| Malignant pleural effusion (MPE) is defined by lung cancer, or other parts of malignancy involving the pleura, or pleural primary tumor-induced pleural effusion, which is one of complications in advanced cancer, especially in lung cancer, accounting for about1/3in MPE occurrences, followed by breast cancer and lymphoma.Clinically, the occurrences of MPE often indicate the tumor spread or progression to advanced, the prognosis is usually poor. A large pleural effusion can cause symptoms of chest tightness, shortness of breath, chest pain and breathing difficulties, severe and even life-threatening, so actively and effectively control the MPE is an important part of the clinical treatment of cancer, which has a great significance on improving symptoms, improving quality of life and prolonging survival of cancer patients.Currently, there are many ways to treat MPE, but due to the toxic side effects of chemotherapy drugs, advanced patients generally also cannot tolerate surgery and other methods, what’s more, the short-term recurrence rate of MPE is still high, so the quality of life of patients is generally low, and the effect is not very satisfied. The traditional Chinese medicine(TCM) play an important role in the treatment of MPE, which is irreplaceable by western medicine. It is the distinguishing feature in treatment of cancer complications, and it has been widely used in clinical practices, especially for the elderly, frail patients, particularly inoperable and intolerant chemotherapy patient, TCM can be used as the preferred method of treatment, the efficacy and safety is higher.Chinese medicine Xiaoshui Formula (XSF) is an experienced prescription used for many years in oncology department of Guang’an men hospital, China academy of Chinese medical sciences. It is mainly composed of Astragalus, Atractylodes, Draba Nemorosa, Jujube, Pericarpium Trichosanthis, Eleutherococcus Senticosus, Plantago seed, Zedoary Turmeric, Cynanchum Paniculatum, Scutellaria Baicalensis, Scutellariae Barbatae, etc., the main function is to Spleen Qi, Xiefei diuresis, remove blood stasis and anticancer. Preliminary clinical studies have found XSF combined with cisplatin intrapleural injection in treatment with65cases MPE, the effective rate was72.3%, and can significantly improve the quality of life of patients, relieve symptoms and improve immune function, prolong the survival time of patients, but its mechanism of action is still not clear.Studies found that the generation and development of MPE is closely related to its local microenvironment, including the interaction between tumor cells and local inflammatory environment, immune cells, stromal cells and related cytokines. Th17cells, newly discovered different from the Thl, Th2-type immune effector cells, play a major role in chronic infections, immune disorders and cancer process. Some scholars have found that compared with peripheral blood, Th17cells at MPE microenvironment significantly increased, and associated with the prognosis of patients.Pharmacological studies have shown that the main drugs of XSF, such as Astragalus, Eleutherococcus Senticosus, Atractylodes, Scutellaria Barbata and other drugs can regulate immune function, improve the tumor microenvironment and improve immune tolerance state of the body, activating lymphocytes and the activity of various cytokines, to achieve antitumor effect. Recent studies have found that the main component of prescription, for example, acanthopanax glycosides, the main component of Eleutherococcus Senticosus; elemene, the main component of Zedoary Turmeric; baicalin, the main component of Scutellaria Baicalensis also can regulate the differentiation of Th17cells. Based on this, we hypothesized that the action mechanism of XSF may be mainly through regulating immune status of MPE microenvironment, especially in the regulation of immune cells and Th17cells cytokines networks aim to achieve prevention and treatment of MPE.1Research ObjectiveIn the early stage of topic, we conducted anoutpatient clinical observation and sum up experiences, then we try to establishe C57BL/6mice lung cancer MPE model, using XSF as an intervention measure, to explore its inhibitory effect on model mice with MPE and its regulatory effect on Th17cells, and related cytokines, proteins, by means of molecular