| Malignant pleural effusion(MPE)is a common complication of advanced cancer,often accompanied by severe symptoms such as dyspnea,pain,cachexia,and decreased physical strength.Once it occurs,the prognosis is poor,with a median survival of 3-12 months.At present,the clinical treatment of MPE is still based on palliative care with limited efficacy,including thoracentesis,pleurodesis,and intrapleural chemotherapy.In the past decade,the pathophysiological of MPE has made important progress and revealed the biological process of MPE formation.Pleural tumors can interfere with pleural fluid drainage,as well as the host vasculature and the immune system,resulting in decreased pleural fluid absorption and increased pleural fluid production via enhanced plasma extravasation into the pleural space.In this process,tumor angiogenesis,increased pleural vascular permeability,tumor growth,and local immune inflammatory microenvironment play an important role.An in-depth analysis of the biological processes of MPE formation also provides a new target for clinical treatment.The Chinese medicine Xiaoshui Formula(XSF)is composed of Semen lepidii,pyrrosia leaf,pepper,paniculate swallowort root,snakegourd fruit,astragalus root,rhizoma alismatis,barbed skullcap herb,ganoderma lucidum,atractylodes,and jujube.The formula has the benefit of spleen and water,and support positive cancer resistance.Previous clinical studies have found that XSF combined with cisplatin for the treatment of malignant pleural effusion can significantly alleviate the symptoms and improve the quality of life.Animal experiments also confirmed that the XSF has a certain role in inhibiting pleural effusion in MPE-bearing mice and can significantly prolong the tumor-bearing survival time of MPE-bearing mice.However,XSF has a weaker effect in inhibiting the proliferation of metastatic tumors,suggesting that the main mechanism for XSF to reduce MPE production is not achieved by inhibiting the growth of metastatic tumors,and other mechanisms may exist.Previous pre-experimental studies found that XSF can reduce the expression of M2 macrophages in MPE.M2 nmacrophages promoting angiogenesis is the key link in the formation of MPE.TAMsautophagy is an important way to regulate macrophage polarization.Therefore,we propose that XSF may interfere with TAMs autophagy,regulate its phenotypic polarization,and thus reduce the formation of MPE.ObjectiveBy observing the effect of XSF on the autophagy and phenotypic polarization of tumor-associated macrophages in MPE,revealing the molecular mechanism of XSF on MPE formation.At the same time,explaining the scientific connotation of XSF in the treatment of MPE,and providing new ideas and scientific theoretical basis for the further application of Chinese medicine in the treatment of MPE.Methods1.The MPE model of Lewis lung cancer in C57BL/6 mice was established by intrapleural injection of Lewis lung cancer cells,and the effect of different Lewis cell concentrations on the model was compared.2.The effects of XSF on body weight,pleural effusion,chest metastases,thoracic tumor burden,and survival time were observed by using live animal imaging techniques.3.The effects of XSF on pleural permeability and angiogenesis in mice were investigated through tail vein injection of Evans blue dye,CD31 and CD34 expression detecting by immunohistochemistry and VEGF expression detecting by Luminex.4.The effect of XSF on the phenotype of macrophage was observed in vivo and in vitro through the expression of CD206,CD 16/32 detecting by Flow cytometry and the expression of IL-10,IL-12 and TNFa detecting by Luminex.5.The effect of XSF on macrophage autophagy was observed in vivo and in vitro through p62 and LC3 expression detecting by Western blot analysis,and MDC and LC3 immunofluorescence.6.Verify that XSF can regulate macrophage phenotype through autophagy by using autophagy inhibitors and agonists;7.Ml and M2 macrophages were established by in vitro induction,and the regulatory effects of XSF on Ml and M2 macrophages were observed.8.Western blot was used to observe the effects of XSF on PI3K/Akt/mTOR pathway and AMPK/mTOR/ULKl pathway in tumor-associated macrophages.Results1.The mice in the high-LLC-concentration group(5.0×106 cells/ml)had a higher molding rate.9 mice in 10 mice developed pleural effusion.At the same time,the pleural effusion volume was 796 ul in 14 days.Compared