沉默凝血因子V基因通过抑制JNK1/2和p38 MAPK信号通路对脓毒症大鼠的保护作用

doi:10.13481/j.1671-587X.20260214

摘要/Abstract

摘要：

目的探讨凝血因子V（FV）对脓毒症大鼠免疫炎症反应和c-Jun氨基末端激酶1/2（JNK1/2）及p38丝裂原活化蛋白激酶（p38 MAPK）信号通路的影响，阐明其可能的机制。方法将150只大鼠分为假手术组、模型组、sh-NC组、sh-FV组和茴香霉素组，每组30只。采用盲肠结扎穿孔（CLP）法构建脓毒症模型。观察各组大鼠造模后5 d的生存率。采用苏木精-伊红（HE）染色法观察各组大鼠肺、肾、肝和脾脏组织病理形态表现，实时荧光定量PCR（RT-qPCR）法检测各组大鼠肺组织中FV mRNA表达水平，Western blotting法检测各组大鼠肺组织中FV、JNK1/2和p38 MAPK信号通路相关蛋白表达水平，酶联免疫吸附试验（ELISA）法检测各组大鼠血清中白细胞介素1β（IL-1β）、白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）水平，流式细胞术检测各组大鼠血液中中性粒细胞凋亡率和T淋巴细胞亚群百分率。结果与模型组和sh-NC组比较，sh-FV组大鼠生存率明显升高（P<0.05）；与sh-FV组比较，茴香霉素组大鼠生存率明显降低（P<0.05）。与假手术组比较，模型组和sh-NC组大鼠肺组织中FV mRNA表达水平均明显升高（P<0.05）；与模型组和sh-NC组比较，sh-FV组大鼠肺组织中FV mRNA表达水平明显降低（P<0.05）。与假手术组比较，模型组和sh-NC组大鼠肺组织中FV蛋白表达水平明显升高（P<0.05），p-JNK1/2/JNK1/2和p-p38 MAPK/p38 MAPK比值明显升高（P<0.05）；与模型组和sh-NC组比较，sh-FV组大鼠肺组织中FV蛋白表达水平和p-JNK1/2/JNK1/2及p-p38 MAPK/p38 MAPK比值明显降低（P<0.05）；与sh-FV组比较，茴香霉素组大鼠肺组织中p-JNK1/2/JNK1/2和p-p38 MAPK/p38 MAPK比值明显升高（P<0.05）。与假手术组比较，模型组和sh-NC组大鼠血清中IL-1β、IL-6及TNF-α水平均明显升高（P<0.05）；与模型组和sh-NC组比较，sh-FV组大鼠血清中IL-1β、IL-6和TNF-α水平均明显降低（P<0.05）；与sh-FV组比较，茴香霉素组大鼠血清中IL-1β、IL-6和TNF-α水平均明显升高（P<0.05）。流式细胞术，与假手术组比较，模型组和sh-NC组大鼠中性粒细胞凋亡率、CD4⁺T淋巴细胞百分率及CD4⁺/CD8⁺T淋巴细胞比值明显降低（P<0.05），CD8⁺T淋巴细胞百分率明显升高（P<0.05）；与模型组和sh-NC组比较，sh-FV组大鼠中性粒细胞凋亡率、CD4⁺T淋巴细胞百分率和CD4⁺/CD8⁺T淋巴细胞比值明显升高（P<0.05），CD8⁺T淋巴细胞百分率明显降低（P<0.05）；与sh-FV组比较，茴香霉素组大鼠中性粒细胞凋亡率、CD4⁺T淋巴细胞百分率和CD4⁺/CD8⁺T淋巴细胞比值明显降低（P<0.05），CD8⁺T淋巴细胞百分率明显升高（P<0.05）。结论沉默FV基因可减轻脓毒症大鼠肺、肝、脾脏和肾脏组织损伤，诱导血液中性粒细胞凋亡，降低血清中炎症因子水平，提高血液中CD4⁺T淋巴细胞百分率和CD4⁺/CD8⁺T淋巴细胞比值，其机制可能与抑制JNK1/2和p38 MAPK信号通路有关。

关键词: 凝血因子V, c-Jun氨基末端激酶1/2, p38丝裂原活化蛋白激酶, 脓毒症, 免疫炎症

Abstract:

