Mechanisms Of Danggui Buxue Decoction On Regulating Erythroid Progenitor Cell Differentiation

BACKGROUND AND STUDY AIMRed blood cells (RBC) are one of the largest numbers of blood cells in human blood, are the main medium of blood transports oxygen and also have immune function. So it is known as red guards in the body. Erythropoiesis is a complex, multi-step process. First, pluripotent stem cells differentiate into erythroid progenitor cells(CFU-E and BFU-E), and then erythoid progenitor cells eventually differentiate into mature red blood cells by multi-step process. There are many kinds of blood cells and cytokines involved.RBC is the main innate immune cells and has an important function in the normal body’s own defense systems. And many diseases are often associated with the changes of the RBC innate immune function, such as oncology, infectious diseases, hematologic and autoimmune diseases etc. The main functions of RBC in the innate immune:enhance the phagocytosis, clear the circulating immune complexes, recognize and carry antigen, immune regulatory effect and act as effector cells etc.RBC transfusion can be used for the treatment of aglobulia in clinic, but is often accompanied by viral and bacterial infections. Intravenous iron also can be used in the treatment of iron deficiency anemia, but can often lead to side effects such as nausea, flushing, hypotension and anaphylaxis etc, severe cases can cause death. Erythropoietin can be used to stimulate production of red blood cells, but the production of neutralizing antibody and other reasons can result in treatment failure. EPO plays a key role in early stage of CFU-E progenitor cells differentiated into mature red blood cells, so numbers of CFU-E progenitor cells limit the capacity of erythropoietin. And EPO as a single therapy in treating aglobulia only regulates one part of erythropoiesis, cannot fundamentally regulate erythropoiesis. Meanwhile, consumption of CFU-E can result in consumption of BFU-E, which results in the failure of BFU-E progenitor cell library and influences the body’s subsequent hematopoietic. It is documented that EPO treatment can lead to serious pure red cell aplasia. So it is necessary to develop erythropoietin drugs that are convenient, multiple targets, whole regulatory, safe and effective.Blood tonic Chinese herbal medicine is superior drug of Chinese medicine. Danggui buxue decoction has obvious curative effects and little side effect, and has been used for clinical applications more than 800 years of history. Traditional Chinese medicine, especially compound traditional Chinese medicine has the characteristics of complex components and diversity targets, which is in accordance with the whole regulatory of traditional Chinese medicine therapy and is the foundation of traditional Chinese medicine therapy. The whole regulatory of traditional Chinese medicine is in accordance with the complexity of erythropoiesis. So traditional Chinese medicine therapy can make up for the defects of single cytokine therapy, can regulate erythroid progenitor cell differentiation by multi-target and multi-channel and also has low cost.In this project, Danggui buxue decoction is raised by Li Gao which is one of the four schools of the Jin-yuan dynasty in the clarification of perplexities about internal and external damage. It has the effect of supplement Qi and engendering blood, is used to treat overexertion injuries, blood weak, Yang Fu on the outside, hot and dry skin, face red, poludipsia and drink, full and large pulse and virtual etc. It has obvious curative effects, and has been used for clinical applications more than 800 years of history. It can play a role in alleviate radio-chemotherapy induced myelosuppression. It is well documented that Danggui buxue decoction can increase the numbers of red blood cells in peripheral blood, and increase the numbers of BFU-E and CFU-E progenitor cells in bone marrow tissue.Traditional Chinese medicine prescription that can promote the production of blood and have been proven by practices is worth vigorously development. On the base of previous studies, this study will first establish a method for quality control of Danggui buxue decoction in order to ensure the result stability and repetition of pharmacological experiment, then using mice as the animal model to study the ability of erythropoiesis and search the potential targets of Danggui buxue decoction. Then using in vitro cellular model to study the molecular mechanisms of Danggui buxue decoction on erythropoiesis. Finally, using spectrum-activity relationship to study the material basis of Danggui buxue decoction. Main content and conclusionPart 1 Establishment of the quality methods of Danggui buxue decoction1. Optimization of HPLC chromatographic conditions of Danggui buxue decotion2. Methodology validation of HPLC chromatographic conditions of Danggui buxue decotion3. Determination of ferulic acid and formononetin content in danggui buxue decoction4. Study on the common peaks and fingerprints of Danggui buxue decoctionMain conclusions:The number and resolution of chromatographic peaks are different when sample is dissolved using different solvent. When using methanol as the solvent, the numbers of chromatographic peaks are more than the other, and the total peak area of chromatographic peak is also the highest. But the chromatographic peak in the hydrophilic area is wider and has