| ObjecitveRadiation therapy(RT)is an important therapeutic methods for thoracic tumors.Radiation-induced lung injury(RILI)is a common adverse reaction of RT.According to previous study,the incidence of RILI is about 20%.However,there is no radioprotective compound with satisfied efficacy.Magnesium isoglycyrrhizinate(MgIG)is the fourth generation of glycyrrhizic acid preparation,which was found to exert strong anti-inflammation,anti-oxidization,cell membrane stabilization,immunity regulation,and other multiple effects.At present MgIG is mainly used for chronic viral hepatitis and acute drug-induced liver injury.Recently it has been proved that MgIG can improve paraquat-/bleomycin-induced lung injury and pulmonary fibrosis.However,it has never been proposed in previous studies about the effects of MgIG on radiation-induced lung injury.The purpose of the current study was to investigate the therapeutic effect and the underlying mechanism of MgIG on radiation-induced lung injury.Methods1.The therapeutic effect of MgIG on radiation induced lung injury1.1 The establishment of animal model: the radiation-induced lung injury mouse model was established by a single dose of 15 Gy60 Co ?-rays exposure to the whole lung.The mice were sacrificed at 4,8 and 12 weeks post-irradiation.1.2 Grouping and treatment: C57BL/7 mice were evenly randomized into five groups:control group,radiation group(IR group),MgIG group,radiation combined with MgIG group(IR?+?MgIG group),and radiation combined with dexamethasone group(IR?+?DXM group),with 30 mice in each group.MgIG(100 mg/kg)or DXM(0.5 mg/kg)or normal saline(20m L/kg)was intraperitoneally injected into mice 2h prior to irradiation and every day post-irradiation.1.3 Measurement:(1)To observe the general condition of mice(activity,mental state,body weight,hair of chest);(2)To evaluate the histological changes through H&E staining and Masson trichrome staining;(3)The expressions of type I collagen,type III collagen,?-SMA,fibronectin,TGF-?1,p-smad2,p-smad3,Nox4,p-p38 and p-AKT in lung tissue were detected by immunohistochemistry;(4)To detect the mRNA level of ?-SMA,fibronectin and TGF-?1 in lung tissue by real-time quantitative PCR;(5)The ELISA method was performed to detect TGF-?1 content in serum;(6)The contents of MDA,SOD activity and GSH-PX activity were measured by biochemical method.2.MgIG regulates p38MAPK-Akt-Nox4 pathway to inhibit radiation-induced human fetal lung fibrolasts-1(HFL-1)cell differentiation2.1 A model of cell differentiation: After being serum-starved in serum-free medium for24 h,HFL-1 cells were incubated for 24 h with MgIG(0.01mg/mL)in F12 K medium with10% FBS,exposed to a single dose of 8 Gy60 Co ?-rays,and then cultured for an additional72 h.2.2 Measurement:(1)Cell proliferation rates were measured by CCK-8 cell proliferation assay;(2)The expression levels of a-SMA,fibronectin,Nox4,p-p38 and p-AKT were examined by western blot and immunofluorescence assays;(3)To measured cellular ROS levels in irradiated cells pretreated with MgIG by flow cytometryResults1.The therapeutic effect of MgIG on radiation induced lung injury1.1 Irradiated mice showed dispirited,slow action,dull skin and epilation.At 2 weeks post-irradiation,the mental conditions of the mice were gradually improved and they became more active.About 8 weeks after irradiation,we can obviously observe the white hair in the chest of the irradiated mice.The aboved mentioned symptoms could be alleviated with the use of MgIG or DXM.1.2 At 4 weeks post-irradiation,hemorrhagic spots were observed in the lung of mouse,and severe lung congestion appeared at 8 and 12 weeks post-irradiation.Treatment with MgIG or DXM could obviously improve it.1.3 The body weight of mice decreased 1 week after irradiation.Compared with control group,the body weight of IR group mice significantly decreased(P<0.05).Compared to IR group,that of MgIG group increased significantly(P<0.05),while DXM treatment group did not show signicant change.After 2 weeks,the body weight of mice in each group graduallyincreased.8 and 12 weeks after irradiation,IR group mice was significantly lower in the weight than control group(P<0.05),and MgIG treatment group was significantly higher than IR group(P<0.05),and DXM treatment group was significantly lower than IR group(P<0.05).1.4 At 8 and 12 weeks post irradiation,inflammatory and fibrotic foci were mainly observed in the lung of mice.Irradiation could induce obvious alveolitis and pulmonary fibrosis.Treatment with MgIG or DXM significantly alleviated the inflammation and fibrosis of the lung of mice,compared to that of IR group(P<0.05).1.5 The protein levels of collagen I,collagen III,?