| BackgroundWith the vigorous development of China's economy,environmental pollution has become a serious problem.Because of the fuel emissions,bad living habits such as smoking and special working environment,which lead the human to inhale various harmful carcinogens unconsciously for a long time.The environmental pollution changed the normal lung tissue survival microenvironment and have serious influence on the normal lung cells growth and metabolism,people could eventually get lung cancer.According to the statistics,in 2013,the number of lung cancer patients in China was the first among men,second only to the breast cancer among women.Lung cancer also accounted for the first of all deaths in cancer.The treatment of lung cancer mainly includes surgery,chemotherapy,radiotherapy and the latest targeted therapy,but each treatment is a double-edged sword.The radiotherapy has an important role in the early and late treatment of lung cancer.However,when the radiotherapy kill the cancer tissue,at the same time,the normal lung tissue in the radiation field has unnecessary damage inevitably,which finally may cause radioactive pneumonia and seriously affect the prognosis of patients.In addition,due to the resistance of tumor cells to radiation,increasing the dose of clinical radiotherapy will aggravate the damage to normal lung tissue.At present,the treatment of clinical radiation pneumonia is to relieve the symptoms of patients mainly by giving glucocorticoid,which can have serious side effects else.So,the purpose of our study is to find a drug that protects normal lung tissue and enhances tumor therapy in radiotherapy simultaneously.Proanthocyanidin is the most antioxidant natural extract,which is far more potent than the commonly used vitamin C and vitamin E.It has huge advantages including a wide range of sources,low extraction cost,low toxicity and good water solubility.In addition,half of the lethal dose of proanthocyanidins in organism is more than 5000mg/kg,which is far greater than the therapeutic dose.Proanthocyanidin can significantly reduce the early inflammatory infiltrates and radioactive pneumonia after irradiation and late radiation pulmonary fibrosis.Proanthocyanidin can reduce the transformation of epithelial interstitial lung tissue which caused by radiation,and reduce inflammation factor TGF-beta 1,ET-1,IFN-gamma,IL-4,and IL-13.Proanthocyanidin has good therapeutic effect in lung tissue inflammation and disease: Proanthocyanidin can effectively reduce the Angle of laminating the lung tissue induced by acute inflammation,relieve acute airway inflammatory response in mice models of asthma,inhibit the formation of pulmonary edema,and the pneumonia induced by bleomycin.In addition,unlike other antioxidants,Proanthocyanidin is a kind of slow-release antioxidants,which can remain 7-10 days in the blood and tissue,and play role of continuous antioxidant for the progressive progression of prevention and control of radioactive lung injury.Proanthocyanidin also has anti-tumor effect.Over the past decade,in vitro cell experiments and animal experiments,proanthocyanidin has proved that proanthocyanidin has a clear inhibitory effect on skin cancer,breast cancer,prostate cancer,head and neck cancer and lung cancer cell proliferation.The researchers also demonstrated that proanthocyanidin had no significant effect on the growth and vitality of normal cells,but only showed a killing effect on tumor cells in vitro experiments.Combined with the above characteristics,proanthocyanidin is a promising drug for clinical application.This study will focus on the bidirectional role of proanthocyanidins in the radiotherapy of lung cancer and its mechanism.ContentsFirstly,the establishment of tumor model in situ lung cancer in mice.1.The method of the establishment of tumor model--in-situ lung injection;2.The upper lobe of the left lung was taken as the growth environment of lung cancer cells,and the cells was fixed by matrix glue.Secondly,the bidirectional effect of proanthocyanidins on lung cancer radiotherapy.1.Detecting the toxicity of proanthocyanidin in mice lung epithelial cell MLE-12 and human normal pulmonary epithelial cell BEAS-2B by CCK8.2.Detecting the bidirectional effect of proanthocyanidins by flow detection and Cloning efficiency,including the radiation protection of normal lung cells and the radiation sensitization of lung tumor cells.3.Proanthocyanidins can inhibit the growth and metastasis of tumor in mice.