【作者】 吴俊；

【导师】 王凯平；

【作者基本信息】 华中科技大学 ， 药剂学， 2018， 博士

【摘要】 当归是治疗贫血的传统中药,当归多糖（Angelica sinensis polysaccharide,ASP）是从当归饮片中分离纯化得到的水溶性大分子多糖。铁调素（hepcidin）是机体铁代谢平衡的核心调控者,hepcidin高表达在炎症性贫血和肾性贫血的发病过程中发挥着重要作用。促红细胞生成素（erythropoietin,EPO）是调控红细胞生成的必需生长因子,EPO缺乏是引起肾性贫血的主要原因。本课题组在前期研究中发现ASP可以调节缺铁性贫血大鼠铁代谢,降低大鼠血清hepcidin水平,改善贫血。近年来在中药治疗肾性贫血的研究中发现,当归及当归补血汤辅助重组人促红细胞生成素（recombinant human erythropoietin,rhEPO）治疗肾性贫血具有疗效好、安全性高的特点,且能显著减少rhEPO的用量。本文旨在研究ASP抑制hepcidin表达的作用机制,探讨ASP对EPO表达的作用,研究ASP对炎症性贫血和肾性贫血的疗效和作用机制,为开发新型炎症性贫血和肾性贫血的治疗药物提供新思路,也对阐明中药当归治疗贫血的作用机理有积极的作用。本文主要研究内容和成果如下:第一部分当归多糖体外抑制hepcidin表达的研究根据本课题组前期建立的当归多糖分离纯化工艺制备ASP,对其糖含量、纯度、分子量、红外图谱等进行质量考察。结果表明提取的ASP性状均一,产率较高,性质稳定,达到了后续实验的要求。采用重组人IL-6细胞因子刺激HepG2细胞hepcidin表达,分别加入50、100、200?g/mL ASP共培养,通过蛋白质免疫印迹法和免疫荧光分别检测STAT3的磷酸化水平和核转录,采用实时荧光定量PCR检测hepcidin mRNA表达。结果表明,ASP显著抑制了IL-6诱导的STAT3磷酸化和核转录,下调了hepcidin mRNA水平,三个浓度ASP对hepcidin mRNA的抑制率分别为58.8%、66.5%、74.7%,呈剂量依赖性。为进一步探究ASP下调IL-6诱导的STAT3激活和hepcidin表达的作用靶点,加入JAK2蛋白抑制剂AZD1480,研究了ASP在加入AZD1480情况下对IL-6诱导的hepcidin表达的影响。结果发现,在加入AZD1480干预后,ASP对IL-6诱导的p-JAK2及hepcidin mRNA表达无显著影响,表明ASP可能通过抑制JAK2蛋白激活下调JAK2/STAT3信号通路,进而抑制hepcidin转录。采用重组人BMP6细胞因子刺激HepG2细胞hepcidin表达,加入BMP I型受体抑制剂LDN-193189干预,观察了200?g/mL ASP在未加入以及加入LDN-193189情况下对p-SMAD1/5/8和hepcidin mRNA表达的影响。研究表明,ASP能显著抑制BMP6诱导的p-SMAD1/5/8和hepcidin mRNA表达,而在加入LDN-193189干预的细胞中,ASP组p-SMAD1/5/8和hepcidin mRNA的表达相较于BMP6组无显著变化,提示ASP可能通过作用于BMP I型受体来下调BMP/SMAD通路,进而抑制hepcidin转录。给予HepG2细胞脂多糖（lypopolysaccharide,LPS）刺激,分别加入50、100、200μg/mL ASP共培养,考察ASP对LPS刺激的p-STAT3、p-SMAD1/5/8、hepcidin mRNA表达的影响。结果表明,与LPS组相比,三个浓度ASP均显著下调了LPS诱导的p-STAT3和p-SMAD1/5/8;100、200μg/mL ASP组hepcidin mRNA水平相较于LPS组均显著降低,抑制率分别为48%和86.6%,而低浓度ASP组无显著性差异。酶联免疫法（ELISA）检测细胞上清液IL-6水平可知,三个浓度ASP明显抑制了LPS刺激的IL-6分泌,抑制率分别为58.8%、66.5%、74.7%,具有剂量依赖性。蛋白质印迹法检测NF-?B、p-I?B?表达表明,ASP阻断了LPS诱导的I?B?的磷酸化及核NF-?B的表达。根据本部分实验结果可知ASP抑制hepcidin表达的机制为:（1）可能通过抑制JAK2蛋白激活下调JAK2/STAT3信号通路,进而抑制hepcidin转录;（2）可能通过阻断BMP6与BMP I型受体相互作用下调BMP/SMAD信号通路,进而抑制hepcidin转录;（3）通过抑制NF-?B信号通路减少IL-6分泌间接抑制hepcidin表达。本研究为后续ASP作为hepcidin抑制剂治疗铁限制性贫血的体内研究奠定了基础。第二部分当归多糖改善炎症性贫血的体内研究采用给大鼠足跖皮下注射0.15 mL结核分枝杆菌浓度为10 mg/mL的弗氏完全佐剂建立炎症性贫血模型,设立双氯芬酸钠、rhEPO、双氯芬酸钠和rhEPO联合给药三个阳性对照组,分别以0.5 g/kg、1.0 g/kg体重剂量给大鼠灌胃ASP溶液治疗4周。实验结束后检测大鼠血红蛋白（Hb）水平和红细胞（RBC）计数。由结果可知,低、高剂量ASP均显著升高了ACD大鼠Hb水平和RBC计数,低剂量ASP组Hb水平和RBC计数分别升高至135.56 g/L、7.74×10¹²/L,高剂量ASP组为145.29 g/L、8.5×10¹²/L。与阳性对照组相比,高剂量ASP对贫血的改善作用与双氯芬酸钠、rhEPO相当,但弱于双氯芬酸钠和rhEPO联合给药。检测大鼠白细胞计数（WBC）和血清TNF-?、IL-6水平可知ASP的治疗明显降低了ACD大鼠WBC计数和血清TNF-?、IL-6水平。此外ASP显著升高了ACD大鼠血清EPO水平。采用普鲁士蓝染色观察大鼠脾脏组织铁沉积,原子吸收光谱法检测脾脏铁和血清铁水平,ELISA检测血清ferritin水平。由结果可知,模型组大鼠脾脏组织显示出明显的铁沉积,脾脏铁含量相较于对照组显著升高了61.4%,血清铁下降了27.2%,表明ACD大鼠组织铁过载,循环铁利用率低;给药ASP治疗后,大鼠脾脏铁沉积得到明显改善,血清ferritin水平降低,血清铁水平升高。检测大鼠血清hepcidin水平和肝脏hepcidin表达可知,给药低、高剂量ASP治疗后血清hepcidin水平分别下降了14.6%和21.3%,肝脏hepcidin表达也显著下调。为探究ASP改善ACD大鼠铁代谢、炎性的具体分子机制,采用蛋白质印迹法检测大鼠相关蛋白表达,包括（1）肝脏调控hepcidin转录的上游JAK2/STAT3信号通路蛋白p-JAK2、p-STAT3、STAT3,BMP/SMAD信号通路蛋白BMP6、p-SMAD1/5/8、SMAD4;（2）脾脏、肝脏铁代谢相关蛋白ferroportin和ferritin;（3）肝脏NF-?B信号通路蛋白IKK?