| Background:1.Chemotherapy resistance is an important factor affecting the prognosis of gastric cancerAccording to the data published by the World Health Organization in 2020,gastric cancer is the fifth most common malignant tumor and the fourth most cancer-related death cause in the world.Chemotherapy-based comprehensive treatment is the main treatment method,but the 5-year survival rate of gastric cancer patients is still less than 30%.Chemotherapy resistance may be an important influencing factor.Therefore,it is important to explore the key mechanism of chemoresistance and seek effective drugs.2.Cancer-associated fibroblasts(CAFs)promote the formation of chemoresistance in gastric cancer,and M2 type tumor-associated macrophages(TAMs)is related to immunosuppression and chemoresistanceCAFs are the most important stromal cells in the tumor microenvironment.Study has shown that chemotherapy can induce the senescence of CAFs while killing cancer cells,and the senescence-associated secretory phenotype(SASP)can promote the occurrence of chemotherapy resistance.TAMs are important immune cells in tumor microenvironment.M1 type TAMs have anti-tumor effect and mainly exist in the early stage of tumor,while M2 type TAMs have a tumorigenic effect,mainly in the middle and advanced stage of tumor.At the same time,M2 type TAMs have the function of immunosuppression and promoting chemoresistance.Various cells in the tumor microenvironment interact and influence each other.So whether senescent CAFs can induce the polarization of TAMs cells to M2 type and thus enhance the immunosuppression and chemoresistance remains to be studied.3.Fuzheng Jiedu formula is an effective prescription for improving the efficacy of chemotherapy for gastric cancer,and its mechanism needs to be further clarifiedFuzheng Jiedu formula(developed as Fuzheng Jiedu oral liquid in our hospital)is an classical hospital preparation used by the oncology department of our hospital to treat gastric cancer.Previous studies have shown that Fuzheng Jiedu formula can increase the completion rate of chemotherapy and improve the 5-year survival rate of patients with stage Ⅲ gastric cancer by 9.1%.In addition,animal experiments showed that Fuzheng Jiedu formula combined with chemotherapy had a significantly better therapeutic effect on gastric cancer model mice than the chemotherapy group alone,which mechanism may be related to regulating the polarization of TAMs in tumor microenvironment from M2 to M1,and reducing the expressions of immunosuppressive factors(IL-10,IL-13,and TGF-β).However,whether Fuzheng Jiedu formula can improve immune suppression and chemotherapy resistance by regulating senescent CAFs remains to be determined.To sum up,this study is intended to explore the new mechanism of Fuzheng Jiedu formula in improving immune suppression and chemotherapy resistance with"gastric cancer cells-senescent CAFs-TAMs",and solve the bottleneck problem of chemotherapy resistance in clinical treatment of gastric cancer.Purposes:1.This study is aimed to observe the effect of senescent CAFs on the immune suppression and chemoresistance using the senescent CAFs model in vitro,and explore the mechanism of Fuzheng Jiedu formula based on the p38MAPK/MK2 pathway and MeCP2/NOX4/PKM2 pathway.2.Another purpose of this study is to observe the effects of senescent CAFs and Fuzheng Jiedu formula on chemotherapy efficacy using the mice model of senescent CAFs/cancer cells-mixed tumor,and verify that Fuzheng Jiedu formula can improve the immune suppression and chemotherapy resistance by regulating "gastric cancer cells-senescent CAFs-TAMs" in vivo.Methods:1.Study on the high expression of SASP of senescent CAFs and the intervention effect of Fuzheng Jiedu formulaWe applied the senescent CAFs model that has been built by our team previously.Based on ELISA,RT-PCR,and immunofluorescence technology,IL-8,TGF-β,and WNT16B were tested,and the intervention effect of Fuzheng Jiedu formula was observed.2.Study on the polarization of M2 type TAMs induced by conditioned medium(CM)of senescent CAFs and the intervention effect of Fuzheng Jiedu formulaWe collected the CM of senescent CAFs and used it to culture TAMs cells.The expression level of CD206,a marker of M2 type TAMs,was detected by flow cytometry,RT-PCR,and immunofluorescence.At the same time,the intervention effect of Fuzheng Jiedu formula on the polarization of M2 type TAMs was observed.In addition,RT-PCR and Western blot were used to explore