Correlation Between Iron Deposition In Hepatic Stellate Cell And Liver Fibrosis And The Effect Of Compound Gan Du Qing

ObjectivesIron deposition and liver fibrosis are closely related.Iron overload has been proved to play an important role in liver fibrosis during its occurrence and development.In the present study,we took hepatic stellate cell(HSC),which is the key in liver fibrogenesis, as a breakthrough point,and established HSC model with iron deposition,to study the influence of iron deposition in HSC onα-SMA protein expression,collagen typeâ… and TGF-β₁ mRNA expression,as well as HSC apoptosis.Rat liver fibrosis models were established with dimethylnitrosamine(DMN),Rat serum ferritin,transferrin and hepatic iron concentration of liver tissue were used to evaluate iron load of rats.Through prussian blue andα-SMA double staining,iron loaded HSC was located,which made clear the connection between iron deposition and liver fibrosis.Furthermore,by studying the mRNA expression of HSC activation related factor TGFβ1,the effect of iron deposition in HSC on the effect of liver fibrosis was investigated.The reseach of correlation between iron deposition in HSC and liver fibrosis was carried out at histopathological,cellular and molecular levels,in order to illustrate the mechanism of iron deposition in HSC promoting liver fibrogenesis.For in vivo and in vitro experiments,herbal decoction Compound Gan Du Qing was adopted to conduct interference on DMN induced rat liver fibrosis model and HSC model of iron deposition,in the attempt to explain the influence of Compound Gan Du Qing on iron deposition promoting liver fibrogenesis based on the therapy of strengthening healthy qi combined with draining dampness and relieving toxin.Methods(一) Cellular experiment:Influence of iron deposition in HSC on liver fibrosis and the effect of Compound Gan Du Qing1.Establishment of iron deposited HSC modelHSC-T6 was taken as the model cell line and cultured in the 6-well plate,with an 1.8cm square cover glass(sterile) put into each well,and cells were divided into the following 3 groups:iron deposited model group,iron deposited model + desferrioxamine group and normal control group,each group repeated for 2 wells.According to the cytotoxicity experiment,25μM concentration of ferric ammonium citrate(FAC) was chosen as the experimental concentration.After cells grew full adherent the wall,the iron deposited model group was added with FAC,the iron deposited model + desferrioxamine group was supplemented with FAC and desferrioxamine at 1:1 mole ratio.The normal control group was normally cultured HSC-T6.After additional incubation for 24 h,prussian blue staining was performed.2.Iron deposition in HSC on the activation and reversion of HSC and the effect of desferrioxamineHSC-T6 cells were divided into normal control group,iron deposited model group, 50μM and 25μM desferrioxamine group.The normal control group was normally cultured HSC-T6,iron deposited model group was the HSC-T6 cells treated with 25μM FAC which was confirmed to be iron deposited,50μM and 25μM desferrioxamine groups were the HSC-T6 cells treated with 50μM and 25μM desferrioxamine respectively.After additional incubation for 24 h,immunohistochemistry was conducted to detect the expression ofα-SMA,and quantitative PCR was performed to examine the mRNA expression of collagen typeâ… and TGF-β₁,TUNEL assay was used to detect HSC-T6 apoptosis,and electronic microscope was adopted to observe the ultrastructure of HSC-T6 cells.3.Effect of Compound Gan Du Qing on iron deposition induced HSC activation and reversionHSC-T6 cells were divided into normal control group,iron deposited model group, and iron deposited model + traditional chinese medicine group.The normal control group was normally cultured HSC-T6 cells,iron deposited model group was the HSC-T6 cells treated with 25μM FAC which was confirmed to be iron overloaded,and the iron deposited model + traditional chinese medicine group was the HSC-T6 cells of iron overload model group treated with Compound Gan Du Qing(using the maximum concentration experimentally confirmed to be nontoxic).After incubation for 24 h,immunohistochemistry was conducted to detect the expression ofα-SMA,and quantitative PCR was performed to examine TGF-β₁ mRNA expression,TUNEL assay was used to detect HSC-T6 apoptosis, and electronic microscope was adopted to observe the ultrastructure of HSC-T6 cells.