Radioprotective Effect And Mechanism Of Myrtol Standardized In Radiation-Induced Lung Injury

BackgroundRadiation-induced lung injury(RILI)is a major common complications exposed in radiotherapy and nuclear accidents.RILI has a great impact on long term quality of life and could result in fatal respiratory insufficiency.With the improvement of living standards,aging population increasing,environmental deterioration,food safety and the disease spectrum change,most of them actually became public health problem,the incidence of cancer is increasing year by year.World annual cancer reports from International Agency for Research says lung cancer is becoming the leading cause of mobility and mortality of malignant tumors which seriously threat our health and lives.In China,the incident of cancer will be over 5million people and over 3.5million people will die from that to 2030.Hence,more and more patients undergo radiotherapy.Even the radiotherapy techniques have improved a lot in recent years.But RILI prevention and treatment are still not resolved.RILI has traditionally divided into early(acute,1-6 months post-RT)pneumonitis and late(chronic,6months)lung fibrosis depending on the time after radiation exposure.The mechanism of radiation-induced lung injury has not been elucidated clearly.Many studies show that radiation causes oxidative damage,a series of abnormal cytokines and MMPs over expression induced by radiation may play an important role.The current clinical treatment for radiation-induced lung injury primarily involve chemical anti-inflammatory agents,immunosuppressive cytokines and antioxidant agents,such as corticosteroids or IFN-γ.These drugs could attenuate acute inflammation for 2-3 months after irradiation,but not effectively mitigate fibrogenic processes in latter stage.Furthermore,these agents could cause significant side-effects,including infections,vomiting,and even death which limits their application in clinics.There are still not effective drug in clinical practice in this time.It is still big dose limiting factor which influences thoracic radiotherapy in clinical practiced.Thus,to develop an effective and low costing radiation lung injury protective agent becomes an increasing urgent.Herbal medicine is very popular in Asian and European countries.It has been widely used in different kinds of disease.Some of them had been proved radiation-protective effects.Among those,Myrtol standardized(CAS-No.8002-55-9)is a phytotherapeutic extract from Myrtus communis,such as Pinusspp(pine),Citrusaurantifolia(lime)and Eucalyptus globulus,Myrtol standardized mainly containing three monoter-penes:1,8-cineole,α-pinene and dlimonene as marker substances.It has been widely used in the treatment of acute and chronic respiratory disease.Furthermore,it has been found that it has anti-inflammatory,antibacterial,antioxidant,anti-allergic,antimicrobial and nasal mucociliary clearing effects.In addition,the LD50 value of Myrtol is greater than 3500 mg/kg of the body weight,far more than treatment concentration(25 mg/kg/day)when administered orally via gastric intubation.Prolonged administration of the Myrtol was well tolerated and was observed no toxicity.In the present study,we demonstrated that oral administration of Myrtol could alleviate radiation-induced lung injury.We also examined the effects of Myrtol treatment on AKT signaling pathway and its downstream MMP-2 and MMP-9 levels in irradiated mice.Contents:Part one:Established Radiation-induced lung injury mice modeMice were use 60Coγ-ray source whole thorax irradiate induced lung injury as mice mode.Mice lung tissue pathological(H&E and Masson stain)and immune-fluorescence observation(EMT marker α-SMA and vimentine)will carry out in 1,2,4,8,16 weeks post-radiation.Part two: Preventive and treatment effects of Myrtol on radiation induced lung injury in mice modelThese results will compare with prednisone+ir group.1、 Myrtol effects on mice bodyweight and lung coefficient.2、 Myrtol effects on mice lung pathological changes.3、 Myrtol effects on mice early pneumonitis.4、 Myrtol effects on mice radiation lung fibrosis in latter stage.5、 Myrtol effects on mice lung HYP content expression.Part three: The mechanisms of Myrtol effects on radiation induced lung injury1、 Serum levels of TGF-β1,TNF-α,IL-1β,IL-6,PGE2 and these cytokines related m RNA expression in mice post-irradiation.2、 Myrtol treatment on AKT signaling pathway and its downstream MMP-2 and MMP-9 levels in irradiated mice.3、 Mice lung tissue MDA content and SOD activity.Method:The present study use mice mode for investigate the Myrtol effect on radiation-induced lung injury and its related mechanism.Part one:Established Radiation-induced lung injury mice modeUse SMMU radiation center 60 Co source γ-ray 16 Gy single does to irradiate C57BL/6 mice whole thorax.We randomly chosen 6 mice in each group were sacrificed at 1,8,16 weeks post-radiation,Lungs were use HE,Masson staining to observed pathological changes and collagen fiber deposition marker(α-SMA and vimentine)of lung tissue detect by immunofluorescence.Part two: Myrtol effect on prevention and treatment of radiation-induced lung injury1、Fibrosis prone mice 8 week-old female C57BL/6 mice with an approximate weight of 18-20 g were obtained from were used and randomly divided into four groups as follows: group 1,non-irradiated control(n = 30);group 2,irradiation(n = 30);group3,irradiation + Myrtol(n = 30,25 mg/kg/day)and