| Objective: Hepatitis B is very common in China and it lifts the risk of hepaticfibrosis and cirrhosis if it can not be controlled. The five-year mortality of the patientswith decompensatory-stage liver cirrhosis is70%-86%. However, liver cirrhosis lackseffective drugs for prevention and its mechanism is still unclear. Normally, iron isdeposited in liver, spleen and bone marrow of the reticuloendothelial system. Recentreports demonstrate that iron overload is common in end-stage cirrhotic diseases, suchas non-alcoholic fatty liver disease and hepatocellular carcinoma. It can also occur inpatients with history of repeated blood transfusions. Iron overload may play a majorrole in multiple chronic liver diseases. Heme oxygenase-1(HO-1) is the primaryrate-limiting enzyme in heme catabolism. It catalyzes the oxidative degradation of hemeinto free iron, carbon monoxide (CO), and biliverdin. HO-1expression is up-regulatedin liver cirrhosis and it may participate in the pathological processes. Our previousstudies showed that over-expression of HO-1could be harmful to the liver in rats withcirrhosis induced by bile duct ligation (BDL). But how iron overload affects thepathogenesis and treatment of patients with hepatic fibrosis is not yet well explained.Clinically, repeated large-volume blood transfusions are sometimes necessary forcirrhotic patients with massive upper gastrointestinal bleeding; in most cases, patientsare transfused with packed red blood cells, which lead to iron overload. Cirrhoticpatients often have hypersplenism due to splenomegaly. Damaged erythrocytes maylead to iron overload. In this study, we regulate the production of iron throughiron-lifting, iron removal, splenectomy, inducing or inhibiting HO-1expression, inorder to investigate the possible mechanism of iron overload in liver cirrhosis. Part IThe study on intervention of iron homeostasisin in livercirrhotic ratsObject: To investigate the effects of the iron deposition on liver fibrosis in ratswith hepatic fibrosis induced by bile duct ligation (BDL).Methods: Healthy male Sprague-Dawley rats were divided randomly into Shamgroup, BDL group, Fe group and deferoxamine (DFX) group. The levels oftransforming growth factor-β1(TGF-β1) and α-smooth muscle actin (α-SMA) weredetected by western blot and RT-PCR,α-SMA was also detected byimmunohistochemistry. The levels of serum iron, serum ferritin and liver iron content(LIC) were also quantified. Liver iron deposition was observed with Perl’s Prussianblue stain. Hepatic fibrosis and its main pathway were assessed using HE andhydroxyproline. The levels of malonaldehyde (MDA) and superoxide dismutase (SOD)in liver were detected.Results: The levels of AST, ALT and TBIL were higher in BDL group than thosein the Sham group, they were significant increased in the Fe group, and decreased in theDFX group (P <0.01). The protein and mRNA expression levels of TGF-β1andα-SMA were increased in the BDL group compared with those of Sham group, andwere much higher in the Fe group (P <0.01). The DFX group showed less expression ofTGF-β1and α-SMA (P <0.01). The levels of serum iron, serum ferritin and LIC werehigher in the BDL group (229.74±9.72μg/dL vs165.4±10.79μg/dL, P <0.01;45.94±0.52μg/L vs39.42±0.38μg/L, P <0.01;440.74±12.58μg/g vs350.15±16.4μg/g,P <0.01), but were lower in the DFX group (181.63±4.34μg/dL vs229.74±9.72μg/dL,P <0.01;42.54±0.33μg/L vs45.94±0.52μg/L, P <0.01;361.55±21.24μg/g vs440.74±12.58μg/g, P <0.01) and were higher in the Fe group compared to BDL group(378.92±35.59μg/dL vs229.74±9.72μg/dL, P <0.01;53.12±0.65μg/L vs45.94±0.52μg/L, P <0.01;593.55±6.66μg/g vs440.74±12.58μg/g, P <0.01). The SOD level wasincreased and MDA level was decreased in the DFX group (P <0.01).Conclusion: Excessive iron could activate oxidative stress and aggravate liverfibrosis. Reducing hepatic iron deposition by DFX may inhibit TGF-β1/Smads pathwayand prevent hepatic fibrosis. Part IIThe study on iron homeostasis by inducing or inhibiting hemeoxygenase-1in liver cirrhotic ratsObject: To investigate the effects of the heme oxygenase (HO)-1/carbonmonoxide system on iron deposition and liver fibrosis in rats with hepatic