(二) Animal experiments:Liver fibrogenesis of iron deposition in HSC in DMN induced rat liver fibrosis and the effect of Compound Gan Du Qing75 of SD rats were randomly divided into 6 groups:blank control group(12),model group(15),model + desferrioxamine group(12),model + colchicine group(12),model + Compound Gan Du Qing large dosage group(12),model + Compound Gan Du Qing small dosage group(12).The method of making rat liver fibrosis model was as follows:prepare 1: 100(V/V) dimethylnitrosamine(DMN) using normal saline as the dilution solution,and perform intraperitoneal injection on rats during the first 3 days each week,at the dosage of 10μL DM-N/kg body weight,for 4 weeks altogether,with the blank control group injected with normal saline.From the second week of making rat models,rats of the model + colchicine group were subjected to colchicine gastric perfusion at a dosage of 0.1mg/kg body weight once daily for 3 weeks;the model + Compound Gan Du Qing large and small dosage groups were gastrically perfused at the dosage of 20g/kg and 5g/kg body weight, respectively,once daily for 3 weeks.The model group and blank control group were both perfused gastrically with normal saline at 10ml/kg once daily for 3 weeks.From the third week,rats of the model + desferrioxamine group were injected intraperitoneally with desferrioxamine at a dosage of 100mg/kg body weight three times a week for 2 weeks. Animal death was recorded from the beginning of making model to the end of the experiment.At the end of the experiment,the eyeballs were extracted and blood was drawn, and the left anterior lobe of the liver was resected,with one portion fixed in Bouin's, another about 50mg liver tissue cleaned and preserved in Trizol at-70℃for further examination.The left liver tissue was preserved at - 70℃.The left lobe of liver tissue was removed and histopathology investigation was conducted by HE,Masson,prussian blue staining,as well as prussian blue andα-SMA double staining.The ultrastructure of liver tissues was observed with electronic microscope.And ELISA assay was carried to examine the concentrations of rat serum ferritin and transferrin.Hepatic iron concentration of rat liver tissue was evaluated by Flame Atomic Absorption Spectrophotometry(FAAS).AU400 Olympus Automatic Biochemistry Analyzer was adopted to detect hepatic function,serum iron and serum lipid.And quantitative PCR was performed to examine the expression of TGF-β₁ mRNA.Results(二) Cellular experiment:Influence of iron deposition in HSC on liver fibrosis and the effect of Compound Gan Du Qing1.Establishment of iron deposited HSC modelAfter Prussian blue staining,HSC-T6 cells nucleus turned red,and blue iron pellet could be observed in the cytoplasm of HSC-T6 cells in iron deposited model group,which could also be detected in the iron deposited model + desferrioxamine group,with significantly decrease of iron deposition.There was no iron pellet deposition in the normal control group. 2.Iron deposition in HSC on the activation and reversion of HSC and the effect of desferrioxamineImmunohistochemistry results showed that obvious activation occurred in HSC-T6 cells of the normal control group,with highα-SMA expression.In iron deposited model group,α-SMA was also highly expressed.And in desferrioxamine group,α-SMA expression of HSC-T6 cells was significantly decreased compared with the normal control group and iron deposited model group,implying that activation of cells was inhibited to certain extent.Collagen typeâ… and TGF-β₁mRNA expression of HSC-T6 cells revealed that collagen typeâ… mRNA expression was significantly enhanced(P＜0.01) in iron deposited model group compared with the control group,whereas there was no significant difference in TGF-β₁ mRNA expression(P＞0.05) between these two groups.Both 50μM and 25μM desferrioxamine could down regulate collagen typeâ… and TGF-β₁ mRNA expression(P＜0.01),and 50μM desferrioxamine was superior to 25μM desferrioxamine(P＜0.01).These results suggested that iron removal therapy could reduce collagen typeâ… and TGF-β₁ expression at transcription level.Pathology of HSC-T6 cells apoptosis showed that none or only a few cells undewent apoptosis in the control group and iron deposited model group,whereas in desferrioxamine group,HSC-T6 cells undewent apoptosis,whose nucleus appeared to be dark brown,and apoptotic bodies could be observed.Observation by electronic microscope demonstrated that in the control group, organelles such as mitochondria and rough endoplasmic reticulum in HSC-T6 cells exhibited normal morphology.However,in iron deposited model group,there appeared a large number of binucleated cells,in which organelles such as mitochondria and endoplasmic reticulum decreased dramatically.And in the desferrioxamine group,HSC-T6 cells underwent cell apoptosis,with typical features of a reduction in cell volume,nuclear chromatin condensation,obvious reduction of organelles such as mitochondria and endoplasmic reticulum,which were substituted by a large amount of vacuoles3.Effect of Compound Gan Du Qing on iron deposition induced HSC activation and reversionImmunohistochemistry results demonstrated that the expression ofα-SMA decreased significantly in HSC-T6 cells of the iron deposited model+traditional Chinese medicine group compared with the normal control group,implying that activation of cells was inhibited to certain extent.TGF-β₁ mRNA expression of HSC-T6 cells exhibited that TGF-β₁ mRNA expression was down regulated at different degrees in the iron deposited model + traditional chinese medicine group(P＜0.05).Results implied that Compound Gan Du Qing could inhibit TGF-β₁ expression at transcription level.Pathology of HSC-T6 cells apoptosis showed that apoptosis occurred in the HSC-T6 cells of the iron deposited model + traditional Chinese medicine group,with the nucleus densely stained dark brown and apoptotic bodies could be detected.Observation by electronic microscope demonstrated that apoptosis occurred in the HSC-T6 cells of the iron deposited model + traditional Chinese medicine group,with typical apoptosis characteristics of reduction in cell volume,nuclear chromatin condensation,obvious reduction of organelles such as mitochondria and endoplasmic reticulum,which were substituted by a large amount of vacuoles.