| Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer death worldwide.At present,surgical excision,chemoembolization,and systemic therapy are the most common treatment methods for HCC,but they have relatively large toxic side effects and drug resistance.Recent studies have found that crosstalk between tumor cells and surrounding immune cells in HCC is an important mechanism to support tumorigenesis and immune escape.In the complex ecosystem of HCC,tumor-associated macrophage(TAM)is the main immune cell in tumor invasion.TAM has plasticity,which can change its phenotype according to different microenvironmental responses,to inhibit or accelerate tumor growth,migration and metabolic reprogramming.Therefore,targeting TAM appears to be an important intervention strategy for regulating the HCC microenvironment.Echinacea purpurea(L.)Moench(Ep),which is native to North America,is one of the most widely used dietary supplements in Europe and the United States.It has the advantages of high safety,low price,and wide bioactivity.Ep is an internationally recognized "immunomodulator",which can enhance the function of macrophages and lymphocytes to exert immune activity,play anti-inflammatory,antiviral,anti-tumor,and liver protection roles.Clinical investigation reports show that Ep is one of the most common adjuvant therapy plants to improve the quality of life of patients with HCC and other cancers.However,there are few basic studies on Ep’s intervention in HCC by relevant scholars,and the effect and mechanism of Ep on HCC microenvironment have not been clarified.Therefore,the following four scientific questions were raised:(1)Does Ep introduced in China have intervention activity on HCC?(2)Is the effect directly on tumor cells? Or by indirectly acting on immune cells to improve immune function?(3)What is the intervention mechanism?(4)What are the effective components? Based on the above scientific issues,the following studies were carried out:(1)Study on quality evaluation of Ep: NMR metabolomics was used to investigate the levels of metabolites in aerial parts and roots of Ep from different areas;The difference of immune activity was investigated by in vitro experiment.The environmental factors affecting metabolites and activities of Ep were studied by correlation analysis.Preliminary study on immune active components of Ep by partial least squares regression analysis.The results showed that soil p H and annual precipitation were the main factors affecting metabolite content and macrophage immune activity of Ep,and the aerial parts of Shandong and Hunan had rich metabolite and strong immune activity.Amino acids and phenolic acids are the active constituents of Ep to play the role of immune regulation.(2)Effect of Ep on immune microenvironment of HCC: The intervention effect of Ep on HCC was studied by using mouse HCC model induced by Hepa1-6 cells.Secondly,the direct effect of Ep on tumor cells was investigated through in vitro experiments.The co-culture model of tumor cells and macrophages was established to evaluate the intervention effect of Ep on HCC microenvironment.In vivo experiments showed that Ep improved tumor growth,migration,and EMT in HCC mice,inhibited the secretion of inflammatory cytokines IL-6 and IL-10,and reduced the macrophage M2 phenotype ratio.In vitro experiments showed that Ep can directly inhibit the activity,migration,invasion,and EMT of tumor cells.Co-culture experiment results showed that Hepa1-6 cells promoted the M2 polarization of macrophages and the secretion of IL-6 and IL-10.Macrophage-mediated Hepa1-6 cells also accelerated the proliferation,migration,invasion,and EMT.Ep interferes with macrophage polarization towards M2,reduces IL-6 secretion,enhances the effect of intervention on the growth of hepatoma cells,suggesting that Ep interferes with macrophage polarization and expression of inflammatory factors to regulate the immune microenvironment of HCC.(3)Study on the mechanism of Ep interferes with the immune microenvironment of HCC: Using the methods of network pharmacology,metabolomics,and experimental verification,to elucidate the mechanism of Ep interferes with the immune microenvironment of HCC.Network pharmacological results indicate that IL-6/STAT3 and PI3K/AKT pathways are potential mechanisms of Ep to regulate macrophages and interfere with HCC microenvironment.Metabolomics results indicate that Ep can significantly interfere with 14 serum and 11 urine metabolites of HCC mice,and phenylalanine metabolism is a key pathway for Ep to interfere with the reprogramming of HCC metabolism.Ep significantly down-regulates the expression of IL-6/STAT3 in tumor tissue through key proteins IL-6,p-JAK1,p-STAT3 and STAT3,reduces the content of p-PI3 K and p-AKT in PI3K/AKT signal,and reduces the level of key protein PAH in phenylalanine metabolism.In vitro experiments,IL-6 was added to simulate the secretion of macrophages under co-culture conditions.The results showed that Ep can block IL-6-induced STAT3 activation,inhibit PI3 K and AKT phosphorylation,regulate phenylalanine metabolism of tumor cells,and play its role in immune regulation.(4)Study on the active components of Ep to interfere with immune microenvironment of HCC: 12 potential active components predicted by network pharmacology were screened by in vitro co-culture experiment,and the results showed that chichoric acid,chlorogenic acid,caftaric acid,2-phenylethyl-glucopyranoside,and isoglycyrrhiza were effective components.The results of molecular docking and validation experiments showed that chichoric acid had good binding activity with the targets related to HCC immune microenvironment,inidcating it was the most important effective component in the intervention of HCC.Chlorogenic acid regulates HCC immune microenvironment by targeting IL-6/STAT3 signals.Caftaric acid interferes with the proliferation and apoptosis of HCC by regulating phenylalanine metabolism and IL-6/STAT3 pathway.2-phenylethyl-glucopyranoside interferes with HCC migration by targeting IL-6/STAT3 and PI3K/AKT pathway.Isoglycyrrhiza interferes with HCC metabolic reprogramming by regulating phenylalanine metabolism.(5)Development and application of Ep complex: The optimum ratio of Ep complex was obtained by cell experiment in vitro.The intervention effects of Ep complex,Ep and Ep complex combined with positive drugs on HCC immune microenvironment were investigated by mouse HCC model.The results showed that Ep complex can significantly reduce the hepatotoxicity caused by 5-FU,improve the inhibition of HCC growth,migration,and EMT,promote the transformation of macrophages to M1 type,promote the release of TNF-α,reduce the secretion of IL-6,regulate the expression of key proteins in IL-6/STAT3 and PI3K/AKT pathways.And its intervention effect on HCC is better than Ep single preparation.Network pharmacological analysis showed that the reason was related to the synergistic effect of multiple components.The combination of Ep and Ep complex with positive drugs can play a synergistic and detoxifying effect,indicating that the combination of Ep and Ep complex is an effective strategy to improve the clinical intervention effect of HCC.In summary,Ep has shown good intervention effect on HCC cells and animal models,which is related to the remodeling of macrophage phenotype in HCC microenvironment,regulation of inflammatory cytokine release,and intervention of phenylalanine metabolism.IL-6/STAT3 and PI3K/AKT signaling are its potential mechanisms of action.Chichoric acid,chlorogenic acid,caftaric acid,2-phenylethyl-glucopyranoside,and isoglycyrrhizin are effective anti-HCC components in the immune microenvironment.Therefore,Ep and its active ingredients may be potential substances for improving immune function,preventing,and treating HCC.The study of Ep complex and its combination with chemical drugs lays a certain experimental foundation for the development of special medical food in China. |
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