The Inhibitory Effect Of Jiedufang On The M2 Polarization Of Macrophages In Hepatocellular Carcinoma Through Suppressing TGF-β And AGE-RAGE Induced-oxidative Stress

Background In China,the mortality of primary liver cancer ranks second among all kinds of malignant tumor diseases,which seriously threatens people’s lives and health and brings a heavy burden to the economy of China.Although various new therapies emerged,its mortality and rates of recurrence and metastasis remain high.Various factors could lead to liver cancer.Traditional Chinese medicine is regarded as one of the major features of cancer prevention and treatment in China based on the holistic principle of syndrome differentiation.Jiedu Fang(JDF),a Chinese medicine compound created by our department in long-term clinical practice,which has achieved good clinical efficacy in the practice of treating primary liver cancer,but its mechanism of action has not been completely figured out.Previous studies have confirmed that JDF can effectively intervene TGF-β-induced hepatoma cell proliferation,invasion and migration as well as epithelial-mesenchymal transformation.Studies have shown that TGF-β,as a broad regulatory factor of hepatoma,can regulate the M2-type polarization of macrophages via multiple kinds of mechanisms.Therefore,could JDF effectively reduce the M2-type polarization of macrophages not only through TGF-βitself,but also TGF-β related pathways? Our previous studies of rat liver cancer model have confirmed that JDF indeed has anti-liver cancer effect by reducing M2-type polarization of macrophages.Hence,this project intends to explore the potential mechanisms of JDF in M2-type polarization via m RNA chip and literature researches.Objective 1.To observe the mechanism of TGF-β and AGE signaling pathways on oxidative stress level and M2 polarization of tumor-related macrophages.2.To clarify whether JDF can inhibit the promotion effect of polarized M2 macrophages on the migration of liver cancer cells through regulating TGF-β and AGE signaling pathways,as well as oxidative stress pathways of downstream tumor-related macrophages.Methods 1.The bioinformatics analysis was conducted to identify the inflammatory differential genes obtained by m RNA chip in the previous study to screen the synergistic of JDF on inflammatory microenvironment of HCC.2.Deep processing of JDF was completed by the Shanghai Ronghe Pharmaceutical Technology Development Company.JDF was dissolved by adding serum-free DMEM medium and stored in the-20 refrigerator.3.The RAW264.7 macrophages were treated with TGF-β,TGF-β+JDF,AGE,TGF-β+AGE,and TGF-β+AGE+JDF.RT-PCR was employed to investigate the m RNA levels of Arg-1,CD163,IL-10 and CD206.The ROS level of macrophages was observed by fluorescence microscope photography,and the Western blotting assay was carried out on RAGE,NADPH oxidase 4(NOX4),p47 phox and phosphorylated extracellular signalregulated kinase(p-ERK)proteins of RAW264.7 macrophages.4.The conditioned medium of RAW264.7 macrophages induced by TGF-β,AGE,TGF-β+AGE,TGF-β+AGE+JDF were collected to stimulate hepatoma cell Hepa1-6.The migration promotion ability of hepatoma cell Hepa1-6 were observed by transwell chamber migration experiment,and E-cadherin,N-cadherin,Vimentin and TWIST1 proteins of hepatoma cell were tested by Western blotting.Results 1.IPA analysis was conducted on 351 different genes related to inflammation,which found that ROS and NOX production-related signaling pathway in macrophages was one of the signaling pathways closely related to the action of JDF.GO enrichment and KEGG pathway analyses revealed that the anti-liver cancer mechanism of JDF may be related to the regulation of oxidative stress of macrophages and the change in AGE-RAGE signaling pathway.2.TGF-β could significantly up-regulate the m RNA transcription levels of Arg-1,CD163 and IL-10 in RAW264.7 macrophages through RT-PCR experiment(P < 0.01),The m RNA levels of Arg-1,CD163 and IL-10 in JDF group were significantly lower than TGF-β group,and the m RNA levels of Arg-1 and CD163 in AGE+TGF-β group were higher in TGF-β group and AGE group respectively.3.TGF-β group and AGE group can increase the ROS activity in RAW264.7macrophages,while TGF-β+AGE can synergistically up-regulate ROS level in macrophages compared with TGF-β or AGE alone.4.Compared with the mock group,TGF-β group and AGE group,Western blotting showed that TGF-β+AGE promoted the expression of p47 phox,NOX4,RAGE and p-ERK proteins in RAW264.7 macrophages(P < 0.001).JDF can significantly inhibit TGF-β+AGEinduced NOX4,p47 phox,RAGE and p-ERK protein expression(P < 0.001).When TGF-βinhibitor,RAGE inhibitor,ROS inhibitor or NOX inhibitor were treated with TGF-β +AGE,the expression of NOX4,p47 phox and RAGE protein were significantly decreased(P <0.001).5.Compared with the mock group,TGF-β group and AGE group,conditioned medium of RAW264.7 macrophages treated with TGF-β+AGE could significantly increase the migration number of Hepa1-6 cells(P < 0.001),and JDF may antagonize the above effects.6.Western blotting showed that the expression of N-cadherin,Vimentin and TWIST1 protein in hepatocellular carcinoma cells was promoted by conditioned medium of RAW264.7 macrophages treated with TGF-β+AGE,while the expression of E-cadherin protein was down-regulated(P < 0.001),and JDF may antagonize the above effects.Conclusion 1.TGF-β and AGE cross-talk through p47phox-NOX-ROS pathway to enhance each other and promoted the polarization of M2 macrophages.2.JDF can down-regulate the polarization of M2 macrophages,and then reverse the tendency of M2 macrophages promoting the migration of HCC cells by inhibiting the effect of TGF-β and AGE on the oxidative stress of macrophages.