(二) Animal experiments:Liver fibrogenesis of iron deposition in HSC in DMN induced rat liver fibrosis and the effect of Compound Gan Du Qing1.Changes of rat serum ferritin and transferrin in DMN induced rat liver fibrosis:As for the concentration of rat serum ferritin and transferrin,we can see that the concentration of rat serum ferritin in the model group has an significant increase(P＜0.05) than in the control group,and for model + desferrioxamine group,the serum ferritin concentration has a decrease,but there is no statistical significance(P＞0.05),whereas when compared with the model group,the serum ferritin concentration of the model + desfen-ioxamine group decrease dramatically with a statistical significance(P＜0.01).As for transferrin,the concentration in the model group has a significant decrease(P＜0.01),and though transferrin concentration could be detected in the model + desferrioxamine group,there was no statistical significance(P＞0.05).However,when compared with the model group,the concentration of transferrin increase dramatically with a statistical significance(P=0.01). Results revealed that in iron overloaded rat models,the increase of iron concentration is followed by the reduction of transfenin level.2.Changes of hepatic iron concentration of liver tissue in DMN induced rat liver fibrosis:Investigation of hepatic iron concentration demonstrated that the hepatic iron concentration increased in both the model group and the model + desferrioxamine group(P＜0.01 or P＜0.05) compared with the control group,and when compared with the model group,hepatic iron concentration has a significant decrease in the model + desferrioxamine group(P＜0.05).3.Histopathology of iron deposition in HSC in rats of DMN induced liver fibrosis: Prussian blue staining demonstrated that,in the model group,the dark blue iron pellet mainly distributed along the space between lobules,and most iron deposit outside cells.For the model + desferrioxamine group,the distribution of iron pellet decreased compared with the model group.And in normal liver tissues,cells are arranged uniformly,with no iron overload.Prussian blue andα-SMA double staining showed that consistent with the dark blue iron pellet distribution,and around the necrosis area,large number ofα-SMA positive HSC cells could be detected,with iron pellet distributed in HSC.4.Effect of desferrioxamine therapy and Compound Gan Du Qing on hepatic function, serum iron and serum lipid in rats of DMN induced liver fibrosis:Examination of hepatic function,serum iron and serum lipid revealed that,ALT in model group increased significantly(P＜0.01),and at meantime,ALB and G levels decreased significantly(P＜0.01).Desferrioxamine,colchicines,large and small dosage of Compound Gan Du Qing could all decrease ALT level,with desferrioxamine,large and small dosage of Compound Gan Du Qing superior to colchicine(P＜0.01 or P＜0.05).As for the decreasing of AST levels,there was no significant difference between desferrioxamine and small dosage of Compound Gan Du Qing,but both are superior to colchicine(P＜0.01 or P＜0.05).For ALB level increasing,Compound Gan Du Qing large dosage was superior to desferrioxamine,colchicine and Compound Gan Du Qing small dosage(P＜0.01 or P＜0.05).And for G levels,desferrioxamine,colchicine and Compound Gan Du Qing small dosage could all decrease G level(P＜0.01 or P＜0.05). Moreover,desferrioxamine could significantly decrease the levels of serum iron and TG(P＜0.01 or P＜0.05),whereas neither colchicine nor large or small dosage of Compound Gan Du Qing could decrease the levels of serum iron,TG or CHOL(P＞0.05).5.Effect of desferrioxamine therapy and Compound Gan Du Qing on histopathology in rats of DMN induced liver fibrosis:By HE staining we can see that the hepatocellular plates in normal control group was in streaky-shape with the central vein as center and distributed radially outward.There was hepatic sinusoid with regular distribution,but no collagen fiber could be seen.In the model group,hepatocytes got denatured and necrotic, and the hepatic lobule structure was destroyed,fibrous tissues proliferated and pseudolobules formed,and hemorrhagic necrosis can be detected around the fibrotic tissues, with dark brown particles(iron) deposited in liver tissues.And for model + desferrioxamine group,model + colchicine group,model + Compound Gan Du Qing large dosage group and model + Compound Gan Du Qing small dosage group,there was decrease at various degrees in necrotic hepatocytes,fibrous tissues and pseudolobules.Masson