Inhibition Of Hepcidin Expression By Polysaccharide From Angelica Sinensis In Normal And ACD Rats

Hepcidin （a25amino-acid antimicrobial peptide produced by hepatocytes） is anegative regulator of iron homeostasis, which is thought to be a principal iron-regulatoryhormone. When hepcidin expression is inhibited, intestinal iron absorption and macrophageiron release increase, more of iron can be absorbed and utilized to hematopoietic. Previousstudies have suggested that orally administered ASP from the roots of Angelica sinensis（Oliv.） Diels once daily for14consecutive days, hepcidin levels of the experimental groupssignificantly decreased. One of our study purposes was to explore whether hepcidininhibition was enhanced with increasing ASP dose. The other aim was to clarify theunderlying mechanism in hepcidin suppression caused by ASP.Clinical testing found that hepcidin levels of patients with chronic inflammatoryanemia （ACD） in vivo significantly added. For the further study whether ASP can treatACD by supressing hepcidin expression, ACD was induced by the subplantar injection of0.1ml Complete Freund’s Adjuvant （CFA）. After intragastric administration of differentdoses of ASP, serum hepcidin, hematological indices, iron status and inflammatory factorsin model rats were detected.Part One Different concentrations of ASP on inhibition of normal rats hepcidinexpression and its molecular processesTraditional hot water extraction was used to extract ASP from A. Sinensis, then mergedtwo water extracts. Ca（OH）2was added into the ASP water to remove protein, tannic acidand other impurities. The filtrate was added sulfuric acid to adjust the pH to5-6, adjustingthe solution pH can not only remove impurities but also meet the oral administration of PHrequirements. Extract put into a60℃oven to dry. The sugar content of angelicapolysaccharide extract was measured by5%phenol-sulfuric acid method. Anhydrousglucose was diluted to prepare the reference solution, the standard curves calculated thatcarbohydrate content of all ASP was higher than70%.48SD male rats were randomly divided into six groups.48male SD rats wererandomly divided into six groups. Negative control group received normal （0.9%） saline. Positive control groups were the recombinant human erythropoietin （rhEPO） treatmentgroups （800and2000U/kg body weight, respectively）. The experiment groups were givenASP （0.3,0.6and1.2g/kg body weight, respectively）. The negative control group andexperimental groups were administered orally once daily for20consecutive days. Thepositive groups were given rhEPO by intraperitoneal injection once daily for3consecutivedays. Blood was taken from the retro-orbital sinus before the first administration and24hafter the last administration. All rats were executed to detect the levels of liver iron.After20-day treatment of ASP at three doses （0.3,0.6and1.2g/kg）, hepcidin levels were reducedsignificantly by19.4%,27.1%and31.2%, respectively. Hepcidin inhibition was enhancedwith increasing ASP dose. The rats intraperitioneally injected with rhEPO （800U/kg and2000U/kg） had significantly decreased hepcidin by24.3%and29.5%, respectively.Compared with negative groups, red blood cells and hemoglobin content of rhEPO groupshad significantly increased. Serum iron was2.71±0.53mg/L in saline group. Serum iron in800U/kg rhEPO and2000U/kg rhEPO was2.08±0.39mg/L and1.70±0.18mg/L. Serumiron and the iron content of the liver in rhEPO groups were significantly reduced comparedwith the negative group.1.2g/kg ASP decreased serum iron to1.68±0.27mg/L.The presentstudy showed that treatment with rhEPO and ASP （1.2g/kg） led to a reduction in ironstorage in the liver and to a significant decrease in the serum levels of iron. Iron isredistributed to meet the need of erythropoiesis.Western blot results showed that ASP down-regulated content of total JAK2protein, sop-JAK2correspondingly decreased. The p-JAK2content of rats