(1)proanthocyanidins inhibited the growth of subcutaneous tumors in mice;(2)proanthocyanidins inhibited the metastasis of tumors in mice;4.The effect of proanthocyanidins on the survival status of the mice with tumor;5.The effect of proanthocyanidins on the tumor size and the weight of the left upper lobe of the mice with tumor;6.The procyanidins reduced the inflammatory exudation of normal lung tissue;7.Procyanidins can reduce the apoptosis after irradiation in the normal tissue of mice,and promote the apoptosis of tumor tissue after irradiation,while inhibiting the proliferation;8.Proanthocyanidin regulates the secretion of Th1/Th2 related cytokines,IL-6 and IFN-?.Thirdly,the research on the mechanism of proanthocyanidins in the radiotherapy of lung cancer.1.Proanthocyanidins can significantly reduce the production of reactive oxygen species in A549 and BEAS-2B cells after irradiation;2.The effect of proanthocyanidins on p-ERK,p-JNK and p-P38 expressions in lung normal cells and tumor cells was different;3.Proanthocyanidins can activate the expression of Sirt6 protein in normal lung cells and tumor cells.4.Detecting the expressions of p-ERK,p-JNK and p-P38 after overexpressing the Sirt6 protein in lung normal cells and tumor cells;5.Detecting the expression of Sirt6 by inhibiting the expression of p-ERK,p-JNK and p-P38 protein.MethodFirstly,establishing the in-situ lung cancer tumor model and the local radiation lung model of the mice1.Injecting the mixture of tumor cells and matrix glue into the left upper lobe of the mouse with insulin syringe,and establishing a tumor model of lung carcinoma in situ in mice;2.Using the fixed mouse mould designed in the laboratory to establish the radiation model of the lung bureau;3.Irradiation method: the dose rate is 1Gy/min and using 25 Gy single irradiation.Secondly,proanthocyanidins had a bidirectional effect on lung cancer radiotherapy.1.Selecting the mice lung epithelial cell MLE-12,lung cancer cell LLC and human normal lung epithelial cells BEAS-2B,lung adenocarcinoma cell A549 for trial;(1)Cell group and treatment: control group,proanthocyanin treatment group,irradiation group,irradiation group plus proanthocyanidins treatment group;(2)The effect of irradiation and proanthocyanidins on cell proliferation: flow cytometry detection and Cloning efficiency;2.Detecting the bidirectional effect of proanthocyanidin on the basis of in-situ lung cancer tumor model.(1)Animal grouping and treatment: Sham operation control group,Sham operation control group with irradiation,a tumor-burdened control group,a tumor-burdened and proanthocyanidins treatment group,a tumor-burdened group with irradiation,a tumorburdened and proanthocyanidins treatment group,each group choosed three male C57 BL / 6 mice about eight-week old;(2)The effects of proanthocyanidins and irradiation on the mice with in-situ tumor: the survival and weight of mice;(3)The effects of proanthocyanidins and irradiation on tumor of in-situ tumor-bearing mice: H&E staining and measuring the weight of the left upper lobe;(4)The detection of normal lung tissue and tumor tissue proliferation and apoptosis with immunofluorescence staining: Ki67 and p53 staining;(5)The effect of procyanidins on immune cytokines in mice: serum cytokines Il-6 and IFN-?were detected by ELISA.Thirdly,the mechanism of proanthocyanidins in the radiotherapy of lung cancer.1.Detecting the removal of reactive oxygen in cells after irradiation: ROS detection kit;2.Testing the effect of proanthocyanidins on the related proteins in the cells after irradiation: Western blot;3.Detecting the effect of proanthocyanidins on normal lung tissue and tumor tissue protein in the mice with in-situ tumor: Western blot;4.The expressions of p-ERK,p-JNK and p-P38 protein were detected by changing the expression of Sirt6 in cells: Sirt6 transfection of plasmid,Western blot;5.The expressions of p-ERK,p-JNK and p-P38 protein were suppressed by selective inhibitors S1805,SP600125 and SB203580,and the expression of