staining exhibited that only a small amount of collagen fibers could be observed in portal areas and the wall of central vein in normal control group.And in the model group,there was obvious collagen proliferation in rat hepatic tissues,and a large number of thick fibrous septa extended inward the hepatic lobules,dividing and enwraping the liver tissue,leading to pseudolobule formation.And in model + desferrioxamine group, model + colchicine group,model +Compound Gan Du Qing large dosage group and model + Compound Gan Du Qing small dosage group,there was obvious decrease in fibrous tissue proliferation,and fibrous septa turned narrow with faint coloring,and pseudolobules decreased as well.By electronic microscope we can detect that in control group,hepatocytes appeared to be polyhedron,with organelles such as mitochondria and rough endoplasmic reticulum exhibiting normal morphology,the nucleus of hepatocytes appeared to be circular or elliptical,the bilayer film was intact,and the nuclear pore was clear.In iron overload model group,large amount of lipid droplet could be observed in hepatocytes,Disse space was filled with large number of red cells,and other changes in hepatocytes structure include mitochondrial vacuolation and endoplasmic reticulum dilatation,appearing to be giant vesicles.In the model + desferrioxamine group,the amount of lipid droplet and red blood cells in hepatocytes reduced greatly,and the nuclear chromatin condensed,the cell volume got reduced,organelles such as mitochondria and endoplasmic reticulum reduced greatly as well.In the model + colchicine group,model + Compound Gan Du Qing large dosage group and model + Compound Gan Du Qing small dosage group,various degrees of reduction of lipid denaturation and bleeding in hepatocytes can all be detected compared with iron overloaded model group.6.Effect of desferrioxamine therapy and Compound Gan Du Qing on liver tissue collagen in rats of DMN induced liver fibrosis:Ridit analysis of iron overloaded rat liver tissue collagen indicated that the average Ridit values of the six groups were unequal or not completely equal(χ²=44.2236,ν=5,P＜0.01).Further rank-sum test by multi sample and multiple comparison(Nemenyi method) showed that compared with the control group, collagen deposition was greatly enhanced in model group,model+colchicine group and model +Compound Gala Du Qing small dosage group(P＜0.05).However,compared with the model group,collagen deposition was significantly decreased in the model + desferrioxamine group and model + Compound Gan Du Qing small dosage group.And compared with the model + desferrioxamine group,there was no significant differences(P＞0.05) in collagen deposition for the model + colchicine group,model + Compound Gan Du Qing large dosage group,and model + Compound Gan Du Qing small dosage group.7.Effect of desferrioxamine therapy and Compound Gan Du Qing on TGF-β₁ mRNA expression in rats of DMN induced liver fibrosis:Expression of TGF-β₁ mRNA in rat liver tissues indicated that TGF-β₁ lnRNA expression was greatly enhanced(P＜0.01) in the model group and the other groups compared with the control group,whereas compared with the model group,TGF-β₁mRNA expression was significantly down regulated(P＜0.01) in the model + desferrioxamine group,model + colchicine group,model + Compound Gan Du Qing small dosage group,and model + Compound Gan Du Qing large dosage group,with desferrioxamine,small and large dosage of Compound Gan Du Qing significantly superior to colchicine(P＜0.01).Between model + Compound Gan Du Qing small dosage group and model + Compound Gan Du Qing large dosage group,there was no significant difference(P＞0.05) in down regulation of TGF-β₁mRNA expression.Conclusions1.Cellular experiment:Confirmed that exogenous iron could deposit in HSC,thus induce HSC activation,and further lead to liver fibrogenesis.By desferrioxamine therapy,HSC activation was suppressed at various degrees,some of which became quiescent or even undewent apoptosis.Compound Gan Du Qing could play the anti-fibrosis role in vitro, whose mechanism might be its down regulation of TGFβ1 expression,inhibition of HSC activation,and inducing HSC to become quiescent or even undewent apoptosis.2.Animal experiment:In rats of DMN induced liver fibrosis,accompanied with collagen deposition and hepatocytes denaturation and necrocytosis,iron not only deposited in liver tissue,but also in HSC.By desferrioxamine therapy,iron deposition in liver tissue had decreased,and the degree of collagen staining and hepatocytes denaturation and necrocytosis was improved significanttly,which showed that iron deposition in liver tissue was one of the stimulatives in DMN induced rat liver fibrosis.The mechanism related to iron deposition in HSC promoting the activation of HSC.Compound Gan Du Qing could protect hepatocytes and improve liver function.It could inhibit the secretion of TGFβ1 at the transcription level,thus suppress HSC activation.It could also prevent the proliferation of collagen fibers in liver.Therefore,Compound Gan Du Qing can play the anti- fibrosis role in vivo.