| The application of ionizing radiation（IR）in medical diagnosis and treatment is increasing annually,and there are an increasing number of opportunities for human beings to be exposed to IR.IR exposure leads to different degrees of radiation injury and even radiation sickness.The radiosensitivity of different organs varies;for example,the intestine is a highly radiosensitive tissue,the lungs are a moderately sensitive tissue,and the brain is a low-sensitivity tissue.Therefore,aiming to study these three key tissues with different radiosensitivities,microglia,intestinal epithelial cells,alveolar epithelial cells and macrophages were selected as target cells.Alveolar epithelial cells and small intestinal epithelial cells are the main target cells injured by IR in the lungs and intestine,respectively.Microglia are important immune cells in the brain,and macrophages are systemic immune cells.The aim of this study was to investigate the protective effects of RH005 on radiation-induced injury to alveolar epithelial cells and small intestinal epithelial cells and the regulatory effects of RH005 on the biological behavior of microglia and macrophages after irradiation.RH005,a multifunctional polypeptide,plays important roles in many physiological and pathological activities,such as promoting angiogenesis and proliferation and inhibiting apoptosis and inflammation.RH005 can also repair damaged myocardium;promote hair follicle regeneration,skin wound healing and corneal repair;inhibit fibrosis;and so on.Previous studies have found that RH005 has a good therapeutic effect on acute radiation injury in mice,but there are few studies on its role at the cellular level after irradiation.This study was mainly carried out to address two aspects.（1）The protective effects of RH005 on radiation-induced injury to alveolar epithelial cells and intestinal epithelial cells were examined:pulmonary epithelial cells（RLE-6TN）and intestinal epithelial cells（IEC-6）were irradiated with 8 Gy 60Co gamma rays,and RH005（10 ng/ml,100 ng/ml or1000 ng/ml）was added immediately after irradiation.Cell proliferation was detected by the CCK-8 method at 6 h,12 h,24 h and 48 h after irradiation.The expression ofγ-H2AX in cells was detected by immunofluorescence staining at 1 h after irradiation to evaluate the effect of RH005,while apoptosis was detected by Annexin V/PI double staining at 6 h after irradiation.（2）The regulatory effects of RH005 on the biological behavior of microglia and macrophages after irradiation were investigated:microglia（BV-2）and macrophages（RAW264.7）were irradiated with 8 Gy 60Co gamma rays,and RH005（10 ng/ml,100 ng/ml or 1000 ng/ml）was added immediately after irradiation.Cell proliferation was detected by the CCK-8method at 6 h,12 h,24 h and 48 h after irradiation,and apoptosis was detected by Annexin V/PI double staining at 6 h after irradiation.In addition,microglial activation was detected by immunofluorescence staining and immunoblotting at 24 h after irradiation.The expression of inflammatory cytokines（TNF-α,IL-1β,IL-4,IL-6,IL-13 and IFN-γ）in cells was detected by a radioimmunoassay to evaluate the effect of RH005.The results of the above experiments were as follows:（1）RH005 had protective effects on radiation-induced injury to alveolar epithelial cells and intestinal epithelial cells.RH005 promoted the proliferation of epithelial cells after irradiation.The proliferation of RLE-6TN cells in each RH005dose group was significantly higher than that in the R group at 48 h after irradiation;the proliferative activity of IEC-6 cells in the M group was significantly higher than that of those in the R group at 24 h after irradiation,and the proliferative activity in the M and H groups was significantly higher than that in the R group at 48 h after irradiation.RH005 alleviated DNA damage in epithelial cells after irradiation.The DNA damage in RLE-6TN cells and IEC-6 cells in each RH005 dose group was significantly less than that in the corresponding cells in the R group at1 h after irradiation.RH005 inhibited epithelial cell apoptosis after irradiation.RLE-6TN cells were irradiated for 6 h,and the number of late apoptotic cells in the M group and the total number of apoptotic cells in each RH005 dose group were significantly lower than those in the R group,while the number of early apoptotic cells in each RH005 dose group and the number of late apoptotic cells in the M group were significantly lower than those in the R group at 6 h after irradiation for IEC-6 cells.（2）RH005 could regulate the biological behavior of microglia and macrophages after irradiation.RH005 inhibited the activation of microglia induced by irradiation.The expression of Iba-1 in BV-2 cells was increased at 24 h after irradiation.After RH005 treatment,at all doses,the expression of Iba-1 in BV-2 cells was decreased.RH005 inhibited the expression of inflammatory cytokines in microglia and macrophages induced by irradiation.The expression of TNF-α,IL-4 and IFN-γwas increased at 24 h after irradiation,while the expression of IL-4 in the L group and the expression of TNF-α,IL-1βand IL-4 in the M group were decreased after RH005 treatment.The expression of TNF-α,IL-1β,IL-4,IL-6,and IL-13in RAW264.7 cells was increased at 24 h after irradiation.After RH005treatment,the expression of TNF-α,IL-1β,IL-4,IL-6,IL-13 and IFN-γin the RH005 group was decreased,but there were no significant differences.Furthermore,RH005 had no significant effects on the proliferation or apoptosis of microglia and macrophages after irradiation.According to the above results,the conclusions were as follows:（1）RH005 had protective effects on radiation-induced injury to alveolar epithelial cells and intestinal epithelial cells,mainly by reducing cell DNA damage,inhibiting apoptosis and promoting cell proliferation after irradiation.（2）RH005 could regulate the biological behavior of microglia and macrophages after irradiation by inhibiting the activation of microglia and reducing the expression of inflammatory cytokines in microglia and macrophages. |
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