The Protective Effect Of Cordyceps Sinensis Extract On Tissues Of ⁶⁰Coγ Ionization Irradiated Mice And Its Mechanisms

Objectives:To study the protective effect of cordyceps sinensis extract（CSE）in spleen and testicular tissues of mice after ⁶⁰Coγ ionization irradiation and its mechanism,and then provide a strong theoretical support for the potential and new irradiation therapy drug.ChapterⅠ:The protective effect of CSE on the damage of spleen tissues in ionization irradiation-induced mice.Methods:C57BL/6 male mice were divided into 5 groups at random,respectively,the non-irradiated blank group（Control group）,the ionization irradiation group（Model group）,CSE low-dose treatment（312.5mg/kg）;middle-dose treatment（625mg/kg）,and high-dose treatment（1,250mg/kg）.A single dose of 8Gy ⁶⁰Coγ ionization irradiation-induced total-body mouse model was established except the Control group.（1）Body weight and spleen index in five groups were analyzed respectively;（2）The level of white blood cell,neutrophil,red blood cell（RBC）,lymphocyte,platelet and monocyte in peripheral blood of Control,Model and CSE-M groups were detected;（3）HE,MPO and TUNEL staining was used to analyze the morphological structure,inflammatory cells infiltration and the number of apoptosis cells in spleen tissues of mice in Control,Model and CSE-M groups respectively;（4）Western blot was used to analyze Bax,Bcl-2,Caspase 3 and Cleaved-Caspase 3 proteins expressions in spleen tissues of mice in Control,Model and CSE-M groups;（5）Flow cytometry was used to analyze the level of immunoglobulins and inflammatory cytokines in serum of mice in Control,Model and CSE-M groups.Results:（1）Compared with the Control group,the levels of body weight,spleen index of mice in model group were significantly decreased which represented the success of model;Compared with the Model group,the above targets were all dramatically increased in mice of CSE-L,CSE-M,CSE-H groups in a dose-dependent manner;（2）Compared with Control group,the level of WBC,lymphocyte,monocyte and platelet in peripheral blood of mice in Model group were markedly reduced.Compared with Model group,the level of WBC,lymphocyte,platelet in CSE-M group were remarkedly enhanced;There were no dramatic difference of RBC and neutrophil among Control,Model and CSE-M groups;（3）The results of HE,MPO,and TUNEL staining showed that the texture became soft,and the level of inflammatory cells infiltration and apoptosis cells in spleen tissues of mice were increased in contrast with that of Control group respectively.The texture became compact,and the level of inflammatory cells infiltration and apoptosis cells in spleen tissues of mice in CSE-M group were decreased in contrast with that of Model group;（4）The result of western blot shown that the expression of Bcl-2 protein in spleen of Model group was markedly reduced,and the Bax and Cleaved-Caspase 3 proteins expressions in Model group were significantly enhanced.At the meanwhile,the above proteins were dramatically reversed comparing with that of Model group;（5）The result of Flow cytometry shown that the levels of Ig A,Ig G1,Ig G2a,Ig G2b,Ig G3 and Ig M were remarkedly decreased,and IL-1α,IL-1β,IL-6,TNF-αlevels were significantly increased compared with that of Control group.Compared with Model group,the level of Ig A,Ig G2a,Ig G2b,Ig M were all dramatically enhanced,and IL-1β,IL-6,TNF-αlevels were significantly reduced.ChapterⅡThe protective effect of CSE on the ionization irradiation-induced damage of monocytes of spleen in mice.Methods:Firstly,the following two contents were separately detedected through separating splen monocytes in groups of part I exprement.（1）CCK-8 was used to analyze the spleen monocytes activities in five groups;（2）Flow cytometry was used to detect the CD3⁺,CD4⁺,CD8⁺and CD19⁺positive cells levels of spleen monocytes in Control,Model and CSE-M groups.Next,the spleen monocytes in ordinary C57BL/6 mice were separated and divided five groups cells:Control group（normal cells）,Model group(4 Gy ⁶⁰Coγ ionization irradiation cells),CSE-L treaement group（31.25mg/m L）,CSE-M treatment group（62.5mg/m L）and CSE-H treatment group（125mg/m L）,and finally were detected following three contents.（1）CCK-8 was used to detect the spleen monocytes activities,separated from normal C57BL/6 mice,in Control,CSE-L,CSE-M and CSE-H groups.