| Colon cancer is one of the most common malignant tumors worldwide.Given the economic and social development and lifestyle changes in our country,the incidence of this disease is increasing rapidly and it has become a "killer" that seriously endangers people's health.The routine treatments for colon cancer are including surgery,chemotherapy,radiotherapy,and targeted therapy.In recent years,the advent of immune checkpoint inhibitors(such as PD-1,PD-L1 antibodies)provide hope for many malignant tumors.But unfortunately,the effectiveness of PD-1 inhibitor in treating colon cancer is very limited.Therefore,the combination of PD-1 inhibitors with other anti-tumor therapy(such as chemotherapy,resection,radiotherapy,or other immunotherapy)has been studied by many studies and some results has been achieved in order to solve the puzzle.As a treasure of our country,chinese medicine plays an important role in the treatment of diseases.The combination of chinese medicine with chemotherapy and radiation therapy have also achieved better results in the treatment of tumors.However,there are no reports on the treatment of colon cancer with chinese medicine combined with PD-1 inhibitors.By establishing a subcutaneous xenograft model induced by the mouse colon cancer cell line CT26,this study first observed whether Gegen Qinlian Decoction(GQD)+ PD-1 inhibitors were effective in treating subcutaneous xenograft model induced by the mouse colon cancer.Subsequently,flow cytology,immunohistochemistry,immunofluorescence and other methods were used to study the effect of combined therapy on tumor immune microenvironment.Finally,the anti-tumor mechanism of GQD combined therapy was explored by 16 SrDNA high-throughput sequencing and metabolomics,which provides a basis for how to improve the efficacy of PD-1 inhibitor in colon cancer.Part 1 Effects of Gegen Qinlian Decoction on immune status of patients with colorectal cancer by pharmacological databaseObjectives: By network pharmacologica,the potential mechanism of GQD in the treatment of colon cancer was found out.And the effects of GQD on the changes of T lymphocyte subsets in peripheral blood of patients with colorectal cancer(CRC)was explored to understand the immune status.Methods:1.Based on pharmacological database,we first screened GQD active compounds and established candidate compound-target network,then seek targets related to colon cancer by GO and KEGG enrichment analysis,further built protein interaction analysis and network map,lastly performed on the core targets in PPI by GO and KEGG enrichment analysis in order to find the key pathways that GQD may play a role in colon cancer.2.From July 1,2018 to January 1,2019,20 patients with colorectal cancer were recruited from the fourth hospital of Hebei Medical University.Peripheral blood of patients with CRC before and after use of GQD was collected,then the ratio of CD8+T cells and CD4+T cells was detected by flow cytometry.Results:1.From GQD,126 candidate compounds,260 potential therapeutic targets,150 colon cancer related targets were obtained,potential 79 core targets were obtained.And it is revealed that the core targets may be involved in a variety of tumorigenesis and T / B cell immune pathways.2.Compared with that before GQD treatment,the proportion of CD4+T cells in peripheral blood lymphocytes of patients with CRC increased significantly(P<0.05),while the proportion of CD8+T cells was not significantly different.Summary:The related target proteins that GQD plays in are mainly involved in various immune-related pathways such as T cells and B cells.GQD also may improve the immune function of patients with CRC.Part 2 Effects of Gegen Qinlian Decoction combined with PD-1 inhibitor on the treatment of colon cancer and immune microenvironmentObjectives: The efficacy of Gegen Qinlian Decoction combined with PD-1 inhibitor on tumour development,immune status and microenvironment of tumor-bearing mouse was explored.Methods:1.The subcutaneous xenograft model induced by mouse colon cancer cell line(CT26)was constructed.There are 8 groups in total,including A group: Low-GQD(L-GQD: 300mg/kg/d)+PD-1 inhibitor group;B group: Middle-GQD(M-GQD: 1500mg/kg/d)+PD-1 inhibitor group;C group: High-GQD(H-GQD: 7500mg/kg/d)+PD-1 inhibitor group;D group: L-GQD group;E group: M-GQD group;F group: H-GQD group;G group: PD-1 inhibitor group and H group: the control group.The tumor volume and tumor growth inhibition rate(TGI)of each group were evaluated.Besides,tumor samples,peripheral blood,and feces were collected from experimental animals.2.Flow cytometry was used to detect the ratio of CD8+T cells and CD4+T cells in peripheral blood of A group,D group,G group and H group in tumor-bearing mice.3.By immunohistochemistry and immunofluorescence,the differences of CD8+T cells in tumor tissues of above 4 groups in tumor-bearing mice were measured.4.ELISA was used to determine the level of IFN-?IL-2?IL-6?IL-17,TGF-? and PD-1 in tumor tissues of above 4 groups.Results:1.In 32 d mice,the tumor volumes in the three groups(A,B,and C)were all smaller than those in H group(P<0.01).The tumor volume in A group is significantly smaller than that in G group(P<0.05).The tumor volume in G group is significantly smaller than that in H group(P<0.01);There was no significant difference in tumor volume between three groups(D,E,and F)and H group;There was no significant difference in the three groups of A,B and C,and it is the same as the three groups of D,E and F.2.In bearing-tumor mouse,compared with H group,the proportion of CD8+T cells in peripheral blood lymphocytes of D