The Effect And Mechanism Of Jinfukang Oral Liquid On Lymphangiogenesis Of Lung Cancer Via Inhibiting SDF-1/CXCR4 Axis

Objective:Lymphatic metastasis is an important way of metastasis of lung cancer,which makes titanic effort for the stage,treatment and prognosis of tumor.The effective inhibition of lymphangiogenesis is one of the treatments of lung cancer.Previous studies have shown that Jinfukang oral liquid can inhibit the growth of tumor by preventing the bone marrow mesenchymal stem cells from transformation into tumor lymphatic vessels,and its mechanism may be related to the suppression of the SDF-1/CXCR4 signal axis.The aim of this study,is to observe the effect of Jifukang oral liquid on the growth of lung cancer and the newborn of lymph tube,and clarify whether Jinfukang oral liquid to achieve anti-tumor effect is based on the inhibition of SDF-1/CXCR4 axis regulation of lymphangiogenesis via transformation of the CXCR4 short hairpin RNA(ShRNA)into bone marrow mesenchymal stem cells.Methods:(1)Extracting primary BMMSC from the mice of C57BL/6 and transfecting into P3 BMMSC using LV.The transfected cells were observed by Laser Scanning Confocal Microscopelaser confocal microscope to find the best transplant time.(2)Real-time fluorescence quantitative PCR was used to detect the inhibitory of the group of NC,CXCR4 shRNA-1,CXCR4 shRNA-2 and CXCR4 shRNA-3 recombinant lentiviral vector on BMMSC CXCR4 mRNA.(3)Immunomagnetic beads were used to isolate hematopoietic stem cells from bone marrow cells and mixed with LV-transfected bone marrow mesenchymal stem cells(HSC 1×105/mouse,BMMSC 2×105/mouse),and inject intravenously the receptor mice which recived ray,and the mouse bone marrow chimerism model was established.Among them 14 were NC recombinant lentiviral vector group and 20 were CXCR4 shRNA recombinant lentiviral vector group.Using laser confocal microscopy to observe the bone marrow smear to observe whether the chimeric model was successfully constructed after 35 days.(4)1×106/mouse Lewis tumor cells were injected axillary on the right side of the bone marrow chimeric mice to construct the model of subcutaneous transplantation of lung cancer in mice.The mice were divided into five groups:NC + JFK group,NC + NS group,CXCR4 shRNA + JFK group,CXCR4 shRNA +CTX group and CXCR4 shRNA + NS group.The rats in NC + JFK group and CXCR4 shRNA+ JFK group were treated with Jinfu kang oral liquid,NC +NS group and CXCR4 shRNA + NS group were treated with normal saline,daily administration of 0.4ml,continuous administration of 21 days;CXCR4 shRNA + cyclophosphamide group,according to the weight of mice,20mg/kg dose every other day intraperitoneal injection of cyclophosphamide injection,a total of 10 times.Mice were sacrificed at the end of the intervention,whichever was subcutaneously transplanted.(5)RT-PCR and Western Blot were used to detect the expression of CXCR4 mRNA and protein in tumor tissues.(6)Preparing by immunohistochemical staining of tumor tissue,laser confocal microscopy was performed on LVYE-1 green fluorescence pictures,bone marrow-derived red fluorescent cell images and blue DAPI stained nuclei pictures.Results:(1)Recombinant CXCR4 shRNA lentiviral vector was transfected into P3 bone marrow mesenchymal stem cells.The morphology and fluorescence intensity of the cells were observed by confocal laser scanning microscopy.The red fluorescence fluorescence intensity is highest and CXCR4 was stable in the cells on day 7 after transfection.(2)The inhibitory efficiency of CXCR4 shRNA lentivirus was detected by RT-PCR.The results showed that CXCR4 shRNA had the highest inhibitory effect on CXCR4 gene in bone marrow mesenchymal stem cells.(3)The mouse bone marrow chimerism model was constructed and the bone marrow images of mice were observed by confocal microscopy at 35 days after transplantation.The results showed that the red fluorescence intensity of bone marrow cells in CXCR4 silent group was lower than that in the control group(P<0.05).(4)The weight of the subcutaneous tumor in mice:NC + JFK group 2.50g,NC + NS group 3.32g,CXCR4 shRNA+JFK group 1.09g,CXCR4 shRNA + CTX group 0.94g and CXCR4 shRNA + NS group 1.92g,the tumor weight of the mice was significantly lower than that of the non-load control group(P<0.05),and the tumor weight of the mice was lower in the control group.The tumor weight of mice in cyclophosphamide group was the lowest,and the difference was statistically significant(P<0.05).(5)RT-PCR and Western blot were used to detect the expression of CXCR4 mRNA and protein in tumor tissue of each group.The results showed that there was no significant difference in the expression of CXCR4 mRNA and protein between the tumor group and the gene silencing group.The levels of CXCR4 mRNA and protein in Jinfukang group were significantly lower than those in the control group(P<0.05).(6)The number of RFP+,LYVE-1+ and RFP+/LYVE-1+ double positive cells in the immunofluorescence sections of the CXCR4 shRNA + NS group were significantly lower than those in the NC+NS group(P<0.01)The dose of RFP+,LYVE-1+ and RFP+/LYVE-1+ decreased(P<0.05).However,in the silence group,Jinfukang and cyclophosphamide had no obvious inhibitory effect on RFP +,RFP +/LYVE-1+ lymphatic cells,though LYVE-1+decreased.Conclusions:(1)Silencing CXCR4 gene of MSCs inhibited the migration of BMMSC to lung tumor tissue and CXCR4 involved in the lymphangiogenesis of tumor tissue and restrained the growth of tumor tissue.(2)The expression of CXCR4 mRNA in tumor tissue can not be degraded by recombinant CXCR4 shRNA lentiviral vector,also not reduce the content of CXCR4 protein in tumor.However,Jinfukang oral liquid has obvious inhibitory effect on CXCR4 mRNA expression in tumor tissue,and reduce its CXCR4 protein content.(3)Jinfukang oral liquid has an apparent effect on the downsize of the Lewis subcutaneous tumor growth.The mechanism might be via inhibition of bone marrow mesenchymal stem cells to lymphatic endothelial cells to achieve anti-tumor effect,and SDF-1/CXCR4 might be one of the channels.