Caffeic Acid Promotes Expansion Of Hematopoietic Stem Cells And Its Reproductive And Developmental Toxicity Study

Part Ⅰ Effect of caffeic acid on expansion and differentiation of hematopoietic stem cells and its mechanismBackground:Caffeic acid,chemically known as 3,4-dihydroxybenzene acrylic acid,is a plant growth regulator widely distributed in plants.At the same time,caffeic acid is the intermediate of many plant components such as chlorogenic acid,artichoke and some coumarins.At present,caffeic acid is one of the most commonly used drugs in clinical practice for blood cell elevation and stopping bleeding.It has many functions,such as hemostasis,leukocyte and platelet elevation,cholagogy and hypolipidemic effect.In addition,many studies have found that it also has anti-viral,antioxidant,anti-inflammatory,anti-tumor,anti-thrombosis,anti-hypertension,anti-fibrosis and immune regulation effects.In recent years,caffeic acid has been widely used in various causes of thrombocytopenia and leukocytopenia,such as bone marrow suppression after radiotherapy and chemotherapy,aplastic anemia and immune thrombocytopenia.Although caffeic acid is widely used in clinical practice in China there is no clear and internationally recognized comprehensive report on its mechanism of leukocyte and platelet elevation.Therefore,the research on the mechanism of caffeic acid provides a reliable theoretical basis for the clinical application and international promotion of caffeic acid.Purpose:To study the mechanism of caffeic acid elevating leukocyte and platelet,to clarify the effect of caffeic acid on expansion and differentiation of cord blood-derived hematopoietic stem cells in vitro,and to explore the difference of caffeic acid on proliferation of various kinds of tissue cells in vivo.Materials and Methods:1.Collect 80-100 mL cord blood of full-term normal pregnancy,sedimentate red blood cells with gelatin,separate mononuclear cells by Ficoll method,and select CD34+cells by immunomagnetic beads method.2.Establish a serum-free expansion medium(SFEM)system of hematopoietic stem cells containing human stem cell factor(SCF),rhTPO and FLT3 ligand(FLT3L).3.After CD34+ cells were seeded into 24-well plate expantion system,caffeic acidwith concentration of 0,10 or 50 ug/mL(defined as E0,E1 and E2)was added to each group respectively.After 6 days of culture,total cells of each well were counted by cell counting instrument,and the proportion of CD34 cells was determined by flow cytometry.4.Cell colony formation experiments were carried out in each group after cell expantion.The expanded cells were seeded into hematopoietic stem cell differentiation system containing or without caffeic acid(defined as DO(caffeic acid-free semi-solid medium)and D1(caffeic acid-containing semi-solid medium),with a density of 500 CFU/mL for 14 days.On day 14,BFU-E,CFU-G,CFU-M,CFU-GM and CFU-GEMM were counted under low power microscopy.5.Cells in each group after expansion were induced to differentiate into granulocytes,erythrocytes,megakaryocytes or lymphocytes.When cultured for the corresponding days,the total number of cells was counted and the corresponding cell surface markers were detected by flow cytometry.In addition,the morphology of megakaryocytes was observed 14 days after megakaryocyte differentiation induction,and the ploidy of megakaryocytes was determined by flow cytometry.6.Oral administration of caffeic acid or excipient was given to mice for 14 days,and blood routine of mice was measured every 3 days.BrdU was injected intraperitoneally from the 7th day.On the 14th day,flow cytometry was used to determine the proportion of bone marrow hematopoietic stem cells to bone marrow nucleated cells and their BrdU uptake capacity.Meantime,the effects of caffeic acid on Lgr5+ intestinal crypt stem cells and Sca-1+ pulmonary epithelial stem cells as well as the uptake capacity of BrdU were measured.Results:1.In the hematopoietic stem cell expantion experiment,after 6 days culture of CD34+ cells,the total number of cells in 10 ug/mL caffeic acid group(8.45±4.89)*105 was significantly higher than that in the control group(6.23±4.24)*105,P=0.006.However,there was no significant difference in the total number of cells between the 50 ug/mL caffeic acid group and the control group.2.In hematopoietic stem cell colony formation experiment,after being cultured in caffeic acid-free semi-solid