Study On The Effects And Mechanism Of Jianpi Yangzheng Xiaozheng Decoction By Regulating The Exosomal PD-L1 Mediated Expansion Of MDSCs In Affecting The Recurrence And Metastasis Of Gastric Cancer

Background:Gastric cancer is one of the most common cancers worldwide and the fourth leading cause of cancer-related deaths.Cancer cells secrete extracellular vesicles,which can regulate the function of immune cells and induce changes in the surrounding environment to form a microenvironment that supports tumor growth and metastasis.Compared with soluble PD-L1 or other forms of PD-L1,exosomal PD-L1 is more stable and has stronger immunosuppressive effect.Although the role of PD-L1 in immune regulation and immune checkpoint inhibitor therapy has been well elucidated,further research is needed to understand the role of exosomal PD-L1 in regulating immune suppression in the gastric cancer tumor microenvironment.Jianpi Yangzheng Xiaozheng decoction is an empirical Chinese herbal formula that can significantly prolong the survival of patients with advanced cancer.Previous studies have shown that Chinese herbal formula containing the main components of Jianpi Yangzheng Xiaozheng decoction can exert anti-cancer effects by reducing PD-L1 expression in gastric cancer.Modified Jianpi Yangzheng Xiaozheng decoction can regulate gastric cancer extracellular vesicles to inhibit tumor growth and metastasis.Therefore,it is speculated that Jianpi Yangzheng Xiaozheng decoction has a regulatory effect on exosomal PD-L1 in gastric cancer.Objectives:Firstly,to observe the expression differences of PD-L1 in serum and exosomes of gastric cancer patients,and the correlation between exosomal PD-L1 and TNM staging of gastric cancer patients.Secondly,to study the effect and regulatory role of Jianpi Yangzheng Xiaozheng decoction on exosomal PD-L1 in gastric cancer,and to explore its possible mechanisms,providing experimental basis for the clinical aplication of this formula.Methods:Clinical Trial1.The study included 92 gastric cancer patients(including stages ⅠA-Ⅳ)and 71 healthy volunteers(NC)who met the criteria,sourced from the physical examination center and oncology department of Jiangsu Provincial Hospital of Chinese Medicine from March 2020 to September 2021.The PD-L1 concentration in serum and exosomes was detected using a human PD-L1 ELISA kit.2.The study included 30 patients who met the criteria,sourced from the physical examination center and oncology department of Jiangsu Provincial Hospital of Chinese Medicine from May 2020 to March 2022.Among them,15 patients were treated with Jianpi Yangzheng Xiaozheng decoctionon the basis of FOLFIRI chemotherapy(treatment group),while the other 15 patients received only FOLFIRI chemotherapy(control group).Fresh whole blood(2 mL)was collected from patients with spleen-stomach qi deficiency syndrome in the control and treatment groups using an anticoagulant tube.The supernatant plasma of 1mL whole blood was taken for ELISA detection.1 mL of fresh whole blood was stored in a refrigerator at 4℃for flow cytometric detection of MDSCs on the same day.Network PharmacologyEffective active ingredients and targets of Jianpi Yangzheng Xiaozheng Decoction were collected from the TCMSP database.Gastric cancer gene expression profile data was downloaded from the Gene Expression Omnibus(GEO)database.Gastric cancer targets were downloaded from the DisGeNET and GeneCards databases and merged with Predicted targets.The intersection of drug target and disease gene was obtained by Venn diagram.The core targets were analyzed and processed using the STRING database to obtain the relevant interaction relationships among these proteins,which were then imported into Cytoscape 3.7.1 software to construct a PPI network.The connectivity degree of the PPI network was analyzed using the CytoNCA plugin of Cytoscape 3.7.2,and core targets were identified.GO molecular function and KEGG pathway enrichment analyses were performed on the core targets using the DAVID 6.8 tool.Cell ExperimentThe toxicity of Jianpi Yangzheng Xiaozheng Decoction on gastric cancer cells was detected using the CCK8 method.The exosomes were extracted by ultracentrifugation,the morphology of exosomes was observed by transmission electron microscope,and the concentration and particle size distribution of exosomes were detected by nano-particle tracking analysis.A stable transfected gastric cancer cell line was established with PD-L1 overexpression(OE).Mouse bone marrow cells