administrated2000U/kgrhEPO significantly reduced, indicating that EPO inhibits phosphorylation process of totalJAK2protein rather than the expression of JAK2. Both ASP and rhEPO significantly lessenp-SMAD1/5/8content, it blocked phosphorylation of dimer synthesis, which make accessto the hepcidin gene promoter nucleus. Finally hepcidin expression was inhibited.Compared with the negative control group, signaling proteins expression of ERK1/2andTMPRSS6in ASP and rhEPO groups had no obvious change. This indicated that ASP andrhEPO suppress hepcidin expression without adjusting Erk1/2or TMPRSS6. Part Two Different concentrations of ASP on inhibition of ACD rats hepcidinexpression and its molecular processesACD was induced by the subplantar injection of0.1ml CFA containing10mg/mlmycobacterium tuberculosis. Injected foot primary swelling appeared significantly. At10days after injection, model rats appeared secondary reaction: the swelling of thenon-injected contralateral foot and forefoot, inflammation of the ear and tail. Comparedwith negative control group, the red blood cells, hemoglobin and serum iron content ofACD model rat decreased to6.06×10¹²/L,92.4g/L and1.78mg/L. While, its white bloodcells, inflammatory cytokine, like IL6and TNF-alpha contents were significantly increasedto2.58×10¹⁰/L,84.10pg/ml and68.34pg/ml. Animal model plantar foot injected withfreund’s complete adjuvant character of ACD.The key of treatment of ACD is to anti-inflammatory therapy and improve anemia.ACD of experimental rats oralled respectively0.5g/kg and1.0g/kg of angelica sinensispolysaccharide every day, continuously for4weeks. Positive control group is to ACD ratgastric10mg/kg of diclofenac sodium each day, for4weeks continuously. Measure thefoot swelling, as well as the detection of blood routine, hepcidin, serum iron andinflammatory factor index. The high dose of ASP group （1.0g/kg） stronger than the lowdose of ASP group （0.5g/kg） to inhibit hepcidin expression in ACD rat, inhibition rateswere43.1%and28.7%,respectively. Compared with model group rats, the contents ofserum hepcidin diclofenac sodium was also significantly reduced by38.0%. Diclofenacsodium inhibited ACD rat primary foot swelling and secondary foot swelling were strongerthan the1.0g/kg ASP group. White blood cell count was2.58×10¹⁰/L in model group rats,giving diclofenac sodium and1.0g/kg ASP, leukocyte content in ACD rats decreasedsignificantly after treatment, was1.59×10¹⁰/L and1.77×10¹⁰/L, respectively. IL-6andTNF-alpha content in rats treated with diclofenac sodium less than it in1.0g/kg ASP group.The foot X-rays and synovial of hematoxylin and eosin （HE） staining showed that thepositive control group （given diclofenac） had joint protection and the most significantlyreduced inflammatory cells, which means that the anti-inflammatory effect of diclofenacstronger than1.0g/kg ASP. RBC count of model group rats was6.06×10¹²/L, diclofenacsodium and1.0g/kg ASP caused a significant rise in content of red blood cells,7.68×10¹²/L and8.16×10¹²/L, respectively. Red blood cell levels in1.0g/kg ASP treatment of ACD rathigher than that of diclofenac sodium group. Compared with model group, the ironmetabolism related indexes such as hemoglobin and serum iron content also increasedsignificantly, suggesting that ASP （1.0g/kg） effectively improved anemia on ACD rats. Thetherapeutic effect of high doses of ASP on ACD rats stronger than low dose group.Western blot results showed that ASP and diclofenac sodium can inhibit the expressionof hepcidin through reduced signal protein JAK2, STAT3and p-SMAD1/5/8content.Signal protein contents of1.0g/kg ASP lower than0.5g/kg ASP. The three proteins contentof high-dose ASP group was lowest, JAK239.5%, STAT38.1%and p-SMAD1/5/842.0%,respectively. Perhaps this was one of the reason that1.0g/kg ASP can be more significantlysuppressed the expression of hepcidin, compared with the diclofenac sodium group.