烟曲霉感染时糖尿病小鼠中性粒细胞经细胞外调节蛋白激酶1/2通路调控NADPH氧化酶2影响细胞内活性氧的研究  

Study on the regulation of NADPH oxidase 2 by extracellular regulated protein kinases 1/2 pathway in neutrophils from diabetic infected with Aspergillus fumigatus and its impact on intracellular reactive oxygen species

作　　者：许向华 赵红伟 付营 Xu Xianghua;Zhao Hongwei;Fu Ying(Department of Cadre Ward Three,General Hospital of Northern Theater Command,Shenyang 110003,China)

机构地区：[1]北部战区总医院干部病房三科,沈阳110003

出　　处：《中国临床实用医学》2024年第5期55-63,共9页China Clinical Practical Medicine

基　　金：辽宁省科学技术计划项目(2019-ZD-1026)。

摘　　要：目的探讨烟曲霉感染时糖尿病小鼠中性粒细胞经细胞外调节蛋白激酶1/2(ERK1/2)通路对NADPH氧化酶2(NOX2)调节进而影响细胞内活性氧的产生。方法选取5周龄雄性C57BL/6小鼠24只,体质量(18±2)g,体质量范围为16~20 g,分为对照组(n=12,正常雄性C57BL/6小鼠)与糖尿病组(n=12,按照2型糖尿病小鼠模型建立)。烟曲霉菌丝分别与对照组和糖尿病组小鼠中性粒细胞进行孵育6 h,比较p47phox、gp91phox和抗磷酸化p47phox(P-p47phox)相对表达量。采用免疫荧光法对中性粒细胞染色,比较gp91phox和P-p47phox的阳性率。烟曲霉菌丝分别与对照组和糖尿病组小鼠中性粒细胞共孵育6 h,在刺激前后分别检测中性粒细胞ERK1/2蛋白和抗磷酸化ERK1/2(P-ERK1/2)相对表达量的表达。对照组与糖尿病组中性粒细胞分别加入和不加入PD98059(20μmol/L),处理30 min,之后每组均加入烟曲霉菌丝刺激6 h,检测P-ERK1/2、P-p47phox和gp91phox的表达及细胞内活性氧相对量,比较上清液中瘤坏死因子-α(TNF-α)与白细胞介素-1β(IL-1β)含量。结果孵育后,糖尿病组+烟曲霉gp91phox(1.040±0.088)高于对照组+烟曲霉(0.810±0.066),糖尿病组+烟曲霉P-p47phox(0.690±0.059)高于对照组+烟曲霉(0.538±0.044),糖尿病组+烟曲霉gp91phox阳性率[(33.9±3.8)%]高于对照组+烟曲霉[(22.0±3.0)%],糖尿病组+烟曲霉P-p47phox阳性率[(28.8±3.3)%]高于对照组+烟曲霉[(18.7±2.6)%],糖尿病组+烟曲霉P-ERK1/2(0.890±0.075)高于对照组+烟曲霉(0.694±0.057),对照组+PD98059+烟曲霉的P-ERK1/2(0.133±0.020)、P-p47phox(0.151±0.033)、gp91phox(0.112±0.019)低于对照组+烟曲霉[(0.712±0.072)、(0.455±0.016)、(0.356±0.066)],糖尿病组+PD98059+烟曲霉的P-ERK1/2(0.379±0.078)、P-p47phox(0.281±0.030)、gp91phox(0.291±0.062)低于糖尿病组+烟曲霉[(0.910±0.084)、(0.606±0.011)、(0.515±0.058)],糖尿病组+PD98059+烟曲霉细胞内活性氧相对量[(2.438±0.261)A.U.]低于糖尿病组+烟曲霉[(3.2ObjectiveTo investigate the regulation of NADPH oxidase 2(NOX2)by neutrophils via extracellular regulated protein kinases 1/2(ERK1/2)pathway in diabetic mice during Aspergillus fumigatus infection,and its effect on the production of intr reactive oxygen species(ROS).MethodsA total of 245-week-old male C57BL/6 mice were selected,body weight(18±2)g,with a body weight of 16 to 20 g,and divided them into control group(n=12,normal male C57BL/6 mice)and diabetes group(n=12,established as a type 2 diabetes mouse model).Aspergillus fumigatus hyphae were incubated with neutrophils from the control and groups for 6 h,and the relative expression levels of p47phox,gp91phox,and phosphorylated p47phox(P-p47phox)were compared.Neutrophils were stained using immunofluorescence,and positivity rates for gp91phox and P-p47phox were compared.Aspergillus fumigatus hyphae were co-incated with neutrophils from the control and diabetes groups for 6 h,and the relative expression levels of ERK1/2 and P-ERK12 were detected before and after stimulation.Neutrophils from the control and diabetes groups were treated with and without PD98059(20μmol/L)for 30 min,then stimulated with Aspergillus fumigatus hyphae for 6 h.The expression of phosphory ERK1/2(P-ERK1/2),P-p47phox,and gp91phox,as well as theacellular ROS levels,were detected.The levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the supernatant were compared.ResultsAfter incubation,the level of gp91phox in diabetes group+Aspergillus fumigatus(1.040±0.088)was higher than that in control group+Aspergillus fumigatus(0.810±0.066),and the level of P-p47phox in diabetes group+Aspergillus fumigatus(0.690±0.059)was higher than that in control group+Aspergillus fumigatus(0.538±0.044),the positive rate of gp91phox in diabetes group+Aspergillus fumigatus[(33.9±3.8)%]was higher than that in control group+Aspergillus fumigatus[(22.0±3.0)%],and the positive rate of P-p47phox in diabetes group+Aspergillus fumigatus[(28.8±3.3)%]was higher than that in control group+Aspergil

关 键 词：糖尿病 中性粒细胞 NADPH氧化酶2 细胞外调节蛋白激酶1/2 活性氧 

分 类 号：R587.1[医药卫生—内分泌] R519[医药卫生—内科学]