、p-I?B?和NF-?B。结果显示,ASP显著下调了ACD大鼠肝脏JAK2/STAT3和BMP/SMAD通路,升高了脾脏、肝脏ferroportin表达,降低了ferritin水平,抑制了NF-?B信号通路的激活。本部分研究表明ASP主要通过抑制hepcidin表达和抗炎两方面改善炎症性贫血,具体机制分别为:（1）通过下调JAK2/STAT3和BMP/SMAD通路抑制hepcidin表达,进而升高脾脏、肝脏ferroportin表达,增加组织铁输出,从而改善大鼠单核巨噬系统铁沉积,提高循环铁利用率;（2）通过抑制NF-κB的激活下调炎性因子的分泌,一方面间接抑制hepcidin表达,另一方面缓解炎性因子对EPO的抑制作用。本部分研究为ASP作为hepcidin抑制剂防治炎症性贫血提供新的理论和实验依据。第三部分当归多糖体外上调EPO表达的研究本实验采用在培养基中加入50?M CoCl₂刺激Hep3B细胞24小时诱导细胞缺氧,加入100、200?g/mL两种浓度ASP共培养,观察ASP对缺氧诱导HIF-1/2?蛋白和EPO mRNA表达的影响。结果显示,与缺氧组相比,两种浓度ASP均显著上调HIF-1（14）2?蛋白以及EPO mRNA的表达,且呈剂量依赖性。检测HIF-1/2?mRNA表达量可知ASP对HIF-1/2?mRNA水平无显著影响。为探究ASP上调HIF-1（14）2?蛋白表达的作用机制,加入放线菌酮处理Hep3B细胞不同的时间,以抑制蛋白合成检测蛋白的半衰期。结果表明,缺氧诱导的HIF-1（14）2?蛋白半衰期约为0.5 h,而200?g/mL ASP的加入显著延长了蛋白的半衰期大约至3 h。分别采用重组人TNF-?和IL-1?刺激Hep3B细胞,加入100、200?g/mL两种浓度ASP共培养,观察ASP对炎性因子抑制EPO表达的影响。由结果可知,100、200?g/mL ASP的干预显著抑制了TNF-?和IL-1?诱导的GATA2、NF-?B激活,缓解了TNF-?和IL-1?对EPO的抑制作用,上调了EPO mRNA表达,且呈剂量依赖性。接下来观察了ASP对TNF-?在缺氧条件下抑制EPO表达的影响,与对照组相比,缺氧明显上调了核NF-?B和GATA2的表达,TNF-?的加入进一步增加了核NF-?B和GATA2的水平,而200?g/mL ASP组NF-?B和GATA2的表达水平明显减少,且显著性低于缺氧对照组水平。检测EPO mRNA结果显示,ASP显著缓解了TNF-?在缺氧条件下对EPO的抑制作用,且与缺氧对照组相比,ASP组EPO mRNA水平更高。本部分的研究结果表明:（1）ASP通过抑制HIF-2?蛋白降解,延长HIF-2?蛋白的半衰期,进而刺激缺氧诱导EPO的表达,也可能通过抑制缺氧诱导的GATA2、NF-?B激活进一步上调EPO表达;（2）ASP可以显著缓解炎性因子TNF-?、IL-1?对EPO的抑制作用,该作用与下调核核转录因子GATA2、NF-?B有关;（3）ASP能显著缓解TNF-?在缺氧条件下对EPO的抑制作用,一方面通过阻断GATA2、NF-?B激活,一方面可能通过上调HIF-2?蛋白表达促进EPO的转录。该部分研究为后续ASP改善肾性贫血的体内研究奠定了基础。第四部分当归多糖改善肾性贫血的体内研究采用给大鼠先喂食含0.75%腺嘌呤饲料4周再喂食正常饲料4周的方法建立慢性肾脏病（CKD）贫血大鼠模型,以rhEPO为阳性对照药物,给予0.5 g/kg、1.0 g/kg ASP治疗6周。实验期间监测大鼠血常规指标包括Hb、RBC和WBC,及肾功能指标血清尿素氮（BUN）和血清肌酐（Scr）。结果表明,CKD大鼠在第4周开始出现贫血,至第6周最为严重,后略有回升,到8周时趋于稳定贫血状态。ASP在给药治疗4周后即第6周时显著上调了Hb水平和RBC计数,随后Hb水平和RBC计数逐渐回升,到第8周时接近正常值,基本纠正了贫血,高剂量ASP作用最显著。由BUN和Scr水平再结合大鼠肾脏组织H&E染色结果可知,ASP的治疗在一定程度上改善了CKD大鼠肾功能,缓解了肾组织病变。此外,ASP显著降低了CKD大鼠WBC水平,抑制了CKD大鼠肾脏和脾脏促炎性因子TNF-?、IL-1?和IL-6 mRNA表达,缓解了大鼠炎症反应。铁代谢相关指标的检测结果表明,ASP降低了CKD大鼠脾脏铁和肝脏铁含量;与模型组相比,EPO组、LASP组和HASP组血清铁水平分别升高了21.7%、44.8%和53.1%。实验结束后检测CKD大鼠EPO水平得知,低剂量ASP和高剂量ASP组血清EPO水平相较于模型组分别升高了1.3倍和1.7倍;ASP的治疗显著上调了CKD大鼠肾脏及肝脏EPO mRNA表达,其中低剂量ASP组分别升高了6.5倍、89.2%,高剂量组分别升高了9.1倍、1.3倍。检测大鼠肝脏hepcidin mRNA表达可知,ASP显著抑制了CKD大鼠肝脏hepcidin表达,LASP组和HASP组的抑制率分别为54.1%、76.8%。为探究ASP刺激CKD大鼠内源性EPO分泌的机制,检测大鼠肾脏、肝脏HIF-1?、HIF-2?、GATA2和NF-?B蛋白表达以及脯氨酸羟化酶PHD1、PHD2和PHD3 mRNA表达。由结果可知,ASP激活了CKD大鼠肾脏、肝脏HIF信号通路,抑制了PHD1/2/3mRNA表达,增加了HIF-1/2蛋白积聚;ASP抑制了CKD大鼠肾脏、肝脏GATA2和NF-?B表达。由结果分析可知ASP刺激CKD大鼠内源性肾脏和肝脏EPO分泌的机制如下:（1）通过抑制HIF-2?蛋白降解,增加HIF-2?蛋白水平上调EPO基因转录;（2）通过抑制NF-?B和GATA2激活,缓解NF-?B和GATA2对EPO基因转录的抑制作用增加EPO表达;（3）可能通过改善炎性,缓解炎性对缺氧诱导HIF信号通路抑制作用间接增加EPO表达。提取CKD大鼠骨髓单个核细胞,研究EPO介导的下游细胞内信号转导。结果表明ASP显著增加了骨髓单个核细胞EPOR mRNA的表达;激活了EPOR下游JAK2/STAT5、PI3K/Akt通路,上调了p-JAK2、p-STAT5、p-PI3K和p-Akt表达;增加了信号通路下游抗凋亡基因Bcl-xL mRNA水平和Bcl-2/Bax比例以及铁代谢相关基因TfR1、ERFE mRNA的表达。以上结果表明ASP的治疗上调了CKD大鼠骨髓单个核细胞EPOR表达,从而增强了骨髓红系细胞对EPO的响应;激活了EPOR下游细胞内JAK2/STAT5、PI3K/Akt信号通路,上调了抗凋亡和铁代谢相关目的基因的表达,从而抑制了红系细胞凋亡,促进其增殖分化,增加了红系细胞对铁的利用,促进血红蛋白的合成。研究ASP改善CKD大鼠铁代谢的机制结果可知,ASP下调了p-STAT3、p-SMAD1/5/8表达;增加了肝脏、脾脏ferroportin蛋白水平;减少了肝脏、脾脏ferritin蛋白水平;抑制了脾脏DMT1和TfR1 mRNA表达。