基于PI3K/Akt/NF-κB信号通路探讨升阳益胃汤对肺癌大鼠免疫力的影响

doi:10.11958/20221626

摘要/Abstract

摘要：

目的探究升阳益胃汤对肺癌大鼠免疫力的影响及其可能作用机制。方法 72只SPF级Wistar大鼠随机抽取12只为对照组，其余60只大鼠采用气管内灌注致癌碘化油溶液法建立肺癌模型，并分为模型组、顺铂组（5 mg/kg）、升阳益胃汤组（14 g/kg）、磷酸肌醇3-激酶（PI3K）激活剂740 Y-P组（10 mg/kg）、升阳益胃汤+740 Y-P组（升阳益胃汤14 g/kg + 740 Y-P 10 mg/kg），每组12只。给予相应的药物干预后，流式细胞术检测外周血T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺占比，计算CD4⁺/CD8⁺比值；酶联免疫吸附试验（ELISA）检测血清炎性因子白细胞介素（IL）-6、肿瘤坏死因子α（TNF-α）和免疫球蛋白（Ig）G、IgA、IgM水平；取肺、脾和胸腺并称质量，计算肺、脾、胸腺指数；HE染色观察肺组织病理学变化；实时荧光定量PCR检测肺组织基质金属蛋白酶-9（MMP-9）和细胞间黏附分子-1（ICAM-1）mRNA表达；Western blot检测肺组织PI3K/蛋白激酶B（Akt）/核因子-κB（NF-κB）通路相关蛋白表达。结果与对照组相比，模型组CD3⁺、CD4⁺占比和CD4⁺/CD8⁺比值、脾和胸腺指数、IgG、IgA、IgM水平降低，CD8⁺占比、肺指数、IL-6和TNF-α水平、肺组织ICAM-1 mRNA和MMP-9 mRNA水平、p-PI3K/PI3K、p-Akt/Akt比值和核NF-κB p65蛋白水平升高（均P<0.05），肺组织左叶增生、鳞状上皮化生、大量肿瘤细胞片状或条索状排列紊乱，伴有较多炎性细胞浸润；与模型组相比，升阳益胃汤组CD3⁺、CD4⁺占比和CD4⁺/CD8⁺比值、脾和胸腺指数、IgG、IgA、IgM水平升高，CD8⁺占比、肺指数、IL-6和TNF-α水平、肺组织ICAM-1 mRNA和MMP-9 mRNA水平、p-PI3K/PI3K、p-Akt/Akt比值和核NF-κB p65蛋白水平降低（均P<0.05），肺组织病变减轻，肺组织肿胀不明显，肺泡囊及上皮细胞结构基本清晰；在使用740 Y-P的基础上，加用升阳益胃汤干预可明显抑制PI3K/Akt/NF-κB信号通路，增强肺癌大鼠免疫力。结论升阳益胃汤可能通过抑制PI3K/Akt/NF-κB信号通路，增强肺癌大鼠T淋巴细胞免疫力。

关键词: 肺肿瘤, 疾病模型，动物, 升阳益胃汤, 免疫力, 磷酸肌醇3-激酶/蛋白激酶B/核因子-κB信号通路

Abstract:

Objective To explore the effect of Shengyang Yiwei Decoction on the immune function of lung cancer rats and its possible mechanism. Methods Twelve of 72 SPF Wistar rats were randomly divided as the control group, and the remaining 60 rats were infused with carcinogenic iodized oil solution into trachea to establish lung cancer models. After modeling, model rats were divided into the model group, the cisplatin group (5 mg/kg), the Shengyang Yiwei Decoction group (14 g/kg), the phosphoinositide 3-kinase (PI3K) activator 740 Y-P group (10 mg/kg) and the Shengyang Yiwei Decoction + 740 Y-P group (Shengyang Yiwei Decoction 14 g/kg + 740 Y-P 10 mg/kg), with 12 animals in per group. After corresponding drug intervention, the proportions of peripheral blood T lymphocyte subsets CD3⁺, CD4⁺ and CD8⁺were detected by flow cytometry, and CD4⁺/CD8⁺ ratio was calculated. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum inflammatory factors interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and immunoglobulin (IgG，IgA，IgM) levels. Samples of lung, spleen and thymus were taken and weighed to calculate the indexes of lung, spleen and thymus. HE staining was performed to observe the pathological changes of lung tissue. Real-time fluorescent quantitative PCR (RT-qPCR) was performed to measure the expression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) mRNA in lung tissue. Western blot assay was performed to measure PI3K/protein kinase B (Akt)/nuclear factor-κB (NF-κB) pathway related protein expression in lung tissue. Results Compared with the control group, CD3⁺, CD4⁺ proportions, CD4⁺/CD8⁺ ratio, spleen index and thymus index，IgG，IgA and IgM levels were decreased in the model group, and CD8⁺ proportion, lung index, IL-6 and TNF-α levels, lung tissue ICAM-1 mRNA and MMP-9 mRNA levels, p-PI3K/PI3K, p-Akt/Akt ratios and nuclear NF-κB p65 protein level increased (P<0.05). The left lobe of lung tissue was hyperplasia, squamous epithelial metaplasia and a large number of tumor cells were arranged disorderly in sheets or strips, accompanied by more inflammatory cell infiltration. Compared with the model group, the Shengyang Yiwei Decoction group showed increased CD3⁺ and CD4⁺ proportions, CD4⁺/CD8⁺ ratio, spleen index and thymus index, IgG，IgA，and IgM levels，and decreased CD8⁺ proportion，lung index，IL-6 and TNF-α levels, lung tissue ICAM-1 mRNA and MMP-9 mRNA levels, p-PI3K/PI3K, p-Akt/Akt ratios and nuclear NF-κB p65 protein level (P<0.05). Lung tissue lesions were reduced, lung tissue swelling was not obvious, and the structure of alveolar sacs and epithelial cells were basically clear. On the basis of 740 Y-P, Shengyang Yiwei Decoction could inhibit PI3K/Akt/NF-κB signal pathway and enhance the immunity of lung cancer rats. Conclusion Shengyang Yiwei Decoction may enhance the immune function of T lymphocytes in lung cancer rats by inhibiting PI3K/Akt/NF-κB signaling pathway.

