骨化三醇通过B细胞受体/PI3K/AKT/NF-κB通路抑制大鼠高原肺水肿的发生

Calcitriol inhibits B-cell receptor/PI3K/AKT/NF-κB pathway to improve high altitude pulmonary edema in rats

目的 :探讨骨化三醇通过B细胞受体/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/核因子κB(NF-κB)通路抑制大鼠高原肺水肿的发生及其机制。方法 :雄性SD大鼠随机分为常氧组、低氧组、低氧+骨化三醇组(0.252μg/kg),连续灌胃给药5 d后,除常氧组外,其余2组均置于模拟海拔6 000 m的低压氧舱内低氧胁迫48h,建立高原肺水肿大鼠模型。干湿比重法测定肺含水量;H-E染色观察大鼠肺组织病理变化,并进行炎症评分;TUNEL法检测肺组织细胞凋亡率;ELISA检测肺组织B细胞激活因子(BAFF)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、干扰素-γ(IFN-γ)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)含量;免疫荧光染色观察肺组织CD21和核因子κB抑制蛋白α(IκBα)的表达;免疫印迹检测肺组织PI3K、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、IκBα、磷酸化IκBα(p-IκBα)的表达。结果 :与常氧组比较,低氧组大鼠肺组织见轻微肺泡隔增宽,炎症细胞浸润;肺含水量升高,肺组织细胞凋亡率、BAFF、TNF-α、IL-6、IFN-γ含量及CD21、p-PI3K、p-AKT表达明显升高;肺组织IL-4、IL-10含量及IκBα、p-IκBα表达明显降低。与低氧组比较,低氧+骨化三醇组炎症细胞浸润减轻;肺含水量降低、肺组织细胞凋亡率、BAFF、TNF-α、IL-6、IFN-γ含量及CD21、p-PI3K、p-AKT表达明显降低;肺组织IL-4、IL-10含量及IκBα、p-IκBα表达明显升高。结论 :骨化三醇可抑制高原肺水肿炎症反应,其作用机制可能与抑制B细胞受体/PI3K/AKT/NF-κB信号通路有关。

Objective:To investigate the mechanism of which calcitriol improves high altitude pulmonary edema(HAPE)in rats by inhibiting the B-cell receptor/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)/nuclear factor-κB(NF-κB)pathway.Methods:Male SD rats were randomly divided into normoxia,hypoxia,and hypoxia+calcitriol groups(0.252μg/kg).After continuous gavage administration for 5 days,except for the normoxia group,the other two groups were placed under simulated hypoxic stress at an altitude of 6000 m for 48 h to establish the HAPE rat model.Lung water content was determined and measured by dry and wet specific gravity methods;H-E staining was performed to observe the pathological changes of lung tissue and inflammation score;TUNEL method was used to detect apoptosis in lung tissue;ELISA was used to detect B cell activating factor(BAFF),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interferon-γ(IFN-γ),interleukin-4(IL-4)and interleukin-10(IL-10)in lung tissue;immunofluorescence staining was used to observe the expression of CD21 and IκBαin lung tissue;Western blotting was performed to detect the expression of PI3K,p-PI3K,AKT,p-AKT,NF-κB inhibitorα(IκBα)and p-IκBαin lung tissues.Results:Compared with the normoxic group,slight alveolar septal widening and inflammatory cell infiltration were seen in lung tissue.Lung water content,lung tissue apoptosis,the content of BAFF,TNF-α,IL-6,IFN-γand the expression of CD21,p-PI3K,p-AKT were significantly increased.The content of IL-4,IL-10 and the expression of IκBα,p-IκBαwere significantly decreased in lung tissue in the hypoxic group.Compared with the hypoxic group,inflammatory cell infiltration was reduced;lung water content,lung tissue apoptosis,BAFF,TNF-α,IL-6,IFN-γcontent and CD21,p-PI3K,p-AKT expression were significantly decreased;IL-4,IL-10 content and IκBα,p-IκBαexpression in lung tissue were significantly increased in the hypoxic+calcitriol group.Conclusion:Calcitriol can inhibit HAPE inflammatory response,and its mechanism of action may be related to the inhibition of B-cell receptor/PI3K/AKT/NF-κB signaling pathway.