The Meachanism Of Zhoushi Gujin Xiaoliu Tang Regulating G-MDSCs To Inhibit The Progression Of Lung Cancer

Lung cancer is one of the malignant tumors that have the fastest incidence and mortality growth.Although a variety of therapeutic methods have been applied in the clinical treatment of lung cancer,the therapeutic effect did not work well,and more effective therapeutic measures need to be developed urgently.In recent years,immunotherapy has been proved to have a good effect in the treatment of a variety of tumor diseases.However,in lung cancer,there are a large number of immunosuppressive cells,which cause great hindrance to the immunotherapy of lung cancer.Among these inhibitory immune cells,myeloid-derivedsuppressorcells(MDSCs)not only play an important role in the inhibition of anti-tumor immune response,but also play a strong role in promoting angiogenesis,which greatly promotes the growth and metastasis of tumors.Therefore,immunotherapy targeting MDSCs is expected to achieve a breakthrough in the treatment of lung cancer.Previous studies have shown that Zhoushi Gujin Xiaoliu Tang(ZSGJXLT),a traditional Chinese medicine compound,can regulate the development and progression of tumors through a variety of mechanisms,and has been shown to have advantages in the clinical treatment of lung cancer,but its role in the regulation of MDSCs remains unclear.Objective:In this paper,the orthotopic transplanted tumor model constructed by Lewis lung carcinoma line(LLC)was used to explore whether ZSGJXLT can delay the progression of lung cancer,and to observe its influence on the number and migration of granulocytic myeloid-driven suppressor cells(G-MDSCs)in vitro and in vivo.The effect of ZSGJXLT on angiogenesis of G-MDSCs and the role of STAT3-related molecular pathway in it were also discussed.Method:1.Effects of ZSGJXLT on tumor growth in mice,the number of G-MDSCs and tumor microvascular density(1)Effects of ZSGJXLT on tumor progression of orthotopic Lewis lung cancer tumors in mice: UPLC-MS/MS was established for the qualitative and quantitative analysis of five immune-related components in ZSGJXLT,in order to control the quality of traditional Chinese medicine compound.Established the orthotopic Lewis transplantation tumor-bearing mice models were randomly divided into groups: Physiological saline group(NS),ZSGJXLT group(0.25g/kg,1g/kg,4g/kg),cisplatin group(DDP(2mg/kg))and respectively treated for 21 days,the biochemical indexes of serum liver and kidney function were observed to monitor the hepatorenal toxicity of the drug.The weight,growth and survival rate of mice were recorded in real time,and the tumor size was monitored by in vivo fluorescence imaging system.(2)The infiltration and phenotypic changes of MDSCs in tumor-bearing mice after ZSGJXLT treatment: FCM was conducted to test the proportions of total MDSCs,G-MDSCs and M-MDSCs in spleens and tumor of tumor-bearing mice in each treatment group.(3)In vivo tumor angiogenesis after ZSGJXLT treatment: The levels of tumor angiogenesis surface marker antigens CD31 and Western-blotting protein VEGFA were detected by IHC.ELISA was used to examine the expression level of VEGF in serum of tumor-bearing mice in each treatment group.Meanwhile the correlation between the expression of VEGF and the proportion of G-MDSCs in the orthotopic tumor model was determined by linear analysis.2.Effects of ZSGJXLT on the quantity and migration of G-MDSCs in vitro The effects of ZSGJXLT on the cell viability of BEAS-2B and GES-1 were detected by CCK8 experiment,so as to screen the safe concentration for subsequent experiments.G-MDSCs in spleen of tumor-bearing mice were isolated by MACS,and the apoptosis of ZSGJXLT with different concentrations in G-MDSCs was detected by flow apoptosis experiment.The expression changes of Bcl-2 and Bax related apoptosis proteins in G-MDSCs were determined by VII Western-blotting.Transwell cell migration test and Perkin Elmer Operetta CLS high-content imaging system were conducted to monitor the orientation of G-MDSCs by Lewis-derived tumor cell culture supernatant(Tumor cell conditioned medium,TCCM)modified by ZSGJXLT.3.Investigation on the angiogenesis promoting function and mechanism of ZSGJXLT on G-MDSCs in vitro Human umbilical vein endothelial cell(HUVEC)was co-cultured with G-MDSCs isolated from MACS,and the changes of tubule formation in HUVEC after ZSGJXLT treatment were observed in vitro.The expression of angiogenesis-related proteins VEGFA,MMP-9 and immune-related molecule Arg-1 and phosphorylation level of STAT3 pathway-related proteins in G-MDSCs stimulated by ZSGJXLT were detected by Western-blotting.Meanwhile,the