The Regulatory Mechanism of LPA-LPA1 Signalling Pathway on Radiation-induced Pulmonary Cellular Senescence

Abstract

Ionizing Radiation (IR) can inflict irreparable DNA damage on exposed cells that may undergo cellular senescence. Cellular senescence is characterized by the permanent loss of the proliferative potential, expression of senescence-associated β-galactosidase (SA-β-gal), and active metabolism with senescence-associated secretory phenotype (SASP) factors secreted. Radiotherapy, via IR, ranks among the most powerful treatments for lung cancers. However, radiation-induced acute and late toxicities to normal lung tissues limits the radiation doses that can be applied to tumors. By virtue of mouse model experiments, our group has established and published that IR promotes the biosynthesis of high levels of lysophosphatidic acid (LPA) in irradiated lungs, which participates in the radiation-induced lung injuries (RILI) via LPA-LPA receptor1 (LPA1) signalling. This study is designed to discover whether selectively block the LPA-LPA1 signalling pathway with AM966 can effectively ameliorate the cellular senescence caused by IR. SPF C57BL/6J mice were exposed to X-rays at the dose of 16Gy/f on the whole thorax. Irradiated mouse lungs were analysed at different points in time by Western blotting, RT-PCR, immunohistochemical methods, flow cytometry, etc. The irradiated mice were treated with AM966 (15mg/kg, qd) by intraperitoneal injection from the 1st day to the 7th day after irradiation to intervene in the early stage of cellular senescence. To explore the mechanisms underlie the IR-induced cellular senescence, HBE cells were exposed to the same radiation dose of 16Gy/f in combination with LPA(10μM) and/or AM966(20μM), which was studied 48 hours after irradiation with methods as used in vivo . The irradiated lung tissue was substantially positive-stained of SA-β-gal from the 14th day after irradiation, which culminated in accompanying with extensive pulmonary fibrosis on the 60th day. The apoptotic and antiapoptotic activity reached their peak on the 7th day simultaneously. The levels of SASP factors, such as IL-1α, IL-6, TGF-β, TNF-α, MMP9 boosted from the 7th day after irradiation, which was reduced significantly by AM966 prescription. For HBE cells, the mRNA expression of LPA1 standed out as the highest expressed among the LPA receptor1-6 48 hours after irradiation, while the mRNA of Autotaxin, a secreted enzyme important for LPA production, was barely detected. More than 90 percent of the HBE cells were arrested in the G2/M phase 48 hours after irradiation and exhibited high activity of SA-β-gal, which was ameliorated significantly by AM966. Additionally, AM66 also reduced the percentage of γH2AX positive HBE cells. AM966 treatment ameliorates the cellular senescence at the early stage in the irradiated mouse lungs. The inhibition of formation of DSBs and SASP factors may partly attribute to its attenuating effects.