益生菌对脓毒症患儿外周血淋巴细胞焦亡的影响及机制

Effect and mechanism of probiotics on pyroptosis of peripheral lymphocytes in children with sepsis

目的 观察益生菌对脓毒症患儿外周血淋巴细胞焦亡的影响,探讨益生菌的免疫调节作用机制。方法 选择2019年1月至2020年12月河南省人民医院儿童重症监护病房收治的109例脓毒症患儿为研究对象,按随机数字表法将患儿分为对照组(n=55)和观察组(n=54),对照组患儿采用常规治疗,观察组患儿在常规治疗的基础上给予复方嗜酸乳杆菌口服片。采集所有患儿的性别、年龄、小儿危重病例评分(PCIS)、序贯器官衰竭估计评分(SOFA)、原发病类型及发生严重脓毒症比例、脓毒症休克比例、血清白蛋白(ALB)、丙氨酸氨基转移酶(ALT)、血肌酐(Scr)水平。治疗8 d后,取患儿外周血分离血浆和淋巴细胞,应用定量反转录聚合酶链反应法检测淋巴细胞焦亡相关分子NOD样受体热蛋白结构域相关蛋白3(NLRP3)、消皮素D(GSDMD)、天冬氨酸特异性半胱氨酸蛋白酶-1(caspase-1)mRNA及微RNA-224-3p(miR-224-3p)表达水平,应用化学荧光法及流式细胞术检测淋巴细胞的焦亡活性及焦亡比例,应用酶联免疫吸附试验法检测患儿血浆中白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)水平,记录2组患儿二次感染率、住院病死率、住院时间和机械通气时间,应用Pearson法分析淋巴细胞中miR-224-3p水平与caspase-1 mRNA水平的相关性,应用logistic进行二次感染影响因素的多因素回归分析。结果 109例患儿中有4例自动出院(对照组1例,观察组3例),最终纳入105例患儿。2组患儿的性别、年龄、PCIS评分、SOFA评分、严重脓毒症比例、脓毒症休克比例、各类原发疾病比例及ALB、ALT、Scr水平比较差异均无统计学意义(P>0.05)。观察组患儿淋巴细胞中NLRP3、GSDMD、caspase-1 mRNA及miR-224-3p水平显著低于对照组(P<0.05)。观察组患儿淋巴细胞内绿色荧光强度显著低于对照组(P<0.05)。观察组患儿外周血淋巴细胞的早期焦亡比例和晚期焦亡比例显著低于对照组(P<0.05)。观察组患儿二次感染率显著低于对照组(χ^(2)=4.543,P<0.05);观察组与对照组患儿住院病死率、住院时间及机械通气时间比较差异无统计学意义(P>0.05)。观察组患儿血浆中IL-1β、IL-18水平显著低于对照组(P<0.05)。脓毒症患儿淋巴细胞中miR-224-3p水平与caspase-1 mRNA水平呈正相关(r=0.623 7,P<0.05)。淋巴细胞焦亡比例(OR=1.597,P<0.05)和住院时间(OR=1.588,P<0.05)是发生二次感染的独立危险因素。结论 脓毒症患儿外周血淋巴细胞中miR-224-3p水平与其焦亡活性密切相关,益生菌可下调miR-224-3p水平,抑制免疫细胞焦亡,缓解全身炎症反应,改善临床预后。

Objective To observe the effects of probiotics on pyroptosis of peripheral lymphocytes in children with sepsis,and to explore the immunoregulatory mechanism of probiotics.Methods A total of 109 septic children admitted to Pediatric Intensive Care Unit,People′s Hospital of Henan University from January 2019 to December 2020 were selected as the research objects,and they were divided into control group(n=55)and observation group(n=54)according to random number table.The children in the control group was treated with regular therapy;the children in the observation group were given compound eosinophil-lactobacillus tablets on the basis of routine treatment.The gender,age,pediatric critical illness score(PCIS),sequential organ failure assessment(SOFA)score,primary disease ratio,severe sepsis ratio,sepsis shock ratio,and serum albumin(ALB),alanine aminotransferase(ALT)and creatinine(Scr)levels of all children were collected.The plasma and lymphocytes were isolated from peripheral blood of children after 8 days of treatment,the expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),gasdermin-D(GSDMD),cysteinyl aspartate specific proteinase-1(caspase-1)mRNA and microRNA-224-3p(miR-224-3p)were detected by quantitative reverse transcription-polymerase chain reaction.The pyroptotic activation and percentages of pyroptotic lymphocytes of children were detected by chemiluminescence and flow cytometry,respectively.The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in plasma were detected by enzyme linked immunosorbent assay.And the incidence of secondary infection,hospital mortality rate,hospitalization stay and duration of ventilation of children in the two groups were also observed.The correlation between the miR-224-3p level in lymphocyte and caspase-1 mRNA level was analyzed by Pearson method.The influencing factor of secondary infection were analyzed by logistic multivariate regression analysis.Results Four patients left hospital voluntarily in 109 patients(1 case in the control group and 3 cases in the observation group).There was no significant difference in sex,age,PCIS score,SOFA score,severe sepsis ratio,sepsis shock ratio,primary disease ratio,levels of ALB,ALT and Scr of children between the two groups(P>0.05).The expression levels of NLRP3,GSDMD,caspase-1 mRNA and miR-224-3p of children in the observation group were significantly lower than those in the control group(P<0.05).The green fluorescence intensity in lymphocytes of children in the observation group was significantly lower than that in the control group(P<0.05).The percentage of pyroptotic cells in early pyroptotic phase and later pyroptotic phase in peripheral lymphocytes of children in the observation group were significantly lower than those in the control group(P<0.05).The secondary infection rate of children in the observation group was significantly lower than that in the control group(χ^(2)=4.543,P<0.05);there were no statistically significant differences in the hospital mortality rate,hospitalization stay,duration of ventilation of children between the observation group and control group(P>0.05).The levels of plasma IL-1βand IL-18 of children in the observation group were significantly lower than those in the control group(P<0.05).The level of miR-224-3p in lymphocytes of children with sepsis was positively correlated with the level of caspase-1 mRNA(r=0.6237,P<0.05).The percentage of pyroptotic lymphocyte(OR=1.597,P<0.05)and hospitalization stay(OR=1.588,P<0.05)were independent risk factors for secondary infection.Conclusion The levels of miR-224-3p were closely associated to pyroptotic activation of peripheral lymphocytes of septic children.Probiotics can decrease miR-224-3p levels,inhibit pyroptosis in immune cells,alleviate systematic inflammatory reaction,improve prognosis of the septic patients.