Study Of The Therapeutic Efficacy Of Angelica Sinensis Polysaccharide And Its Underlying Mechanism In The Treatment Of Anemia Of Inflammation And Anemia In Chronic Kidney Disease

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-?B,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.