the relevant mechanism based on p38MAPK/MK2 pathway.3.Study on the chemotherapy resistance of MKN-45 gastric cancer cells promoted by senescent CAFs-CM and the intervention effect of Fuzheng Jiedu formulaWe collected the CM of senescent CAFs and used it to culture MKN-45 cells.The effects of senescent CAFs-CM on chemosensitivity of gastric cancer cells were investigated by CCK-8 assay,wound healing assay,and colony formation assay.The intervention effect of Fuzheng Jiedu formula on chemotherapy resistance caused by senescent CAFs-CM was observed,and its mechanism was explored based on MeCP2/NOX4/PKM2 pathway by RT-PCR and Western blot.4.Study on the anti-chemoresistance and TAMs-regulating effects of Fuzheng Jiedu formula on the mice model of senescent CAFs/cancer cells-mixed tumorA total of twenty-five CB17-SCID mice were inoculated with gastric cancer cells alone or mixed with senescent CAFs/gastric cancer cells,and intervened by 5-FU and/or Fuzheng Jiedu formula.The specific grouping is as follows:the control group,the 5-FU group,the mixed-inoculation group,the mixed inoculation+5-FU group,the mixed inoculation+5-FU+Fuzheng Jiedu formula group.After 14 days of intervention,samples were taken and the following indicators were tested:(1)Tumor weight,tumor volume and tumor inhibition rate,and the expression of MeCP2,NOX4,PKM2 by RT-PCR and Western blot.(2)Using ELISA and RT-PCR methods to detect the expression level of SASP factors IL-8,TGF-β,and WNT16B in the mouse serum and tumor tissue.(3)Use flow cytometry and immunofluorescence methods to detect the expression of M2 type TAMs in mouse spleen and tumor tissue.Combining RT-PCR and Western blot methods,detect the mRNA and protein expression levels of the p38MAPK/MK2 pathway.Results:1.Study on the high expression of SASP of senescent CAFs and the intervention effect of Fuzheng Jiedu formulaThe results of ELISA,RT-PCR,and immunofluorescence showed that:(1)Compared with the control group,the SASP related factors including IL-8,TGF-β,and WNT16B in the senescent CAFs group,were at a high expression level(P<0.05).(2)Compared with the blank serum group,the serum of Fuzheng Jiedu formula could significantly inhibit the SASP related factor including TGF-β and WNT16B in senescent CAFs(P<0.05).The expression level of IL-8 was lower than that of the blank serum group,but the difference was not statistically significant(P>0.05).2.Study on the polarization of M2 type TAMs induced by senescent CAFs-CM and the intervention effect of Fuzheng Jiedu formula(1)The results of flow cytometry,immunofluorescence,and RT-PCR showed that:①Compared with the control group,the M2 type TAMs cell marker,CD206,in the senescent CAFs-CM group was at a high expression level,with a statistically significant difference(P<0.05),suggesting that senescent CAFs can promote the polarization of TAMs cells towards M2 type.②Compared with the blank serum group,the serum of Fuzheng Jiedu formula could reduce the expression level of CD206(the cell marker of M2 type TAMs),and improve the polarization of M2 type TAMs caused by senescent CAFs.(2)The results of RT-PCR and Western blot showed that compared with the blank serum group,the serum of Fuzheng Jiedu formula could reduce the expression levels of p38MAPK and MK2,with a statistically significant difference(P<0.05).It is suggested that the mechanism of Fuzheng Jiedu formula in improving the polarization of M2 type TAMs induced by senescent CAFs may be achieved by regulating the p38MAPK/MK2 pathway.3.Study on the chemotherapy resistance of MKN-45 gastric cancer cells promoted by senescent CAFs-CM and the intervention effect of Fuzheng Jiedu formula(1)The results of CCK-8 assay,wound healing assay,and colony formation assay showed that:① Compared with the 5-FU group,the cell inhibition rate was significantly increased,and the number of cloned cells and rate of cell migration were significantly reduced(P<0.05)in the senescent CAFs+5-FU group,which indicates that senescent CAFs can reduce the sensitivity of tumor cells to chemotherapy and promote the formation of chemotherapy resistance.②Compared with the blank serum group,the serum of Fuzheng Jiedu formula can increase the rate of cell inhibition,reduce the number of cell clones and rate of cell migration(P<0.05),which indicates that Fuzheng Jiedu formula can inhibit the proliferation and migration ability of tumor cells,improve the chemotherapy resistance caused by senescent CAFs.