shoulder peak, and the symmetry and resolution is worse than the other. The chromatographic peak height using 5% and 50% methanol as solvent is lower than 95% methanol as solvent in the hydrophobic area, and the number of chromatographic peak is fewer using 5% methanol as solvent, and affects the sensitivity. The sensitivity is not affected when using 50%methanol as solvent. The purpose of establishing the chromatographic conditions is to detect more compounds. So 50% methanol is chosen as the solvent.The resolution of Chinese medicine ingredient can be affected by different chromatographic column. By comparing the chromatogram of Danggui buxue decoction, when using Agilent TC-C18 as the chromatographic column the peak retention time is longer and the resolution of hydrophilic drug components is higher than the other columns. The resolution of hydrophobic drug components in the three columns can meet the requirement. Synthesizes the above analysis, Agilent TC-C18 is chose as the chromatographic column.The elution capability of different mobile phase is also different. The elution capability of acetonitrile is better than methanol, so the retention time is shorter. Compared with the other three elution system, when using methanol-acetic acid elution system as the elution solvent the numbers of chromatographic peak in the Ⅱ area are more, and the resolution are higher and the symmetry is better. But in the Ⅲ area, the peak resolution using acetonitrile is better than using methanol. However, using methanol cannot affect the detection of chromatographic peak. Synthesizes the above analysis, methanol-acetic acid elution system is chosen as the mobile phase.Due to the different elution capability of mobile phase, the resolution between the peak and peak is different. According to the resolution between the peak and peak, we choose different gradient elution method to improve separation efficiency.In order to control product quality, the analytical methods must have a high accuracy and reliability, so study on methodology validation of analytical methods is necessary. The stability test showed that chromatographic fingerprint derived from different points has higher similarity compared with mutual mode, the relative standard deviation (RSD) of the relative peak retention time is less than 0.3%, and the RSD of the relative peak height is less than 5%(except 6 and 11 peaks). So the test solution is stable within 24 hour. The precision test showed that chromatographic fingerprint by repeated 6 times has higher similarity compared with mutual mode, the RSD of the relative peak retention time is less than 0.8%, and the RSD of the relative peak height is less than 5%. So the precision of analytical method is good. The replication experiment showed that chromatographic fingerprint derived from 6 samples has higher similarity compared with mutual mode, the RSD of the relative retention time is less than 1.5%, and the RSD of the relative peak height is less than 5%. So the replication of analytical method is good.By comparing fingerprint of 6 danggui buxue decoction, a total of 17 common peaks are selected. And the RSD of common peaks retention time is less than 0.1%. So the 17 common peaks are the mutual peaks.Part 2 Mechanisms of Danggui buxue decoction on regulating erythroid progenitor cell differentiation1. Study on the erythropoiesis and the targets of Danggui buxue decoction2. Establishment of a cellular model in vitro3. Mechanisms of Danggui buxue decoction on increasing number of bone marrow stromal cell4. Mechanisms of Danggui buxue decoction on regulating erythroid progenitor cell differentiationMain conclusions:Danggui buxue decoction can promote erythropoiesis in mice. Compared with model group, the number of reticulocytes in the group treated with high dose danggui buxue decoction was higher remarkably (P<0.05), and the number of RBC in the group treated with Danggui buxue decoction is no significant difference. Thus, this paper concludes that DBD can promote the production of reticulocytes in mice, but cannot promote the production of RBC. This might be associated with life cycle of mature RBC in peripheral blood.Compared with model group, the number of CFU-E in the group treated with medium and high dose DBD was higher remarkably (P<0.05), the number of BFU-E in the group treated with high dose DBD was higher remarkably (P<0.05), the percent of erythroid cells in bone marrow in the group treated with medium and high dose DBD was higher remarkably (P<0.05). These results show that DBD can promote the production of RBC by regulating the proliferation and differentiation of erythroid progenitor cells.The growth state of bone marrow stromal cell is observed by cultured bone marrow cell of different groups in vitro. The results showed that the cells grew with attaching on the plate on the third day out and the cells are covered about a quarter of 96-well plates in the normal group and DBD group, but the number of adherent cells is less in the model group. On the sixth day, the adherent cells are covered about a half of 96-well plates in the group treated with high dose DBD. On the ninth day, the clone of fibroblast could be found in the group treated with high dose DBD.Analysis of cell cycle showed that the percent of G2M phase cells is 8%,7.4% and 7.6% in low dose, medium dose and high dose group, respectively. And the percent of G2M phase cells has no