左金丸通过TIPE1/PI3K/Akt信号通路抑制肿瘤相关巨噬细胞M2极化拮抗结直肠癌OA

Objective:To investigate the mechanisms of Zuojinwan in the treatment of colorectal cancer from the perspective of inhibiting M2 polarization of tumor-associated macrophages.Methods:A colorectal cancer mouse model was established by azoxymethane(AOM)/dextran sulfate sodium(DSS).Body weight was recorded,disease activity index(DAI)and tumor number were calculated,and hematoxylin-eosin(HE)staining was used to observe colonic pathological changes.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-6(IL-6),transforming growth factor-β(TGF-β),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)in colonic homogenates.Immunohistochemistry was used to detect proliferating cell nuclear antigen(PCNA)expression in the colon,and immunofluorescence was used to detect mannose receptor(CD206)expression in macrophages.In vitro,M2 macrophages were induced using IL-4/IL-13,and macrophages were treated with different concentrations of Zuojinwan-containing serum.CD206 and differentiation cluster 86(CD86)were analyzed by flow cytometry.A CT26 and RAW264.7 macrophage co-culture system was established,and the migration and proliferation abilities of CT26 cells were detected using Transwell assays and trypan blue exclusion.Finally,proteomics was used to screen key proteins and signaling pathways involved in Zuojinwan-mediated inhibition of M2 polarization of tumor-associated macrophages in colorectal cancer,and Western blot was used for validation.Results:In animal experiments,compared with the blank group,mice in the model group showed significantly decreased body weight(P<0.05),significantly increased DAI scores(P<0.01),and significantly increased tumor number(P<0.01),with severe colonic carcinogenesis and lymphatic metastasis.The protein expression levels of PCNA and CD206 were significantly increased(P<0.01),and the levels of IL-6,IL-1β,TNF-α,and TGF-β in colonic homogenates were significantly increased(P<0.01).Compared with the model group,mice in the Zuojinwan intervention group showed significantly higher body weight(P<0.05),significantly decreased DAI scores(P<0.01),and significantly reduced tumor numbers(P<0.01).The protein levels of PCNA and CD206 were significantly decreased(P<0.05),and the levels of IL-6,IL-1β,TNF-α,and TGF-β in colonic homogenates were significantly decreased(P<0.05).In the CT26 and RAW264.7 co-culture system in vitro,compared with the blank group,co-culture with M2-type RAW264.7 macrophages significantly increased CT26 cell migration rate and proliferation number(P<0.01).Compared with the M2 group(M2-type macrophages),the proportion of CD86 was significantly increased in the Zuojinwan-containing serum group(P<0.01),while the CT26 cell migration rate and proliferation number were significantly decreased in the 8%Zuojinwan-containing serum group(P<0.01).Proteomics analysis indicated that tumor necrosis factor-α-induced protein 8-like protein 1(TIPE1)may be a key target through which Zuojinwan regulates macrophage phenotypic transformation to inhibit colorectal cancer.The phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway showed the highest enrichment.Compared with the blank group,the levels of TIPE1,p-PI3K,and p-Akt in M2 macrophages were significantly increased(P<0.05,P<0.01).After treatment with Zuojinwan-containing serum,the levels of TIPE1,p-PI3K,and p-Akt were significantly decreased(P<0.05,P<0.01).Conclusion:Zuojinwan may inhibit colorectal cancer proliferation and migration by suppressing M2 polarization of tumor-associated macrophages,which may be associated with inhibition of the PI3K/Akt signaling pathway in tumor-associated macrophages.