由结果分析可知ASP改善CKD大鼠组织铁沉积,升高循环铁水平的分子机制总结如下:（1）ASP通过下调STAT3和SMAD信号通路抑制了CKD大鼠肝脏hepcidin表达,进而上调了大鼠脾脏和肝脏ferroportin的水平,最终增加了大鼠脾脏铁和肝脏铁的输出;（2）ASP可能通过改善炎性抑制了CKD大鼠ferritin表达,从而减少了大鼠肝脏、脾脏铁的储存;（3）ASP可能通过改善炎性减少了CKD大鼠DMT1和TfR1 mRNA水平,从而减少了巨噬细胞对铁的摄入。本部分研究表明ASP通过刺激内源性肾脏、肝脏EPO产生升高了血清EPO水平,增加了骨髓单个核细胞EPOR mRNA表达,进而激活了EPOR下游细胞内信号转导,促进了红细胞生成;通过抑制hepcidin表达和改善炎性动员了组织铁的利用,升高了血清铁水平,增加了红细胞生成所需要的铁原料的供应,最终纠正了肾性贫血。本文为开发新型肾性贫血的治疗药物提供新思路,也揭示了中药当归辅助治疗肾性贫血的机理。更多还原

【Abstract】 The root of Angelica sinensis（Oliv.）Diels（A.sinensis）has been used to treat anemia for thousands of years in Chinese traditional medicine.Angelica sinensis polysaccharide（ASP）,a water-soluble acidic polysaccharide,was isolated and purified from the root of A.sinensis.Hepcidin is the central regulator of system iron metabolism.Hepcidin excess plays a vital role in the pathogenesis of anemia of chronic disease（ACD）and anemia of chronic kidney disease（CKD）.Erythropoietin（EPO）,the primary driver of erythropoiesis,is the hormone essential for maintaining the survival,proliferation and differentiation of erythroid progenitor cells in the bone marrow.Relative deficiency of erythropoietin（EPO）is the predominant cause of anemia in chronic kidney disease（CKD）.Previous study indicated that ASP could regulate iron metabolism,reduce serum hepcidin levels and reverse anemia in rats with iron deficiency anemia.Recent clinical evidence showed that a combined treatment of Danggui Buxue Decoction or A.sinensis and recombinant human erythropoietin（rhEPO）for renal anemia,which could improve the therapeutic efficacy of rhEPO without adverse event observed,and reduce the dose of rhEPO.The primary purpose of this study is to investigate the underlying mechanism for the inhibitory effects of ASP on hepcidin expression,assess the effects of ASP toward EPO expression,and study the therapeutic efficacy of ASP and its underlying mechanism in the treatment of ACD and anemia of CKD.Section I:Investigation of the inhibitory effects of ASP on hepcidinexpression in vitroThe polysaccharide from A.sinensis was isolated and purified based on our previous protocol.The percentage of total sugar,purity,molecular weight and infrared spectra of ASP were determined for identification.Results showed that uniform,high yield and stable ASP was prepared,which met the requirements of follow-up experiments.HepG2 cells were pre-treated with ASP（50,100,200mg/mL）for 16 h,and then exposed to 50 ng/mL IL-6 for another 8 h.The phosphorylation and nuclear translocation of STAT3 were measured by Western Blotting and Immunofluorescence Staining,respectively.Results indicated that ASP significantly decreased the phosphorylation and nuclear translocation of STAT3 induced by IL-6.And the presence of ASP reduced hepcidin mRNA by 58.8%,66.5%and 74.7%,respectively,in a dose-dependent manner.To further explore the mechanisms