-SMA,fibronectin,TGF-?1,p-smad2,p-smad3,Nox4,p-p38 and p-AKT in the lung of irradiated mice significantly increased,which significantly decreased by MgIG or DXM treatment.1.6 At 12 weeks post irradiation,the mRNA expression of ?-SMA in the lung of mice was significantly increased(P<0.05)compared with MgIG or DXM group.However,no significant difference was observed in fibronectin mRNA expression.1.7 Comparing to control group,the MDA content of the lung increased(P<0.05)and the activity of SOD and GSH-PX decreased(P<0.05)in IR group 4,8,12 weeks post-irradiation.After treatment with MgIG or DXM,the MDA content at each time point was significantly lower than that of IR group(P<0.05),while the activity of SOD and GSH-Px increased significantly(P<0.05).1.8 At 4,8,12 weeks after irradiation,the level of TGF-?1 in serum and TGF-?1 mRNA expression in the lung significantly increased(P<0.05),and treatment with MgIG or DXM significantly reduced them(P<0.05 vs IR group).1.9 Between MgIG treatment group and DXM treatment group,there was a significant difference in body weight,the mRNA expression(?-SMA)at 12 weeks post irradiation in the lung,and SOD activity 8 weeks after irradiation,but no significant difference was observed in the degree of alveolitis and fibrosis,and the level of TGF-?1 in serum.2.MgIG regulates p38MAPK-Akt-Nox4 pathway to inhibit radiation-induced HFL-1cell differentiation2.1 Irradiation did not significantly inhibited HFL-1 cells proliferation(P>0.05 vs Control group),which was also not affected by pretreatment with MgIG(0.0001 ~0.01mg/mL),and MgIG(0.1mg/mL)could significantly inhibit cell growth(P<0.05 vs IR group).2.2 After irradiation,the expression of ?-SMA significantly increased compared with the control HFL-1 cells.When cells were pre-treated with MgIG for 24 h before irradiation,the expression of ?-SMA was reduced in a dose-dependent manner.2.3 The expression levels of ?-SMA and fibronectin proteins were measured by Western blot and immunofluorescent assays and MgIG significantly inhibited ?-SMA and fibronectin protein expression(P<0.05).2.4 The mean fluorescence intensity(MFI)in HFL-1 cells significantly increased after irradiation(P<0.05).When HFL-1 cells were pretreated with MgIG for 24 h before ?-ray irradiation,the cellular ROS production was significantly reduced compared with the model group(P<0.05)2.5 At 72 h after irradiation,the protein expression level of Nox4 significantly increased in the IR group compared with control group(P<0.05),and the level of Nox4 protein decreased in MgIG group(P<0.05 vs IR group).The phosphorylation of p38 MAPK and Akt increased at 1 h after irradiation,and pretreatment with MgIG could decrease the level of phosphorylation of p38 MAPK and Akt(P<0.05).2.6 Pretreated with Nox4,p-p38 and p-AKT inhibitors,the expression of ?-SMA and fibronetin significantly decreased 72 h after irradiation(P<0.05 vs IR group).Compared with IR group,the expression of ?-SMA and fibronetin significantly decreased in the cells treated by MgIG(P<0.05).2.7 Pretreated with siNox4,the expression of Nox4 in HFL-1 cells decreased(P<0.05 vs IR group without siNox4)and MgIG could inhibit the protein expression of ?-SMA and fibronetin in the cells pretreated by siNox4.Conclusions1.A mice model of radiation-induced lung injury was established successfully2.MgIG exhibited a protective effect on radiation-induced lung injury,which may be involved in regulating oxidative stress response,reducing the expression of TGF-?1 and inhibiting smad2/3 pathway or p38/AKT/Nox4 pathway.3.The inhibitory effect of MgIG on HFL-1 cell differentiation involves the inhibition of p38MAPK-Akt-Nox4 pathway. |
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