GPR146经P-JNK通路对肺动脉高压小鼠血管重塑的影响

doi:10.3877/cma.j.issn.1674-6902.2023.04.002

目的

分析G蛋白偶联受体146(orphan G protein-coupled receptor 146, GPR146)经磷酸化氨基末端蛋白激酶(phosphorylated c-Jun N-terminal kinase, P-JNK)通路对肺动脉高压(pulmonary hypertension, PH)肺血管重构(pulmonary vascular remodeling, PVR)致肺血管压力升高的影响。

方法

构建PH模型，采用SD大鼠10只，分为对照组和SuHx组，每组5只；采用小鼠20只，分为Control组、SuHx组、SuHx+SiNC组、SuHx+SiGPR146组，共4组。测量每组右心室收缩压(right ventricular systolic pressure, RVSP)和右心室肥厚指数(right ventricular hypertrophy index, RVHI)。免疫荧光(immunofluorescence, IFC)染色观察GPR146表达，免疫印迹检测GPR146、P-JNK与增殖细胞抗原(proliferating cell nuclear antigen, PCNA)表达。

结果

与Control组相比，SuHx组大鼠肺组织GPR146升高(P<0.05，t＝4.742)。GPR146主要在α-actin染色血管中膜层平滑肌细胞表达；SuHx组小鼠肺组织GPR146升高，SuHx+SiGPR146组GPR146比SuHx+SiNC组低(P<0.05，F＝8.576)。SuHx组小鼠肺组织P-JNK与PCNA升高，干扰GPR146后P-JNK与PCNA表达降低(P<0.05，F＝6.048，25.55)；SuHx组小鼠RVSP和RVHI高于Control组，干扰GPR146后SuHx+SiGPR146组小鼠RVSP和RVHI低于SuHx+SiNC组；SuHx组小鼠肺血管壁厚度比Control组厚；敲除GPR146后，SuHx+siGPR146组小鼠肺血管壁厚度小于SuHx+siNC组(P<0.05，F＝4.106)；HYP+SiGPR146组PASMCs中GPR146下调(P<0.05，F＝6.907)，与SiNC组相比，HYP+SiNC组PASMCs中P-JNK与PCNA表达上调，敲除GPR146后HYP+SiGPR146组中P-JNK与PCNA蛋白相较于HYP+SiNC组下调(P<0.05，F＝7.436，33.68)。

结论

GPR146经P-JNK通路促进肺动脉平滑肌细胞(PASMCs)增殖，加剧PH进展，可为预防和治疗PH可行靶点。

关键词: G蛋白偶联受体146, 血管重塑, 肺动脉平滑肌细胞, 磷酸化氨基末端蛋白激酶

Objective

To investigate the effect of G protein-coupled receptor 146 (GPR146) on pulmonary vascular pressure elevation induced by pulmonary vascular remodeling(PVR) in pulmonary hypertension (PH) via phosphorylated c-Jun N-terminal kinase(P-JNK) pathway.

Methods

Ten SD rats were used to establish PH models and were divided into control group and SuHx group, with 5 rats in each group. Twenty mice were divided into Control group, SuHx group, SuHx+ SiNC group and SuHx+ SiGPR146 group. Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were measured. The expression of GPR146 was detected by immunofluorescence (IFC) staining, and the expressions of GPR146, P-JNK and proliferating cell nuclear antigen (PCNA) were detected by Western Blot.

Results

Compared with Control group, GPR146 in lung tissue of SuHx group was increased(P<0.05, t=4.742). GPR146 was mainly expressed in α-actin stained vascular mesenchyme smooth muscle cells. GPR146 in SuHx group was increased, and GPR146 in SuHx+ SiGPR146 group was lower than SuHx+ SiNC group (P<0.05, F=8.576). The expression of P-JNK and PCNA in lung tissue of SuHx group was increased, and the expression of P-JNK and PCNA was decreased after interference with GPR146(P<0.05, F=6.048, 25.55); The RVSP and RVHI of SuHx group were higher than those of Control group, and the RVSP and RVHI of SuHx+ SiGPR146 group were lower than those of SuHx+ SiNC group after interference with GPR146. The pulmonary vascular wall thickness in SuHx group was thicker than that in Control group. After GPR146 was knocked out, the pulmonary vascular wall thickness in SuHx+ siGPR146 group was lower than that in SuHx+ siNC group(P<0.05, F=4.106); GPR146 in PASMCs of HYP+ SiGPR146 group was down-regulated (P<0.05, F=6.907). Compared with SiNC group, P-JNK and PCNA expressions in PASMCs of HYP+ SiNC group were up-regulated. After GPR146 knockout, P-JNK and PCNA proteins in HYP+ SiGPR146 group were down-regulated compared with those in HYP+ SiNC group(P<0.05, F=7.436, 33.68).

Conclusion

GPR146 promotes the proliferation of pulmonary artery smooth muscle cells (PASMCs) through the P-JNK pathway and aggravates the progression of PH, which can be a feasible target for the prevention and treatment of PH.

Key words: Orphan G protein-coupled receptor 146, Vascular remodeling, Pulmonary artery smooth muscle cells, Phosphorylated c-Jun N-terminal kinase

图1 PH大鼠肺组织中GPR146蛋白表达。注：每组小鼠肺组织匀浆Western Blot实验。Control：对照组；SuHx：缺氧+Sugen5416组，n＝3, **P<0.01

图2 PH大鼠肺组织免疫荧光结果。注：Control：对照组；SuHx：缺氧+Sugen5416组。比例尺＝100 μm

图3 GPR146缺乏对小鼠肺组织P-JNK和PCNA的影响。注：各组小鼠肺组织匀浆Western Blot实验。Control：对照组；SuHx：缺氧+Sugen5416组；SuHx+siNC：缺氧+Sugen5416+阴性对照组；SuHx+siGPR146：缺氧+Sugen5416+GPR146基因敲除组。n＝4，*P<0.05，**P<0.01，***P<0.001

图4 GPR146缺乏对PH小鼠RVSP和RVHI的影响。注：Control：对照组；SuHx：缺氧+Sugen5416组；SuHx+siNC：缺氧+Sugen5416+阴性对照组；SuHx+siGPR146：缺氧+Sugen5416+GPR146基因敲除组。n＝3，*P<0.05，**P<0.01，***P<0.001

图5 GPR146缺乏对PH小鼠肺血管重塑的影响(HE染色)。注：Control：对照组；SuHx：缺氧+Sugen5416组；SuHx+siNC：缺氧+Sugen5416+阴性对照组；SuHx+siGPR146：缺氧+Sugen5416+GPR146基因敲除组。比例尺＝50 μm。n＝3，*P<0.05

图6 GPR146对PASMCs中P-JNK和PCNA的影响。注：各组PASMCs Western Blot实验。SiNC：常氧+阴性对照；Hyp+siNC：缺氧+阴性对照；Hyp+siGPR146：缺氧+GPR146基因敲除。n＝3，*P<0.05，**P<0.01，***P<0.001

图7 GPR146过表达对PASMCs中P-JNK的影响。注：各组PASMCs Western Blot实验。pcDNA：pcDNA3.1(+)载体；pcDNA-GPR146：GPR146过表达质粒。n＝3，*P<0.05，**P<0.01

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Effects of GPR146 on vascular remodeling in mice with pulmonary hypertension via P-JNK pathway

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Effects of GPR146 on vascular remodeling in mice with pulmonary hypertension via P-JNK pathway