| Objective: In the early stage of the research,we found that Chang Weiqing can inhibit colon cancer with oxaliplatin,and can reduce the expression of cytokines such as VEGF and TGF-β.In the course of clinical tumor treatment,chemotherapy will change the tumor microenvironment and even promote the polarization of M2 macrophages.M2 macrophages have the effect of promoting tumor progression,leading to the reaction of chemotherapy and weakening the antitumor effect of chemotherapy drugs.Because traditional Chinese medicine has the advantage of “enhanced and attenuated” by adjuvant radiotherapy and chemotherapy,on this basis,we have established Balb/c mice by inducing human peripheral blood THP-1 cells into M2 macrophages in vitro.Subcutaneous xenograft model was used to observe whether Chang Weiqing decoction has a synergistic effect on oxaliplatin-treated subcutaneous xenografts of colon cancer cells.To explore whether gastrointestinal clearing can affect the development of colon cancer after chemotherapy by inhibiting the polarization of M2 macrophages.Methods: 1.In vivo establishment of Balb/c mouse subcutaneous xenograft model（CTRL group）,respectively,given Chang Weiqing（CWQ group）,oxaliplatin intraperitoneal injection（OXA group）,and combination of Chang Weiqing and oxaliplatin（CWQ+OXA group）,the inhibitory effect of Chang Weiqing on colon cancer was observed by plotting tumor growth curve,tumor weighing,and small animal living imaging.Western blot and immunohistochemistry were used to detect the expression of P53,MMP2,KI67 and other proteins in subcutaneous xenografts of Balb/c mice.2.Western blot and immunohistochemistry were used to detect the expression of Bcl-2,VEGF,P53,MMP2,Ki67 and other proteins in subcutaneously transpl anted tumors of Balb/c mice.3.Stable M2 macrophage cell line was constructed in vitro,and M2 macrophag e marker CD163 was identified by RT-PCR and flow cytometry.4.Flow cytometry and immunofluorescence single staining were used to detect the effect of gastrointestinal clear on M2 macrophage polarization and its polarization pathway STAT6.5.To investigate the effects of gastrointestinal clearing on apoptosis and cytokine secretion of M2 macrophages by cytotoxicity assay,flow cytometry and enzymelinked immunosorbent assay.Results: 1.Tumor weight of tumor-bearing mice decreased significantly after four weeks of treatment,from large to small,followed by control group（1.58±0.87g）,Ch ang Weiqing group（0.77±0.35g）,oxaliplatin group（0.55±0.42g）,combined gr oup（0.32±0.24g）.The inhibition rate of gastrointestinal clear group was 51.27%（inhibition rate =（control group average mass-experimental group average mass）/ control group average mass × 100%）,oxaliplatin group inhibition rate was 65.19%,combined group inhibition The tumor rate is 79.75%;small ani mal in vivo imaging showed that the fluorescence intensity of the combined group was 3.68×1010±9.10×109[p/s/cm2/sr]/[μW/cm2] compared with the oxalipl atin group is reduced to 5.06×109 ± 4.32×109 [p/s/cm2/sr] / [μW/cm2].2.The results of Western blotting showed that the expression of p53 in the Chang Weiqing group,oxaliplatin group and the combined group was significantly higher than that in the control group（P<0.05）,and there was no significant change in the expression of Bcl-2,VEGF and MMP2（P >0.05）;Immunohistochemistry showed that Ki67 expression was significantly decreased in the combined group compared with the control group（P<0.05）.3.RT-PCR results showed that the M2 macrophage markers IL10（26.81±9.68）,Arg-1（3.03±0.73）and CD163（16.91±2.79）were significantly expressed in the M2 group compared with the M0 group.Elevation;flow cytometry results showed that CD163-positive cells after polarization increased from（2.55±2.44%）to（82.45±5.36%）compared with uninduced THP-1.Together,both demonstrate successful induction of stable M2 macrophages in vitro.4.Flow cytometry results showed that the control group（82.77±4.44%）,the gastrointestinal group（76.13±5.43%）,the oxaliplatin group（64.67±7.62%）,and the combined group tumor-bearing mice（51.47）±3.84%)M2 type macrophage markers F4/80,CD163 double positive proportion decreased in sequence;immunofluorescence single staining and Western blotting results showed that compared with the control group,the combined group M2 macrophage marker CD163 The expression of the cells decreased significantly（P<0.05）.The results of immunofluorescence single staining showed that the fluorescence intensity of STAT6 in the M2 macrophage polarization pathway was significantly decreased compared with the control group（P<0.001）.5.The results of cytotoxicity and flow cytometry showed that gastrointestinal clearing had certain killing and apoptosis effects on M2 macrophages in a dose-dependent manner.The results of enzyme-linked immunosorbent assay showed that compared with the control group,the combination was compared.After treatment,VEGF decreased from 51.95±11.32 pg/ml to 38.96±3.59 pg/ml（P<0.05）.Compared with oxaliplatin group,TGF-β decreased from 601.50±39.09 pg/ml to 457.80± compared with oxaliplatin group.40.11 pg/ml（P<0.05）.Conclusion: 1.Chang Weiqing can synergize with oxaliplatin to inhibit subcutaneous tumor growth in Balb/c mice 2.Chang Weiqing can inhibit the polarization of M2 macrophages in the tumor microenvironment;3.Intestinal clearing promotes the apoptosis of M2 macrophages and affects the secretion of cytokines VEGF and TGF-β,and promotes the sensitivity of oxaliplatin. |
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