Objective To discuss the effect of coagulation factor V （FV） on the immune inflammatory response and the c-Jun N-terminal kinase 1/2 （JNK1/2） and p38 mitogen-activated protein kinase （p38 MAPK） signaling pathways in the septic rats， and to clarify its possible mechanism. Methods A total of 150 rats were divided into sham operation group， model group， sh-NC group， sh-FV group， and anisomycin group， with 30 rats in each group. The sepsis model was established by cecal ligation and puncture （CLP） method. The survival rates of the rats in various groups within 5 d after modeling were observed. Hematoxylin-eosin （HE） staining was used to observe the pathomorphology of lung， kidney， liver， and spleen tissues of the rats in various groups； real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression levels of FV mRNA in lung tissue of the rats in various groups； Western blotting method was used to detect the expression levels of FV， JNK1/2， and p38 MAPK signaling pathway-related proteins in lung tissue of the rats in various groups； enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in serum of the rats in various groups； flow cytometry was used to detect the apoptotic rates of neutrophils and the percentages of T lymphocyte subsets in blood of the rats in various groups. Results Compared with model group and sh-NC group， the survival rate of the rats in sh-FV group was significantly increased （P<0.05）； compared with sh-FV group， the survival rate of the rats in anisomycin group was significantly decreased （P<0.05）. Compared with sham operation group， the expression levels of FV mRNA in lung tissue of the rats in model group and sh-NC group were significantly increased （P<0.05）； compared with model group and sh-NC group， the expression level of FV mRNA in lung tissue of the rats in sh-FV group was significantly decreased （P<0.05）. The Western blotting results showed that compared with sham operation group， the expression levels of FV protein in lung tissue of the rats in model group and sh-NC group were significantly increased （P<0.05）， and the ratios of p-JNK1/2/JNK1/2 and p-p38 MAPK/p38 MAPK were significantly increased （P>0.05）； compared with model group and sh-NC group， the expression level of FV protein and the ratios of p-JNK1/2/JNK1/2 and p-p38 MAPK/p38 MAPK in lung tissue of the rats in sh-FV group were significantly decreased （P<0.05）； compared with sh-FV group， the ratios of p-JNK1/2/JNK1/2 and p-p38 MAPK/p38 MAPK in lung tissue of the rats in anisomycin group were significantly increased （P<0.05）. The ELISA results showed that compared with sham operation group， the serum levels of IL-1β， IL-6， and TNF-α of the rats in model group and sh-NC group were significantly increased （P<0.05）； compared with model group and sh-NC group， the serum levels of IL-1β， IL-6， and TNF-α of the rats in sh-FV group were significantly decreased （P<0.05）； compared with sh-FV group， the serum levels of IL-1β， IL-6， and TNF-α of the rats in anisomycin group were significantly increased （P<0.05）. The flow cytometry results showed that compared with sham operation group， the apoptotic rates of neutrophils， the percentage of CD4⁺ T lymphocytes， and the ratios of CD4⁺/CD8⁺ T lymphocytes of the rats in model group and sh-NC group were significantly decreased （P<0.05）， while the percentages of CD8⁺ T lymphocytes were significantly increased （P<0.05）； compared with model group and sh-NC group， the apoptotic rate of neutrophils， the percentage of CD4⁺ T lymphocytes， and the ratio of CD4⁺/CD8⁺ T lymphocytes of the rats in sh-FV group were significantly increased （P<0.05）， while the percentage of CD8⁺ T lymphocytes was significantly decreased （P<0.05）； compared with sh-FV group， the apoptotic rate of neutrophils， the percentage of CD4⁺ T lymphocytes， and the ratio of CD4⁺/CD8⁺ T lymphocytes of the rats in anisomycin group were significantly decreased （P<0.05）， while the percentage of CD8⁺ cells was significantly increased （P<0.05）. Conclusion Silencing FV gene can alleviate the damage of lung， liver， spleen， and kidney tissues， induce the apoptosis of blood neutrophils， reduce the serum levels of inflammatory factors， and increase the percentage of CD4⁺ T lymphocytes and the ratio of CD4⁺/CD8⁺ T lymphocytes in blood of the septic rats. Its mechanism may be related to the inhibition of JNK1/2 and p38 MAPK signaling pathways.

Key words: Coagulation factor V, c-Jun N-terminal kinase 1/2, p38 mitogen-activated protein kinase, Sepsis, Immune inflammation

中图分类号:

R364.5

王镜媛,陈芳,刘艳存,李士欣,寿松涛. 沉默凝血因子V基因通过抑制JNK1/2和p38 MAPK信号通路对脓毒症大鼠的保护作用[J]. 吉林大学学报(医学版), 2026, 52(2): 418-428.

Jingyuan WANG,Fang CHEN,Yancun LIU,Shixin LI,Songtao SHOU. Protective effect of silencing coagulation factor V gene on septic rats by inhibiting JNK1/2 and p38 MAPK signaling pathways[J]. Journal of Jilin University(Medicine Edition), 2026, 52(2): 418-428.

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Group	Survival number
Group	(t/d) 0	1	3	5
Sham operation	10（100.00）	10（100.00）	10（100.00）	10（100.00）
Model	10（100.00）	8（80.00）^*	5（50.00）^*	2（20.00）^*
Sh-NC	10（100.00）	9（90.00）	5（50.00）^*	3（30.00）^*
Sh-FV	10（100.00）	10（100.00）^#	8（80.00）^△#	6（60.00）^△#
Anisomycin	10（100.00）	8（80.00）^○	6（60.00）^○	3（30.00）^○