with the other two groups,there were more tumor tissues in the pleural cavity and also more pleuralmetastases with statistically significant difference.2.The volume of malignant pleural effusion in the XSF group was significantly lower than that in the control group;however,there was no significant difference between XSF group and the control group in pleural metastases.The living animal imaging showed that there was no significant difference in fluorescence values between the groups on 3,7,10th day.The fluorescence value of the XSF group on the 14th day was slightly lower than that of the control group with statistically significant difference.At the 14th day,XSF group weight was significantly better than that of the cisplatin group and the model control group;The survival time of the XSF group was better than that of the control group with statistically significant difference.3.The Evans blue ratio of pleural effusion and serum in the XSF group was lower than that in the control group.The VEGF level in pleural effusion was also lower than that in the control group.Immunohistochemistry showed that the expression of CD31 and CD34 was also lower than that in the control group with statistically significant difference.4.The CD206 level of macrophages in mouse thoracic cavity was decreased and the expression of CD 16/32 was increased in XSF group with statistically significant difference;the expression of IL-10 in the MPE in XSF group was decreased,IL-12 level increased.TNF-a level decreased with statistically significant difference;in vitro co-culture model,macrophage CD206 level decreased,CD 16/32 expression increased,IL-10 level decreased and IL-12 expression increased in XSF group with statistically significant difference;5.The p62 protein expression of cells in MPE decreased and the ratio of LC3-Ⅱ/Ⅰincreased in XSF group.There was no difference of p62 protein expression,LC3-Ⅱ/Ⅰratio and immunofluorescence of LC3 in pleural tumor tissue between XSF group and control group.In the in vitro co-culture model,the p62 protein expression of macrophages in XSF group decreased,the ratio of LC3-Ⅱ/Ⅰ increased,and the immunofluorescence value of MDC was higher than that in the control group with statistically significant difference;6.Compared with XSF group,the expression of CD 16/32 in macrophages of XSF+ rapamycin group increased,and that of CD206 decreased;while the expression of CD16/32 decreased and CD206 increased in XSF+3-MA group with statistically significant difference;the IL-10 expression in XSF+ rapamycin group was lower with statistically significant difference,and there was no significant difference in IL-12;the IL-10 expression in XSF+3MA group also increased,and the level of IL-12 decreased,the difference was significant.7.There was no difference in the expression of CD206,CD16/32,and IL-10 and IL-12 in M1 macrophage between XSF group and control group;Macrophage CD206 expression was decreased in M2 macrophage of XSF group,the expression of CD 16/32 was increased,the expression of IL-10 in the culture supernatant was decreased,and the expression of IL-12 was increased,and the difference was statistically significant;There was no statistically significant difference in p62 protein level,LC3 protein level and MDC fluorescence value in M1 macrophage between XSF group and control group.The expression of p62 protein in MO and M2 macrophages was decreased,the ratio of LC3-LC3-II/I was increased,and the immunofluorescence value of MDC was higher than that in the control group.The differences were statistically significant.8.P-AMPK,p-ULK1(Ser555)expression of tumor-associated macrophages in XSF group increased,and the expression of p-AKT,p-mTOR and p-ULKl(Ser757)decreased.There was no significant difference in the expression of AMPK,ULK1,mTOR and AKT.Conclusion1.XSF can inhibit the production of MPE in mice,improve the body weight of mice,and prolong the survival period of mice,but has limited effects on thoracic cavity tumors;its inhibition of MPE production may be related to the reduction of pleural permeability and inhibition of angiogenesis;2.XSF can regulate the phenotypic polarization of TAMs by up-regulating the level of autophagy,and promote the conversion of M2 TAMs to Ml in MPE;3.The role of XSF in promoting the regulation of TAMs phenotype by autophagy may be related to the regulation of AMPK/mTOR/ULK1 and PI3K/AKT/mTOR pathways. |
