Research On The Effect And Molecular Mechanism That Ziyin Huatan Recipe Inhibits Angiogenesis Of Gastric Cancer Through Regulating Macrophage Polarization

Objective: As one of the most common malignant tumors of the digestive tract in the world,tumor invasion,metastasis,and angiogenesis are important factors for the rapid development of gastric cancer.In recent years,the tumor environment(TME)has been one of the highlights in the field of gastric cancer therapy.Studies have shown that tumor-associated macrophages(TAMs)in TME play a crucial role in the development of tumors.Macrophages show heterogeneity in different micro-environments.According to surface markers and secreted cytokines,they can be divided into two subgroups,one is named as M1 type macrophages(classically activated type)and the other is named as M2 type macrophages(Alternatively activated type).Exosomes play an important role in signal transmission and material transmission between surrounding normal cells and tumor cells in TME.Based on the theory of phlegm syndrome of gastric cancer,Chinese medicine compound named Ziyin Huatan Recipe(ZYHT)was developed by the Department of Traditional Chinese Medicine of Changzheng Hospital for advanced gastric cancer.ZYHT has a positive clinical effect improving patients’ quality of life and prolonging survival rate.Preliminary experimental studies have shown that ZYHT has inhibitory effects in gastric tumor cells proliferation and angiogenesis,but we have not clearly identified its specific mechanism.This study intends to explore whether ZYHT can inhibit gastric cancer angiogenesis by regulating the polarization of macrophages through series of experiments in vivo and in vitro.And study whether exosomes play a key role in regulating it,and its possible regulatory mechanism.The study provides data support for elucidating the scientific principles of effective prescriptions for the treatment of gastric cancer.Methods:Animal experiment: We establish a subcutaneous transplantation tumor model of MFC(Mouse Fore-stomach Carcinoma Cell)tumor-bearing mice,and randomly divide615 mice into different groups including blank control group;low,medium and high dose groups of ZYHT;macrophages-removal group and the high dose of ZYHT combined with the macrophages-removal group.1.We use immunohistochemistry(IHC)and immunofluorescence(IF)experiments to observe the effect of ZYHT on the micro-vessel density(MVD)of tumor tissue in tumor-bearing mice.2.Explore the regulatory effect of ZYHT on immune factors in blood of mice by using cytokine antibody chip,and screening the cytokines related to macrophage polarization.3.Observe the effect of ZYHT on the abundance of M2 macrophages in peripheral blood and neoplastic tissues of tumor-bearing mice through FCM,IHC,and IF experiments.Cell experiment: This study uses Bone Marrow Derived Macrophages(BMDMs)and the macrophage cell line RAW264.7 as the research objects.1.Use Western blot(WB)and q-PCR to detect the effects of ZYHT on M2 type of BMDMs and RAW264.7 by screening the expression of STAT3,PPAR-γ,SOCS1,STAT1,JAK2 and SOCS3 which achieved by the chip analysis.2.We set up groups including control group;supernatant from M2 type of BMDMs group;exosomes inhibitor GW4869 group;supernatant from M2 type of BMDMs combined with GW4869 group and exosomes derived from M2 type of BMDMs group.By using WB,ELISA and Transwell methods,we detect the expression levels of angiogenesis-related factors VEGFA,VEGFC,VEGFR2 and b FGF in Murine gastric cancer cell MFC and human gastric cancer cell MKN-45 and cell supernatant in each group.3.Extract exosomes from M2 type of BMDMs after adding low,medium and high doses of ZYHT.Transwell experiment was used to observe the effect of the exosomes on the migration and invasion of MFC and MKN-45 cells.Through tube formation assay of HUVECs,we observe the influence of exosomes co-cultured with MFC and MKN-45 cells by ZYHT on angiogenesis.Use WB,ELISA and q-PCR to observe the changes in the protein expression and secretion of VEGFA,VEGFC,VEGFR2 and b FGF in MFC and MKN-45 cells and cell supernatants after using exosomes extracted from M2 type of BMDMs intervened by ZYHT.4.Use WB to detect the protein expression of p-65,IκBαand My D88 related to angiogenesis and VEGF gene transcription after extracting exosomes derived from M2 type of BMDMs intervened by ZYHT.Results:Animal experiments: 1.Immunohistochemistry and immunofluorescence assay showed that ZYHT reduced the microvessel density in the tumor tissue of the subcutaneous transplanted tumor and inhibited the formation of microvessels in the tumor.2.After analyzing the peripheral blood of mice with cytokine antibody chip,it was found that secretion of pro-inflammatory factors such as IL-6,TNF-α,IL-1β related to M1 macrophage was higher than the control group,and anti-inflammatory factors such as TGF-β1 and IL-10 related to M2 type of macrophage were lower than the control group.3.Flow cytometry,IHC and IF assay showed that ZYHT reduced the expression of reduced both M0 type of macrophage marker F4/80 and M2 type of macrophage marker CD206 in tumor tissues of tumor-bearing mice.Cell experiment: 1.The results of WB and q-PCR showed that ZYHT inhibited the protein expression of STAT3,PPAR-γ and SOCS1,and up-regulated the protein expression of STAT1,JAK2 and SOCS3 in comparison with NC group.2.The results of WB,ELISA and Transwell assay showed that M2 type of BMDMs inhibited the expression of VEGFA,VEGFC,VEGFR2 and b FGF,of which exosomes derived from M2 type of BMDMs played a major role.3.The results of the Transwell assaay showed that with the continuous increasing of the concentration,the exosomes extracted after intervened by ZYHT on M2 type of BMDMs had increasingly inhibited the migration and invasion of gastric cancer MFC and MKN-45 cells.The tube formation assay of HUVECs proved that the exosomes extracted from M2 type of BMDMs by ZYHT significantly inhibited the tube formation ability of HUVECs.The results of WB,ELISA and q-PCR showed that exosomes extracted from M2 type of BMDMs intervened by ZYHT reduced the expression of VEGFA,VEGFC,VEGFR2 and b FGF in MFC and MKN-45 cells and cell supernatants.4.WB results showed that exosomes extracted from M2 type of BMDMs intervened by ZYHT inhibited the expression of p-65 and My D88 in MFC and MKN-45 cells,and up-regulated the expression of IκBα.Conclusion: 1.M2 macrophage polarization plays an important role in the course of oncogenesis and progression in gastric cancer.The anti-tumor effect in gastric cancer of ZYHT was related to the inhibition of M2 macrophage polarization,which might be carried out by inhibiting the SOCS1/STAT3/PPAR-γ signaling pathway and activating the STAT1/JAK2/SOCS3 signaling pathway.2.Macrophage-derived exosomes promoted the migration,metastasis and angiogenesis in gastric cancer.Zyin Huatan Recipe can exert its effect on the invasion,metastasis and angiogenesis of gastric cancer by inhibiting the M2 polarization of macrophages.The mechanism of action is related to the exosomes secreted by macrophages.