by which ASP suppressed IL-6-induced STAT3 activation and hepcidin expression,we examined the effects of ASP on IL-6-induced hepcidin expression in the presence of AZD1480,an inhibitor of JAK2.Results demonstrated that ASP did not significantly modulate JAK2 phosphorylation and hepcidin expression compared with the IL-6-treated group in AZD1480-treated cells.These results suggested that ASP could exert inhibitory effects on IL-6-induced STAT3 activation and hepcidin transcription possibly through inhibiting JAK2 activation.Next,we examined the effects of ASP（200mg/mL）on BMP6-mediated SMAD1/5/8phosphorylation and hepcidin induction in the presence of LDN-193189,an inhibitor of BMP type I receptor.Results showed that p-SMAD1/5/8 and hepcidin mRNA levels were significantly increased after BMP6（50 ng/mL）challenge,but attenuated by ASP.LDN-193189 effectively blocked SMAD1/5/8 phosphorylation and hepcidin induction,and no significant changes were observed with ASP treatment in the presence of LDN-193189.Taken together,these results suggested that ASP was able to inhibit SMAD1/5/8phosphorylation and hepcidin activation induced by BMP6,and this effect might be through inhibiting the binding of BMP6 to BMP type I receptor.We proceeded to examine the effects of ASP on LPS-induced p-SMAD1/5/8,p-STAT3and hepcidin mRNA expression.HepG2 cells were stimulated with lippolysaccride in the presence of ASP（50,100,200mg/mL）for 24 h.Compared with LPS group,ASP dramatically decreased p-SMAD1/5/8 and p-STAT3 expression;hepcidin mRNA expression were dose-dependently reduced by ASP at the dose of 100,200mg/mL and the inhibition rate was 48%and 86.6%,respectively.We next analyzed the IL-6 content in cell culture supernatants by ELISA.ASP at the dose of 50,100,200mg/mL dose-dependently ameliorated IL-6 secretion evoked by LPS and the inhibition rate was 58.8%,66.5%and74.7%,respectively.Moreover,immunoblot assays indicated that ASP significantly inhibited p-IkBaexpression and nuclear translocation of NF-kB.These results indicated that ASP inhibited inflammatory hepcidin through attenuating NF-kB activation and IL-6 secretion beyond directly targeting JAK2/STAT3 and BMP/SMAD signaling.This study laid the foundation for the subsequent in vivo study of ASP as a hepcidin inhibitor in the treatment of iron-restricted anemia.Section II:Study of the therapeutic efficacy of ASP and its underlyingmechanism in the treatment of ACD in ratsThe rat model of ACD was inoculated on day 0 with a subcutaneous injection of 0.15mL of complete Freund’s adjuvant（CFA）containing 10 mg/mL of mycobacteria into the paw of the left hind limb of each rat.For ASP experiments,CFA-injected rats were treated with an intragastrical administration of ASP at two dosages（0.5 or 1.0 g/kg BW）for 4 weeks.Another CFA-injected rats were treated with recombinant human erythropoietin（rhEPO）,Diclofenac sodium alone or a