Protective Effects And Mechanisms Of Astragaloside Ⅳ（AST-Ⅳ） On The Mice Lung Injury Induced By ¹²C⁶⁺ Irradiation

Lung is one of the most sensitive organs to radiation and it often is damaged by the radiation of X-ray,γ-ray,heavy ion that caused by the therapy process of tumors of lung,esophagus,breast and lymphatic system,bone marrow transplantation and nuclear accidents.Radiation-induced lung injury is a common and critical problem that limits the doses of delivered in radiotherapy.Therefore,it is extremely important to study the molecular mechanism of radiation-induced lung injury,and further develop effective drugs for the prevention and treatment of radiation-induced lung injury.The first part of this paper reported our recent findings on the differences among the mice survival,lung histopathological changes,DNA damage,apoptosis and expression of TGF-β and TNF-α and evaluated the heavy ion radiation-induced mice lung injury after 7 days of carbon ion beam radiation(0,2,4,6 Gy)on the whole lung of healthy kunming mice.Secondly,the effects of Astragaloside Ⅳ on heavy ion radiation-induced lung injury and fibrosis were evaluated by the detection of mice bone marrow micronucleus,DNA damage,apoptosis and cell cycle of mice lung cells,and the analysis of the histopathological changes of mice lung tissue after fed orally different doses of Astragaloside Ⅳ before radiation.Lastly,the potential molecular mechanisms of Astragaloside IV on the protection of heavy ion radiation-induced mice lung injury and fibrosis was analysed by the activity of antioxidant enzyme(determined by measuring the SOD、MDA、GSH and CAT),the expression of MMP-2 and MMP-9(assayed by enzyme-linked immunosorbent assay),TGF-Pand TNF-a(analyzed by immunohistochemistry),ATR gene in DNA damage checkpoint(tested by RT-PCR technology),key protein APE-1 of DNA damage repair,oxidative stress adjustment factor Nrf-2,key protein Bax and Bcl-2 of cell apoptosis(detected by western blotting).These results will provide experimental evidence for the clinical applications of Astragaloside Ⅳ.The main results are as follows:1.Carbon ion beams radiation induces dose dependent increase of double-strand DNA damage as well as apoptosis,and increases the expression of TGF-β and TNF-αin mice lung tissue after 7 days of carbon ion beam radiation.In the all treatment groups,6 Gy radiation induces the most serious damage to the lung tissue with visible alveolar structural destroyed,inflammatory cell infiltrates and hemorrhage,alveolar septal thickening and so on.2.The antioxidant capacity of the lung tissue significantly enhanced in group of the mice which are feed with different doses of Astragaloside Ⅳ for 7 days before radiation with 4Gy 12C6+.In addition,Astragaloside Ⅳ intragastric pretreatment also significantly reduced the double-strand DNA damage,apoptosis and bone marrow micronucleus rate,while promoted proliferation of lung tissue cells,accelerated repair of heavy ion beam radiation-induced mouse lung tissue damage and delayed radiation-induced cell cycle arrest.The histopathology results also showed that Astragaloside Ⅳ intragastric pretreatment could significantly alleviate heavy ion beam radiation-induced lung injury,such as interstitial lung congestion,bleeding and edema,alveolar wall thickening.3.Pretreatment with Astragaloside Ⅳ by gastric perfusion could inhibited the expression of key proteases MMP-2 and MMP-9,reduced the concentration of hydroxyproline and enhanced the antioxidant capacity of mice lung tissue in a dose-dependent manner.The results detected by the immunohistochemistry showed that the expressions of TGF-β and TNF-α were down-regulated and the tan and brown particles were decreased.4.The results of molecular biology detection indicated that the astragaloside Ⅳintragastric pretreatment could down-regulate the expression of member protein(ATR)in DNA damage checkpoint,DNA damage repair key protein APE-1 and the expression of the key apoptosis promoting proteins Bax,and up-regulate the expression of the oxidative stress adjustment factor Nrf-2 and the key protein Bcl-2 with the function of inhibit the apoptosis.These results indicate that:(1).There is a dose effect relationship between carbon ion beams radiation and damage in tissue,cell and molecular of mice lung.This may be related to the accumulation of active oxygen.(2).Pretreatment with Astragaloside IV can effectively reduce the radiation-induced cell cycle arrest,DNA damage and apoptosis in lung cells and tissue.(3).Astragaloside Ⅳ can protect lung injury by inhibiting hydroxyproline synthesis,MMP and collagen expression,and reducing inflammation and fibrosis of the lung tissue.(4).The protection mechanism of Astragaloside IV for heavy ion beams radiation-induced lung injury may related with scanvenging free radicals,enhancing antioxidant enzyme activities and up-regulating Nrf-2 to strengthen the anti-oxidative and self-repair capability of living body,inhibiting MMP and APE-1 expression,promoting the repair of DNA damage,inhibiting TGF-β and TNF-α expression,and inhibiting apoptosis by regulating the expression of Bax and Bcl-2.