fibrosisinduced by bile duct ligation (BDL).Methods: Male Sprague-Dawley rats were divided randomly into a Sham group,BDL group, cobalt protoporphyrin (CoPP) group and zinc protoporphyrin (ZnPP) group.The levels of HO-1were detected using western blot, RT-PCR andimmunohistochemistry. The protein and mRNA levels of nuclear factor-E2-relatedfactor2(Nrf2) were measured. The serum iron, and liver iron content (LIC) were alsoquantified. Liver iron were also visualized by Perl’s Prussian blue stain. The plasmalevels of hepcidin and serum transforming growth factor-β1(TGF-β1) were measuredby ELISA. Hepatic fibrosis and its main pathway were assessed using HE and VanGieson’s stain. The levels of malonaldehyde (MDA) and superoxide dismutase (SOD)in liver were detected.Results: The levels of AST, ALT and TBIL were higher in BDL group than in theSham group (P <0.01), they were significant increased in the CoPP group (P <0.01),and lower in the ZnPP group (P <0.01). The protein and mRNA expression levels ofHO-1and Nrf2were increased in the BDL group compared with the Sham group andwere much higher in the CoPP group (P <0.01). The ZnPP group showed lowerexpression of HO-1and Nrf2(P <0.01). The levels of serum iron and LIC wereenhanced in the BDL group compared to Sham group (229.74±9.72μg/dl vs165.4±10.8μg/dl, P <0.01), but were lower in the ZnPP group (315.79±12.0ng/ml vs292.35±11.81ng/ml, P <0.01) and were higher in the CoPP group compared to BDLgroup (275.73±10.13ng/ml vs292.35±11.81ng/ml, P <0.01). Hepcidin levels werehigher (315.79±12.0ng/ml vs292.35±11.81ng/ml, P <0.01), whereas SOD levels wereincreased and MDA levels were decreased in the ZnPP group (P <0.01). The ZnPPgroup also showed inhibited TGF-β1expression, as well as obviously attenuated liverfibrosis.Conclusion: Reducing hepatic iron deposition by inhibiting HO-1activity maybethough the Nrf2/Keap1pathway could be helpful in improving hepatic fibrosis andregulating PVP. Part IIIThe study on iron homeostasis and portal vein pressure bysplenectomy in liver cirrhotic ratsObject: To find out whether splenectomy in cirrhotic rats induced by bile ductligation (BDL), through the HO/CO pathway, could slow development of liver fibrosis.Methods: Thirty-two male Sprague-Dawley rats were divided randomly into Sham,BDL, splenectomy groups. Serum alanine aminotransferase (ALT), aspartateaminotransferase (AST) and total bilirubin,(TBIL) were detected. The level of HO-1was detected by western blot and reverse transcription polymerase chain reaction.Serum carboxyhemoglobin (COHb), iron, and portal vein pressure (PVP) were alsoquantified. Liver iron was measured by atomic absorption spectrometry withacetylene-air flame atomization. HO-1in liver and spleen were localized byimmunohistochemistry. Collagen I in spleen was detected by Van Gieson’s stain. Liverand spleen iron were visualized by Perl’s Prussian blue stain. Hepatic fibrosis wasassessed using hematoxylin and eosin (H&E) staining. Enzyme-linked immunosorbentassay (ELISA) was used to detect serum transforming growth factor-β1(TGF-β1).Malonaldehyde (MDA) and glutathione (GSH) levels were measured.Results: The levels of AST, ALT and TBIL were significant higher in BDL groupthan in Sham group (P <0.01), and they were lower in Splenectomy group compared toBDL group (P <0.01). HE stain showed that HO-1mainly expressed in Kupffer cells.The results show that liver, spleen, and serum levels of HO-1, COHb and iron weregreatly enhanced in the BDL group compared with Sham (P <0.01). They were reducedfollowing splenectomy (P <0.01). Hydroxyproline (HYP), TGF-β1and PVP levelswere higher in BDL group compared to Sham group, and HYP (153.2±12.4μg/g vs173.4±18.4μg/g, P <0.01), TGF-β1(4.6±0.28ng/ml vs9.86±0.72ng/mL, P <0.01),and PVP (10.31±0.98mmHg vs15.81±1.66mmHg, P <0.01) were lower in thesplenectomy group compared to BDL group. MDA levels lower in the splenectomygroup compared to BDL group (P <0.01).Conclusion: Our study shows that splenectomy reduces iron and CO levels in partby reducing HO-1expression, decreased portal pressure, and slightly decreased hepatic fibroproliferation. |
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