combination of Diclofenac sodium and rhEPO as positive controls.At the end of experiments,the mean hemoglobin（Hb）concentrations and red blood cell（RBC）counts of ACD rats were determined.Results indicated that both low-dose and high-dose of ASP treatment resulted in a dramatic elevation of Hb concentrations and RBC counts.And the Hb concentrations and RBC counts in low-dose of ASP group rose to 135.56 g/L and 7.74×10¹²/L,and that in high-dose of ASP group rose to145.29 g/L and 8.5×10¹²/L.Compared with the positive control groups,the effect of high-dose ASP on anemia was comparable to that of diclofenac sodium and rhEPO but weaker than that of combination of diclofenac sodium and rhEPO.Detection of white blood cell counts（WBC）,and serum TNF-aand IL-6 levels in rats showed that treatment with ASP significantly reduced WBC counts and serum TNF-a,IL-6 levels in ACD rats.In addition,ASP significantly increased serum EPO levels.Iron deposition in the spleen of rats were examined by Prussian blue staining.And iron levels in spleen and serum were detected by atomic absorption spectroscopy.Results demonstrated that the spleen displayed evident iron accumulation,spleen iron levels rose by 61.4%and serum iron were reduced by 27.2%,indicating iron sequestration in spleen and limited availability of iron for erythropoiesis.Of note,this iron retention was also ameliorated by ASP treatment,serum iron levels were markedly elevated and serum ferritin levels were dramatically declined with ASP administration.Furthermore,treatment with ASP substantially reduced serum hepcidin levels by 14.6%and 21.3%,respectively,and suppressed hepatic hepcidin expression.To explore the specific molecular mechanism of ASP in improving iron metabolism and inflammation in ACD rats,immunoblot assays were used to detect related proteins expression including:（1）upstream JAK2/STAT3 and BMP/SMAD pathways related proteins regulating hepcidin transcription,p-JAK2,p-STAT3 and STAT3 as well as BMP6,p-SMAD1/5/8 and SMAD4;（2）iron-regulated proteins in spleen and liver,ferroportin and ferritin;（3）NF-kB signaling related proteins,IKKa（11）、p-IkBaand NF-kB.Results showed that ASP significantly downregulated JAK2/STAT3 and BMP/SMAD pathways in the liver of ACD rats,increased ferroportin and reduced ferritin expression in liver and spleen,and inhibited the activation of NF-kB signaling.The current work revealed that ASP corrected inflammatory anemia through suppressing hepcidin expression and ameliorating inflammation,the specific mechanism was as follows:（1）Inhibiting hepcidin expression through down-regulating JAK2/STAT3 and BMP/SMAD pathways,subsequently increasing ferroportin expression and iron efflux in spleen and liver,thereby alleviating iron sequestration within cells of the reticuloendothelial system and improving iron availability;（2）Reducing the secretion of inflammatory cytokines by inhibiting