| Objective:Objective to investigate the effect of Xihuang Pill on 4T1 tumor bearing mice in situ tumor,the improvement of blood stasis symptoms,the inhibition of breast cancer lung metastasis,and the changes of important components and biomarkers related to the pre metastasis microenvironment in blood and lung tissue,so as to clarify the effect of Xihuang Pill on breast cancer lung metastasis and its related mechanism.Method:1.In this study,five female BALB/c mice were randomly selected and subcutaneously injected with 4T1 breast cancer cells to produce tumor in situ.When the tumor grew to about0.8cm in diameter,the mice were killed,and the tumor blocks with appropriate size were cut into 2mm3 and transplanted into the fourth pair of breast fat pads of other mice Model control group,XH group and ADM group,25 rats in each group.Objective to evaluate the progress of blood stasis syndrome in mice and analyze the improvement effect of Xihuang Pill on blood stasis syndrome in tumor bearing mice.The tumor was weighed,the length and width of tumor were measured,the tumor volume and inhibition rate were estimated,and the effect of Xihuang Pill on tumor in situ was observed.2.The lung metastasis model of breast cancer in BALB/c mice was established by subcutaneous transplantation of 4T1 breast cancer tumor;Three methods including naked eye observation,he staining and immunohistochemistry were used to evaluate the lung metastasis and determine whether the lung metastasis model of breast cancer in mice was successfully constructed.The expression levels of MMP9 and S100A8/A9 were detected to study the occurrence time of lung metastasis in different groups.3.Quantitative flow cytometry was used to detect the number and proportion of MDSCs in blood and lung of mice with lung metastasis of breast cancer.Western blot was used to detect the expression of MMP9 and NF-κB in lung tissue before lung metastasis.Result:1.Subcutaneous inoculation of 4T1 breast cancer cells in BALB/c female mice model:local tumor formation was observed 5 days after subcutaneous inoculation,and the tumor growth was in good condition,and the tumor formation rate was 100%.The inhibitory effect of Xihuang Pill on tumor in situ:(1)The score of blood stasis syndrome in mice: on the 14 th day after subcutaneous inoculation of tumor,the model group had obvious symptoms of blood stasis syndrome,the score of blood stasis syndrome in model group was 9.00±1.00,and the score of blood stasis syndrome in Xihuang Pill group was 6.00±1.00,and the score of blood stasis syndrome in adriamycin group was 7.00±1.00;Compared with the model group,the blood stasis area of tongue in Xihuang Pill group was less than 1/3,and there was no vein blood stasis,obvious bleeding and ecchymosis(P<0.05);The blood stasis syndrome of adriamycin group was more obvious,and the difference was not statistically significant(P>0.05).On the 20 th day,The score of blood stasis syndrome in model group was13.33±0.58,and the score of blood stasis syndrome in Xihuang Pill group was 10.33±1.16,and the score of blood stasis syndrome in adriamycin group was 11.33±0.58;Compared with the model group,the blood stasis syndrome of Xihuang Pill group was significantly reduced(P<0.01);There was no significant difference between Xihuang Pill group and adriamycin group(P>0.05).(2)Tumor volume: on the 8th day of inoculation,The tumor volume of model group was(42.50±6.61)mm3,and the tumor volume of Xihuang Pill group was(29.33±4.62)mm3,and the tumor volume of doxorubicin group was(26.17±5.10)mm3;Compared with the model group,the tumor volume of Xihuang Pill group and adriamycin group was smaller,and the difference was statistically significant(P<0.05).On the 14 th day after inoculation,the tumor volume of the model group was(262.50±54.44)mm3,and the tumor volume of Xihuang Pill group was(138.00±19.08)mm3,and the tumor volume of doxorubicin group was(123.17±23.51)mm3;Compared with the model group,the tumor volume of Xihuang Pill group and adriamycin group was significantly reduced(P<0.01).(3)Tumor weight: on the 17 th day of inoculation,the weight of tumor in the model group was(0.504±0.156)g,and the weight of tumor in Xihuang Pill group was(0.159±0.025)g,and the weight of tumor in adriamycin group was(0.145±0.017)g,Compared with the model group,the weight of tumor in Xihuang Pill group and adriamycin group were significantly reduced(P<0.01),but there was no significant difference between Xihuang Pill group and adriamycin group(P>0.05).