Edffect Of GCH1 Regulating Tetrahydrobiopterin Biosynthesis Pathway On The Progression Of Radiation-induced Lung Injury And Its Mechanism

Objective Radiation therapy of thoracic malignancies(lung cancer,esophageal cancer,breast cancer,etc.)or accidental nuclear accidents can easily lead to radiation-induced lung injury.Tetrahydrobiotrexate(BH4)is an important cofactor of all NOSs and a determinant of NOS function.GTP cyclohydrolase I(GCH1)is a key enzyme in the synthesis of BH4.This study investigated the role of GCH1/BH4 in radiation-induced lung injury and the mechanism.Methods In vitro experiments:The changes of BH4 before and after ionizing radiation exposure were detected by UV-visible spectroscopy.Western blotting was used to detect the changes of GCH1 content in HELF and BEAS-2B cells before and after ionizing radiation exposure.CCK-8 based assay and colony formation assay were utilized to detect whether GCH1/BH4 affect the proliferation of irradiated lung cells.Lactate dehydrogenase release was detected by lactate dehydrogenase cytotoxicity assay.Nitric oxide fluorescent probe was utilized to measure NO content in irradiated lung cells after overexpression of GCH1.DCF-DA assay was used to detect whether GCH1/BH4 affect ROS levels in irradiated lung cells.After GCH1 was knocked down.distribution and the activity of Smad2/3 and the expression of fibrotic Marker proteins were detected by immunofluorescence,luciferase and Western Blot.In vivo experiments:a model of radiation-induced lung injury by unilateral pulmonary irradiation in C57 mice was established.Ad-GCH1 and BH4 aqueous solutions were injected immediately after ionizing radiation.ROS.MDA and NO levels,and protein expression of TGF-β/Smads pathway and EMT pathway were measured one week later.After three months,the lung injury of C57 mice was detected by H&E staining.Masson staining.A model of radiation-induced lung injury by unilateral pulmonary irradiation in SD rats was established.Ad-GCH1 and BH4 aqueous solutions were injected immediately after ionizing radiation.The weight of the rats was monitored,and the lung injury of SD rats after ionizing radiation was observed three months later.Results In vitro experiments:UV-visible spectroscopy and Western blotting results showed that BH4 changed after ionizing radiation,and its key synthetase GCH1 content decreases.CCK-8 based assay and colony formation assay showed that GCH1/BH4 facilitated the proliferation of irradiated lung cells.LDH assay showed that GCH1/BH4 decreased LDH release of irradiated lung cells.Overexpression of GCH1 could restore the intracellular NO content.GCH1/BH4 reduced the level of ROS induced by ionizing radiation in lung cells.Knockdown of GCH1 with siRNA in HELF could increase promote Smad2 nucleation,Smad2/3 activity and increase the expression of fibrotic Marker proteins.In vivo experiments:a model of radiation-induced lung injury by unilateral pulmonary irradiation in C5 7 mice was established,and lung tissue was collected one week later.The results showed that overexpression of GCH1 and exogenous administration of BH4 through vein tail injection could reduced the levels of radiation-induced ROS and MDA and restore the NO content in the exposed lung tissue,and overexpression of GCH1 and administration of BH4 could inhibit the TGF-β/Smads pathway and EMT pathway.Lung tissues were resected three months after ionizing radiation,and the results showed that the GCH1/BH4 alleviated radiation-induced lung injury.A model of radiation-induced lung injury by unilateral pulmonary irradiation in SD rats was established,and lung tissue was collected three months later.The results showed that the rats with overexpression of GCH1 and tail vein injection of BH4 had higher body weight than the control group,and the lung injury was alleviated to some extent.Conclusion This study illustrates the key role of GCH1/BH4 axis in radiation-induced lung injury,which may provide a novel strategy for the clinical treatment of this disease.