the activation of NF-κB,which contributed both to indirectly suppressing hepcidin expression,and to rescuing the inhibition of EPO production by inflammatory cytokines.This part of the study provided a new theoretical and experimental basis for ASP as a hepcidin inhibitor against inflammatory anemia.Section III:Investigation of the stimulatory effects of ASP on EPOexpression in vitroTo investigate the effects of ASP on hypoxia-induced HIF-1/2aand EPO expression,Hep3B cells were treated with 50mM CoCl₂ for 24 hours to mimic the hypoxic conditions and incubated with ASP（100mg/mL,200mg/mL）for 24 h.Results indicated that ASP significantly up-regulated the expression of HIF-1/2aprotein and EPO mRNA in a dose-dependent manner.Analysis of HIF-1aand HIF-2amRNA expression demonstrated that neither HIF-1anor HIF-2amRNA expression was augmented by ASP treatment under hypoxic conditions.For assessing protein stability,the cells were treated with cycloheximide that inhibited protein synthesis for indicated time.Results showed that the half-life of HIF-1aand HIF-2aprotein were both prolonged to 3 h in the presence of 200mg/mL ASP,whereas that in the absence of ASP was 0.5 h.To assess the effects of ASP on inflammation-inhibited EPO expression,we then used recombinant human TNF-aand IL-1bfor EPO suppression in Hep3B cells,which were co-cultured with 100,200mg/mL ASP.Results demonstrated that ASP significantly inhibited the activation of GATA2 and NF-kB induced by TNF-aand IL-1b,and abrogated the suppression of EPO mRNA in a dose-dependent manner.Next,we evaluated the effects of ASP on the inhibition of EPO by TNF-aunder hypoxic conditions.Results indicated that hypoxia induced GATA2 and NF-kB expression relative to normoxia,and the addition of TNF-afurther enhanced GATA2 and NF-kB expression.By contrast,ASP at the dose of200mg/mL completely abolished the induction of GATA2 and NF-kB activation and partially suppressed the induction by hypoxia compared with normoxia.Consistently,ASP reversed the inhibition of EPO by TNF-aunder hypoxia and further augmented hypoxia-induced EPO.In conclusion,results of this section revealed that:（1）ASP stimulated hypoxia-induced EPO expression by inhibiting the degradation of HIF-2 protein and prolonging the half-life of HIF-2 protein,thereby prompting EPO transcription,and possibly by blocking hypoxia-induced GATA2 and NF-kB activation;（2）ASP could reverse the decrease in EPO induced by TNF-aand IL-1bby blocking NF-kB and GATA2 activation;（3）ASP could rescue the inhibition of EPO expression by TNF-aunder hypoxia through blocking the activation of GATA2 and NF-kB,and probably through up-regulating the expression of HIF-2aprotein.This part of the study laid the foundation for the subsequent in vivo studies of ASP in treating renal anemia.Section IV:Evaluation of the therapeutic efficacy of ASP toward anemiaof CKD in rats and its