PI3K-Akt-mTOR/PFKFB3 Pathway Mediated LPS-induced Aerobic Glycolysis And Collagen Synthesis In Lung Fibroblasts

Background Pulmonary fibrosis is an important pathological process of sepsis-associated ARDS.It is characterized by abnormal proliferation and activation of lung fibroblasts,accumulation of large amounts of extracellular matrix,and collagen deposition.Lipopolysaccharide(LPS),a component of gram-negative bacillus endotoxin,plays an important role in sepsis-induced pulmonary fibrosis,but the detailed mechanisms have not been fully elucidated.This experiment intends to explore the mechanism of LPSinduced lung fibroblasts abnormal metabolic process by establishing a LPS-induced pulmonary fibrosis model,and clarify the function of the PI3K-Akt-m TOR /PFKFB3 pathway in it,which lays a foundation for the early prevention and treatment of pulmonary fibrosis.Methods1.Part I: Human lung fibroblasts were stimulated with LPS.The expression of PFKFB3 and collagen I were detected by Western blot or Immunofluorescence.OCR and ECAR were measured using Seahorse bioscience.Colorimetric method was used to detect the levels of lactate in cell culture supernatants.2.Part II: An aerobic glycolysis inhibitor 2-DG,a PFKFB3 inhibitor 3PO and sh RNA lentivirus,PI3K-Akt-m TOR inhibitors Rapamycin and Ly294002 were used to pretreat before LPS stimulation,and Western blot was used to detect the phosphorylation of PI3K-Akt-m TOR signaling pathway,the expression of PFKFB3 and collagen I;Seahorse was used to detect the level of OCR and ECAR;Colorimetric method was used to measure the production of lactate.The pulmonary fibrosis model was established by LPS intraperitoneal injection for five consecutive days.HE、Masson staining and immunohistochemical staining of α-SMA were performed in lung tissue sections.PFKFB3、α-SMA、collagen I and TGF-β1 protein expression of lung tissues were detected by Western Blot.Detection of hydroxyproline content in lung tissues as well as lactate content in plasma and bronchoalveolar lavage fluid(BALF).PFKFB3 inhibitor 3PO was performed intraperitoneally,following by establishing a pulmonary fibrosis model according to the above method,and comparing the changes of the above indicators.Results1.Part I: In human lung fibroblasts,LPS stimulation caused an increased in PFKFB3 expression,a decreased in OCR,an increased in ECAR,an increased in the production of lactic acid,and an increased in the expression of collagen I.2.Part II: Pretreatment with aerobic glycolysis inhibitor 2-DG reversed collagen I synthesis induced by LPS;Pretreatment with PFKFB3 inhibitor 3PO and sh RNA lentivirus inhibited aerobic glycolysis and collagen I synthesis induced by LPS;PI3KAkt-m TOR inhibitors Ly294002 and Rapamycin reversed LPS-induced upregulation of PFKFB3 expression,enhanced aerobic glycolysis,and collagen I synthesis,respectively.Vivo experiments confirmed that LPS continuous intraperitoneal injection can cause pulmonary fibrosis in mice,accompanied by upregulation of PFKFB3 and enhanced aerobic glycolysis in lung tissues.Intraperitoneal pre-injection of 3PO can reverse LPS-induced pulmonary fibrosis and lung tissue aerobic glycolysis in mice.ConclusionLPS can induce aerobic glycolysis and cause pulmonary fibrosis in lung fibroblasts.LPS promotes collagen I synthesis in the lung fibroblasts through aerobic glycolysis via activation of PI3K-Akt-m TOR/PFKFB3 pathway in LPS-induced pulmonary fibrosis.