Study On Expression Of The Related Genes Of Mitochondrial Apoptotic Pathway In Radiation Induced Bystander Effect And The Intervention Of Gui Qi Yi Yuan Ointment

Objective: To discuss the expression of related genes of mitochondrial apoptosis pathway and the effect of Gui Qi Yi Yuan Ointment on genetic expression of cytochrome C,Caspase-9,Caspase-3,Bax and Bcl-2 in mitochondrial apoptosis pathway during X-ray and heavy ion radiation induced bystander effect.Methods: The study is divided into two parts.Part one is about the expression change of the related genes of the mitochondrial apoptotic pathway in the bystander effect in vivo induced by different doses of X-ray and the effect on the intervention of Gui Qi Yi Yuan Ointment.70 male Wistar rats were randomly divided into ten groups,the normal control group,and the radiation group of 2Gy 6h,the radiation group of 2Gy 24 h,the radiation group of 2Gy 48 h,the radiation group of 4Gy 6h,the radiation group of 4Gy 24 h,the radiation group of 4Gy 48 h,the radiation group of 8Gy 6h,the radiation group of 8Gy 24 h,the radiation group of 8Gy 48 h.In addition to the normal control group,the right lung of the rats of the rest groups was respectively radiated by 2,4,8Gy X-ray.And the rest of the body was covered with lead shielding.At 6h,24 h and 48 h respectively after radiation,7 rats of each radiation group were executed.The variations of mRNA expression of Cytochrome C,Caspase-9,Caspase-3,Bax and Bcl-2 in the right lung,left lung and left kidney tissue were detected with quantitative polymerase chain reaction(Q-PCR).Later 36 male Wistar rats were randomly divided into three groups,the normal control group,the radiation group and the Chinese medicine group.Before radiation,the Chinese medicine group was given Gui Qi Yi Yuan Ointment(3.28g·kg-1·d-1)with gavage for two weeks.The normal control group and the radiation group were given the equal quantity of normal saline with gavage for two weeks.After two weeks,the right lung of the rats of the radiation group and the Chinese medicine group was radiated by 8Gy X-ray only once.The normal control group was not radiated.At 24 h after radiation,all rats were put to death.The expressions of protein and mRNA of Cytochrome C,Caspase-9,Caspase-3,Bax and Bcl-2 in the right lung,left lung and left kidney tissue were detected with Q-PCR,immunohistochemically staining and Western Blot.The changes of the pathological morphology of the lung and kidney tissue were tested with H.E.staining.The count of leukocyte,erythrocyte,hemoglobin and thrombocyte was tested with fully automatic animal blood corpuscle analyzer.Part two is about the function of Gui Qi Yi Yuan Ointment on regulating and controlling genetic expression of the related genes of the mitochondrial apoptotic pathway in the heavy ion radiation induced bystander effect in vivo.42 male Wistar rats were randomly divided into seven groups,the normal control group,the radiation group 6h,the radiation group 12 h,the radiation group 24 h,and the Chinese medicine group 6h,the Chinese medicine group 12 h,the Chinese medicine group 24 h.Before radiation,every Chinese medicine group was given Gui Qi Yi Yuan Ointment(3.28g·kg-1·d-1)with gavage for two weeks.The normal control group and every radiation group were given the equal quantity normal saline with gavage.After two weeks,the right lung of the rats of every radiation group and every Chinese medicine group was radiated by 2Gy heavy ion only once.The normal control group was not radiated.At 6h,12 h,24h after radiation,all rats were put to death.The pathological morphology changes of the lung and kidney tissue were tested with H.E.staining.The count of leukocyte,erythrocyte,hemoglobin and thrombocyte was tested.The levels of IL-6,IL-1?,TGF-?1,IL-8 and TNF-? in serum were measured by ELISA.The expressions of protein and mRNA of Cytochrome C,Caspase-9,Caspase-3,Bax and Bcl-2 in the right lung,left lung and left kidney tissue were detected with Q-PCR,immunohistochemically staining and Western Blot.Results: Part one: the m RNA expression of Cytochrome C,Caspase-9 and Caspase-3 