脓毒症患者血清糖酵解代谢物变化及对T淋巴细胞功能的影响

Changes of serum glycolytic metabolites and their effects on T lymphocyte function in patients with sepsis

目的探讨脓毒症患者血清糖酵解代谢物变化及对T淋巴细胞功能的影响。方法选取2022年8月至2023年12月德驭医疗马鞍山总医院收治的54例脓毒症患者和同期54例健康志愿者作为研究对象,采用乳酸检测试剂盒(比色法)说明书检测两组患者血清中糖酵解代谢物乳酸的含量;采用酶联免疫吸附法(ELISA)检测两组患者血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-10的表达水平。H9人淋巴细胞系分为乳酸处理组和乳酸未处理组,向乳酸处理组细胞中加入8 mmol/L的乳酸,乳酸未处理组则加入等体积的生理盐水,孵育24 h。采用细胞增殖检测试剂盒(MTT法)检测增殖活力;采用流式细胞术检测T淋巴细胞亚群比例变化;采用荧光定量聚合酶链反应(PCR)分析TNF-α、IL-1β、IL-10 mRNA表达水平。结果脓毒症患者血清中乳酸水平[(5.51±1.14)mmol/L]明显高于对照组[(2.01±0.66)mmol/L],差异有统计学意义(t=18.74,P<0.05)。脓毒症患者血清中TNF-α[(240.38±58.50)pg/ml]和IL-1β[(117.39±16.35)pg/ml]水平明显高于对照组[(78.52±14.87)、(35.89±8.34)pg/ml],IL-10[(35.43±8.58)pg/ml]水平明显低于对照组[(99.21±14.76)pg/ml,差异有统计学意义(t=18.96、31.40、26.42,P<0.05)。乳酸处理组细胞(0.74±0.04)的吸光度值明显低于乳酸未处理组(1.24±0.08),差异有统计学意义(t=13.56,P<0.05)。乳酸处理组细胞的CD8+T淋巴细胞比例[(27.07±1.18)%]明显高于乳酸未处理组[(23.70±0.98)%],CD4+T淋巴细胞比例[(29.52±0.74)%]、CD4+/CD8+T比值(1.09±0.04)明显低于乳酸未处理组[(34.92±2.81)%、(1.47±0.12)],差异有统计学意义(t=5.40、4.55、7.24,P<0.05)。乳酸处理组细胞的TNF-α(1.18±0.08)和IL-1β(1.32±0.07)mRNA水平明显高于乳酸未处理组(0.47±0.04、0.59±0.06),IL-10(0.56±0.06)mRNA水平明显低于乳酸未处理组(1.05±0.09),差异有统计学意义(t=19.90、18.51、11.68,P<0.05)。结论脓毒症患者血清中糖酵解代谢物乳酸水平显著增加。乳酸水平的升高可通过抑制T淋巴细胞的增殖和分化,调控炎性因子的水平,最终导致细胞免疫功能受损。

ObjectiveTo investigate the changes of serum glycolytic metabolites and their effects on T lymphocyte function in patients with sepsis.MethodsA total of 54 patients with sepsis admitted to our hospital from August 2022 to December 2023 and 54 healthy volunteers during the same period were selected as the study objects.Lactic acid detection kit(colorimetric method)was used to detect the content of serum lactic acid in the two groups.The expression of tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-10 in serum of the two groups was detected by enzyme-linked immunosorbent assay(ELISA).H9 human lymphoid cell lines were divided into lactic acid treated group and untreated group.8 mmol/L of lactic acid was added to the lactic acid treated group,and equal volume of normal saline was added to the the untreated group,and H9 cells were incubated for 24 h.Cell proliferation assay kit(MTT method)was used to detect proliferation activity.The proportion of T lymphocyte subsets was detected by flow cytometry.The mRNA expression of TNF-α,IL-1βand IL-10 was analyzed by fluorescence quantitative polymerase chain reaction(PCR).ResultsThe lactic acid level in sepsis patients[(5.51±1.14)mmol/L]was significantly higher than that in control group[(2.01±0.66)mmol/L,t=18.74,P<0.05].The levels of TNF-α[(240.38±58.50)pg/ml]and IL-1β[(117.39±16.35)pg/ml]in sepsis patients were significantly higher than those of control patients[(78.52±14.87),(35.89±8.34)pg/ml],whereas the IL-10[(35.43±8.58)pg/ml]level significantly lower than the control group[(99.21±14.76)pg/ml,t=18.96,31.40,26.42,P<0.05].The absorbance in lactic acid treated group(0.74±0.04)was significantly lower than in untreated group(1.24±0.08,t=13.56,P<0.05).The percentage of CD8+T lymphocytes[(27.07±1.18)%]in lactic acid treated group was significantly higher than that in untreated group[(23.70±0.98)%].The percentage of CD4+T lymphocytes[(29.52±0.74)%]and CD4+/CD8+T ratio(1.09±0.04)in lactic acid treated group were significantly lower than those in untreated group[(34.92±2.81)%,1.47-0.12,t=5.40,4.55,7.24,P<0.05].The mRNA expression of TNF-α(1.18±0.08)and IL-1β(1.32±0.07)in lactic acid treated group was significantly higher than that in untreated group(0.47±0.04,0.59±0.06),whereas IL-10(0.56±0.06)was significantly lower than that of untreated group(1.05±0.09,t=19.90,18.51,11.68,P<0.05).ConclusionThe serum levels of lactic acid increased significantly in patients with sepsis.The increase of lactic acid can regulate the level of inflammatory factors by inhibiting the proliferation and differentiation of T lymphocytes,and ultimately lead to impaired cellular immune function.