坏死性小肠结肠炎患儿外周血白细胞自噬相关蛋白表达及临床意义

Autophagy-related protein expression in peripheral leukocytes and clinical significance in children with necrotizing enterocolitis

目的探讨坏死性小肠结肠炎(necrotizing enterocolitis,NEC)患儿外周血白细胞中自噬相关蛋白微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、Beclin1的表达及临床意义。方法采用前瞻性研究方法,收集2017年1月至2019年9月在山西省人民医院住院的NEC患儿及正常对照组患儿外周血,分离白细胞,采用Western Blot检测白细胞中LC3Ⅱ、LC3Ⅰ、Beclin1表达量,计算LC3Ⅱ/Ⅰ比例;ELISA法检测血清白细胞介素6(interleukin-6,IL-6)、白细胞介素4(interleukin-4,IL-4)及肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)含量;检测C反应蛋白(C-reactive protein,CRP)水平。记录NEC组肠鸣音消失持续时间、便潜血持续时间、持续禁食时间。比较两组患儿白细胞中LC3Ⅱ/Ⅰ比例、Beclin1表达量及血清细胞因子含量,分析上述指标与NEC患儿病情严重程度的相关性。结果共纳入NEC组33例,对照组36例。(1)NEC组外周血白细胞中自噬相关蛋白LC3Ⅱ/Ⅰ比例及Beclin1表达量高于对照组[2.04(1.65,2.65)比1.00(0.97,1.03),1.87(1.16,2.02)ng/ml比0.88(0.77,0.96)ng/ml],差异有统计学意义(P<0.05)。(2)NEC组血清IL-4、IL-6及TNF-α表达量均高于对照组[3.0(2.3,3.6)ng/L比1.6(1.3,1.9)ng/L,10.0(7.7,12.1)ng/L比3.8(2.1,4.8)ng/L,32.0(26.5,37.5)ng/L比11.5(9.8,12.6)ng/L],差异有统计学意义(P<0.05)。(3)自噬相关蛋白LC3Ⅱ/Ⅰ比例和Beclin1表达量与CRP、肠鸣音消失持续时间、便潜血持续时间、持续禁食时间成正相关(P<0.05);IL-4与CRP、肠鸣音消失持续时间、便潜血持续时间、持续禁食时间均无相关性(P>0.05);IL-6和TNF-α与肠鸣音消失持续时间、便潜血持续时间、持续禁食时间、CRP成正相关(P<0.05)。(4)自噬相关蛋白LC3Ⅱ/Ⅰ比例、Beclin1表达量与IL-4、IL-6及TNF-α含量均成正相关(P<0.05)。(5)NEC组中,高自噬水平患儿CRP水平高于低自噬水平患儿,肠鸣音消失持续时间、便潜血持续时间和持续禁食时间长于低自噬水平患儿(P<0.05)。结论NEC患儿外周血白细胞中发生自噬,自噬水平与细胞因子成正相关,自噬程度与NEC病情相关。

Objective To study the expression and clinical significance of microtubule-associated protein 1 light chain 3(LC3)and Beclin1 in peripheral leukocytes of children with necrotizing enterocolitis(NEC).Method Pediatric patients admitted to Shanxi Province People's Hospital from January 2017 to September 2019 were prospectively collected.Leukocytes were separated from peripheral blood of infants in NEC group and those in control group.Western Blot was used to detect the expression of LC3Ⅱ,LC3Ⅰ,Beclin1 in white blood cells.The serum levels of interleukin-6(IL-6),interleukin-4(IL-4)and tumor necrosis factor-α(TNF-α)were measured by ELISA.C-reactive protein(CRP)was also measured.The duration of disappearance of bowel sounds,fecal occult blood,and fasting time of infants in the NEC group was recorded.The ratio of LC3Ⅱto LC3Ⅰ,Beclin1 expression and serum cytokine content in white blood cells of two groups of infants were compared,and the correlation between the above indexes and the severity of NEC was analyzed.Result A total of 33 cases in the NEC group and 36 cases in the control group were included.The ratio of autophagy protein LC3ⅡtoⅠand Beclin1 expression in peripheral leukocytes of NEC group were higher than those of control group[2.04(1.65,2.65)vs.1.00(0.97,1.03),1.87(1.16,2.02)ng/ml vs.0.88(0.77,0.96)ng/ml],the difference was statistically significant(P<0.05).The expression levels of serum IL-4,IL-6 and TNF-αin the NEC group were higher than those in the control group[3.0(2.3,3.6)ng/L vs.1.6(1.3,1.9)ng/L,10.0(7.7,12.1)ng/L vs.3.8(2.1,4.8)ng/L,32.0(26.5,37.5)ng/L vs.11.5(9.8,12.6)ng/L],the difference was statistically significant(P<0.05).The ratio of autophagy protein LC3Ⅱto LC3Ⅰand Beclin1 expression were positively correlated with the value of CRP,bowel sounds disappearance duration,fecal occult blood duration,and continuous fasting time(P<0.05).There was no correlation between IL-4 and CRP,duration of disappearance of bowel sounds,duration of fecal occult blood,and duration of continuous fasting(P>0.05).IL-6 and TNF-αwere positively correlated with the duration of bowel sound disappearance,the duration of fecal occult blood,the duration of fasting,and CRP(P<0.05).The ratio of autophagy protein LC3Ⅱto LC3Ⅰand Beclin1 expression were positively correlated with the level of IL-4,IL-6 and TNF-α(P<0.05).In the NEC group,the CRP level of children with high autophagy levels was higher than that of children with low autophagy levels,the duration of disappearance of bowel sounds,the duration of fecal occult blood and the duration of fasting were also longer than those of children with low autophagy levels(P<0.05).Conclusion Autophagy occurs in peripheral leukocytes in children with NEC.The level of autophagy is positively correlated with cytokines,and the degree of autophagy is related to the severity of NEC.