Effect Of Human Interleukin-8 On CCl₄-induced Liver Fibrosis In Mice And PDGF-BB-induced HSC-T6 Cell Activation And The Partial Mechanism Study

BackgroundLiver fibrosis is a process involving abnormal accumulation of extracellular matrix（ECM）and scarring caused by various chronic liver injuries.There are many factors that cause liver fibrosis,such as viral hepatitis,alcoholic liver disease,autoimmune hepatitis,non-alcoholic steatohepatitis and cholestatic liver disease.If liver fibrosis is not effectively controlled,it can further develop into cirrhosis and hepatocellular carcinoma,which can be seriously life-threatening.In recent years,the mechanisms of liver fibrosis have been extensively studied.Previous studies have shown that hepatic stellate cells（HSCs） play a key role in the development and progression of liver fibrosis,as activated HSCs secrete large amounts of ECM.Under physiological conditions,HSCs maintain ECM homeostasis and store vitamin-A.When the liver is injured,quiescent HSCs differentiate into myofibroblasts,characterized by a marked upregulation of α-smooth muscle actin（α-SMA）and type I collagen（COL1α1）.Liver fibrosis has had a more profound impact on global public health.However,due to the complexity of the etiology of liver fibrosis,there is no good clinical treatment available.Previous studies have shown that serum levels of interleukin-8 （IL-8） are significantly elevated in patients with liver fibrosis and that increased IL-8 is also associated with significant fibrosis,suggesting a close relationship between IL-8 and the development of liver fibrosis.The phosphatidylinositol-3 kinase（PI3K）/Akt and hypoxia-inducible factor 1α（HIF-1α） signaling pathways have been shown to be activated and play an important role in a variety of diseases.Notably,IL-8 can affect a variety of diseases by activating the PI3K/Akt/HIF-1α signaling pathway.The aim of this experiment was to observe whether hIL-8 can have an effect on liver fibrosis through PI3K/Akt/HIF-1α signaling pathway by delivering human IL-8（hIL-8） to the liver of liver fibrosis mice as well as PDGF-BB-induced HSC-T6 cells.By understanding the role of IL-8 in liver fibrosis mice in vivo and in PDGF-BB-induced HSC-T6 cells,it may provide some new therapeutic ideas for the treatment of liver fibrosis.MethodDue to the absence of the IL-8 gene in mice.In the in vivo animal experimental design,the LV5-hIL-8 lentiviral vector was delivered to the mice in this study by the caudal vein hydrodynamic injection.The mice were then divided into five groups:（1）control group;（2）model group injected with CCl₄;（3）LV5-NC group injected with lentiviral empty vector;（4）LV5-hIL-8 group injected with human IL-8 lentiviral vector;（5）LV5-hIL-8+LY294002 group added with PI3K/AKT signaling pathway inhibitor LY294004.In the design of in vitro cell experiments,plasmid transfection technology was used to express hIL-8 in HSC-T6 cells.The cells were then divided into 5 groups:（1）control group;（2）PDGF stimulation group;（3）pEX-3-NC group transfected with empty plasmid vector;（4）PDGF+p EX-3-hIL-8 group transfected with human IL-8 plasmid vector;（5）PDGF+pEX-3-hIL-8+LY294002 group added with LY294004.Then,the protein and mRNA expression levels of PI3K/Akt/HIF-1α signaling pathway and liver fibrosis-related indicators were detected by biochemical kits,histopathological research methods,Western Blot and qRT-PCR.ResultsAnimal experiments showed that hIL-8 could activate the PI3K/Akt/HIF-1α signaling pathway in the liver of CCl₄-induced liver fibrosis mice and enhance the expression levels of downstream liver fibrosis-related indicators.The addition of LY294002 inhibited the activation of hIL-8.In addition,cellular assays showed that hIL-8 could also activate PDGF-induced PI3K/Akt/HIF-1α signaling pathway in HSC-T6 cells and enhance the expression levels of downstream liver fibrosis-related indicators.The addition of LY294002 inhibited the activating effect of hIL-8.ConclusionThe hIL-8 cytokine could aggravate CCl₄-induced liver fibrosis in mice and enhance the expression levels of liver fibrosis-related indicators in PDGF-induced HSC-T6 cells via PI3K/Akt/HIF-1α signaling pathway.