The Role And Mechanism Of GGPPS On Bleomycin-induced Pulmonary Fibrosis In Mice

Background and Objective:Idiopathic pulmonary fibrosis(IPF)is a chronic and progressive pulmonary interstitial fibrosis disease of unknown causes,with more than 5 million patients worldwide.IPF is commonly found in middle-aged and elderly people,with poor prognosis and high mortality.The average survival time after diagnosis is 2-4 years.Most patients eventually die of respiratory failure.Geranylgeranyl pyrophosphate synthase(GGPPS)is an important branch enzyme in the mevalonate metabolic pathway.It mainly catalyzes famesyl pyrophosphate(FPP)to geranylgeranyl pyrophosphate(GGPP).Abnormal expression of GGPPS will disturb the balance between the famesylation and geranylgeranylation of proteins.Studies have confirmed that GGPPS participates in the occurrence of inflammation,tumor,infertility,liver fibrosis,and other diseases,and is crucial to the development of the fetal lung.At present,it is not clear whether GGPPS participates in the process of pulmonary fibrosis.Therefore,our research aims to explore the role of GGPPS in the formation of pulmonary fibrosis and seek the possible mechanism.Methods:1.We constructed a bleomycin-induced pulmonary fibrosis model in mice and detected the expression of GGPPS in wild type(WT)mouse lung tissue at indicated various time points by immunohistochemisry and Western blot 2.The alveolar epithelium-specific GGPPS knockout mice were used to establish a model,and the effects of GGPPS knockout on pulmonary inflammation and fibrosis,proliferation and activation of pulmonary fibroblasts were detected by qRT-PCR,immunohistochemistry,immunofluorescence,Western blot,Masson staining,ELISA and other methods,and the role of TGF-?/BMP signaling pathway in GGPPS regulation of pulmonary fibrosis was explored.3.Rescue experiment was conducted to observe the effect of GGPP supplementation in advance on the degree of pulmonary fibrosis and the TGF-?/BMP signaling pathway induced by GGPPS knockout in mice.Results:1.In the process of bleomycin-induced lung injury and pulmonary fibrosis in mice,GGPPS expression was up-regulated:24 hours after bleomycin administration,GGPPS expression in wild-type mice lung tissue slightly increased,significantly increased on the 7th day,about 2 times,and remained high until the 21st day,suggesting that GGPPS might play a role in the formation of pulmonary fibrosis.2.GGPPS deletion promoted the increase of extracellular matrix deposition and aggravated the degree of pulmonary fibrosis:Compared with WT mice,the content of hydroxyproline in lung homogenates of GGPPS knockout mice was significantly increased.Masson staining showed more blue collagen deposition,higher pulmonary fibrosis score,and the mRNA and protein levels of type I collagen and fibronectin were significantly increased.GGPP supplementation in advance could effectively prevent pulmonary fibrosis induced by GGPPS knockout in mice.3.GGPPS deletion aggravated pulmonary inflammation:Compared with WT mice,the total number of cells and the proportion of neutrophils in bronchoalveolar lavage fluid(BALF)of GGPPS knockout mice increased on the 7th day after modeling,the total protein level and IL-1 level rose.4.GGPPS deletion promoted the activation and proliferation of lung fibroblasts:Immunohistochemistry WB,and PCR results all showed that SMA expression in lung tissue of GGPPS knockout mice was increased,and immunofluorescence double staining also showed that the number of myofibroblasts in lung tissue of GGPPS knockout mice was significantly increased.5.GGPPS regulated pulmonary fibrosis through the TGF-1/BMP-4 signaling pathway:After GGPPS deletion,TGF-1 expression increased,p-Smad2 level increased,while p-Smad1/5 level decreased,TGF-1/Smad2 signaling pathway was activated,while BMP-4/Smad1/5 signal was inhibited,and the signal balance between TGF-1 and BMP was destroyed.After the GGPP supplement,the balance between TGF-1 and BMP was restored.Conclusion:Our study confirms that GGPPS plays a protective role in the formation of pulmonary fibrosis.GGPPS deletion promotes the process of pulmonary fibrosis by activating the TGF-1 signal and inhibiting the BMP-4 signal.