biology techniques. It provide clinical and experimental basis for the further development and application of TCM, and promote the application of TCM in the treatment of MPE.2Contents And MethodsClinical Observation:collecting21cases of patients with malignant pleural effusion in outpatient, only give XSF (Spleen Qi, Xiefei diuresis) with differential therapy, the pleural effusion remission, KPS score changes and improvement of clinical symptoms were observed after treatment. Experimental study:Lewis lung carcinoma cells were injected into pleural cavity to established C57BL/6mice lung cancer MPE model. Three days later, all mices were randomly divided into five group: XSF group, DDP group, XSF+DDP group, Control group, Blank group, medication for10days. On the14th day, each group of10mice were sacrificed, and the remaining mice were observed for survival.During the medication, respectively, in the third, eighth,13th day, using small animal in vivo imaging system to observe and weighed once every other day. When mice killed, the blood and MPE were collected, meanwhile measured the volume of pleural effusion, counted the chest wall metastases, and weighted metastases.Their peripheral blood and MPE was obtained to detect the expression of Th17cells and Treg cells by flow cytometry, and the expression of IL-6、IL-17A、IL-21、IL-23, and TGF-(31by CBA or ELISA. Using Western Blot to detect the expression levels of STAT-3protein of metastatic tumor tissue, and the data were analyzed by SPSS18.0statistical software.3Results(1)The21cases of pleural effusion in patients with total effective as71.43%; KPS score increased and stable rate was80.95%; clinical benefit rate, dyspnea was91.67%, coughing was85.00%, chest pain was75.00%, fatigue was77.78%, anorexia was87.50%. and there is no significant adverse reactions.(2) Lewis lung carcinoma cells were injected into pleural cavity to established C57BL/6mice lung cancer MPE model. We observed model mice was generally in poor condition, pleural effusion were mostly bloody, pleural effusion rate was high, the chest wall metastasis were also more. (3) When using small animal in vivo imaging system to observe effects of different drugs intervention on MPE model mice, the eighth day imaging, XSF compared with the Control group, P>0.05, no significant difference; while XSF+DDP and DDP were compared with the Control group, P<0.05. The13th day imaging, the fluorescence intensity values of each group:XSF+DDP<DDP<XSF<Control group; XSF, DDP, XSF+DDP compared with Control group, respectively, P<0.05; and XSF+DDP compared with DDP and XSF group, P<0.05.(4) Different drugs intervention on weight change in mice, the7th day, compared with Blank group, the body weight of mice in each group began to decline, P<0.05; body weight compared to the rest of the group:XSF+DDP<DDP<XSF<Control group. The11th day, compared with Blank group, the body weight of mice in each group significantly decreased, P<0.01; XSF compared with DDP group, XSF compared with XSF+DDP group, both P<0.01. The14th day, compared with Blank groups, each group of mice was significantly decreased in body weight, P<0.01; XSF, DDP, XSF+DDP group pairwise comparisons, all P<0.05.(5) Different drugs intervention on survival of mice, the mice survival time:XSF group was longest (22.00[95%CI=18.90-25.19]), XSF+DDP group was second (21.00[95%CI=20.29-21.71])5DDP group was shortest(19.00[95%CI=16.98-20.02]), XSF and XSF+DDP, respectively, compared with the Control group, there were significant differences (P=0.007<0.01, P=0.006<0.01); additionally, XSF and DDP group, P=0.043<0.05, there were also significant differences. XSF group life extension rate (15.79%) is greater than the XSF+DDP group (10.51%), while the DDP group did not improve the life span of mice.(6) Different drugs intervention on amount of pleural effusion in mice, XSF+DDP group was the smallest (177±64ul), Control group was the maximum (715±61ul); each experimental group compared with the Control group, P value were all less than0.01; XSF+DDP compared with DDP and XSF group, respectively, have a significant statistically difference (P=0.047<0.05, P=0.013<0.05).