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

Abstract

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 collagen Ⅰ synthesis 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 collagen Ⅰin 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. Key words: Lung; Lipopolysaccharide; Fibroblasts; 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3; Aerobic glycolysis; Pulmonary fibrosis