significant difference compared with model group (P>0.05). Cell adhesion assay showed that the cell number of aggregation in the group treated with medium dose and high dose DBD were higher remarkably than model group (P<0.05). Through above the outcome of animal experiment, we speculated that the pathway of DBD regulating erythroid progenitor cells differentiation is to send signals for erythroid progenitor cells differentiate into mature red blood cells by enhancing the mutual contact between erythroid progenitor cells and bone marrow stromal cellsThe growth state of bone marrow stromal cell can be affected by cells seeding density and culture medium volume. Bone marrow stromal cell did not grow when the inoculation density is 1×105cell/well. The cells are covered about 30 percent of 96-well plates when the inoculation density is 3×105cell/well. The cells are covered about 90 percent of 96-well plates when the inoculation density is 5×105cell/well. When the inoculation density is 3×105cell/well, the number of adherent cells in 150 μL and 200μL culture system is remarkably less than in 100μL culture system. However, when the inoculation density is 5×105cell/well, the number of adherent cells had no significant difference in all groups. When the inoculation density is 3×105cell/well, the number of adherent cell in conditioned medium group is remarkably higher than common medium (P<0.05). However, when the inoculation density is 5×105cell/well, the number of adherent cell in conditioned medium group has no significant difference compared with common medium. The results suggest that conditioned medium include the growth factors that are necessary for the growth of bone marrow stromal cells in vitro. The growth factors are secreted by bone marrow stromal cells in vitro, can promote the growth of bone marrow stromal cells. So we can use conditioned medium to low-density culture of bone marrow stromal cells in vitro.DBD can promote the number of adherent bone marrow stromal cells in vitro. The effects show a dose-dependent relationship with concentration in low dose concentration. The number of adherent cells increases with the increase of drug concentration. However, the effect declines with the further increase of drug concentration. The effect is most pronounced when the drug concentration is 1000 μg/ml. The growth curve experiment showed that the slope of growth curve has no significant difference between control and DBD group. The cell cycle experiment showed that the percent of G1 phase, S phase and G2M phase cells has no significant difference between control and DBD group. So the cell cycle of the adherent cell cannot be affected by DBD. However, the percent of G1 phase cells in DBD group is significant less than in control group (P<0.05), and the percent of S phase and G2M phase cells in DBD group is significant higher than in control group (P<0.05). The administration time experiment showed that DBD could not promote the growth of bone marrow stromal cells when the drug was added four days later. The above results show that DBD promotes the increase of bone marrow stromal cells number by enhanced the adherent capability of bone marrow stromal cells, rather than through stimulated proliferation. But DBD can promote the proliferation of suspension cells which may be associated with the communication between cells.CFU experiments show that DBD cannot act directly on erythroid progenitor cells. In order to regulate erythroid progenitor cell differentiation, DBD need the help of other cells and cell factors. CFAC experiments show that the clone number in DBD group is significant higher than in control group (P<0.05), and the size of clone in DBD group is larger than in control group. The above results show that with the help of bone marrow stromal cells, DBD can promote the formation of erythroid progenitor cells clone and can promote the proliferation of erythroid progenitor cells. Synthesizes the above analysis, we speculate the possible mechanisms of DBD regulate erythroid progenitor cells differentiate into mature RBC:DBD promote the expression of adhesion molecules in bone marrow stromal cells, and adhesion molecules can enhance the communication between bone marrow stromal cell and erythroid progenitor cell, which can lead to send signals of regulating erythroid progenitor cells proliferation and differentiation.Part 3 Material basis of Danggui buxue decoction on regulating erythroid progenitor cells differentiation1. Pharmacodynamic effects of different batches of DBD2. Spectrum-effect relationship between pharmacodynamic effects and fingerprints of different batches of DBDMain conclusion:The numbers of adherent bone marrow stromal cells are different in different batches of DBD. The number of adherent bone marrow stromal cells in GG group is significant higher than in other groups. Study on the spectrum-effect relationship by intuitive comparison and correlation coefficient method. By compared the fingerprint of GG group with other groups, we found the potential material foundation of DBD regulating erythroid progenitor differentiation. By analyzing the correlation between chromatographic peak area and pharmacodynamics effects, we found the potential material foundation of DBD regulating erythroid progenitor differentiation. The above results show that the chromatographic peak related with pharmacodynamics effects focus on hydrophilic region.