| Purpose:The co-culture model of CAF and MDA-MB-231 cells was established by using the Transwell system.The effects of serum containing Danzhi Xiaoyao Powder on the growth and metastasis of breast cancer MDA-MB-231 cells by regulating CAF and its molecular mechanism were discussed.It is revealed that Danzhi Xiaoyao Powder may inhibit breast cancer by regulating CAF pathway.Material and method:45 SPF male SD rats(250±10)g,after 7 days of adaptive breed ing,the rats were randomly divided into three groups according to the random number table method,the rats were randomly divided into three groups: namely the blank group,Danzhi Xiaoyao Powder group,Xihuang Pill group,each group with 15 rats.The equivalent dose of Danzhi Xiaoyao Powder is 8.99 g·kg-1 in rat,the equivalent dose of Xihuang Pill is 0.55g·kg-1.The blank group was given physiological saline.Each group was administered continuously for 7 days,once a day.One hour after the last dose,anesthesia was performed and blood was taken from the abdominal aorta.After standing at room temperature for 2 hours,the serum was separated using a low-temperature centrifuge.The blood was centrifuged for 10 minutes,at 4°C,1500 rpm·min-1.Collect the same serum and inactivate it at 56℃ for 30 min.The inactivated serum was dispensed into 1.5 m L EP tubes and stored at-80°C.Conditioned medium was used to induce the conversion of HMF cell to CAF,and the expression ofα-SMA was detected by immunofluorescence to identify the model.CCK-8 method was used to detect the effects of serum containing Danzhi Xiaoyao Powder on the proliferation of MDA-MB-231 cells by regulating CAF at 12 h,24h,48 h and 72 h.Cell invasion test was used to detect the effects of serum containing Danzhi Xiaoyao Powder on the invasion ability of MDA-MB-231 cells by regulating CAF.Cell adhesion test was used to detect the effects of serum containing Danzhi Xiaoyao Powder on the adhesion of MDA-MB-231 cells by regulating CAF.Flow cytometry was used to analyse cell apoptosis and cell cycle of MDA-MB-231 cells by regulating CAF with serum containing Danzhi Xiaoyao Powder.Western blot was used to detect the effects of serum containing Danzhi Xiaoyao Powder on the expression of HIF-1α,MCT1,MCT4,Caveolin-1,CXCR4,CD47,MIF,IL-6,IL-8 and collagen Ⅰ in MDA-MB-231 cells by regulating CAF.Results:1.Identification of CAFImmunofluorescence results showed that α-SMA was rarely expressed in HMF cell and highly expressed in CAF(P<0.01).2.Effects of serum containing Danzhi Xiaoyao Powder on proliferation of breast cancer MDA-MB-231 cells by regulating CAFCCK-8 results showed that the MDA-MB-231 cells proliferation was most significantly suppressed after 72 h of intervention with 15% serum containing Danzhi Xiaoyao Powder by regulating CAF(P<0.01).Therefore,the choice of 15% serum containing Danzhi Xiaoyao Powder for 72 h will be the next experimental condition.3.Effects of serum containing Danzhi Xiaoyao Powder on invasion of breast cancer MDA-MB-231 cells by regulating CAFThe results of the Cell invasion experiment showed that the number of cells that penetrated the basement membrane of each group was as follows: the tumor group(24.80±2.51),co-culture group(68.13±6.99),Danzhi Xiaoyao Powder group(42.33±4.32),Xihuang Pill group(30.60±3.33).Compared with the tumor group,the invasive ability of breast cancer cells in co-culture group was significantly enhanced(P<0.01);compared with co-culture group,the invasive ability of breast cancer cells in Danzhi Xiaoyao Powder group was significantly reduced(P<0.01);compared with co-culture group,the invasion ability of breast cancer cells in Xihuang Pill group was significantly reduced(P<0.01).4.Effects of serum containing Danzhi Xiaoyao Powder on adhesion of breast cancer MDA-MB-231 cells by regulating CAFThe results of cell adhesion experiment showed that the number of cell adhesion in each group was: the tumor group(25.00±1.97),co-culture group(63.67±3.80),Danzhi Xiaoyao Powder group(49.80±3.86),Xihuang Pill group(28.93±2.27).Compared with the tumor group,the cell adhesion ability of co-culture group was significantly enhanced(P<0.01);compared with co-culture group,the cell adhesion ability of Danzhi Xiaoyao Powder group was significantly decreased(P<0.01);compared with co-culture group,the cell adhesion ability of Xihuang Pill