Isoorientin-2"-O-α-L-arabinopyranosyy Attenuates Hepatic Fibrosis Induced By Porcine Serum In Rats Via Inactivation Of NF-κB Signaling Pathway

Objective:Liver fibrosis is a wound-healing response to various chronic liver injuries.It is a pivotal and necessary stage to cirrhosis,which leads to lethal complications and high mortality.Contrary to the traditional view that cirrhosis is an irreversible disease,recent evidence show that hepatic fibrosis is reversible.Thus,how to inhibit hepatic fibrosis and to find a safe and effective medicine to reverse hepatic fibrosis has become the focus in this field.In this study,a saponin was isolated from Gypsophila elegans and was identified as isoorientin-2"-O-α-L-arabinopyranosyl(10A),and the protective effect and underlying mechanism of IOA on liver fibrosis induced by porcine serum in rats would be explored.Method:Hepatic fibrosis was induced by intraperitoneal injection with porcine serum in male Sprague-Dawley(SD)rats.In brief,a total of 90 male rats were divided into two groups:Ⅰ group(75 animals)and Ⅱ group(15 animals).The rats in the Ⅰ group were intraperitoneally injected with 0.5 ml of porcine serum twice per week for 16 weeks,and the animals in the Ⅱ group received an equivalent normal saline.Then,the rats in the Ⅰ groups were randomly divided into five groups including porcine serum-treated model group,positive control group(PDTC-treated group),high-,medium-and low-dosages of IOA-treated groups.In addition,the Ⅱ group was served as a normal control group.Next,in addition to porcine serum,the rats in the PDTC-treated group received PDTC 150 mg/kg/d intragastrically;the animals in the high-,medium-and low-dosages of IOA-treated groups were orally administrated IOA 100,50,25mg/kg/d,respectively;and the rats in the model control group and the normal control group were given intragastric an equivalent normal saline.After a treatment for 8 weeks,the animals were killed.Serum samples were collected into tubes.Liver samples were dissected out and washed immediately with ice cold saline to remove excessive blood.Subsequently,the liver samples were divided into two parts for different use,with one immediately stored at 80℃ for future analysis,and another excised and fixed in 10%formalin solution for H&E and Masson staining.The activities of serum ALT,AST,ALB and GLB were measured by an automatic biochemical analyzer.The levels of serum HA,LN,Hyp,MDA,SOD,PⅢNP,Col-Ⅰ,Col-Ⅳ and hepatic SOD,MDA,TNF-α,IL-1,IL-10 and TGF-β1 were assayed according to the instructions of the commercially available kits,respectively.The expressions of hepatic NF-κBβ50,TNF-α,MMP-9,TIMP-Ⅰ and ICAM-1 were determined using a method of immunohistochemistry.The expressions of Col-Ⅰ,Col-Ⅲ,NF-κB p65,IκB-αand TGF-β1 mRNA in liver were analyzed by RT-PCR.And the expressions of hepatic NF-κB p65,phospho-NF-κB p65,NF-κB p50,IκB-α,phospho-IκB-α,IKK-β and Col-Ⅰ were detected by western blot immunoassayResult:1.Construction of the porcine serum-induced liver fibrosis modelIn this study,the liver fibrosis was induced by porcine serum for 16 weeks in SD rats.Our results revealed that the liver index was higher in the model control group than in the normal control group.The serum levels of ALT and AST were increased in the model group compared with the normal control group,but had no significant difference.Moreover,in the model control group,severe changes were observed in liver morphology,including fatty degeneration,necrosis and infiltration of inflammatory cells.These results indicated that the liver fibrosis model in rats was successfully established.2.Protective effect of IOA on porcine serum-induced hepatic fibrosis in ratsCompared with the model control group,treatment with IOA(100,50,25mg/kg)or PDTC(150 mg/kg)markedly reduced the liver index and decreased the activities of serum ALT and AST,significantly increased the content of serum albumin and the ratio of A/G(ALB/GLB).Furthermore,IOA markedly decreased the contents of LN,HA and Hyp as well as the generation and deposition of hepatic collagen.These results indicated that IOA has evident inhibition on porcine serum-induced hepatic fibrosis.3.Inhibitory effect of IOA on CollagenThe serum Col-Ⅰ,Col-Ⅳ and PⅢNP were measured by commercial ELISA kits according to the manufacturer’s instructions.Our data revealed that the serum Col-Ⅰ,Col-Ⅳ and PⅢNP levels in the model group were significantly higher compared to those in the normal control group.While treatment with IOA markedly inhibited these up-regulations of Col-Ⅰ,Col-Ⅳand PⅢNP.The results of RT-PCR showed that the expression of Col-Ⅰ and Col-Ⅲ mRNA in model control group was increased compared with the normal control group,treatment with IOA significantly decreased the expression of Col-Ⅰ and Col-Ⅲ mRNA.Furthermore,the results of western blot showed that IOA significantly decreased the expression of Col-Ⅰ protein.These results further confirmed the inhibition of IOA on hepatic fibrosis.4.Antioxidant effect of IOAThe hepatic SOD activities significantly decreased after porcine serum administration.In contrast,IOA significantly increased the enzyme activities in a dose-dependent manner.In addition,the hepatic MDA level increased significantly in the model group.Treatment with IOA effectively decreased the MDA level.These results indicated that IOA could reduce oxidative damage in liver induced by porcine serum.5.Inhibitory effect of IOA on inflammatory cytokinesThe inflammatory cytokines including hepatic IL-1,IL-10,TNF-α and TGF-β1 were measured by commercial ELISA kits according to the manufacturer’s instructions.Our data revealed that the hepatic IL-1,TNF-α and TGF-β1 levels in the model group were significantly higher compared to those in the normal control group.These up-regulations of IL-1,TNF-α and TGF-β1 were markedly inhibited by treatment with IOA.In addition,the IL-10 content decreased considerably after porcine serum administration,whereas the decrease was reversed by IOA treatment.Similarly to the ELISA test results,IOA significantly suppressed the expression of TNF-α protein and TGF-β1 mRNA.These results confirmed the inhibitory effect of IOA on inflammatory cytokines.6.Effect of IOA on NF-κB signaling pathwayNF-κB,a dimeric transcription factor,involves in the regulation of many key genes that play important role in inflammatory response.In this study,the immunohistochemistry result revealed that administration of porcine serum markedly increased nuclear NF-κB p50 content,however,treatment with IOA significantly decreased nuclear NF-κB p65 content.The result of RT-PCR showed that IOA suppressed the expression of NF-κB P65 mRNA,while increased the expression of IκB-α mRNA.In addition,the expressions of hepatic NF-κB p65,phospho-NF-KB p65,NF-κB p50,IκB-α,phospho-IκB-α,IKK-β were detected by western blot immunoassay.Our data revealed that the hepatic NF-κB p65,phospho-NF-κB p65,NF-κB p50,phospho-IκB-α and IKK-β levels in the model group were significantly higher compared to those in the normal control group.These up-regulations of NF-κB p65,phospho-NF-κB p65,NF-κB p50,phospho-IκB-α and IKK-β were markedly inhibited by IOA treatment.In addition,the IκB-α content decreased considerably after porcine serum administration,whereas the decrease was reversed after treatment with IOA.These results indicated that IOA has evident inhibition on porcine serum-induced hepatic fibrosis due to blocking the NF-κB signaling pathway.7.Effect of IOA on the expression of intercellular cell adhesion molecule(ICAM-1)In this study,the immunohistochemistry result revealed that administration of porcine serum markedly induced increase of nuclear ICAM-1 content,however,treatment with IOA significantly decreased nuclear ICAM-1 content.8.Effect of IOA on the expression of matrix metalloproteinase(MMP-9)and tissue inhibitor of metalloproteinases(TIMP-1)In this study,we determined hepatic MMP-9 activity and TIMP-1 using immunohistochemistry.The results showed that expression of TIMP-1 increased considerably after porcine serum administration,whereas the increase was reversed after treatment with IOA.Moreover,compared to the model control,treatment with 100,50 or 25 mg/kg IOA significantly increased the expression of MMP-9.This result indicated that IOA could promote the degradation of ECM by maintaining the dynamic balance between MMPs and TIMPs.Conclusion:IOA can significantly alleviate the hepatic injury and fibrosis induced by porcine serum.The mechanism of the protective action of IOA may contribute to the inhibitions of inflammatory response and HSCs activation via inactivation of NF-κB signaling pathway,and the decrease in ECM excessive deposition via regulation of matrix metalloproteinase.