changes of secretion levels of inflammatory molecules IL-6 and TGF-β were analyzed by ELISA.Results:(1)The mass concentrations of Adenine 、 Adenosine 、 Ginsenoside(Rg1)、Ginsenoside(Re)、Ginsenoside(Rb1)measured by UPLC-MS/MS were 0.21,6.68,11.99,4.82 and 0.15mg/g respectively.The indexes of liver and kidney function of mice in the cisplatin group were significantly elevated compared with those in the normal group and the tumor-bearing group.ZSGJXLT had no obvious side effects on liver and kidney toxicity.Compared with the normal group,the body weight of mice in the cisplatin group decreased significantly on the 12 th,15th,18 th and 21 st days.ZSGJXLT did not affect the body weight growth of tumor-bearing mice.The tumor-inhibiting rates of ZSGJXLT(0.25,1 and 4g/kg)were 27.63%,45.16% and 56.77% respectively.In vivo fluorescence imaging system also monitored tumor regression and significantly prolonged survival.(2)FCM results indicated that the proportion of total MDSCs cells in spleens and tumor tissue of mice treated with ZSGJXLT was decreased compared with the control group.The infiltrating proportion of G-MDSCs in spleens and tumor tissue of mice was dropped in a concentration-dependent manner.The infiltration rate of M-MDSCs cells in spleen also showed a downward trend,while the ratio of infiltrating M-MDSCs cells in tumor tissue had no statistically significant change trend.(3)IHC results revealed that the density of tumor microvessels was inhibited by different doses of ZSGJXLT significantly;Western-blotting results displayed that the protein expression of VEGFA in tumor tissue was decreased in a concentration-dependent manner by ZSGJXLT.ELISA assay showed that the concentration of VEGF in tumor-bearing mice serum was significantly higher than that in normal C57BL/6 mice;after treatment with ZSGJXLT,the concentration of VEGF in tumor-bearing mice was significantly lower than that in tumor-bearing mice.The results of linear regression analysis showed that there was a positive correlation between the concentration of serum VEGF and G-MDSCs in tumor tissue.(4)CCK8 results showed that BEAS-2B,GES-1 was treated with different doses of ZSGJXLT in vitro after 24 h,the change of cell number was not statistically significant compared with the control group.FCM results showed that ZSGJXLT stimulation at the corresponding concentration of the above experiment could induce apoptosis in G-MDSCs,and apoptosis-related proteins were detected in G-MDSCs cells by Western-blotting,and the level of Bax/Bcl-2 protein was obviously up-regulated.Transwell detected that the migration number of G-MDSCs treated with TCCM was higher than that of the untreated group,while the migration of G-MDSCs was inhibited under the conditioned medium of ZSGJXLT.Perkin Elmer Operetta CLS high-content imaging system was conducted to track the migration trajectory of G-MDSCs,and the data demonstrated that the mean square displacement and migration distance of G-MDSCs cultured in the conditioned medium of TCCM were increased and longer than that of G-MDSCs in the untreated group.The cell migration was strikingly inhibited after cultured in TCCM conditional medium with ZSGJXLT.In the above experiment,the concentrations of TGF-β and IL-6 in the supernatant of tumor cells were significantly higher than those in normal culture medium,but ZSGJXLT could inhibit their expression.(5)The experimental results of in vitro angiogenesis of HUVEC showed that the number of circular vascular structures in the co-culture system of G-MDSCs and HUVEC increased,and the circular vascular structure of HUVEC declined in a concentration-dependent manner after ZSGJXLT exposure under the co-culture system.The results of Western-blotting detection illustrated that the expression levels of VEGFA,MMP-9 and Arg-1 in G-MDSCs were markedly inhibited and the phosphorylation level of proteins related to STAT3 pathway decreased after the intervention of ZSGJXLT,while the concentrations of TGF-β and IL-6 in the supernatant of G-MDSCs cell were down-regulated by ELISA test.Conclusion:(1)ZSGJXLT can inhibit the amplification and accumulation of G-MDSCs,thereby partially eliminating the immunosuppression and delaying the tumor development of Lewis tumor-bearing mice.(2)ZSGJXLT may reduce the density of tumor microvessels by inhibiting the pro-angiogenesis function of G-MDSCs.(3)In vitro,by down-regulating the phosphorylation level of STAT3 pathway protein,ZSGJXLT can effectively inhibit the synthesis and secretion of various angiogenic factors and inflammatory factors of G-MDSCs,and inhibit the angiogenic response to effectively control the growth and metastasis of tumors.