Sirt6 was detected: Western blot.ResultsFirstly,establishing the in-situ lung cancer tumor model and the local radiation lung model of the mice successfully.1.Successfully establishing a tumor model in mice,and the tumor growth was consistent with the experimental requirements;2.Successfully establishing a local irradiation model of the lung of the tumor mice.Secondly,proanthocyanidins play a bidirectional role on the radiotherapy of tumor cells.1.Normal lung cells and tumor cells in mice: detecting by flow cytometry showed that proanthocyanidins could reduce the apoptosis of mice lung epithelial cell MLE-12 after irradiation;Through the experiment of Cloning efficiency,proanthocyanidin had radiation protective effect on the MLE-12,and had radiosensitive effect on mice lung cancer cell LLC.2.Normal human lung and tumor cells: through the detection of flow cytometry,proanthocyanidins can reduce the apoptosis of human normal lung epithelial cells BEAS-2B and promote the apoptosis of lung adenocarcinoma cells A549;Through the experiment of Cloning efficiency,proanthocyanidin has radioprotective effect on BEAS-2B cells,and has radiosensitive effect on A549 cells.Thirdly,detecting the bidirectional effect of proanthocyanidins in the mice with in-situ tumor.1.By recording the survival period of the tumor-burden mice,it was found that the proanthocyanidins treatment with irradiation group survived longer than the pure irradiation group.2.Through the measurement of tumor in the lung of mice,it is found that the growth of the tumors in the left upper lobe of the mice are inhibited by the treatment of proanthocyanidins,and the effect was more obvious than that of the pure irradiation group.3.Detecting the proliferation related protein Ki67 and the apoptosis related protein p53 respectively by immunofluorescence staining.In normal mice lung tissue,compared with the pure radiation group,the expression of p53 in the proanthocyanidins plus radiation group is significantly reduced;In the tumor tissues,the expression of Ki67 in the treatment group was significantly lower than that in the control group,while the expression level of p53 was significantly increased compared with that in the proanthocyanidins plus radiation group.4.Detecting the serum cell factors in tumor-burdened mice by ELISA.Compared with the control group,proanthocyanidins plus radiation group could significantly inhibit the expression of IL-6 and improve IFN-gamma,which can promote the tumor inhibition,reduce inflammatory injury.Thirdly,the mechanism of proanthocyanidins in the radiotherapy of lung cancer.1.The proanthocyanidins can significantly remove the free radicals produced in human lung cancer cells A549 and normal lung epithelial cells BEAS-2B after radiation.2.In human lung cancer cell A549,proanthocyanidin obviously improved the expression of p-JNK protein after irradiation;In human normal pulmonary epithelial cell BEAS-2B,proanthocyanidin can significantly inhibit the increase of p-JNK,p-ERK and p-P38,which were related to the removal of reactive oxygen free radicals.3.In human lung cancer cell A549,proanthocyanidin can change the expression of pJNK,p-P38 by increasing the expression of Sirt6 protein,and promote the radiosensitivity.ConclusionWe found that on a cellular level,proanthocyanidins had radiosensitive effect on mice lung cancer cells LLC and human lung cancer cells A549,and had radioprotective effect on mice normal lung cells MLE-12 and human normal lung epithelial cells BEAS-2B.In human lung cancer cell A549,proanthocyanidin can change p-JNK,p-P38 protein by improving the expression of Sirt6 protein,promoting the radiation sensibilization.In human normal pulmonary epithelial cell BEAS-2B.Proanthocyanidin may inhibit the increase of pJNK,p-ERK and p-P38 by scavenging the reactive oxygen free radicals,and reduce the inflammatory effect.Through the establishment of the radiotherapy of lung cancer in situ model in mice,we found that proanthocyanidin can improve the survival of mice,change the expression of serum levels of cytokines,reduce the radiation damage of normal tissue of the lung in mice,and have the function of the radiotherapy sensitization in tumor. |
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