in the right lung,left lung and left kidney tissue of the 2Gy,4Gy,8Gy radiation group up-regulates obviously at 6h,24 h,48h after radiation.While the mRNA expression of Bcl-2 down-regulates obviously at 6h,24 h,48h after radiation in the right lung and left lung.In comparison with the normal control group,there is statistically significant difference(P<0.05).In the right lung,the expression of Bax mRNA is significantly increased in the other radiation groups except the 8Gy radiation group 48 h.In the left lung,the expression of Bax mRNA is significantly increased in each radiation group.In the left kidney,the expression of Bax mRNA is significantly increased in the other groups except the 2Gy radiation group 6h.Compared with the normal control group,the difference is statistically significant(P<0.05).The expression of Bcl-2 mRNA is decreased at all time points after radiation except the 8Gy radiation group 6h and the difference is statistically significant(P<0.05)compared with the normal control group.At 24 h after the right lung of the rats is radiated by 8Gy X-ray only once,the alveolar walls of the right lung are thickened.And there has a little exudate and inflammatory cell infiltrate in the alveolar cavity of the left lung.But the left kidney has no pathological variations.The count of leukocyte,erythrocyte,hemoglobin and thrombocyte reduces obviously.The expressions of protein and mRNA of Cytochrome C,Caspase-9,Caspase-3 and Bax in the right lung,left lung and left kidney tissue obviously up-regulate.While the expression of protein and mRNA of Bcl-2 down-regulates obviously at 24 h after radiation.In comparison with the normal control group,there is statistically significant difference(P<0.05).Compared with the radiation group,the lung pathological damage of the Chinese medicine group mitigates distinctly.The count of leukocyte,erythrocyte,hemoglobin and thrombocyte increases obviously.The expressions of protein and mRNA of Cytochrome C,Caspase-9,Caspase-3 and Bax in the right lung,left lung and left kidney tissue of the Chinese medicine group obviously down-regulate.The expression of protein and mRNA of Bcl-2 up-regulates visibly.There is statistically significant difference(P<0.05).Part two:After the right lung of the rats is radiated by 2Gy heavy ion,in comparison with the normal group,the count of leukocyte,erythrocyte,hemoglobin and thrombocyte of the radiation group decreases obviously at 12 h after radiation(P<0.01).The level of IL-6?IL-1??TGF-?1?IL-8 in serum rises overtly at 12 h after radiation(P<0.01).The level of TNF-? in serum rises overtly at 6h after radiation(P<0.01).HE staining shows the right lung has a little inflammatory cell infiltration and alveolar walls capillary hemorrhage at 6h after radiation.With the passage of time,the pathological damage worsens gradually.The alveolar walls are thickened.And there has exudate and inflammatory cell infiltrate in the alveolar cavity.Alveolar walls capillary is of hyperemia and hemorrhage.The left lung has no pathological variations at 6h,12 h after radiation.At 24 h after radiation,there has a little exudate and inflammatory cell infiltrate in the alveolar cavity.The left kidney has no obvious pathological change at 6h,12 h and 24 h after radiation.Compared with the normal control group,the amount of mRNA expression of Cytochrome C,Caspase-9,Caspase-3 and Bax in the right lung,left lung and left kidney of the radiation group obviously increases at 6h,12 h and 24 h after radiation.While the amount of protein expression of Cytochrome C,Caspase-9,Caspase-3 and Bax of the radiation group obviously increases at 24 h after radiation.There is statistically significant difference(P<0.01).The amount of protein and m RNA expression of Bcl-2 in the right lung of the radiation group obviously weakens at 24 h after radiation.But in the left lung and left kidney of the radiation group it obviously weakens at 6h,12 h and 24 h after radiation.Compared with the normal control group,there is statistically significant difference(P<0.01).Pathological damage of the lung tissue of every Chinese medicine