NLRC3在低氧性肺动脉高压中的作用及机制

萧颖畴; 蔡志贵; 王婧; 罗颖

doi:10.12125/j.chj.202412140

摘要:

目的探讨NLRC3介导的免疫调节在低氧性肺动脉高压（HPH）中的作用及可能机制。

方法野生型和 NLRC3 敲除(NLRC3^−/−)小鼠随机分为对照组、NLRC3^−/−组、低氧（Hypoxia）组、Hypoxia+NLRC3^−/−组。其中，Hypoxia组和Hypoxia+NLRC3^−/−组置于低压低氧实验舱中，Control组和NLRC3^−/−组置于常压常氧环境中，维持6周。检测各组小鼠肺血管重构、右心室压力、右心室肥厚、NLRC3表达、炎性细胞因子表达和NF-κB信号活化；培养野生型和NLRC3^−/−小鼠原代骨髓间充质细胞（BMDMs），经巨噬细胞集落刺激因子（macrophage-stimulating factor，M-CSF）诱导后分为对照组、NLRC3^−/−组、低氧组、Hypoxia+NLRC3^−/−组。Hypoxia组和Hypoxia+NLRC3^−/−组置于低氧培养箱中刺激24 h或48 h，Control组和NLRC3^−/−组置于常氧培养箱。检测各组NLRC3、炎性细胞因子表达和NF-κB信号活化水平。

结果与Control组相比，Hypoxia组肺组织和BMDMs中NLRC3表达均显著降低（P<0.01）；血管壁厚度比值（WT%）、血管壁面积比（WA%）、右心室收缩压（RVSP）、右心室肥厚指数（RVHI）显著增加（P<0.01），α-SMA阳性细胞增多；肺组织中IL-1β、IL-18表达增加（P<0.05）并且磷酸化p65/总p65（p-p65/p65）比值升高（P<0.01）；肺组织中F4/80阳性细胞增多；BMDMs中IL-1β、IL-18表达增加且p-p65/p65比值升高（P<0.01）。与NLRC3^−/−组相比，Hypoxia+NLRC3^−/−组小鼠的WT%、WA%、RVSP、RVHI增加（P<0.01），α-SMA阳性细胞增多，F4/80阳性细胞明显增多；肺组织与BMDMs中IL-1β、IL-18表达和p-p65/p65比值增加（P<0.01）。与Hypoxia组相比，Hypoxia+NLRC3^−/−组小鼠的WT%、WA%、RVSP、RVHI增加（P<0.01），α-SMA阳性细胞增多；肺组织中IL-1β、IL-18表达和p-p65/p65比值增加（P<0.01）； F4/80阳性细胞亦明显增多；BMDMs中IL-1β、IL-18表达增加（P<0.01），p-p65/p65比值升高（P<0.05）。

结论 NLRC3在HPH小鼠的巨噬细胞中通过负性调节NF-κB信号活化，抑制炎症反应。

Abstract:

AIM To investigate the role and underlying mechanism of NLRC3 in modulating hypoxic pulmonary hypertension (HPH) through immune regulation.

METHODS Wild-type (WT) and NLRC3 knockout (NLRC3^−/−) mice were randomly divided into four groups: control, NLRC3^−/−, hypoxia, and hypoxia+NLRC3^−/−. Mice in the hypoxia and hypoxia+NLRC3^−/− groups were exposed to hypobaric hypoxia for 6 weeks, while those in the control and NLRC3^−/− groups were maintained in normobaric normoxia. Pulmonary vascular remodeling, right ventricular pressure (RVSP), right ventricular hypertrophy index (RVHI), NLRC3 expression, inflammatory cytokine levels, and NF-κB signaling activation were evaluated. Cultivate wild-type and NLRC3^−/−mouse primary bone marrow mesenchymal cells (BMDMs), induce them with macrophage colony-stimulating factor (M-CSF), and divide them into control group, NLRC3^−/− group, hypoxia group, and Hypoxia+NLRC3^−/− group. The Hypoxia group and Hypoxia+NLRC3^−/−group were stimulated in a low oxygen incubator for 24 or 48 hours, while the Control group and NLRC3^−/− group were placed in a normoxic incubator. Detect NLRC3, inflammatory cytokine expression, and NF - κ B signaling activation levels in each group.

RESULTS Compared with the Control group, the expression of NLRC3 in lung tissue and BMDMs in the Hypoxia group decreased (P<0.01), and the ratio of vascular wall thickness (WT%), vascular wall area ratio (WA%), right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) increased (P<0.01), and α-SMA positive cells increased; The expression of IL-1β and IL-18 in lung tissue increased (P<0.05) and the ratio of p-p65/p65 increased (P<0.01); The expression of IL-1β, IL-18 and the ratio of p-p65/p65 in BMDMs increased (P<0.01). Compared with NLRC3^−/− group, WT%, WA%, RVSP, RVHI and α-SMA positive cells were increased in Hypoxia+NLRC3^−/−group (P<0.01); The expression of IL-1β, IL-18 and the ratio of p-p65/p65 in lung tissue and BMDMs increased (P<0.01). Compared with the Hypoxia group, WT%, WA%, RVSP and RVHI in the Hypoxia+NLRC3^−/−group increased (P<0.01), and α-SMA positive cells increased; The expression of IL-1β, IL-18 and p-p65/p65 ratio in lung tissue increased (P<0.01); The expression of IL-1β and IL-18 in BMDMs increased (P<0.01), and the ratio of p-p65/p65 increased (P<0.05).

CONCLUSION NLRC3 negatively regulates NF-κB signaling activation in macrophages of mice with HPH, thereby inhibiting the inflammatory response.

NLRC3在低氧性肺动脉高压中的作用及机制

Role and mechanism of NLRC3 in hypoxic pulmonary hypertension