group 4 irradiation + prednisone(n = 30,5 mg/kg/day).Mice were kept under standard laboratory conditions(22 ± 2°C,55 ± 10% humidity and 12-12 h/light-dark cycle),during which sterilized food and water were supplied ad libitum.Mice were allowed to acclimate from animal center for 1 week prior to treatment.Myrtol standardized was given orally a week before irradiation at a dose of 25 mg/kg/day and maintained(25 mg/kg/day)until 4 weeks after irradiation.Prednisone was administered orally 5 mg/kg daily post irradiation for 4 weeks.2、The body weight was measured at 1,2,4,8 and 16 weeks post-irradiation.The ratio of the lung wet weight(mg)to body weight(g)(lung weight/bw)was used as the lung coefficient3、Lung sections stained with H&E and Masson trichrome were carried out with alveolitis score and ashcroft score,respectively.4、Use immunofluorescence analysis was performed to identify the expression of α-SMA and vimentin,mesenchymal cell marker in the lung tissue.5、The concentration of hydroxyproline was measured by hydroxyproline(Hyp)assay kit according to the manufacturer’s protocol.Part three: The mechanisms of Myrtol effects on radiation induced lung injury1、After measuring body weight,cardiac puncture was performed to obtain about 500 μL blood.The blood was allowed to clot at room temperature for 1.5-2 h and then centrifuged at 3,500 rpm,4°C for 15 minutes.The serum was collected and stored at-80°C for latter enzyme-linked immunosorbent assay(ELISA).Serum levels of TGF-β1,TNF-α,IL-1β,IL-6 and PGE2 were determined by using the commercially available ELISA kit according to the manufacturer’s instructions and use RT-PCR detected IL-1β\IL-6\TGF-β1\TNF-α\PGEs m RNA expression.2、Mice Lung tissue AKT、p-AKT、MMP-2\-9 Protein expression were detect by western blot.3、 Malondialdehyde(MDA)content and SOD activity in lung tissue were measured using commercialized chemical assay kits according to the manufacturer’s protocolResults:Part one: Successfully establishing radiation-induced lung injury mice modelThe pathological changes of lung tissue stained with H&E,Masson,immune-fluorescence EMT marker α-SMA and vimentine were consistent with previously reported.Part two: Myrtol effect on prevention and treatment of radiation-induced lung injuryTreated with Myrtol standardized,but not prednisone,significantly reduced lung coefficient and collagen deposition in lung tissues compare with IR group(P<0.05 or P<0.01),while attenuated histological damages induced by irradiation.Histological alveolitis score and ashcroft score had significantly difference with IR group(P<0.05 or P<0.01).Immunofluorescence analysis also revealed elevation of vimentin and α-SMA in the alveoli after a single dose of 16 Gy.We also found that Myrtol dramatically decreased the Hyp content and alleviated the lung tissue fibrosis formation in 4,8,16 weeks compare with IR group(P<0.05 or P<0.01).Even though the treatment of prednisone could reduce early radiation-induced pneumonitis.However,it did not show inhibition of the latter fibrosis formation.Part three: The mechanisms of Myrtol effects on radiation induced lung injuryMyrtol standardized also reduced the production of MDA,while increased the level of SOD.It was also observed that Myrtol standardized could obviously inhibited TGF-β1 and a series of pro-inflammatory cytokines including TNF-α,IL-1β,IL-6,PGE2 compare with IR mice(P<0.05 or P<0.01).These cytokines m RNA expression also significantly inhibits by Myrtol compare with IR group mice(P<0.05 or P<0.01).On the other side it also downgrade of AKT activation and it may block MMP-2/-9 expression in lung tissue,reduced lung structure damage.Discussion:RILI is a major common complications exposed in radiotherapy.We have detected mice bodyweight,lung coefficient,histological,immunofluorescence analysis and HYP content.We found that Myrtol can be an effective agent for attenuating the lung injury induced by irradiation,including the early pneumonitis and late fibrosis formation.Radiation induced a series of pro-inflammatory cytokines,Matrix metalloproteinases-2/-9 high expression and oxidant injury may mainly contributes to pneumonitis and fibrosis formation.Myrtol could inhibited TGF-β1 and a series of pro-inflammatory cytokines including TNF-α,IL-1β,IL-6,PGE2.It can also inhibit the related m RNA expression.AKT signaling pathway plays a critical role in radiation induced lung injury,Myrtol treatment could inhibited AKT activation and down-regulated the matrix metalloproteinase-2 and matrix metalloproteinase-9 expression.Besides,Myrtol also reduced the production of MDA,while increased the level of SOD.In prednisone group,even though the early pneumonitis was ameliorated,the collagen disposition remained unchanged in latter times.Conversely,long term administration of prednisone showed some severe side effects including weight loss,osteoporosis,infection complications.Conclusion:In conclusion,the present results have shown that Myrtol treatment provides persistent antioxidant,anti-inflammatory,and anti-fibrosis effects that protect against radiation-induced injury.These findings suggest that Myrtol might suppress EMT process through inhibiting of AKT activation.Myrtol might has great potential to be used in treating radiotherapy complications in terms of protecting from radiation-induced lung injury.The present study shows that Myrtol standardized can be an effective agent for attenuating the lung injury induced by irradiation.