(4)Tumor inhibition rate: compared with the model group,the tumor inhibition rate of adriamycin group was 53.08% on the 14 th day after inoculation,and the tumor inhibition rate of Xihuang Pill group was 52.77% on the 17 th day after inoculation.2.Lung metastasis in mice:(1)HE staining results showed that metastasis had occurred in the model group before the 14 th day,and breast cancer lung metastasis had occurred in the Xihuang Pill group and adriamycin group before the 17 th day.(2)Observation of lung metastasis in mice: compared with before metastasis,small tumor nodules were observed in the whole lung after metastasis in the three groups,and the lung became hard and dark red.The number of pulmonary metastatic nodules in the model group was(21.00±2.65),and the number of pulmonary metastatic nodules in Xihuang Pill group was(12.33±1.53),and the number of pulmonary metastatic nodules in adriamycin group was(15.33±2.52),Compared with the model group,Xihuang Pill group and adriamycin group could reduce the number of lung metastatic nodules of breast cancer in mice(P<0.05).There was no significant difference between the two groups(P>0.05).(3)The results of immunohistochemistry showed that compared with the model group,The expression of MMP-9 in lung tissue was significantly decreased in Xihuang Pill group and adriamycin group(P<0.0001);The expression of S100A8/A9 in Xihuang Pill group was significantly decreased(P<0.0001),and the expression of S100A8/A9 in adriamycin group was lower than that in model group(P<0.001).3.Expression of microenvironment related biomarkers in lung metastasis of breast cancer:(1)results of flow cytometry: the proportion of MDSCs in the blood of mice showed that the proportion of MDSCs in the model group was 51.4%,64.6% and 79.1% before,during and after metastasis,respectively;The proportion of MDSCs in Xihuang Pill group was 22.7%,53.3% and 62.7% respectively;The proportion of MDSCs in adriamycin group was 24.6%,54.5% and 67.8%,respectively;Compared with the model group,Xihuang Pill can significantly reduce the proportion of MDSCs in the blood of breast cancer mice before,during and after metastasis;At the same time,the proportion of MDSCs in Xihuang Pill group was always lower than that in adriamycin group(P<0.05).The proportion of MDSCs in the lung of mice showed that the total expression of MDSCs in the model group was 51.9%,55.4% and 79.7% before,during and after metastasis,respectively;The expression of MDSCs in Xihuang Pill group was 49.1%,61.2% and 67.0% respectively;The percentage of MDSCs in doxorubicin group was 51.4%,53.6% and 65.7% respectively.Compared with the model group,Xihuang Pill had certain inhibitory effect on MDSCs before metastasis(P<0.01);In metastasis,the inhibitory effect was poor(P>0.05);After metastasis,the proportion of MDSCs in Xihuang Pill group decreased(P<0.01).Compared with the two treatment groups,the effect of Xihuang Pill group was better than that of doxorubicin group before metastasis(P<0.05),and the effect of doxorubicin group on inhibiting the proportion of MDSCs in lung was better from metastasis to the end(P<0.05).(2)Western blot detection of protein expression: compared with the MMP9 in the model group,the expression of MMP9 in the Xihuang Pill group decreased before metastasis(P<0.05).When metastasis occurred,there was no significant difference between Xihuang Pill group and model group(P>0.05);After metastasis,the expression of MMP9 decreased(P<0.01).Compared with NF-κB in model group Compared with the control group,the expression in Xihuangwan group was significantly lower before metastasis(P<0.0001);When metastasis occurred,there was no significant difference between Xihuangwan group and model group(P>0.05);After metastasis,the expression in Xihuang Pill group decreased significantly(P<0.001).Conclusion1.Xihuang Pill not only has a certain inhibitory effect on breast cancer in situ tumor,but also can effectively improve the symptoms of blood stasis syndrome and improve the quality of life of tumor bearing mice.2.Xihuang Pill can reduce the proportion of MDSCs in blood and the accumulation of MDSCs in lung tissue before lung metastasis.3.Xihuang Pill may reduce the expression of MMP9,S100A8/A9 and NF-κB in lung tissue expression level,inhibit the metastasis ability of tumor cells,prolong the time of metastasis,reduce the degree of lung metastasis of breast cancer. |
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