（2）CCK-8 was used to detect the spleen monocytes activities,separated from normal C57BL/6 mice,in above five groups;（3）Flow cytometry was used to analyze the levels of CD3⁺,CD4⁺,CD8⁺and CD19⁺positive cells in Control,Model,CSE-M groups,which were separated from normal C57BL/6 mice.Results:Firstly,following two concludings were obtained through separating spleen monocytes in groups of part I exprement.（1）Compared with Control group,the activity of spleen monocytes of ionization irradiated mice in Model group was remarkedly decreased.Compared with the Model group,the activity of spleen monocytes in CSE-L,CSE-M and CSE-H groups were significantly increased.（2）Compared with Control group,The level of CD3⁺,CD4⁺,CD8⁺and CD19⁺cells of irradiated mice were remarkedly reduced in spleen of mice in Model group;Compared with the Model group,the above positive cells were all markedly increased in spleen tissues of mice in CSE-M group.Next,the following three conclusions were obtained through separating spleen monocytes in ordinary C57BL/6 mice.（1）Compared with Control group,the cell activity of CSE（31.25mg/m L,62.5mg/m L,125mg/m L）administration was remarkedly increased;（2）Compared with the Control group,the activity of 4Gy ⁶⁰Coγ ionization irradiation-induced spleen monocytes were dramatically reduced;Compared with Model group,CSE（62.5mg/m L,125mg/m L）administration dramatically enhanced the activities of ionization irradiation-induced spleen monocytes.（3）Compared with Control group,the rate of CD3⁺and CD19⁺in Model group were remarkedly decreased;Compared with Model group,CSE（62.5mg/kg）administration dramatically enhanced the level of CD3⁺and CD19⁺cells.Chapter III The protective effect of CSE on the damage of testicular tissues in ionization irradiation-induced mice.Methods:（1）The malondialdehyde（MDA）and superoxide dismutase（SOD）activity levels in testicular tissues of mice in five groups in the chapter I were detected;（2）HE,MPO and TUNEL staining were used to analyze the morphological structure,inflammatory cells infiltration and the number of apoptosis cells in testicular tissues of mice respectively in Control,Model and CSE-H groups;（3）The expressions of apoptosis-related genes and proteins were analyzed respectively.Results:（1）The results of HE,MPO,and TUNEL staining shown respectively that the texture became soft,morphological change,cavity empty,and the level of inflammatory cells infiltration and apoptosis cells in testicular tissues of mice were increased in contrast with that of Control group.The texture became compact,and the level of inflammatory cells infiltration and apoptosis cells in testicular tissues of mice were decreased in contrast with that of Model group.（2）Compared with Control group,the level of SOD activity,anti-apoptosis relative m RNA expressions of GSH-PX,CAT,GPX and Bcl-2,anti-apoptosis relative protein（Bcl-2）expression in testicular tissues of mice in Model group were significant down-regulated,and the level of pro-apoptosis relative m RNA expression of Bax and Caspase 3,pro-apoptosis relative protein expressions（Bax,Cleaved-Caspase 3）in testicular tissues of mice in Model group were remarkedly up-regulated.Compared with Model group,the SOD activity,anti-apoptosis proteins and genes expressions（in part III）in testicular tissues of mice in CSE-H group were dramatically enhanced,and MDA level,pro-apoptosis genes and proteins expressions（in part III）in testicular tissues of mice in CSE-H group were significantly reduced.Conclusion:1.CSE effectively increased the level of survival rate,body weight,spleen index and WBC,lymphocyte,platelet in peripheral blood in ⁶⁰Coγ ionization irradiation-induced mice;2.CSE effectively regulated the activity and CD3⁺,CD4⁺,CD8⁺and CD19⁺cells in spleen monocytes,immunoglobulins and inflammatory cytokines in serum of mice after ⁶⁰Coγ ionization irradiation which enhanced the adaptive immunity of mice.3.CSE dramatically regulated the MDA level and SOD activity in testicular tissues of mice after⁶⁰Coγ ionization irradiation which increased antioxidant ability.4.CSE effectively protected the damage of morphological structures,and notably reduced the Inflammatory infiltrating cells and TUNEL-positive cells in spleen and testicular tissues of mice5.CSE effectively reduced the cell apoptosis in spleen and testicular tissues of mice after ⁶⁰Coγ ionization irradiation which regulated the Bcl-2 and Bax proteins expressions,and inhabited the activation of the downstream Caspase 3 pathway.