underlying mechanisms of actionFor the establishment of CKD model,rats were fed a 0.75%adenine supplemented diet for4 weeks followed by a regular diet for 4 weeks.For ASP experiments,the adenine-fed rats were treated with an intragastrical administration of ASP at two dosages（0.5 or 1.0 g/kg BW）for 6 weeks,starting from week 2.One group of adenine-fed rats were treated with a weekly intraperitoneal injection of rhEPO as positive control.Hematological parameters,Hb,RBC and WBC,and renal functional makers,serum urea nitrogen（BUN）and serum creatinine（Scr）were monitored throughout the experimental period.Results showed that there was a dramatic decrease of Hb and RBC at week 4 that reached a nadir at week 6 and persisted to week 8 with Hb 106 g/L and RBC 5.27×10¹²/L.Compared with untreated animals,ASP-treated rats developed less severe anemia,exhibited markedly elevated Hb and RBC levels as early as at week 6 and recovered completely at week 8.And the high-dose of ASP was the more effective.Results of BUN and Scr levels as well as H&E staining of kidneys indicated that treatment with ASP improved renal function and attenuated renal histopathological damage in CKD rats.After ASP treatment,rats showed a prominent reduction in the number of WBCs.Moreover,ASP treatment dose-dependently suppressed TNF-a（11）IL-1band IL-6mRNA expression in kidney,and this effect was paralleled by corresponding changes in the spleen.We next assessed the effects of ASP on iron metabolism and found that iron contents in spleen and liver were dramatically reduced by ASP treatment.In comparison to model group,serum iron levels in EPO group,low-dose and high-dose of ASP groups were increased by 21.7%,44.8%and 53.1%,respectively.At the end of the experiment,the EPO levels in CKD rats were detected.Results indicated that serum EPO levels in low-dose of ASP and high-dose of ASP groups were increased by 1.3-fold and 1.7-fold,respectively,relative to model group.Moreover,treatment with ASP dose-dependently stimulated EPO mRNA in kidney and liver,and EPO mRNA levels in LASP group were increased by 6.5folds and 89.2%,respectively,while that in HASP group were 9.1 folds and 1.3 folds.Detection of hepatic hepcidin mRNA expression in rats showed that two doses of ASP significantly decreased hepatic hepcidin mRNA expression as compared to that in untreated CKD rats,and the inhibition rates were 54.1%and 76.8%,respectively.To study the mechanisms underlying the induction of EPO by ASP,we analyzed the protein expression of HIF-1a（11）HIF-2a（11）GATA2 and NF-kB as well as the mRNA expression of PHD1,PHD2 and PHD3 in kidney and liver of rats.Results revealed that ASP activated HIF signaling pathway in kidney and liver,inhibited the expression of PHD1/2/3 mRNA,and increased the accumulation of HIF-1/2aprotein and repressed GATA2 and NF-kB expression.The mechanism underlying the stimulation of endogenous