(2)The results of RT-PCR and Western blot showed that compared with the blank serum group,the serum of Fuzheng Jiedu formula could reduce the expression levels of MeCP2,NOX4,and PKM2,with a statistically significant difference(P<0.05).It is suggested that the mechanism of Fuzheng Jiedu formula in improving chemotherapy resistance caused by senescent CAFs may be achieved by regulating the MeCP2/NOX4/PKM2 pathway.4.Study on the anti-chemoresistance and TAMs-regulating effects of Fuzheng Jiedu formula on the mice model of senescent CAFs/cancer cells-mixed tumor4.1 Study on the effect of senescent CAFs on chemosensitivity and the intervention effect of Fuzheng Jiedu formula(1)The results of tumor weight,volume,and inhibition rate showed that:①The tumor weight and volume of the group of inoculating gastric cancer cell alone(the control group,the 5-FU group)were smaller than those of the mixed inoculation group(the mixed-inoculation group,the mixed inoculation+5-FU group),and the difference was statistically significant(P<0.05),indicating that senescent CAFs have a tumor promoting effect.②Compared to the 5-FU group,the mixed inoculation+5-FU group showed a significant increase in tumor weight and volume(P<0.05),and a significant decrease in tumor inhibition rate,indicating that senescent CAFs can cause chemotherapy resistance.③Compared with the mixed inoculation+5-FU group,the tumor weight and volume of the mixed inoculation+5-FU+Fuzheng Jiedu formula group were significantly reduced(P<0.05),and the tumor inhibition rate was significantly increased(P<0.05).(2)The results of RT-PCR and Western blot showed that:Compared to the mixed inoculation+5-FU group,the expression levels of MeCP2,NOX4,and PKM2 in tumor tissue of the mixed inoculation+5-FU+Fuzheng Jiedu formula group decreased significantly(P<0.05).It was confirmed from the vivo experiments that the mechanism of Fuzheng Jiedu formula to improve chemotherapy resistance may be achieved through regulating the MeCP2/NOX4/PKM2 pathway.4.2 Detection of SASP related factors and the intervention effect of Fuzheng Jiedu formulaThe results of ELISA and RT-PCR showed that:(1)In the serum,compared to the 5-FU group,TGF-β and IL-8 in the mixed inoculation+5-FU group are at a high expression level,and the difference is statistically significant(P<0.05).In the tumor tissue,compared to the 5-FU group,IL-8,TGF-β and WNT16B in the mixed inoculation+5-FU group are at a high expression level(P<0.05).(2)Compared with the mixed inoculation+5-FU group,IL-8,TGF-β and WNT16B in the serum and tumor tissue of the mixed inoculation+5-FU+Fuzheng Jiedu formula group are significantly reduced(P<0.05).4.3 Study on the expression of M2 type TAMs and the intervention effect of Fuzheng Jiedu formula(1)The results of flow cytometry and immunofluorescence showed that:①Compared to the group of inoculating gastric cancer cell alone(the control group,the 5-FU group),the expression level of CD206 in the spleen in the mixed-inoculation groups(the mixed-inoculation group,the mixed inoculation+5-FU group,the mixed inoculation+5-FU+Fuzheng Jiedu formula group)was significantly increased,and the difference was statistically significant(P<0.05).②Compared with the mixed inoculation+5-FU group,CD206 in the spleen and tumor tissue in the mixed inoculation+5-FU+Fuzheng Jiedu formula group was significantly reduced(P<0.05).(2)The results of RT-PCR and Western blot showed that:Compared with the mixed inoculation+5-FU group,the expression levels of p38MAPK and MK2 in tumor tissue of the mixed inoculation+5-FU+Fuzheng Jiedu formula group decreased significantly(P<0.05),which was confirmed that the mechanism of Fuzheng Jiedu formula in improving the polarization of TAMs cells to M2 type may be achieved through regulating the p38MAPK/MK2 pathway.Conclusions:1.Fuzheng Jiedu formula can downregulate the expression of SASP factors(WNT16B,IL-8,TGF-β).2.Fuzheng Jiedu formula can improve the polarization of M2 type TAMs caused by senescent CAFs,alleviate immune suppression,and its mechanism is related to regulating the p38MAPK/MK2 pathway.3.Fuzheng Jiedu formula can alleviate the chemotherapy resistance of gastric cancer cells to 5-FU caused by senescent CAFs,which mechanism is related to regulating the MeCP2/NOX4/PKM2 pathway.To sum up,Fuzheng Jiedu formula can improve immune suppression and chemotherapy resistance by regulating the "gastric cancer cells-senescent CAFsTAMs cells" in the tumor microenvironment. |
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