PFKFB3参与脂多糖诱导肺成纤维细胞及肺组织有氧糖酵解的观察性研究

目的:观察脂多糖(lipopolysaccharide,LPS)诱导肺成纤维细胞及肺组织有氧糖酵解关键酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)表达及其与有氧糖酵解的关系,探讨在LPS诱导肺纤维化过程中肺成纤维细胞和肺组织有氧糖酵解的潜在机制。方法:将人胚肺成纤维细胞MRC-5细胞系采用随机数字表法分为PBS对照组(PBS组)和LPS组(每组3孔),Western blot检测LPS刺激细胞6h后PFKFB3表达情况,同时免疫荧光显示PFKFB3在细胞内的定位情况;于LPS刺激后48 h采用海马细胞能量代谢仪检测细胞耗氧率(oxygen consumption rate,OCR)和产酸率(extracellular acidification rate,ECAR),并采用比色法检测有氧糖酵解产物乳酸产生情况,同时Western blot检测LPS刺激48 h后Ⅰ型胶原蛋白合成情况。将24只C57BL/6小鼠按随机数字表法分为生理盐水对照组(C组)、LPS组(L组),每组12只,L组、C组连续5d分别腹腔注射5 mg/kg LPS、等容量生理盐水;每组各6只于造模后第7天无痛处死小鼠,取血浆和肺组织,Western blot和免疫荧光检测各组肺组织中PFKFB3表达情况,比色法检测各组小鼠血浆中乳酸的含量;剩余小鼠于造模后第28天取肺组织,一侧肺通过Western blot检测肺组织Ⅰ型胶原蛋白合成情况,另一侧肺做石蜡切片进行病理学检测。结果:与PBS组比较,LPS刺激细胞6h后PFKFB3表达明显升高(P<0.05);LPS刺激细胞48 h后,与PBS组比较,LPS组细胞耗氧率降低、产酸率增加,代谢产物乳酸含量明显升高(P<0.05),同时细胞Ⅰ型胶原蛋白合成显著增加(P<0.05)。与C组比较,L组小鼠腹腔注射LPS 7 d后肺组织中PFKFB3表达明显升高(P<0.05),血浆乳酸含量明显升高(P<0.05);LPS注射28 d后,L组小鼠Ⅰ型胶原蛋白表达明显升高(P<0.05),肺组织出现明显纤维化。结论:在LPS诱导的肺纤维化过程中,LPS可诱导肺成纤维细胞和肺组织中PFKFB3蛋白表达,该过程与其有氧糖酵解过程相关,PFKFB3的表达上调可能是LPS诱导肺成纤维细胞和肺组织有氧糖酵解和肺纤维化的关键环节。

An observational study on the involvement of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 in aerobic glycolysis in lung fibroblasts and lung tissue induced by lipopolysaccharide

Objective To observe the effects of lipopolysaccharide(LPS)on the expression of a key enzyme during aerobic glycolysis,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3),and its relationship with aerobic glycolysis,so as to explore the potential mechanism of aerobic glycolysis in lung fibroblasts and lung tissue during LPS-induced pulmonary fibrosis.Methods Human embryonic lung fibroblasts(MRC-5 cell line)were divided into two groups according to the random number table method(n=3):a PBS control(PBS)group and an LPS group.After LPS stimulation for 6 h,the expression of PFKFB3 was detected by Western blot,while the intracellular localization of PFKFB3 was determined by immunofluorescence.The oxygen consumption rate(OCR)and the extracellular acidification rate(ECAR)were mea-sured by the Seahorse Extracellular Flux Analyzer,and the colorimetric method was used to detect the production of lactic acid,a product of aer-obic glycolysis.Meanwhile,the synthesis of collagenⅠwas detected by Western blot after LPS stimulation for 48 h.Twenty-four C57BL/6 mice were divided into two groups according to the random number table method(n=12):a normal saline control group(group C),and an LPS group(group L).Groups L and C were intraperitoneally injected with 5 mg/kg LPS or an equal volume of normal saline for five consecutive days.Six mice of each group were sacrificed on Day 7 after modeling to obtain the plasma and lung tissue.The expression of PFKFB3 in lung tissue of each group was detected by Western blot and immunofluorescence,and the colorimetric method was used to detect the content of lactic acid in the plasma of mice in each group.Lung tissues were collected from the remaining mice on Day 28 after modeling,where a lung was collected to detect collagenⅠsynthesis by Western blot,while the other lung were taken to prepare paraffin sections for pathological examination.Results Compared with the PBS group,the expression of PFKFB3 in lung fibroblasts significantly increased after LPS stimulation for 6 h(P<0.05).After LPS stimulation for 48 h,compared with the PBS group,the LPS group presented a decreased OCR,an increased ECAR,and a re-markably increased amount of lactic acid(P<0.05),with significantly increased synthesis of collagenⅠin the cells(P<0.05).Compared with group C,the expression of PFKFB3 in lung tissue significantly increased after intraperitoneal injection of LPS into the mice of group L for seven days(P<0.05),with a significantly increased content of lactic acid in the plasma(P<0.05).After LPS injection for 28 days,the mice in group L presented significantly increased expression of collagenⅠ(P<0.05),with obvious fibrosis in lung tissue.Conclusions LPS can induce the ex-pression of PFKFB3 in lung fibroblasts and lung tissue during LPS-induced pulmonary fibrosis,which is related to aerobic glycolysis.The upreg-ulated expression of PFKFB3 may be a key step of LPS-induced aerobic glycolysis and pulmonary fibrosis in lung fibroblasts and lung tissue.