| 摘要: |
| 目的：探讨MAL基因敲除小鼠肝郁型哮喘模型受体酪氨酸激酶肝配蛋白A型受体2(ephrin type-a receptor 2，EphA2）、信号转导和转录激活因子-3（acute-phase response factor 3，STAT3）、p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase，p38MAPK）、白细胞介素-6（interleukin-6，IL-6）、白细胞介素-8（interleukin-8，IL-8）的表达及柴朴汤干预的影响。方法：C57BL/6 遗传背景的野生型小鼠16只、MAL基因敲除小鼠32只按随机数字表法分别分为野生对照组（A 组）、野生哮喘组（B组）、敲基因对照组（C组）、敲基因哮喘组（D 组）、敲基因肝郁型哮喘组（E 组）、柴朴汤干预敲基因肝郁型哮喘组（F 组），每组各8只。B、D、E、F组腹腔注射卵清蛋白加氢氧化铝混合液致敏，A、C组注入0.9%氯化钠注射液;肝郁造模结束后的第 1~7 天，B、D、E、F组雾化吸入1%的卵清蛋白与0.9%氯化钠注射液混合溶液激发哮喘，A、C组雾化吸入同等量的0.9%氯化钠注射液，每次雾化30 min，连续7 d。F组于每次雾化前30 min予0.2 mL柴朴汤药液灌胃，其他组予等量0.9%氯化钠注射液灌胃。末次雾化结束24 h后脱颈处死小鼠，取肺组织行苏木精-伊红（Hematoxylin and Eosin，HE）染色及过碘酸希夫（Periodic Acid-Schiff，PAS）染色，观察病理变化，分别采用逆转录实时荧光定量聚合酶链式反应（Reverse Transcription quantitative Polymerase Chain Reaction，RT-qPCR）和蛋白质免疫印迹（Western Blot，WB）检测小鼠肺组织中EphA2、STAT3、p38MAPK、IL-6、IL-8蛋白及其mRNA的表达。结果：1）HE染色结果。A组支气管壁结构形态正常;B、C、D组均可见管腔增厚、气道狭窄，可见炎症细胞浸润，C组管腔有少许断裂;E、F组管腔破坏严重，可见明显边缘不齐、管腔增厚、上皮细胞增生及炎症细胞浸润。2）PAS染色结果。A组无明显黏液分泌;B组气道壁少许黏液分泌;C组较A组管腔增厚，可见黏液分泌;D组可见明显气道壁增厚、伴有管腔狭窄，大量杯状细胞增生及黏液分泌;E、F组管腔不规整，均可见明显黏液栓及上皮细胞增生。3）RT-qPCR结果。与B组比较，D组EphA2、p38MAPK、IL-6、IL-8 mRNA表达显著降低（P＜0.01），2组STAT3 mRNA表达差异无统计学意义（P＞0.05）;与D组比较，E组EphA2、STAT3、p38MAPK、IL-6、IL-8 mRNA表达均降低（P＜0.05）;与E组相比，F组EphA2 mRNA 表达上调（P＜0.01），2组其余各项指标差异均无统计学意义（P＞0.05）。4）WB检测结果。B组EphA2、STAT3、p38MAPK、IL-6、IL-8 蛋白的表达高于A、C组（P＜0.05），E、F组各项指标差异无统计学意义（P＞0.05）。结论：1）MAL基因敲除影响哮喘小鼠肺组织中EphA2、STAT3、p38MAPK、IL-6、IL-8的表达;2）MAL基因可能是柴朴汤治疗肝郁型哮喘的主要作用靶点之一，作用机制可能与EphA2、STAT3/p38MAPK信号通路有关。 |
| 关键词: 柴朴汤 支气管哮喘 肝郁型 MAL基因敲除小鼠 EphA2 STAT3 p38MAPK |
| DOI： |
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| Effect of Chaipu decoction on the expression of ephrin type-A receptor 2，acute-phase response factor 3，p38 mitogen-activated protein kinase，and inflammatory factors in an MAL gene-knockout mouse model of asthma with stagnation of liver Qi |
| WANG Yiyan,TANG Hongting,HUANG Yan |
| (Hunan University of Chinese Medicine，Changsha 410208，Hunan，China;Changde First Hospital of Traditional Chinese Medicine，Changde 415000，Hunan，China;The First Affiliated Hospital of University of South China，Hengyang 421000，Hunan，China) |
| Abstract: |
| Objective：To investigate the expression of ephrin type-A receptor 2 (EphA2)，acute-phase response factor 3 (STAT3)，p38 mitogen-activated protein kinase (p38MAPK)，ainterleukin-6 (IL-6)，and interleukin-8 (IL-8) in an MAL gene-knockout mouse model of asthma with stagnation of liver Qi，as well as the interventional effect of Chaipu decoction.Methods: A total of 16 wild-type C57BL/6 mice and 32 MAL gene-knockout mice were divided into wild-type control group (group A)，wild-type asthma group (group B)，gene-knockout control group (group C)，gene-knockout asthma group (group D)，gene-knockout asthma with stagnation of liver Qi group (group E)，and gene-knockout asthma with stagnation of liver Qi+Chaipu decoction intervention group (group F) using a random number table，with 8 mice in each group.The mice in groups B，D，E，and F were given intraperitoneal injection of ovalbumin and aluminum hydroxide for sensitization，and those in groups A and C were given 0.9% sodium chloride injection;on days 1-7 after modeling of