renal and hepatic EPO secretion by ASP was as follows:（1）Inhibiting the degradation of HIF-2aprotein,increasing the accumulation HIF-2aprotein,thus up-regulating EPO transcription;（2）Attenuating the induction of NF-kB and GATA2,thereby reversing the inhibition of EPO transcription by inflammation;（3）Improving inflammation and rescuing inflammatory inhibition on HIF signaling pathways,thus indirectly increasing EPO production.To further determine the effects of EPO restoration with ASP treatment on downstream EPOR signaling systems,we analyzed EPOR expression,the downstream JAK2/STAT5and PI3K/AKT signaling and their target gene in bone marrow-derived mononuclear cells（BM-MNCs）.Results indicated that ASP treatment significantly enhanced EPOR mRNA,resulted in a trend towards increased expression of p-JAK2 and p-STAT5 as well as p-PI3K and p-Akt and led to a significant rise of Bcl-xL mRNA,Bcl-2/Bax ratio as well as TfR1and Fam132b mRNA.Accordingly,we could demonstrate that ASP might enhance bone marrow response to endogenous EPO by increasing EPOR expression,and cause the proliferation and terminal differentiation of erythroid precursors by suppressing apoptosis of erythroid precursors and increase the utilization of iron for hemoglobin synthesis by upregulating iron-related gene expression.Investigation of the mechanisms underlying the effects of ASP on iron metabolism revealed that ASP tremendously lowered p-STAT3 and p-SMAD1/5/8 levels,increased ferroportin protein levels in liver and spleen,decreased ferritin protein levels in liver and spleen and inhibited DMT1 and TfR1 mRNA expression in spleen.Based on these results,the mechanism of ASP in improving iron deposition and increasing circulating iron levels in CKD rats was summarized as follows:（1）ASP inhibited hepcidin expression in CKD rats by down-regulating STAT3 and SMAD pathways,thereby upregulating ferroportin protein levels,ultimately increasing iron efflux from spleen and liver;（2）ASP inhibited ferritin expression in CKD rats by attenuating inflammation,thereby reducing iron storage in liver and spleen;（3）ASP reduced DMT1 and TfR1 mRNA levels in CKD rats through attenuating inflammation,thereby reducing iron uptake by macrophages.In summary,the present data indicated that ASP increased serum EPO levels by stimulating endogenous renal and hepatic EPO production,enhanced EPOR mRNA expression and activated EPO-mediated downstream intracellular signal transduction,thus stimulating erythropoiesis.Furthermore,by inhibiting hepcidin expression and reducing inflammation ASP mobilized iron from spleen and liver,increases iron availability,consequently restoring iron supply for hemoglobin synthesis,and finally correcting anemia.This study provided new ideas for the development of new therapeutic drugs for renal anemia,and revealed the mechanism of adjuvant treatment of renal anemia with A.sinensis.更多还原