The Role Of RKIP In The Pathogenesis Of Immune Hepatic Fibrosis

ObjectiveHepatic fibrosis is a common pathological process resulted from various chronic hepatic injuries,which is characterized by the excessive production and deposition of extracellular matrix(ECM).Hepatic stellate cells(HSCs)are recognized as the primary cellular source of ECM components in chronic liver disease,and play a critical role in the development and maintenance of liver fibrosis.Previous studies have suggested that inhibition of the activation,proliferation and migration of HSCs may be an attractive anti-fibrotic therapy.Recently,increasing evidences have confirmed that Raf-1/MEK/ERK1,2 signaling pathway plays an important role in hepatic fibrogenesis due to its ability to regulate the activation and proliferation of HSCs.Raf inhibiting protein kinase(RKIP)is an inhibitory factor of this signaling pathway,but it is unclear whether RKIP has also an important role in this kind of chronic liver desease.In this study,immune liver fibrosis was induced by porcine serum in rats and primary HSCs were extracted from rat liver to investigate the potential role of RKIP in the pathogenesis of liver fibrosis.Briefly,the distribution and content of RKIP and the activation of ERK protein in liver in different stages of liver fibrogenesis were detected to explore the correlation between the activation of Raf-1/MEK/ERK1,2 signaling pathway and the expression and phosphorylation levels of RKIP.In addition,the cell viability,expression levels of RKIP,ERK1/2,p-ERK1/2 and collagen were examined using MTT assay,RT-PCR and Western blot,respectively,to investigate the effects of RKIP on HSCs proliferation and apoptosis.MethodsPart Ⅰ:Animal experimentsHepatic fibrosis was induced by intraperitoneal injection with porcine serum in male Sprague-Dawley(SD)rats.In brief,a total of 88 male rats were divided into two groups:model group(60 animals)and normal control group(28 animals).The rats in the model group were intraperitoneally injected with 0.5 ml of porcine serum twice per week for 18 weeks,and the animals in the control group received an equivalent normal saline.At 8 wk,11 wk,14 wk,18 wk,7 and 15 rats were selected randomly from normal control group and model group,respectively.The blood was collected from rat’s eyeball and rats were sacrificed to obtain livers.A portion of liver was fixed in 4%paraformaldehyde and the other was stored in-80℃.The serum ALT and AST levels were detected by automatic biochemical analyzer,the contents of the serum collagen Ⅰ(COL-Ⅰ),precollagen Ⅲ(HPC Ⅲ),HA,LN and the liver precollagen Ⅲ(HPCⅢ),HA,LN were detected by ELISA test kits.The level of liver fibrosis was examined by using HE and Masson trichome staining.The protein expressions of RKIP,p-RKIP,ERK1/2 and p-ERK1/2 were detected by immunohistochemistry,and the mRNA expression of RKIP,COL-Ⅰ,COL-Ⅲ,ERK1 and ERK2 in liver specimen were determined by RT-PCR assay.In addition,the protein expression of RKIP,p-RKIP,p-ERK1/2,ERK1/2,MEK,P-MEK,COL-Ⅰ,COL-Ⅲ and α-SMA were detected by Western blot.Part Ⅱ:Cell experimentsThe rat primary hepatic stellate cells were extracted by density gradient centrifugation and the cell activation condition was determined by immunohistochemistry.HSCs was then treated with IL-1β or Locostatin(an inhibitor of RKIP).Subsequently,HSCs proliferation was detected by MTT assay.The expressions of RKIP,ERK-1,COL-Ⅰ and COL-Ⅲ mRNA were tested by RT-PCR analysis.The protein expression levels of RKIP,ERK1/2,p-ERK1/2,COL-Ⅰ,COL-Ⅲ and a-SMA were detected by Western blot.Results1.Construction of the immune hepatic fibrosis animal modelSerological results showed that the levels of serum ALT,AST,HA,LN,COL Ⅰ and HPC Ⅲ in the model group were higher than those of the normal control group.HE and Masson staining showed the pathological changes in the model group,including inflammatory cell infiltration,thick fibrotic septa connecting portal tracts and increased collagen fibres.The RT-PCR and Western Blot results showed that the mRNA and proteins expressions of COL-Ⅰ and COL-Ⅲ were significantly increased in the model group compared to the normal control group(P<0.05 or P<0.01).These results indicated that the liver fibrosis model was successfully established in rats and the degree of fibrosis was aggravated with the prolonged time of the administration of porcine serum.2.The expression of Raf-1/MEK/ERK1,2 pathway in liver fibrosisImmunohistochemistry results showed that the expressions of ERK and p-ERK were higher than those of the model group compared with the normal control group during the process of liver fibrogenesis.The RT-PCR assay revealed that intraperitoneal injection with porcine serum resulted in a significant increase in the mRNA expressions of ERK1 and ERK2 in a time-dependent manner.Similarly,Western blot results showed a higher expression of ERK,p-ERK,p-Raf or p-MEK in the model group compared with the normal control group.And the longer administration of porcine serum,the higher expressions of Raf and MEK proteints were observed(P<0.05 or P<0.01).In short,with the development of hepatic fibrosis,the expression and phosphorylation levels of Raf-1,MEK and ERK1,2 were significantly increased,indicating that hepatic fibrogenesis was closely related to the activation of Raf-1/MEK/ERK1,2 signaling pathway.3.The expression of RKIP in liver fibrosisThe immunohistochemistry results showed that there was a high expression of RKIP and a low expression of p-RKIP in the normal control group.In contrast,low expression of RKIP and high expression of p-RKIP were seen in the model group.Furthermore,the results of the RT-PCR and the Western blot also revealed that the mRNA and protein expressions of RKIP were significantly decreased compared to the normal control(P<0.05 or P<0.01).All the results mentioned above suggested that lower-expression of RKIP might result in the activation of Raf-1/MEK/ERK1,2 signaling pathway.4.The experimental results in vitroCompared with the normal control,the cells appreciation rate in IL-1β-stimulated group was 80.2%,Locastatin+IL-1β group was 25.1%and Locastatin group was 7.7%.The RT-PCR analysis revealed that there was not statistical difference in RKIP mRNA expression among the normal control group,the IL-11β stimulation group,the Locostatin+IL-1β group and the Locostatin group.The mRNA expression levels of ERK-1,COL-Ⅰ and COL-Ⅲin the latter three groups were higher than those of the normal control group significantly(P<0.05 or P<0.01).Western blot assay showed that the protein expressions of RKIP,ERK,p-ERK,COL-I and COL-III were significantly elevated by IL-1β treatment for 48 h.Moreover,there was significant decrease in the expressions of RKIP,COL-I and COL-III,but significant increase in the levels of p-ERK and a-SMA in the HSCs incubated with Locostatin for 48 h.In addition,after HSCs was sequentially treated with Locostatin for 1 h and IL-βfor 47 h,the RKIP and p-ERK proteins were detected.The results showed a lower level of RKIP but a higher level of p-ERK in the Locostatin+IL-1β group as compared with the normal control group.These results suggested that lower-expression of RKIP might result in the proliferation of HSCs via activation of Raf-1/MEK/ERK1,2 signaling pathway.ConclusionOur study indicates that the activation of Raf-1/MEK/ERK 1,2 signaling pathway is closely related to the decrease in RKIP protein expression and the increase in RKIP phosphorylation,suggesting that RKIP may play a critical role in liver fibrosis.That is,inhibiting Raf-1/MEK/ERK 1,2 signaling pathway through over-expression of RKIP may be an important target for the treatment of liver fibrosis.