The Protective Effects And Partial Mechanism Of Kv1.3 Mediated Macrophage Function On Hepatic Fibrosis In Mice

Hepatic fibrosis refers to the abnormal proliferation of fibrous connective tissue in the liver under the continuous stimulation of various chronic inflammation or injury.Now,the formation mechanism of hepatic fibrosis is a variety of pathogenic factor and leads to liver cell injury,which activates liver Kupffer cells(KCs),platelets,Hepatic sinus endothelial cells and liver cells,then hepatic stellate cells(HSCs)are under the combined effects of a variety of cytokines and chemical neurotransmitters,finally,the HSCs are activated and differentiate into myofibroblasts.Macrophages play a crucial role in chronic liver injury,studies have shown that macrophages have different subtypes,which are important regulating factors in the progress of liver fibrosis,macrophages play a different function in the progression and regression in liver injury,in addition,secretion of cytokines from macrophages or Kupffer cells is one of the important regulatory factors in liver fibrosis progression.Furthermore,migration and infiltration of macrophages from peripheral blood into liver is a key factor in the progression of liver inflammation and fibrosis.As a member of the voltage-dependent potassium channel family,voltagedependent potassium channel subtype 1.3(Kv1.3)is involved in the occurrence and development of various diseases,such as fibrosis,and is closely related to the regulation of immune cell function.Kv1.3 is expressed in many tissues and cells,especially in immune cells.Kv1.3 was originally found in T lymphocytes,which plays an important role in regulating activity and differentiation of T cells.The blockers of Kv1.3 as a potential treatment for hepatic fibrosis has not been reported,voltage-dependent potassium ion channel 1.3(Kv1.3)-specific blocker margatoxin(Mg TX)was applied on hepatic fibrosis models to observe the effects of Mg TX on the macrophage polarization,cytokine secretion.RAW264.7 cell lines was incubated with Mg TX,RNA-seq was used to find the target associated with migration of macrophages,and to observe the effects of the target on infiltration into the liver.To study the effects and mechanism of Mg TX on alleviating liver fibrosis through regulating the functions of macrophages: polarization,cytokine secretion and migration,the following research contents were carried out:The electrophysiological characteristics and kinetic parameters of peritoneal macrophages were recorded by perfusion in normal mice.The types and channel subtypes of the voltage-dependent potassium channels were detected by recording the current of macrophages using the non-selective blocker tetramethylamine(TEA),the selective inhibitor(Mg TX)or replacing the potassium ions in the intracellular solution with cesium ions.The results showed that the current of peritoneal macrophages in the normal mice showed the characteristic of outward rectification currents,and the current intensity increased with the depolarization of cells,and the currents could be effectively supressed by TEA and Mg TX.When intracellular K+ was substituted with Cs+ in the intracellular solution,the outward currents basically disappeared.It is suggested that voltagedependent potassium ion channels are mainly expressed on peritoneal macrophages,and such voltage-dependent potassium ion channels exist on peritoneal macrophages in the form of Kv1.3 subtype.Effects and mechanisms of Mg TX on hepatic fibrosis mice was observed and studied.The model of liver fibrosis in mice was established.After intraperitoneal injections with Mg TX,the severe steatosis,immune cell infiltration and degree of fibrosis caused by CCL4 were observed by hematoxylin-eosin(HE),Masson,Sirius Red and immunohistochemical staining.Using immunohistochemical method and WB observed the expression changes of α-SMA,CollagenⅠ,MMP12,MMP13,Periostin(POSTN),δ-catenin,CCL2 and Kv1.3 in the liver tissue.In order to further understand the Mg TX decreased the expression levels of these protein involved in hepatic fibrosis associated with macrophage/kupffer cells,RAW264.7 cell line as the research object was studied,RAW264.7 cell lines was induced to M1,M2 phenotype,WB method is used to detect the expression change of hepatic fibrosis related protein(MMP12,MMP13,POSTN,CCL2,Kv1.3 and δ-catenin)changes in M1 and M2 phenotype.In order to study the changes of serum cytokine levels in the hepatic fibrosis mice treated with Mg TX,Enzyme-Linked Immunosorbent Assay(ELISA)was used to determine the levels change of important cytokines(IL-1β,IL-6,IL-20,TNF-α,IL10,TGF-β)in the serum of hepatic fibrosis mice injected with Mg TX group.In order to investigate whether Mg TX alleviates