| Anemia is one of the most common symptoms in clinic, and it is accompanied by many diseases. In2010,32.9%of the world’s population suffered from anemia in various degrees, and millions of people died of diseases caused by anemia every year. Thus, the treatment of anemia is very crucial. As a famous traditional Chinese medicine, Fufang E’jiao Jiang (FEJ) was used to replenish qi and nourish blood in clinic. Its exact curative effect in replenishing blood has gained more and more attention. In recent years, progress has been made in studying the therapeutic effect of FEJ on anemia both in animal models and clinical application. However, these studies only observed the overall therapeutic effect of FEJ on anemia without in-depth and systematic researches on its activity, and much less research on the underlying mechanisms. The further development and application of FEJ were limited. Based on previous studies, we established three kinds of animal models with common anemias, which are renal anemia, iron deficiency anemia, myelosuppressed anemia. After FEJ treatment, the pharmacological effects of FEJ in correcting anemia were investigated by the methods of modern pharmacology. Moreover, the action mechanisms at molecular level were explored as well. The main contents of this study are summarized as follows.1Therapeutic effects of FEJ on renal anemia rats and its action mechanismsThe renal anemia rat model was established using adenine. After the model was established, all rats were randomly divided into8groups:normal control group, model control group, low-, medium-, and high-dose FEJ (3,6,12mL/kg-d, respectively) groups, erythropoietin (EPO,200U/kg every three days) positive control group, E’jiao (EJ) group, and all of the components in FEJ but E’jiao (WEFF) group. After successive administration for24days, the levels of red blood cell count (RBC), hemoglobin (Hb) and hematokrit (Hct) in peripheral blood were measured and the levels of blood urea nitrogen (BUN), serum creatinine (Scr), EPO, superoxide dismutase (SOD), reduced glutathione (GSH), glutathion peroxidase (GSH-Px) in serum were also analyzed, as well as the erythrocyte fragility, in each group. The pathologic morphology of kidney tissue was examined by HE staining. The mRNA levels of EPO in renal cortex and EPO receptor (EPOR) in bone marrow nucleated cells (BMNC) were determined by real-time quantitative PCR (RT-PCR).The results showed that, medium-and high-dose FEJ significantly increased the levels of RBC, Hb and Hct in peripheral blood, significantly reduced the quantities of BUN and Scr, as well as significantly increased the levels of SOD, GSH and GSH-Px in serum (P<0.05). Moreover, with the increase of the doses of FEJ in the three FEJ treatment groups, the level of EPO in serum had a tendency to increase gradually. The erythrocyte fragility was significantly decreased in low-, medium-and high-dose FEJ groups (P<0.05). Medium-and high-dose FEJ could improve the pathologic morphology of kidney tissue evidently, and up-regulate the mRNA levels of EPO in renal cortex and EPOR in BMNC (P<0.05). Furthermore, EJ could significantly increase the levels of SOD, GSH in serum and the mRNA level of EPOR in BMNC of model rats (P<0.05). WEFF could significantly increase the level of GSH in serum and reduce the erythrocyte fragility (P<0.05).The results suggest that FEJ could treat renal anemia rats effectively on the whole, which may be attributed to multiple factors, ways and targets. In the process that FEJ treats renal anemia, EJ and WEFF, which make up FEJ, may supplement each other. The therapeutic effects of FEJ on renal anemia rats are probably attributed to two mechanisms. Firstly, FEJ enhances the function of internal free radical scavenging system, postpones the development of renal lesions and decreases the erythrocyte fragility. Secondly, FEJ up-regulates the mRNA levels of EPO in renal cortex and EPOR in BMNC, possibly elevates the level of internal EPO, and eventually improves bone marrow erythroid hematopoiesis. The two pathways may also supplement one another to make a virtuous circle, which improve renal anemia eventually.2Therapeutic effects of FEJ on iron deficiency anemia rats and its action mechanismsThe rat model with iron deficiency anemia was established by feeding the rats with a low iron content diet and followed by bleeding. After the model was successfully established, all rats were randomly divided into11groups:normal control group, model control group, FEJ (5mL/kg·d) group, FeSO4(10mg/kg-d Fe) group, low-, medium-, high-dose FEJ (2.5,5,10mL/kg-d, respectively) plus FeSO4combination groups, EJ (250mg/kg-d) group, EJ plus FeSO4combination group, WEFF (5mL/kg-d) group, WEFF plus FeSO4combination group. After successive administration for21days, the contents of iron in FEJ, EJ solution (50mg/mL) and WEFF were measured by atomic absorption spectrometry. The levels of RBC, Hb, Hct in peripheral blood were measured and heart index, liver index, spleen index were calculated as well. The contents of serum iron (SI), serum ferritin (SF), serum transferrin (sTRF), serum transferrin receptor (sTfR) and EPO in the serum of model rats were measured by ELISA. The mRNA levels of divalent metal transporter-1(DMT-1), metal transporters-1(MTP-1), hephaestin (Hp) in the duodenum and the mRNA level of Hp in livers were measured by RT-PCR.The results showed that, the contents of iron in FEJ, EJ solution (50mg/mL) and WEFF were108.871μg/ml,0.496μg/ml,46.115μg/ml, respectively. FEJ, FeSO4and low-, medium-, high-doses of FEJ plus FeSO4treatments could significantly increase the levels of RBC, Hb, Hct in peripheral blood, decrease heart index, spleen index, increase liver index, elevate the content of SI, lower the contents of sTRF, sTfR (P<0.05). The therapeutic effect of