were induced to differentiate into MDSCs.The exosomes extracted from the supernatant of gastric cancer MFC cells(NC-exo,100 μg/mL)and the exosomes from MFC cells overexpressing PD-L1(PD-L1-OE-exo,100 μg/mL)were added to the culture medium respectively.After incubation at 37℃ for 4 days,flow cytometry was used to detect changes in the proportion of MDSCs induced by exosomal PD-L1 in C57BL/6 mice.Animal experimentMice from 615 line(6-8 weeks old,weighing 18-22g)were injected with 2×106 MFC cells in the right axilla.After 7 days,the mice were randomly divided into four groups:model group,low-dose group of Jianpi Yangzheng Xiaozheng Decoction(13.20g/kg,O.1mL/10g oral gavage),high-dose group of Jianpi Yangzheng xiaozheng decoction(26.39g/kg,0.1mL/10g oral gavage),and the exosome secretion inhibitor GW4869 group(2.5μg/g intraperitoneal injection).On the 21st day,blood and tumor tissues were collected.ELISA was used to detect the level of exosomal PD-L1,and immunofluorescence was used to detect the expression of MDSCs and PD-L1 in tumor tissue.The histopathology was observed by hematoxylin and eosin staining,the proportion of MDSCs in tumor tissue and blood was detected by flow cytometry,and the level of immunosuppressive inflammatory factors was detected by ELISA.Results:1.There was no significant difference in serum PD-L1 levels between gastric cancer patients and healthy controls,but the plasma exosomal PD-L1 levels were significantly higher in the patient group(P<0.05).2.The level of exosomal PD-L1 in plasma was significantly higher than that in serum(P<0.05),and the PD-L1 level in exosomes derived from gastric cancer was positively correlated with clinical stage(the later the TNM stage,the higher the PD-L1 level).3.The Jianpi Yangzheng Xiaozheng Decoction" effectively reduced the abundance of tumor-derived exosomal PD-L1(P<0.05)and the expression level of PMN-MDSCs in the peripheral blood of ⅡA-ⅢB stage gastric cancer patients(P<0.05).4.In various types of tumor-infiltrating immune cells from the TISIDB database,the expression of MDSCs was positively correlated with that of PD-L1.There was a positive correlation between exosomal PD-L1 and polymorphonuclear MDSCs in advanced gastric cancer patients.5.Network pharmacology results indicated that PD-L1 might be a key target of "JianPi Yangzheng Xiaozheng Decoction" for treating gastric cancer,and IL-6 was a key target in this formula for gastric cancer.6.The inhibitory effect of Jianpi Yangzheng Xiaozheng Decoction on PD-L1 in MKN74 cells and tumor-bearing mice might be mediated through the IL-6/JAK2/STAT3 pathway.7.Jianpi Yangzheng Xiaozheng Decoction reduced the expression level of tumor-derived exosomal PD-L1 in MFC cells and a xenograft gastric cancer mouse model.8.Exosomes extracted from the supernatant of MFC cells significantly induced the differentiation of MDSCs when co-cultured with bone marrow cells from C57BL/6 mice(P<0.001).The exosomes derived from MFC cells overexpressing PD-L1 and co-cultured with bone marrow cells showed that PD-Ll-OE-exo mediated the expansion of MDSCs(P<0.05).9.High dose Jianpi Yangzheng Xiaozheng Decoction could reduce the proportion of CD11b+Gr-1+MDSCs(P<0.05)and the level of exosomal PD-L1(P<0.05)in tumor tissue and blood of mice model of gastric cancer xenotransplantation.Jianpi Yangzheng Xiaozheng Decoction could inhibit the transfer of exosomal PD-L1 from gastric cancer to MDSCs,and reduce the differentiation and expansion of exosomal PD-Ll-induced CD11b+Gr-1+MDSCs in MFC and xenogeneic gastric cancer mice(P<0.01).10.Jianpi Yangzheng Xiaozheng Decoction could reduce the levels of immunosuppressive factors iNOS,Arg-1,TGF-β,IL-10 and IL-6 in 615 strain mice,reshape the tumor microenvironment of gastric cancer and control the progression of gastric cancer.Conclusion:The proportion of PD-L1 in exosomes of patients with gastric cancer is much higher than that in serum.Exosomal PD-L1 reflects the disease status of patients with gastric cancer.Exosomal PD-L1 from gastric cancer may be the key factor to control the differentiation and amplification of MDSCs.Jianpi Yangzheng Xiaozheng decoction can alleviate the progression of gastric cancer by inhibiting the transfer of exosomal PD-L1 to bone marrow cells,inhibiting the expansion of MDSCs,and reshaping the immunosuppressive tumor microenvironment,which provides experimental evidence for the clinical application of Jianpi Yangzheng Xiaozheng decoction in the treatment of gastric cancer through PD-L1.