stagnation of liver Qi，the mice in groups B，D，E，and F were given aerosol inhalation of the mixed solution of 1% ovalbumin and 0.9% sodium chloride injection to stimulate asthma，and those in groups A and C were given aerosol inhalation of an equal volume of 0.9% sodium chloride injection，for 30 minutes each time and 7 consecutive days.The mice in group F were given 0.2 mL of Chaipu decoction by gavage at 30 minutes before each time of aerosol inhalation，and those in the other groups were given an equal volume of 0.9% sodium chloride injection by gavage.At 24 hours after last aerosol inhalation，the mice were sacrificed by neck dislocation，and HE staining and PAS staining were performed for lung tissue to observe its pathological changes，and RT-qPCR and Western blot were used to measure the mRNA and protein expression levels of EphA2，STAT3，p38MAPK，IL-6，and IL-8 in lung tissue.Results: HE staining showed that the mice in group A had normal structure and morphology of tracheal wall;the mice in groups B，C，and D had thickening of tracheal lumen，airway stenosis，and inflammatory cell infiltration，while those in group C had slight rupture of tracheal lumen;the mice in groups E and F had severe destruction of tracheal lumen，with uneven edges，thickening of tracheal lumen，epithelial cell hyperplasia，and inflammatory cell infiltration.PAS staining showed that group A had no obvious mucus secretion and group B had a small amount of mucus secretion on tracheal wall;compared with group A，group C had thickening of tracheal lumen with the presence of mucus secretion;group D had obvious thickening of tracheal lumen，lumen stenosis，goblet cell hyperplasia，and mucus secretion;groups E and F had irregular tracheal lumen，with marked mucus plug and epithelial cell hyperplasia.RT-qPCR showed than compared with group B，group D had significant reductions in the mRNA expression levels of EphA2，p38MAPK，IL-6，and IL-8 (P < 0.01)，while there was no significant difference in the mRNA expression of STAT3 between the two groups;compared with group D，group E had significant reductions in the mRNA expression levels of EphA2，STAT3，p38MAPK，IL-6，and IL-8 (P < 0.05);compared with group E，group F had a significant increase in the mRNA expression level of EphA2 (P < 0.01)，and there were no significant differences in the other indicators between the two groups (P > 0.05).Western blot showed that group B had significantly higher protein expression levels of EphA2，STAT3，p38MAPK，IL-6，and IL-8 than groups A and C (P < 0.05)，and there were no significant differences in these indicators between groups E and F (P > 0.05).Conclusion: MAL gene knockout affects the expression of EphA2，STAT3，p38MAPK，IL-6，and IL-8 in lung tissue of mice with asthma，and the MAL gene might be one of the main action targets for Chaipu decoction in the treatment of asthma with stagnation of liver Qi，which might be associated with the EphA2 and STAT3/p38MAPK signaling pathways. |
| Key words: Chaipu decoction bronchial asthma stagnation of liver Qi MAL gene-knockout mice ephrin type-A receptor 2 acute-phase response factor 3 p38 mitogen-activated protein kinase |