FEJ plus FeSO4treatment was the best among all the groups. The therapeutic effect of FeSO4treatment was better than that of FEJ, while FEJ is better than EJ and WEFF. With the increase of the doses of FEJ in the three FEJ plus FeSO4combination groups, the level of SI in serum had a tendency to increase gradually while the level of SF had a tendency to decrease gradually. Moreover, in high-dose FEJ plus FeSO4combination group and WEFF group, the mRNA levels of DMT-1, MTP-1, Hp in the duodenum were higher than those of other medication administration groups.The results suggest that FEJ could ameliorate iron deficiency anemia to some extent, but its therapeutic effect was not as good as that of FeSO4. When treating iron deficiency anemia, FEJ combined with FeSO4may has a synergistic effect. The therapeutic effect of FEJ on iron deficiency anemia may be attributed to facilitating the absorption and utilization of iron, which is used to synthetize Hb. Furthermore, in the process that FEJ treats iron deficiency anemia, EJ and WEFF, which make up FEJ, may supplement each other. Besides providing a very small amount of iron, the action mechanisms of FEJ in treating iron deficiency anemia may be as follows. Not only can FEJ up-regulate the mRNA levels of DMT-1, MTP-1, Hp to facilitate the absorption of iron in the duodenum, but it also can reduce the level of ferritin (stored iron) in the body and increase the contents of SI and sTRF. Then the quantity of iron that gets into proerythroblasts in bone marrow is elevated, which is used to produce Hb and erythrocytes.3Therapeutic effects of FEJ on myelosuppressed anemia mice and its action mechanismsThe myelosuppressed anemia mouse model was induced by60Co y radiation, cyclophosphamide and chloramphenicol. After the model was established, all mice were randomly divided into7groups:normal control group, model control group, low-, medium-, and high-dose FEJ (5,10,20mL/kg·d, respectively) groups, EJ (500mg/kg-d) group, WEFF (10mL/kg·d) group. The levels of white blood cell count (WBC), RBC, Hb, Hct, platelets in peripheral blood and the number of BMNC were counted. Body weight and the thymus and spleen indices were also measured. The numbers of burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), colony-forming unit granulocyte-monocyte (CFU-GM) and colony-forming unit-fibroblast (CFU-F) were measured by methylcellulose medium culture in vitro. The ratio of hematopoietic stem cells (HSC) in BMNC, cell cycle and apoptosis of BMNC were determined by flow cytometry. The bone marrow histology of femoral bone was examined by HE staining. The levels of transforming growth factor-β (TGF-β), tumor necrosis factor-a (TNF-a), EPO, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3(IL-3) and interleukin-6(IL-6) in serum were measured by ELISA. The mRNA levels of IL-1β, IL-3and IL-6in spleen were detected by RT-PCR. In addition, bone marrow stromal cells (BMSC) were cultured in vitro followed by treatment with different doses of FEJ (2.5,5,10uL/mL), EJ (250μg/mL) and WEFF (5μL/mL) for48h. Then the levels of IL-6, GM-CSF and stem cell factor (SCF) in the conditioned media, as well as their mRNA levels in BMSC, were determined by ELISA and RT-PCR, respectively.The results showed that, medium-and high-doses of FEJ significantly increased the levels of WBC, RBC, Hb, Hct in peripheral blood and the number of BMNC, and reversed the loss of body weight and the atrophy of thymus and spleen (P<0.05). In medium-and high-doses of FEJ groups, the quantities of BFU-E, CFU-E, CFU-GM and CFU-F in bone marrow were also significantly increased (P<0.05), and the femoral bone marrow morphology was improved obviously. High-dose FEJ could significantly increase the ratio of HSC in BMNC, promote bone marrow cells entering the proliferative cycle phase (S+G2/M) and prevent cells from proceeding to the apoptotic phase (P<0.05). Medium-and high-doses of FEJ could significantly increase the levels of GM-CSF, IL-3in serum and the mRNA levels of IL-1β, IL-3in spleen (P<0.05). High-dose FEJ could significantly decrease the level of TGF-β in serum (P<0.05). Medium-and high-doses of FEJ could significantly increase the levels of SCF and GM-CSF in the BMSC conditioned media and their mRNA levels in BMSC in vitro (P<0.05). High-dose FEJ could also significantly increase the level of IL-6in the BMSC conditioned media and the mRNA level of IL-6in BMSC in vitro (P<0.05). In addition, in terms of blood routine, the number of BMNC, the quantities of BFU-E, CFU-E, CFU-GM and CFU-F, the levels of cytokines associated with hemopoiesis in serum in vivo, and the effects on the expression of cytokines associated with hemopoiesis in BMSC in vitro, EJ could improve these indicators to the extent very close to that of medium-dose FEJ. But the therapeutic effect of EJ is not as good as that of medium-dose FEJ. In WEFF group, the proliferation index of BMNC was elevated significantly (P<0.05).The results suggest that FEJ had therapeutic effect on myelosuppressed anemia mice. FEJ could improve the myelosuppression and enhance bone marrow hemopoietic function significantly. In the process that FEJ treats myelosuppressed anemia, EJ and WEFF, which make up FEJ, may supplement each other. The effects of FEJ on facilitating bone marrow hemopoietic function are probably attributed to:(i) improving bone marrow hematopoietic microenvironment through enhancing the forming capacity of CFU-F and improving bone marrow pathology;(ii) facilitating the cell proliferation and preventing BMNC from apoptosis;(iii) stimulating the expression of IL-ip, IL-3, IL-6, SCF and GM-CSF and inhibiting the expression of TGF-β. The three pathways may also supplement one another to improve myelosuppressed anemia in the end. |
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