group mitigates obviously in comparison with every radiation group.The count of leukocyte,erythrocyte,hemoglobin and thrombocyte of the Chinese medicine group 12 h exceeds the radiation group12 h.And the levels of IL-6,IL-1?,TGF-?1 and IL-8 in serum of the Chinese medicine group 12 h are below the radiation group 12 h obviously.The content of TNF-? of the Chinese medicine group 6h was significantly lower than that in radiation group 6h.There is statistically significant difference(P<0.01).The protein expression of Cytochrome C,Caspase-9,Caspase-3 and Bax in the right lung,left lung,and left kidney of the Chinese medicine group at 24 h after radiation were significantly lower than those in the radiation group at 24h(P<0.01).The mRNA expression levels of Cytochrome C,Caspase-9 and Bax in the right lung of the Chinese medicine group at 6h and 24 h were significantly lower than those in the radiation group at the same time point.The mRNA expression of Caspase-3 of the Chinese medicine at 12 h and 24 h was significantly lower than that in the same time radiation group,the difference was statistically significant(P<0.05).The mRNA expression of Cytochrome C in the left lung of the Chinese medicine group at 6h,12 h and 24 h were significantly lower than those in the radiation group at the same time point.The mRNA expression of Caspase-9 and Bax of the Chinese medicine group was significantly lower than that of the radiation group at 12 h and 24 h.The m RNA expression of Caspase-3 of the Chinese medicine group at 24 h was significantly lower than that in radiation group(P<0.01).The mRNA expression of Caspase-3 in the left kidney of the Chinese medicine group at 6h,12 h and 24 h were significantly lower than those in the radiation group at the same time point.The mRNA expression of Cytochrome C and Caspase-3 of the Chinese medicine group at 12 h and 24 h was significantly lower than that of the radiation group at the same time point(P<0.01).The mRNA expression of Bax of the Chinese medicine group at 24 h was significantly lower than that in the radiation group.The difference was statistically significant(P <0.01).The amount of protein and mRNA expression of Bcl-2 in the right lung of the Chinese medicine group 24 h is above the radiation group 24 h obviously.The amount of protein and mRNA expression of Bcl-2 in the left lung and left kidney of all Chinese medicine groups is above all radiation groups obviously.And there is statistically significant difference(P<0.01).Conclusions: After the right lung of the rats was radiated by different dose of X-ray and heavy ion,the genetic expression of Cytochrome C,Caspase-9,Caspase-3,Bax and Bcl-2 of the mitochondrial apoptotic pathway in the right lung is abnormal.And the variation is very prominent.Among them the expressions of Cytochrome C,Caspase-9,Caspase-3 and Bax obviously up-regulate.While the expression of Bcl-2 obviously down-regulates.The left lung and left kidney tissue that are not radiated also have the similarity variation of genetic expression.It indicates that the unirradiated tissues also make the response to the radiation of X-ray and heavy ion.And the radiation induced bystander effect is produced in vivo.Between every radiation dose and every time point,there is no apparent effect difference.By delaying the decreased expression of Bcl-2,Gui Qi Yi Yuan Ointment can inhibit the increased expression of Cytochrome C and Bax.So it can control the activation and expression of Caspase-3 and Caspase-9 in the downstream pathway.Thus it can prevent the lung and kidney cell apoptosis and defend the damage caused by X-ray and heavy ion radiation induced bystander effect in vivo.Gui Qi Yi Yuan ointment can inhibit the production of TGF-?1 in serum,and then cut down the expression and release of IL-6,TNF-?,IL-8 and IL-1?.As a result it can alleviate the pathologic damage of the lung.The local lung irradiation can also damage the body's hematopoietic system.Gui Qi Yi Yuan ointment can delay the decline of the count of white blood cell,red blood cells,platelet and hemoglobin.So it can protect the peripheral blood.