 ± 1. 68） %（ P〈0. 05）compared with those in WT hypoxic mice. Conclusion Chronic hypoxia-induced development of HPH is closely associated with increased pulmonary LIGHT expression,which promotes cell growth,upregulates IL-6,and increases the production of pulmonary interstitial macrophages possibly by activating HVEM under hypoxic condition.