| Objectives:SYKT is a natural medicine originating from an ancient prescription of the Dai nationality in Yunnan.It is selected from the Bayesian Sutra.It is made up of 8 herbs such as ginger,blood exhaustion,licorice,poria,sand kernel,Sanqi,and Angelica.Previous studies have shown that SYKT has the effects of stimulating bone marrow hematopoiesis and protecting myocardial damage caused by anthracene chemotherapy drugs.Clinical practice has found that lung cancer patients who jointly use SYKT can benefit from survival and quality of life in radiotherapy,but the mechanism of their effects has not yet been studied in depth.This study will discuss the radiation sensitization and protection effects of SYKT,and the mechanism of its molecular action.Methods:Firstly,a retrospective analysis was made of 289 patients with NSCLC of III A/III B who were eligible for admission.Evaluate the curative effect and side effects,compare the differences between the groups with progressives free survival(PFS)and overall survival(OS).Secondly,survival rate of NSCLC cells after the SYKT treatment was calculated by the MTT assay.The radiosensitization of SYKT(0.5 g/mL and 1 g/mL)on cell line NCI-H460 and the radioresistant cell line NCI-H460R was studied by MTT assay and clone formation assay.The protein expression levels of iNOS,Cyclin B1 and CDC2 was determined by Western-blot,and the expression of NO was measured by Griess method.Cell cycle and apoptotic rate of NSCLC cell line NCI-H460 were accessed by flow cytometry assay.BrdU was also applied to detected the cell proliferation after the irradiation with or without SYKT treatment.Thirdly,C57BL/6 mice were treated with radiotherapy with or without SYKT.Bone marrow hematopoietic function was evaluated by measuring peripheral blood cells,bone marrow CD34+and CD44+cell count and apoptosis rate.The degree of immune organ injury was assessed by detecting the apoptosis rate of spleen and thymus cells,spleen and thymus index.Apoptosis was detected by flow cytometry(FCM).The enzyme-linked immunoassay(ELISA)was used to determine the level of reactive oxygen radicals(ROS)in cells.Finally,culture of MTSCc,C57/B6-SPL in vitro.The activation of p53,p38 and JNK in the cells were detected by Western-blot.PFT-α,SB203580 and SP 600125 were used as specific inhibitors of p53,p38 and JNK.The cytoplasm and mitochondrial protein were extracted by mitochondrial isolation kit,and the expression of Bax and cytochrome C in cytoplasm and mitochondria were detected by Western-blot.The expression of apoptosis-associated proteins caspase-3 and ADP nucleic acid polymerase(PARP)was detected by Western-blot.The expression of proteins Ku70,Ku80 and DNA-PKcs mRNA were detected by Real time-PCR.The expression of DNA related proteins Ku70,Ku80 and DNA-PKcs were detected by Western-blot.Results:1.The median follow-up time was 31 months(2-67m),1 year,2 years,3 years,5 years survival rate was 84.79%,64.41%,30.35%,18.37%in observation group,80.38%,58.86%,27.85%,14.46%in control group,respectively.The median OS was 27.3m(1-65m)VS 23.1m(1-59m)(P=0.01).The median PFS 10.8m(1-29.8m)in the observation group and the median PFS 9.6m(1-25.6m)in the control group(P=0.491).The local control rate was 74.81%(98/131)contrast 56.96%(90/158)between two groups(P=0.002).There were 21 cases(16.03%)in observation group and 43 cases(27.22%)in control group occured Ⅲ ° above myelosuppression(P=0.023).The KPS and weight improvement of the observation group were better than that of the control group(P<0.05).After 1 month of treatment,the immune function decreased obviously in both groups.The decrease in control group was more obvious than that in the observation group(P=0.001).2.The IC10 value of SYKT for NCI-H460 cells was 1.03 g/mL.After 1 g/mL SYKT treatment,the radiosensitivity of NCI-H460R cells was enhanced.The level of iNOS in the cells was found decreased after irradiation.We also found that SYKT could enhance the expression of iNOS and NO in radiation resistance cells,and inhibit the expression level of cyclin B1 and CDC2.The results of cell cycle and apoptosis assay demonstrated that the cell cycle was arrested in G2/M phase and the level of apoptosis increased when combining β-irradiation with SYKT.3.The lowest survival rate was found in the radiation group and the combined survival rate of SYKT was better than that in the radiation group(P=0.031).The number of peripheral blood cells and bone marrow CD34+and CD44+cells decreased significantly in radiation group compared with the combination group(P=0.022).The apoptosis rate of bone marrow in radiation group increased significantly compared with the combination group(P=0.035).The spleen index and thymus index of the radiation group were lower than that of the combination group,the difference was statistically significant(P=0.023,P=0.037).The apoptosis of splenocytes and thymocytes in the radiated group was lower than that in the combination group(P=0.039,P=0.041).In addition,ROS levels in bone marrow cells,spleen and thymus cells increased significantly,and were proportional to apoptosis rate.In combination with SYKT,ROS production decreased and apoptosis rate decreased.In addition,the measurement of subcutaneous tumor volume and apoptosis rate in mice are no difference between RT group and RT+SYKT group.4.In MTSCc,C57B6-SPL cells,the preapoptosis proteins p53,p38,JNK activated and Bax transposed after radiation treatment,mediated the apoptosis signaling pathway dependent on caspase and caused apoptosis of MTSCc,C57B6-SPL cells.In combination with SYKT,the expression of activated p53,p38 and JNK decreased,Bax translocation decreased,cytochrome C release decreased,apoptosis-related protein caspase-3 and PARP decreased,and apoptosis of MTSC,C57B6-SPL cells was obviously inhibited.Contrast with PFT,SB203580,SP600125,SYKT effect is accurate.These results prompt that SYKT can inhibit the activation of p53 and MAPKs and prevent the pathway of apoptosis,then protective the cells.At the same time,the expression of DNA damage repair protein Ku70,Ku80,DNA-PK cs mRNA increased than combined with SYKT group.Conclusions:1.Combined radiotherapy with SYKT can improve prognosis of LA-NSCLC and increase local control rate.SYKT can improve myelosuppression and immune function also.2.SYKT might promote the expression of NO by promoting the expression of iNOS after radiotherapy,and then regulate the cell apoptosis and cell cycle through arresting the cell cycle in the G2/M phase.Moreover,the effects of SYKT on the radiosensitization of NSCLC should be further investigated in clinical application.3.The results showed that SYKT could relieve radiation-induced myelosuppression and immunotoxicity in mice.It can reduces apoptosis in bone marrow and immune cells by inhibiting ROS production in cells.Combined with SYKT may be a new,safe and effective adjuvant treatment for myelosuppression and immune damage in radiotherapy patients.4.We found that the apoptosis of MTSCc and C57B6-SPL cells was induced by activating the signal pathway of apoptosis mediated by p53 and JNK-p38.SYKT can effectively inhibit this process and thus provide radiation protection. |
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