Effect of tetrahydrobiopterin (BH4) metabolic pathway on the progression of radiation-induced lung injury and its mechanism

• 1、School of Radiation Medicine and Protection,School of Medicine,Soochow University,Suzhou 215123
• 2、Anshan Cancer Hospital,Anshan,114033

Abstract：Objective: Radiation-induced lung injury is a common complication of radiation therapy for thoracic malignancies, including radiation pneumonitis and radiation-induced pulmonary fibrosis. This study was to investigate the role and mechanism of the tetrahydrobiopterin (BH4) metabolic pathway in lung injury caused by ionizing radiation. Methods: Ultraviolet-visible spectroscopy was utilized to detect whether ionizing radiation disrupt the structure of BH4. Western blot analysis was performed to detect the change of BH4 rate-limiting enzyme GCH1 in lung cells before and after irradiation. The effects of GCH1 and BH4 on the proliferation and LDH release of irradiated lung cells were detected by CCK-8 assay, clone clonogenic assay and LDH assay. Using ROS generation assay and nitric oxide fluorescent probe to detect intracellular ROS and NO levels in irradiated lung cells overexpressing GCH1. Establishing a mouse model of radiation-induced lung injury by unilateral pulmonary irradiation, after 20 Gy of X-ray exposure, mice received tail vein injection of control adenoviurs (Ad-NC), GCH1-overexpression adenovirus (Ad-GCH1), PBS or BH4. Seven days after irradiation, the mice were sacrificed and lung tissues were collected to detect ROS, MDA and NO levels. And Western blotting was used to detect the expression of TGF-β1/Smad and EMT pathway-related proteins. Three months later, lung tissue was taken to observe the lung injury after irradiation. Results: Ultraviolet-visible spectroscopy and Western blotting showed that BH4 changed after irradiation, and its key enzyme GCH1 content decreased. CCK-8 assay, clone clonogenic assay and LDH assay showed that GCH1 promoted the proliferation and decreased the LDH release in irradiated lung cells. ROS generation assay and NO determination assay showed that intracellular ROS levels decreased and NO levels increased after GCH1 overexpression in exposed lung cells. A model of unilateral lung injury was established in C57 mice, and lung tissue was taken after 7 days. The results showed that the levels of ROS and MDA in lung tissues of the group overexpressing GCH1 and injecting BH4 were decreased, while the levels of NO were increased. GCH1 and BH4 inhibited TGF-β1/Smad and EMT pathway. Andpulmonary fibrosis was alleviated in the groups with GCH1 overexpression and BH4 injection by tail vein. Conclusion: Overexpression of GCH1 can restore BH4 and NO content in irradiated lung cells, reverse the inhibition of NOS uncoupling caused by ionizing radiation, clear ROS caused by ionizing radiation, promote cell proliferation and reduce cell damage. In animal experiments, injection of BH4 or overexpression of GCH1 alleviates radiation-induced lung injury. Taken together, the results of this study clarify the critical role of GCH1/BH4 in radiation-induced lung injury and may provide a new strategy for the treatment of this disease.

Keywords： ionizing radiation radiation-induced lung injury GCH1 tetrahydrobiopterin (BH4)