(7) Different drugs intervention on the number of chest metastasis in mice, the number of metastases in XSF+DDP group was the minimum (2.5±1.0), Control group was up to9.3±0.6. Each experimental group compared with the Control group, P values were all less than0.01; XSF+DDP compared with XSF group, P<0.05; DDP compared with XSF group, also showed differences (P<0.05).(8) Different drugs intervention on chest wall tumor weight in mice, the weight of DDP group metastases was lightest (111.0±43.9mg), XSF+DDP group followed (297.6±144.5mg), XSF group was heaviest (569.2±152.1mg). Each experimental group compared with the Control group, P values were all less than0.01; XSF+DDP and DDP group were compared with the XSF group, both P<0.01; XSF+DDP and DDP group, also has differences (P=0.01<0.05).(9) The levels of MPE Th17cells in each group of mice, XSF group was the highest (1.46±0.37%), significantly higher than the DDP and Control group, the differences were significant (P=0.01<0.05, P=0.005<0.01), however, compared with XSF+DDP group, no significant difference (P=0.453>0.05); XSF+DDP group was compared with DDP and Control group, respectively, there was statistically significance (P=0.048<0.05, P=0.026<0.05). The levels of peripheral blood Thl7cells in each group of mice, each experimental group and Control group were higher than Blank group (0.17±0.07%), P values were all less than0.05. While XSF group (0.89±0.19%), XSF+DDP group (0.74±0.22%) compared with Control group, respectively, had significant difference (P<0.05); XSF group was significantly higher than DDP group (0.60±0.31%), the difference was statistically significant (P<0.05).(10) The levels of MPE Treg cells in each group of mice, Control group was the highest (13.94+2.89), XSF groups was the lowest (6.47±1.71%); XSF was significantly lower than the DDP and Control group, the differences were statistically significant (P=0.007<0.01, P<0.01); additionally, XSF+DDP compared with DDP and Control group were also statistically significant (P=0.0480.05, P=0.003<0.01). The levels of peripheral blood Treg cells in each group of mice, each experimental group and Control group were significantly higher than Blank group (0.40±1.36%), P values were all less than0.05. The Control group (3.12±0.53%) compared with the experimental group were significantly higher, P values were all less than0.01, with statistically significance. In each experimental group, XSF group was highest. XSF+DDP group was the lowest, when compared, there were significant differences, P=0.036<0.05.(11) The expression levels of MPE cytokines in each group of mice, IL-6expression: the MFI value of XSF group was the highest (349.12±56.48), XSF+DDP group was second (135.77+19.8), pairwise comparison of each group had obvious differences, P<0.01. IL-17A expression:the MFI values of XSF+DDP group (1.64±1.01) was significantly higher than XSF, DDP and Control group (P=0.027<0.05, P=0.005<0.01, P<0.001). IL-21expression:the MFI values of DDP group (6.32±1.49) was significantly higher than XSF, XSF+DDP and Control group, P values were all less than0.01; what’s more, XSF+DDP and Control group compared, P=0.024<0.05, also exhibited differences. IL-23expression:the MFI values of XSF+DDP group was highest (1.73±0.83), respectively, compared with the XSF and Control group, were statistically significance (P<0.01, P=0.006<0.01); the XSF was the lowest group (1.45±0.69), compared with the DDP group, P=0.004<0.01. TGF-β1expression:the MFI values of XSF+DDP group was the lowest (8.84±4.74), the highest was Control group (42.67±1.78). XSF, DDP and XSF+DDP group compared with Control group, respectively, with a statistically significance (P<0.01, P<0.01, P<0.01); XSF+DDP and DDP group, P=0.006<0.01, there were also significant differences.(12) The expression levels of peripheral blood cytokines in each group of mice, IL-6expression:XSF+DDP group was the highest (6.98±6.47), Blank group was the lowest (2.48±1.62); XSF, XSF+DDP and Control groups were compared with Blank group, P values were all less than0.01; XSF, DDP and XSF+DDP group were compared with the Control group, P values were also less than0.01; in addition, XSF+DDP were compared with DDP and XSF group also showed a difference (P<0.01). IL-17A expression, XSF+DDP group (4.18±1.78) was significantly higher than XSF, DDP, Control, and Blank group, respectively, P<0.01; additionally, XSF compared with Control and Blank group, respectively, had a significant difference (P=0.002<0.01, P=0.001<0.01,). IL-21expression:XSF+DDP group (5.19±1.05) was significantly higher than XSF, DDP, Control, and Blank group (P=0.0410.05, P=0.023<0.05, P<0.01, P=0.003<0.01); moreover, DDP group was compared with Blank and Control group, there was statistically significance (P=0.002<0.01, P=0.001<0.01,). IL-23expression:XSF+DDP group was highest (1.98±1.90), Control group was the lowest (1.46±0.60); XSF, XSF+DDP, and Control group were compared with Blank group, there showed a statistically significance (P=0.019<0.05, P=0.013<0.05, P=0.004<0.01); DDP and XSF+DDP group were compared with Control group, also had significant differences (P<0.01, P<0.01); DDP and XSF+DDP group compared with XSF group, respectively, also showed a significant difference (P<0.01, P<0.01). TGF-β1expression:Blank was the lowest group (30.15±10.77), compared with the other groups, P values were all less than0.05; in each experimental groups, Control group was highest (42.67±1.78), compared with XSF, DDP, and XSF+DDP group, respectively, P values were less than0.01; XSF+DDP group was the lowest in each experimental group, compared with XSF group, P<0.01.(13) The expression levels of STAT-3protein in metastases in each group of mice, XSF+DDP group was the highest (1.770±0.534), XSF group followed (1.473±0.137); XSF and XSF+DDP group were compared with the Control group, the differences were significant (P=0.014<0.05, P=0.014<0.05); compared with DDP and XSF group, P=0.021<0.05, with statistical significance; compared with DDP and XSF+DDP group, P=0.021<0.05,also with statistical difference.4Conclusion(1)XSF shows a good effect and safety, when it was simply used to be the main prescription in differential treating malignant pleural effusion, and can improve the quality of life for patients, improve clinical symptoms. Its advantages were obvious.(2) Lewis lung carcinoma were injected into pleural cavity to establish a C57BL/6mouse model of lung cancer MPE, its pleural fluid formation rate was better. It was a high quality model.(3) XSF can reduce MPE and inhibit pleural metastases; moreover, combined with chemotherapy, it showed a more pronounced inhibition.(4) XSF can significantly alleviate the weight loss of model mouse, and prevent the occurrence of cachexia through enhancing immune function; while reducing the effects of chemotherapy in mouse body weight, increase resistance to chemotherapy and improve efficacy.(5) XSF can significantly prolong overall survival time of MPE model mouse, and it shows a more obvious advantage compared to in combination with cisplatin chemotherapy; whereas cisplatin did not benefit the survival time of mice.(6) XSF is weak in terms of the role of inhibition of proliferation of metastasis tumor, suggesting that the mechanism of XSF reducing MPE may not inhibit the growth of metastases tumor, there may be other complex mechanisms.(7) When using flow cytometry to assay the levels of Thl7and Treg cells in MPE and peripheral blood, we found that the levels of Th17and Treg in MPE was significantly higher than peripheral blood. Furthermore, the levels of peripheral blood Th17and Treg cells in control group were significantly higher than in normal group.(8) We found that XSF can regulate Th17and Treg cells in MPE and peripheral blood of model mouse, mainly through up-regulating the expression Th17cells and down-regulating the expression Treg cells, mediate the imbalance of Th17/Treg cells in microenvironment, reducing immunosuppression in MPE microenvironment to enhance the tumor immunity.(9) We found that XSF alone or in combination with chemotherapy may up-regulate the expression of IL-6, IL-17A, IL-23, and down-regulate the expression of TGF-β1in MPE and peripheral blood by CBA and Elisa assay. And these changes may induce an initial Th0cells into Th17cells in local and systemic, which play a role in promoting tumor immunity; also may reduce ThO cells to Treg cells, which makes sense to reduce tumor immune suppression.(10) We found that XSD alone or in combination with chemotherapy can significantly improve the expression of STAT-3protein through western blot assay. It can promote Th17cells differentiation, proliferation, and promote cytokine expression, so as to play a role in the treatment of MPE. |
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