group,G group and A group increased significantly(P<0.05),and the difference between the combined group and the H group was the most obvious(P<0.05).However,the proportion of CD4+T cells was not significant different.3.Compared with H group,the proportion of CD8+T cells in tumor tissue of A group increased significantly by immune-histochemistry(P<0.05).There was no difference in the proportion of CD8+T cells in mouse tumor tissue between two groups(D group and G group)compared with H group.4.Compared with othors group(D group,G group,H group),the fluorescence intensity of CD8+T cells in tumor tissue of A group increased by immune-histochemistry.5.The levels of IFN- and IL-2 in A group were significantly higher than those in H group(P<0.05).The levels of PD-1 in A group was significantly lower than H group(P<0.05).The levels of IL-17,IL-6 and TGF-? did not differ significantly between the groups.Summary:GQD combined with PD-1 inhibitor was found to enhance the antitumor effect on tumor-bearing mice.GQD also combined with PD-1 inhibitor can also improve the immune status of peripheral blood and reshape the tumor immune microenvironment.Part 3 Gut microbial and metabolomics analysis of combination GQD and PD-1 inhibitor in colon cancer xenograft modelObjectives: Based on 16 S rDNA sequencing of feces in tumor-bearing mice and analysis of peripheral blood metabolomics,the differential gut microbial and related metabolites were screened after combined treatment.Methods:1.16 S rDNA sequencing were performed on feces collected from tumorbearing mice before anti-PD-1 treatment(GQD group: A group + D group;Non-GQD group: G group +H group)and after anti-PD-1 treatment(A group,D group,G group and H group).The different analysis,including OTU,PCA,species diversity,and species were compared between groups,genus,and species.2.By removing blood from the eyeballs,the serum of tumor-bearing mice in 4 groups(A group,D group,G group and H group)after PD-1 inhibitor treatment was collected.Ultraperformance liquid chromatography-tandem mass spectrometer(UPLC-MS)was performed to screen polar small molecules and lipid differential metabolites to obtain signal pathways that differential metabolites may participate in.Results:1.The 16 S r RNA sequencing data of mice faeces before anti-PD-1 immunotherapyWe obtained a total of 2629590 effective sequences(the average sequence length of 252.6091528),664258502 effective bases,and 263 OTUs.GQD group vs.Non-GQD group,there were 9 different genus and 17 different species.By linear discriminant analysis of effect size(LEf Se),Erysipelotrichaceae were enriched in GQD group,while Peptococcaceae and Bacteroidales were enriched in Non-GQD group.2.The 16 S r RNA sequencing data of mice faeces after anti-PD-1 immunotherapyWe obtained a total of 2692571 effective sequences(the average sequence length of 252.5071873),679969282 effective bases,and 318 OTUs.D group vs.H group,there are 17 different genus and 29 different species.G group vs.H group,there are 7 different genus and 17 different species.A group vs.H group,there are 13 different genus and 26 different species.Interestingly,based on the Wilcoxon rank-sum test,when we compared the phylogenetic composition of common bacterial taxa at the species level,we found that s_Bacteroides_acidifaciens and s_uncultured_organism_g_norank_f_Bacteroidales_S24-7_group were enriched in the A group and s_uncultured_bacterium_g_norank_f_Bacteroidales_S24-7_group and s_uncultured_Bacteroidales_bacterium_g_norank_f_Bacteroidales_S24-7_group were enriched in the H group.A group vs.G group,there are 10 different genus and 12 different species.s_Bacteroides_acidifaciens were enriched in the A group and cterodales_bacterium_g_norank_f_Bacteroidales_S24-7_group were en-riched in the A group3.The effect of plasma metabolome on combination GQD and PD-1 inhibitorIn this experiment,2554 negative ion compounds and 3504 positive ion were measured.A total of 391 differential metabolites were obtained in D group compared with the H group;a total of 308 differential metabolites were obtained in G group compared with the H group;a total of 322 differential metabolites were obtained in A group compared with the H group,of which 158 specific metabolite mainly affected the metabolic pathways of glycerophospholipid metabolism and sphingolipid metabolism.Through hierarchical clustering of differential metabolites,the more abundant metabolites in A group mainly include Lyso PC(20:3(5Z,8Z,11Z)),vignatic acid B,Lyso PE(0:0 / 22:4(7Z,10 Z,13Z,16Z)),Lyso PE(0:0/20:2(11Z,14Z))? PI(20:4(8Z,11 Z,14Z,17Z)/ 16:0)and he less abundant metabolites in the same group,mainly including(1a S,5R,6R,9a R)-6-[2,6-dideoxy-2-(methylamino)],isopeonidin 3-rutinoside,glycerol 2-(9Z,12Zoctadecadienoate),tetraethylene glycol ? pentadecanoylglycine.Summary:GQD combined with PD-1 inhibito enhance the anti-tumor effects by improve the structure of the gut microbiota and certain specific metabolites.Conclusion:1.The related target proteins that GQD plays in are mainly involved in various immune-related pathways such as T cells and B cells.GQD also may improve the immune function of patients with CRC.2.GQD combined with PD-1 inhibitor was found to enhance the antitumor effect on tumor-bearing mice.GQD also combined with PD-1 inhibitor can also improve the immune status of peripheral blood and reshape the tumor immune microenvironment.3.GQD combined with PD-1 inhibito enhance the anti-tumor effects by improve the structure of the gut microbiota and certain specific metabolites. |
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