| Background:Lung cancer is the most common-seen malignant tumor in the world,at the same time that the morbidity and lethality of it is ascending quickly day after day.By the classification of lung cancer,NSCLC accounts for 80%～85%of the totality.Nowadays,the idealist and most effective therapy of NSCLC is still the surgery,but 30%-70%of the whole surgery patients may be dead by the cause of relapse and metastasis.The main reason of the death attributes to the halfway surgery that causes by minor metastasis occurred before the treatment.According to the newest NCCN Guide,adjuvant chemotherapy would be taken of NSCLC after the surgery.The recommended drug of NSCLC is the platinum-based chemotherapy at the stage of II and IIIA,that lots of large sample clinical studies have already confirmed its curative effect.However,concerned its side effect,Chinese Traditional medicine is the better treatment,because of the advantage of restricting relapse and metastasis,extending disease free survival,releasing patients’ symptom.The pathogenesis of the cancer is complex,but it could be summarized by these 6 parts:Qi,Blood,phlegm,poison,and weakness.For the large part of the lung cancer patients,at the time when they found their disease,it had be the middle and advanced stage-of the cancer.For that reason,with the healthy qi become weak,and the syndrome of weakness of qi and yin remains prominent.Therefore,Traditional Chinese Medicinal herbs of supplementing qi and nourishing yin method for non-small cell lung cancer would be the better way of treatment,and which combined with chemotherapy should be the most effective and common-used treatment.Fei-liu-ping ointment(FLP)as the most widely used and studied Chinese medicine with supplementing qi and nourishing yin method,the curative effect of it has been recognized,but the molecular mechanism study of it has not been lucubrated generally.Found by the latest research,tumor microenvironment can change the potential of tumor metastasis,which means the method of regulating and controlling tumor microenvironment from the micro-level entirety has been taken seriously gradually.The core of tumor microenvironment is immunity and inflammation,in which cancer and non-cancer components play a different role of improving or inhibiting metastasis.Then how FLP take part of this procedure is what we need to research afterwards.Consequently,the experimental section of this research will establish the model of tumor cell co-culture with immunity and inflammation environment firstly,and make FLP into drug serums.Then observe whether the FLP drug serums have bi-directional regulation for the cancer(LL/2-luc-M3 8)and non-cancer(DC,RAW264.7)components respectively through the key signaling pathway PI3K/AKT/NF-κB,in order to prevent the lung cancer metastasis.Purpose:1.To analysis and evaluate the curative effect,security and application prospect of the use of FLP for the NSCLC patients.2.By selecting the representative drug by supplementing qi and nourishing yin method of FLP to execute the research,attempt to explore the bi-directional regulation for the cancer and non-cancer components respectively through the key signaling pathway PI3K/AKT/NF-κB,in order to explain the mechanism of the lung cancer metastasis.Methods:1.In the first part of the research,we made a systematic review to systemically evaluate the effectiveness and safety of traditional Chinese medicinal herbs of supplementing qi and nourishing yin method for NSCLC.We searched RCT trails or researches articles on the CNKI,CBM,VIP,the Cochranelibrary website,and PubMed databases.Meta-analysis was performed for homogeneous studies using RevMan 5.0 software,and evaluate the effectiveness and safety of supplementing qi and nourishing yin herbs.2.In the second part of the research,we select the FLP medicine as the receptiveness of supplementing qi and nourishing yin method,and make it into drug serums to make in vitro and in vivo experiments respectively.(1)Study the influence of immune function of Lewis lung cancer tumor-bearing mice treated by FLP detected by flow cytometry:Inspect the regulation function of FLP to DC and TAM subtype in spleen of Lewis lung cancer tumor-bearing mice in vivo experiments.(2)Establishing myelogenous DC in C57BL/6 mice,detect the biological characteristics of DC by the way of light and electron microscope,flow cytometry.Then make FLP into drug serums,for the preparation of vitro experiments.(3)By flow cytometry,to observe the impact of FLP serums with non-cancer(DC,RAW264.7)component in the tumor microenvironment:the influence of FLP serums to the DC phenotype of C57BL/6 mice,and the cell amount with phenotype of RAW264.7.