| Background: Hepatocellular carcinoma(HCC)is a malignant tumor with high recurrence rate and high mortality,which is one of the major burdens of global health.Hepatectomy is the main treatment methods for early HCC,which resulting in ischemia reperfusion injury(IRI)becoming an inevitable complication of clinical liver surgery.The molecular mechanisms and signal pathways involved in liver IRI are multi-directional,including inflammatory stress injury,mitochondrial injury,immune microenvironment disorder,etc.Currently,existing clinical data support that liver IRI is one of the independent risk factors for postoperative HCC recurrence,and prove that the disorder of immune microenvironment exerts a non-negligible role in the process of HCC recurrence.Therefore,this study focused on exploring the correlation between the liver immune microenvironment disorder induced by liver IRI and the HCC recurrence after surgery,and the mechanisms involved in the regulation of immune cells were deeply studied.Methods: Liver IRI model was constructed in C57BL/6J mice,and liver tissue and peripheral blood samples were collected to detect the inflammatory activation and the changes of immune microenvironment;Naive CD4+T cells from normal mice and IRI mice were extracted for transcriptomic sequencing to clarify the influence of IRI on the gene expression profile of immune cells,and screened out the potential regulatory transcription factor FOXO1,and verify the expression trend of FOXO1 in human IRI samples and mice IRI models;In order to clarify the effect of FOXO1 in IRI and the change of Th17 cell proportion,the mice model of FOXO1 overexpression was established by Resveratrol,and then IRI model was performed,and the expression of tumor stemness,epithelial-mesenchymal transition(EMT)factor and related factors in formation of tumor pre-metastasis microenvironment were detected.The HCC recurrence model was established in IRI mice,and the correlation between IRI and HCC recurrence was clarified,and the function of FOXO1 in this process was illustrated.Naive CD4+T cells were extracted and cultured in vitro under the condition of Th17 cell polarization,and co-cultured with human HCC cells and Human Umbilical Vein Endothelial Cells(HUVECs)to explore the effect on proliferation,migration,invasion abilities of HCC cells and angiogenesis.Furthermore,Th17 cells were obtained through in vitro polarization,and adoptive transfer experiments were conducted in mice to further verify the role of Th17 cells in promoting HCC recurrence.Results: 1.The IRI model verified that liver IRI can activate liver inflammatory response and imbalance of Th17/Treg cells.Through the detection of liver samples both in mice and human IRI model,it was found that the expression of inflammatory factors increased significantly,and H&E staining presented that liver necrosis areas were increased,while the biochemical indicators,such as serum ALT and AST,were increased dramatically;Meanwhile,the imbalance of Th17/Treg cells was verified by qPCR and flow cytometry,which indicated that the proportion of Th17 cells increased and the proportion of Treg cells decreased.2.Through transcriptome sequencing analysis of Naive CD4+T cells,the gene expression profile changed dramatically after undergoing IR stress,and the differentially expressed genes(DEGs)were more inclined to Th17 cells polarization,while the polarization of Treg cells was inhibited;In addition,these DEGs were regulated by the transcription factor FOXO1.Furthermore,we verified the expression trend of FOXO1 at mRNA and protein levels.3.By constructing mice model of FOXO1 overexpression with Resveratrol,we verified that FOXO1 can exert a protective role in the process of liver IRI by reducing the expression of inflammatory factors and reversing the imbalance of Th17/Treg cell ratio.4.It was verified that FOXO1 expression was reduced and Th17 cells infiltration was increased in HCC recurrence models;Moreover,the correlation between liver IRI and HCC recurrence has been verified;Through qPCR and WB technology,it has been illustrated that liver IRI can promote HCC recurrence by activating EMT program,cancer stemness,and participating in the formation of caner pre-metastasis microenvironment.5.Naive CD4+T cells were extracted and cultured in vitro under the condition of Th17 cells polarization,and co-cultured with HCC cells and HUVECs.It was indicated that Th17 cells can enhance the ability of HCC cells to migrate,invade and proliferate,and can promote angiogenesis;At the same time,the adoptively transfer experiment of Th17 cells in vivo demonstrated the effect of Th17 cells in promoting HCC recurrence.Conclusion: this paper systematically demonstrated the increased proportion of Th17 cells stimulated by hepatic IRI have ability to reshape the liver microenvironment by activating EMT program,cancer stemness pathway,promoting the formation of pre-metastatic microenvironment and angiogenesis,making it more prone to the colonization of residual HCC cells and ultimately contribute to HCC recurrence.And FOXO1 has ability to stabilize the liver microenvironment and reduce HCC recurrence by inhibiting the polarization and function of Th17 cells,which may be used as a potential therapeutic target to alleviate IRI-induced HCC recurrence. |
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