the progression of liver fibrosis by regulating the phenotype of macrophages,RAW264.7 cell lines were induced into two subtypes M1 and M2,and the expression changes of surface markers of M1 and M2 phenotype were detected by Real-time quantitative PCR and western blot after Mg TX treatment.Previous studies have shown that the M1 and M2 subtypes of macrophages are related to the STAT1/STAT6 signaling pathway.In order to verify whether the Mg TX regulated the macrophage phenotype through the STAT1/STAT6 signaling pathway,western blot method was used to detect the level changes of phosphorylated STAT1/STAT6 in the M1 and M2 subtypes with Mg TX treatment.The experimental results showed that the Mg TX can alleviate the pathological characteristics of hepatic fibrosis induced by CCL4,decreased the expression of α-SMA and CollagenⅠin the tissue of hepatic fibrosis,significantly decreased level of protein expression related hepatic fibrosis(MMP12,MMP13,POSTN,CCL2,Kv1.3 and δ-catenin)and regulated cytokines in the serum related hepatic fibrosis(IL-1β,IL-6,IL-20,TNF α,IL-10,TGF-β),In addition,Mg TX decreased the expression of M1 phenotype markers(i NOS,CCL2,TNF-α and IL-1β)by regulating the phosphorylation level of STAT1,and increased the expression of M2 phenotype markers(IL-10,CD163,Arg-1 and MRC-2)by regulating the phosphorylation level of STAT6,resulted in delaying the progression of hepatic fibrosis.Therefore,it is suggested that Mg TX may play an antifibrosis role by regulating the phenotype switch and cytokines secretion in macrophages.In the early stage of hepatic fibrosis,the migration and infiltration of peripheral macrophages into the liver is an important factor to promote hepatic fibrosis.Therefore,we further explored that the mechanism of Mg TX might reduce the migration of peripheral macrophages into the liver.In order to find out Mg TX targets on migration of macrophages,RNA-seq profiling analysis was carried out and showed that the most obvious change by Mg TX treatment was downregulation of δ-catenin and related signaling pathways(Rho GTPase),real-time quantitative PCR and WB were used to verify the consistency between the expression of δ-catenin regulated by Mg TX and the results of RNA-seq profiling analysis,and to observe whether the expression of δ-catenin has dose dependence with Mg TX,and its function of δ-catenin was further studied.In order to study the effects of Mg TX on the migration function in macrophages through reducing the expression of δ-catenin,wound-healing and transwell assays were applied on RAW264.7 cell lines with δ-catenin knocked-down or over-expressed,respectively to verify the effects of Mg TX on migration function of macrophage.In the results of RNAseq,the downstream signaling pathway of δ-catenin is Rho GTPases,and Rho GTPases signaling pathway is related to cell migration function.The family of Rho GTPases,including Rho A,Rac,and Cdc42 were measured by RT-q PCR to determine the relationship between Rho A with δ-catenin overexpression,δ-catenin knock-down or Mg TX treatment.The relationship between δ-catenin and Rho A was further verified by RT-q PCR and WB.The RNA-seq profiling analysis results showed that the expression of δ-catenin could be significantly down-regulated by Mg TX,the migration ability of macrophages could be significantly inhibited by Mg TX treatment or δ-catenin knockdown and overexpression of δ-catenin could improve the migration function of macrophages,and the relationship between δ-catenin and Rho A were positively correlated.Thus,the macrophages migration was inhibited by reducing the expression of δ-catenin and through the downstream signaling pathway of Rho A,thus,may reduce the number of peripheral macrophages infiltrating into the liver,which could be an important mechanism for Mg TX to alleviate the progression of hepatic fibrosis.In this study,voltage-dependent potassium channel subtype(Kv1.3)was found and mainly expressed by patch clamp technique on peritoneal macrophages,in addition,Kv1.3 was highly expressed in hepatic fibrosis tissues in mice.Intraperitoneal injection Mg TX of Kv1.3 channel inbibitor in hepatic fibrosis mice can significantly decreased the expression of Kv1.3 and alleviated the pathological features of hepatic fibrosis in mice,suggesting that Kv1.3 is closely related to the progression of hepatic fibrosis,and inhibition of Kv1.3 can play a protective role on hepatic fibrosis in mice.The protective mechanism of Mg TX in liver is as follows: 1.Margatoxin may mitigate CCL4 induced hepatic fibrosis in mice via macrophage polarization,cytokine secretion and STAT signaling.2.Blockage of Kv1.3 regulates macrophage migration in acute liver injury by targeting δ-catenin through Rho A signaling.