(4)Through the Western blot and real-time quantitative PCR,explore the impact on expression of the cancer and non-cancer components respectively through the key signaling pathway PI3K/AKT/NF-κB in tumor microenvironment.Firstly establish the tumor immune microenvironment,observe the impact of FLP serums on the expression of PI3K/AKT/NF-κB in the myelogenous DC with the LL/2-luc-M38 cell.Secondly establish the tumor inflammatory microenvironment,observe the impact of FLP serums on the expression of PI3K/AKT/NF-κB in the RAW264.7 with the LL/2-luc-M38 cell.Finally,to research the bi-directional regulation by FLP for the cancer and non-cancer components through the key signaling pathway PI3K/AKT/NF-κB.Results:1.From the result of meta-analysis,the herbs by supplementing qi and nourishing yin method combined with chemotherapy together was superior to chemotherapy alone in terms of recent survival rate,quality of life,the improvement of symptom,and the control of digestive or marrow side effects.2.The regulation function of FLP to DC and TAM subtype in spleen of Lewis lung cancer tumor-bearing mice by flow cytometry:DC was lower in the group of FLP and Model,while in Model group,the amount of DC descended gradually,from 4.26%to 2.59%.In the day of 14,21,and 28 in the FLP group,the amount of DC was 5.39%,4.83%,4.15%.While the sum of macrophage kept in high level both in the Model and FLP group,as well as the M1 and M2 hypotype with M1/M2.In the Model group,the amount of M1 macrophage increased(8.59%to 25.98%)with the M2 macrophage(0.92%to 5.42%),while M1/M2 decreased from 9.34 to 4.79.In the FLP group,the amount of M1 M2 macrophage at a high value by the day of 21,was 23.16%and 2.77%,with the ratio of M1/M2 was 7.86,8.36,9.62.3.The influence of FLP serums to the DC phenotype of C57BL/6 mice by flow cytometry:In the group of FLP serums,CD11c/MHC-II accounted for 70.59%,CDllc/CD80 accounted for 66.01%,which had obvious differences of the blank serum group(BS)(P<0.01).The influence of FLP serums to the amount of macrophage RAW264.7 by flow cytometry:In the group of FLP-S,F4/80-CD16/32 accounted for 12.14%,F4/80-CD206 accounted for 2.31%;while in the group of BS,the amount of them was declined obviously(P<0.05),but with the higher ratio of M1/M2.4.The impact of FLP serums on the expression of PI3K/AKT/NF-κB for the cancer and non-cancer components in the tumor immune microenvironment through the Western blot and real-time quantitative PCR in the model of tumor cell co-culture with immunity environment:In the LL/2-luc-M38,FLP-S could inhibit the expression of the protein and gene of PI3K,AKT,and NF-κB,in which the inhibition of NF-κB was remarkable(P<0.01).In DC,FLP-S could ascend the expression of the protein and gene of PI3K,AKT,and NF-κB,in which the ascent of PI3K was remarkable(P<0.001).The impact of FLP serums on the expression of PI3K/AKT/NF-κB for the cancer and non-cancer components in the tumor immune microenvironment through the Western blot and real-time quantitative PCR in the model of tumor cell co-culture with inflammatory environment:In the LL/2-luc-M38,FLP-S could inhibit the expression of the protein and gene of PI3K,AKT,and NF-κB,in which the inhibition of PI3K and NF-κB was remarkable(P<0.01).At the same time,FLP-S could inhibit the expression of mRNA gene of PI3K,AKT,NF-κB.In RAW264.7,the protein expression of PI3Kwas declining,while AKT and NF-κB was ascending in FLP-S group(P>0.05).Meanwhile the gene expression of PI3K,AKT and NF-κB ascended in varying degrees,and only the mRNA expression of NF-κB was ascending remarkably(P<0.01).Conclusion:1.The therapeutic effect of traditional Chinese medicinal herbs of supplementing qi and nourishing yin method is positive,especially combined with chemotherapy together is superior to chemotherapy alone in terms of recent survival rate,quality of life,the improvement of symptom,and the control of digestive or marrow side effects.Therefore,the TCM herbs of supplementing qi and nourishing yin are worthy to popularize in the future.2.The the most prominent characteristic of FLP for the immune function regulation of Lewis lung cancer tumor-bearing mice was able to balance the amount of DC and macrophage,in order to maintain a stable status.3.FLP serums could enhance the quantity of myelogenous DC,and promote them development.From the direction of RAW264.7 activation made by FLP serums,the mechanism was not only inhibiting M2 macrophage,but also the M1 macrophage.When FLP regulated M1 and M2,it inhibited the M2 macrophage highlighted.4.FLP serums had the bi-directional regulation for the cancer and non-cancer components respectively through the key signaling pathway PI3K/AKT/NF-κB in tumor microenvironment,but with different regulatory molecules would divide different action advantages.In the tumor inflammatory environment,pathway NF-κB played the key role in regulation.NF-κB had the role of inhibition for the cancer components and promotion in non-cancer components,while also had the inhibition of the components what had the function of promoting in non-cancer components.Finally from the overall results of the research,the regulation of FLP serums in tumor microenvironment may be executed by the function of balance the cancer and non-cancer components. |
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