The Effect And Mechanism Of JDF On Recruitment And Differentiation Of MDSCs Induced By HSCs

BackgroundPrimary liver cancer is closely related to hepatic fibrosis/cirrhosis,and activation and proliferation of hepatic stellate cells(HSCs)are a key link in the formation of hepatic fibrosis/cirrhosis.Myeloid-derived suppressor cells(MDSCs)show great immunosuppressive and tumor-promoting function in the tumor microenvironment(TME)through multiple mechanisms.HSCs are stromal cells unique to the liver,and they not only hold the key to occurrence and development of hepatic fibrosis/ cirrhosis but also play a crucial role in the process of occurrence and development of hepatocellular carcinoma(HCC).At the same time,activated HSCs can also play a role in immune regulation via recruiting MDSCs.The TCM oncology department of our hospital prepared “Jinmao Jiedu Formula(JDF)” based on decades of clinical experience and proved its definite therapeutic effect in the long-term clinical application.However,its mechanism has not been fully elucidated.ObjectiveTo explore the influence of Jinmao Jiedu Formula on recruitment and differentiation of MDSCs induced by HSCs and its mechanism,thus providing a theoretical basis for Jinmao Jiedu Formula exerting its anti-HCC effects by regulating TME.Methods1.The hepatic cirrhosis model was established by intraperitoneal injection of dimethylnitrosamine(DMN)5 mg/kg for adult male C57 mice(20 ± 2 g),and the orthotopic transplantation tumor of HCC was prepared after four weeks.Intragastric administration of JDF(12.74 g/kg every day)was performed in the JDF group after modeling.The mice were executed after two weeks of intragastric administration.Then the expression of α-SMA in the hepatic tissue was observed with immunohistochemistry,and the quantity and distribution of CD11b+Gr1+MDSCs in the tumor tissue were observed with immunofluorescence.2.The primary bone marrow and spleen cells of the mice were extracted,and JS1cells(HSCs of the mice)were intervened with TGF-β(10 ng/ml)and JDF(1 mg/ml)to obtain HSC-conditioned media(HSC-CM)with different treatments.The HSC-CM was used to intervene in the primary bone marrow or spleen cells for five days.The influence of JDF on differentiation of MDSCs and secretion of cytokines induced by HSCs in vitro was observed with flow cytometry and PCR technology.The primary spleen cells of the mice were extracted.Magnetic beads method was adopted to separate CD11b+Gr1+MDSCs from the spleen cells of the mice,and they were put in the upper chamber of Transwell,while the HSC-CM with different treatments was added in the lower chamber.After six hours,flow cytometry was used to count the migration quantity.3.The overexpression of cytokines in the supernatant of activated HSCs was detected via cytokine chips and further verified with fluorescence quantitative RT-PCR to determine the effect of JDF on overexpression of cytokines in activated HSCs.4.The primary spleen cells of the mice were extracted.CXCL2(5 ng/ml),HSC-CM,and SB225002(10 μM)were used to jointly intervene for five days,and flow cytometry was applied to detect the proportion of CD11b+Gr1+MDSCs.Transwell chamber was adopted to analyze the influence of the above intervention methods on migration of MDSCs,and flow cytometry was used to count the migration quantity.5.The primary spleen cells of the mice were extracted.CXCL2(5 ng/ml)and JDF(1 mg/ml)were used to jointly intervene for five days,and flow cytometry was applied to detect the proportion of CD11b+Gr1+MDSCs.Transwell chamber was adopted to analyze the influence of the above intervention methods on migration of MDSCs,and flow cytometry was used to count the migration quantity.6.The primary spleen cells of the mice were extracted.They were pretreated with activated HSC-CM,SB225002(10 μM),and JDF(1 mg/ml)for 60 min,and then treated with CXCL2(5 ng/ml)for 60 min.The level of p-ERK and p-AKT was detected with Western blot.Results1.In the orthotopic transplantation tumor model of HCC mice,the expression of α-SMA in the DMN group increased significantly compared with the control group,while the expression of α-SMA in the hepatic tissue of the JDF group decreased significantly compared with the DMN group.The quantity of CD11b+GR1+double stained cells in the tumor tissue of the DMN group increased significantly compared with the control group,while the quantity of CD11b+GR1+double stained cells in the tumor tissue of the JDF group decreased significantly compared with the DMN group.2.The proportion of CD11b+Gr1+MDSCs in the primary bone marrow cells of the activated HSC-CM group was remarkably higher than that of the control group(P<0.05),while the proportion of differentiated CD11b+Gr1+MDSCs in the JDF group was remarkably lower than that of the activated HSC-CM group(P<0.01).The proportion of CD11b+Gr1+MDSCs in the primary spleen cells of the activated HSC-CM group was also dramatically higher than that of the control group(P<0.001),while the proportion of CD11b+Gr1+MDSCs in the JDF group was dramatically lower than that of the activated HSC-CM group(P<0.001).The results of Transwell chamber revealed that the migration quantity of CD11b+Gr1+MDSCs in the JDF group was obviously lower than that of the activated HSC-CM group(P<0.05).In addition,compared with the activated HSC-CM group,the m RNA expression level of Arg-1 and IL-10 in the MDSCs of the JDF group obviously reduced(P<0.05).3.The cytokine chip detection found that the increase of CXCL2 was the highest among all the cytokines secreted by the activated HSCs.The fluorescence quantitative RT-PCR showed that the m RNA expression level in the activated HSCs was apparently higher than the control group(P<0.01),and the m RNA expression level of CXCL2 in the JDF group was apparently lower than the activated HSC group(P<0.01).4.Compared with the control group,the proportion of CD11b+Gr1+MDSCs in the primary bone marrow cells of the CXCL2 group markedly increased(P<0.001),and compared with the CXCL2 group,the proportion of CD11b+Gr1+MDSCs markedly decreased(P<0.01)and the migration quantity of MDSCs also markedly reduced(P<0.01)in the CXCL2+SB225002 group.5.The proportion of CD11b+Gr1+MDSCs in the HSC-CM+SB225002 group was evidently lower than that in the HSC-CM group(P<0.001),and meanwhile the migration quantity of MDSCs in the HSC-CM+SB225002 group was also evidently lower than that in the HSC-CM group(P<0.001),thus proving that blocking the CXCL2-CXCR2 pathway can inhibit the recruitment and differentiation of MDSCs induced by activated HSCs.Compared with the CXCL2 group,the proportion of differentiated CD11b+Gr1+MDSCs in the primary bone marrow cells clearly decreased(P<0.001)and the migration quantity of MDSCs also clearly reduced(P<0.01)in the JDF group.6.The Western blot indicated that compared with the control group,the expression level of p-AKT and p-ERK in the CXCL2 group and the unactivated HSC-CM group significantly increased;however,compared with the CXCL2 group,the expression level of p-AKT and p-ERK in the SB225002 group and the JDF group significantly decreased.The expression level of p-AKT and p-ERK in the activated HSC-CM group was obviously higher than that in the unactivated HSC-CM group.The expression level of p-AKT and p-ERK in the SB225002 group and the JDF group was obviously lower than that in the activated HSC-CM group.ConclusionJinmao Jiedu Formula can restrain the quantity of CD11b+GR1+MDSCs in the orthotopic transplantation tumor model of HCC mice and inhibit the recruitment and differentiation of MDSCs induced by activated HSCs in vitro,and its effects may be related to the blocking of CXCL2-CXCR2-ERK and CXCL2-CXCR2-AKT pathways.