Key words: lung neoplasms, disease models, animal, Shengyang Yiwei Decoction, immunity, phosphoinositol 3-kinase/protein kinase B/nuclear factor- κB signal path

中图分类号:

R734.2，R286.91

图/表 8

参考文献 27

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组别	CD3⁺/%	CD4⁺/%
对照组	72.64±7.31	65.23±7.12
模型组	48.19±5.24^a	42.18±6.49^a
顺铂组	36.30±4.07^b	30.43±4.80^b
升阳益胃汤组	59.57±7.16^bc	54.90±6.32^bc
740 Y-P组	32.24±4.35^b	27.60±4.28^b
升阳益胃汤+740 Y-P组	45.61±5.29^d	39.08±3.56^d
F	82.799^＊＊	80.689^＊＊
组别	CD8⁺/%	CD4⁺/CD8⁺
对照组	27.34±3.25	2.38±0.17
模型组	38.11±5.16^a	1.10±0.13^a
顺铂组	46.56±5.2^b	0.65±0.10^b
升阳益胃汤组	32.31±6.85^bc	1.68±0.15^bc
740 Y-P组	49.24±5.40^b	0.55±0.09^b
升阳益胃汤+740 Y-P组	40.83±3.62^d	0.96±0.12^d
F	41.064^＊＊	344.714^＊＊

组别	CD3⁺/%	CD4⁺/%
对照组	72.64±7.31	65.23±7.12
模型组	48.19±5.24^a	42.18±6.49^a
顺铂组	36.30±4.07^b	30.43±4.80^b
升阳益胃汤组	59.57±7.16^bc	54.90±6.32^bc
740 Y-P组	32.24±4.35^b	27.60±4.28^b
升阳益胃汤+740 Y-P组	45.61±5.29^d	39.08±3.56^d
F	82.799^＊＊	80.689^＊＊
组别	CD8⁺/%	CD4⁺/CD8⁺
对照组	27.34±3.25	2.38±0.17
模型组	38.11±5.16^a	1.10±0.13^a
顺铂组	46.56±5.2^b	0.65±0.10^b
升阳益胃汤组	32.31±6.85^bc	1.68±0.15^bc
740 Y-P组	49.24±5.40^b	0.55±0.09^b
升阳益胃汤+740 Y-P组	40.83±3.62^d	0.96±0.12^d
F	41.064^＊＊	344.714^＊＊

组别	肺指数	脾指数	胸腺指数
对照组	2.75±0.34	4.91±0.57	2.56±0.31
模型组	4.83±0.56^a	2.52±0.33^a	2.04±0.27^a
顺铂组	3.50±0.41^b	2.03±0.28^b	1.69±0.22^b
升阳益胃汤组	3.62±0.49^b	3.40±0.36^bc	2.45±0.30^bc
740 Y-P组	5.87±0.62^b	1.81±0.25^b	1.42±0.24^b
升阳益胃汤+740 Y-P组	4.90±0.53^d	2.39±0.30^d	1.81±0.23^d
F	62.721^＊＊	120.021^＊＊	33.981^＊＊

组别	肺指数	脾指数	胸腺指数
对照组	2.75±0.34	4.91±0.57	2.56±0.31
模型组	4.83±0.56^a	2.52±0.33^a	2.04±0.27^a
顺铂组	3.50±0.41^b	2.03±0.28^b	1.69±0.22^b
升阳益胃汤组	3.62±0.49^b	3.40±0.36^bc	2.45±0.30^bc
740 Y-P组	5.87±0.62^b	1.81±0.25^b	1.42±0.24^b
升阳益胃汤+740 Y-P组	4.90±0.53^d	2.39±0.30^d	1.81±0.23^d
F	62.721^＊＊	120.021^＊＊	33.981^＊＊

组别	IL-6	TNF-α
对照组	34.26±6.45	24.45±4.20
模型组	61.47±8.39^a	56.29±7.86^a
顺铂组	50.15±7.62^b	43.31±6.45^b
升阳益胃汤组	39.22±6.18^bc	32.06±5.29^bc
740 Y-P组	93.40±10.34^b	81.52±8.37^b
升阳益胃汤+740 Y-P组	70.83±9.56^d	62.48±7.56^d
F	85.268^＊＊	115.121^＊＊