group was significantly decreased(P<0.01).5.Effects of serum containing Danzhi Xiaoyao Powder on cell apoptosis of breast cancer MDA-MB-231 cells by regulating CAFThe results showed that compared with the tumor group,the apoptosis rate of co-culture group was significantly decreased(P<0.01);compared with co-culture group,the apoptosis rate of Danzhi Xiaoyao Powder group was significantly increased(P<0.01);compared with co-culture group,the apoptosis rate of Xihuang Pill group was increased significantly(P<0.01).6.Effects of serum containing Danzhi Xiaoyao Powder on cell cycle of breast cancer MDA-MB-231 cells by regulating CAFThe results showed that the cell ratio of the tumor group in G1 phase was(55.88±1.29)%;co-culture group(39.30±1.88)%,compared with the tumor group,the difference was statistically significant(P<0.01).The cell ratio of Danzhi Xiaoyao Powder group in G1 phase was(44.27±0.64)%,compared with co-culture group,the difference was statistically significant(P< 0.05).The cell ratio of Xihuang Pill group in G1 phase was(50.05±2.60)%,compared with co-culture group,the difference was statistically significant(P<0.01).The cell ratio of the tumor group in S phase was(30.25±0.53)%.The cell ratio of co-culture group in S phase was(46.86±0.62)%,compared with the tumor group,the difference was statistically significant(P<0.01).The cell ratio of Danzhi Xiaoyao Powder group in S phase was(41.46±0.88)%,compared with co-culture group,the difference was statistically significant(P<0.01).The cell ratio of Xi huang Pill group in S phase was(34.96±0.76)%,compared with co-culture group,the difference was statistically significant(P<0.01).7.Effects of serum containing Danzhi Xiaoyao Powder on expression of HIF-1α、MCT1、MCT4、Caveolin-1 in breast cancer MDA-MB-231 cells by regulating CAFWestern blot results showed that compared with the tumor group,the expressions of HIF-1α、MCT1、MCT4 protein in co-culture group were both increased(P<0.01),and the expression level of Caveolin-1 protein was decreased(P<0.01);compared with co-culture group,the expressions of HIF-1α、MCT1、MCT4 protein in Danzhi Xiaoyao Powder group were both decreased(P<0.01),and the expression level of Caveolin-1 protein was increased(P<0.01);compared with co-culture group,the expressions of HIF-1α、MCT1、MCT4 protein in Xihuang Pill group were both decreased(P<0.01),and the expression level of Caveolin-1protein was increased(P<0.01).8.Effects of serum containing Danzhi Xiaoyao Powder on expression of IL-6、IL-8、CD47、MIF in breast cancer MDA-MB-231 cells by regulating CAFWestern blot results showed that compared with the tumor group,the expressions of IL-6、IL-8、CD47、MIF protein in co-culture group were both increased(P<0.01);compared with co-culture group,the expressions of IL-8、CD47、MIF protein in Danzhi Xiaoyao Powder group were both decreased(P<0.01),IL-6 protein in Danzhi Xiaoyao Powder group was decreased(P<0.05);compared with co-culture group,the expressions of IL-6、IL-8、CD47、MIF protein in Xihuang Pill group were both decreased(P<0.01).9.Effects of serum containing Danzhi Xiaoyao Powder on expression of CXCR4、collagen Ⅰin breast cancer MDA-MB-231 cells by regulating CAFWestern blot results showed that compared with the tumor group,the expressions of CXCR4、collagen Ⅰ protein in co-culture group were both increased(P<0.01);compared with co-culture group,the expressions of CXCR4、collagen Ⅰ protein in Danzhi Xiaoyao Powder group were both decreased(P<0.01);compared with co-culture group,the expressions of CXCR4、collagen Ⅰ protein in Xihuang Pill group were both decreased(P<0.01).Conclusion:1.Serum containing Danzhi Xiaoyao Powder can regulate CAF to inhibit the proliferation,invasion and adhesion of MDA-MB-231 cells,and promote MDA-MB-231 cells apoptosis and block MDA-MB-231 cells in G1 phase.2.Serum containing Danzhi Xiaoyao Powder can decrease the expressions of HIF-1α、MCT1、MCT4、CD47、MIF、CXCR4、IL-6、IL-8、collagen Ⅰ,increase the expression of Caveolin-1protein,this reminds that serum containing Danzhi Xiaoyao Powder can inhibit breast cancer MDA-MB-231 cells growth and metastasis by regulating CAF,this may be related to the regulation of abnormal lactic acid metabolism,changes in inflammatory factors,ECM,EMT imbalance in hypoxic environment. |
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