medium,the colony formation ability and the proportion of different types of colony in 10 μg/mL caffeic acid group(EIDO)and control group had no significant difference.After culture in caffeic acid-containing semi-solid medium,there was no significant difference in colony formation ability and colony proportion between 10 μg/mL caffeic acid group(E1D1)and control group(E0D1),and there was no significant difference between caffeic acid-free semi-solid medium group and caffeic acid-containing semi-solid medium group.3.In megakaryocyte induction experiment,hematopoietic stem cells in caffeic acid group or control group were cultured in caffeic acid-free differentiation system.On the 7th day,the percentage of CD34+hematopoietic progenitor cells in caffeic acid group(El)was(20.91±2.9)%,which was significantly higher than that in control group(E0),(11.89±2.3)%,P=0.013.The proportion of immature megakaryocytes was significantly higher and the proportion of mature megakaryocytes was significantly lower than that in the control group.On the 14th day,the difference between the 10 ug/mL caffeic acid group(E1)and the control group(E0)disappeared.The cells in the 50 ug/mL caffeic acid group(E2)were not in good condition under the microscope,so no further detection was performed.It is considered that the high concentration of caffeic acid could inhibit the cells to some extent.In addition,hematopoietic stem cells with or without caffeic acid during the expansion process were cultured in the differentiation induction system,and there was no significant difference among the groups.4.In erythrocyte induction experiment the percentage of CD235a positive cells in the control group,the expansion dosing group,the differentiation dosing group and the amplification and differentiation dosing group on the 14th day were 93.1±5.1,92.4±2.8,91.4±3.9 and 95.7±1.9(%),respectively.The P values between the latter three groups and the control group were 0.763,0.158 and 0.463,respectively.There was no significant difference in the total number of cells between groups.5.In granulocyte induction experiment the percentage of CD15 positive cells in the control group,the expansion dosing group,the differentiation dosing group and the amplification and differentiation dosing group on the 14th day were 83.9±6.1,82.7±8.9,84.0±9.6 and 86.2±10.6(%),respectively.The P values between the latter three groups and the control group were 0.543,0.978 and 0.584,respectively.There was no significant difference in the total number of cells between groups.6.In T lymphocyte induction experiment,the percentage of CD3 positive cells in the control group,the expansion dosing group,the differentiation dosing group and the amplification and differentiation dosing group on the 11th day were 88.6±4.6,92.4±2.8,91.4±3.9 and 95.7±1.9(%),respectively.The P values between the latter three groups and the control group were 0.271,0.532 and 0.199,respectively.There was no significant difference in the total number of cells between groups.7.In B lymphocyte induction experiment the percentage of CD19 positive cells in the control group,the expansion dosing group,the differentiation dosing group and the amplification and differentiation dosing group on the 14th day were 9.5 ± 0.8,9.9±2.2,8.6±0.8 and 11.2±1.0(%),respectively.The P values between the latter three groups and the control group were 0.823,0.134 and 0.203,respectively.There was no significant difference in the total number of cells between groups.8.After administration of caffeic acid in mice,there was no significant change in blood routine parameters.After 14 days of administration,there was no significant difference in the proportion of LSK hematopoietic stem cells in bone marrow between caffeic acid group and control group.The uptake of BrdU by hematopoietic stem cells was detected by flow cytometry.The uptake rate of BrdU by bone marrow mononuclear cells in caffeic acid group was significantly higher than that in control group.There was no significant difference in G2/M phase ratio of bone marrow mononuclear cells between the two groups.The proportion of Lgr5+ intestinal crypt stem cells and Sca-1+ pulmonary epithelial stem cells and the uptake ability of BrdU in histiocytes of mice showed no significant difference between caffeic acid group and control group.Conclusions:1.Caffeic acid can promote the expansion of human umbilical cord blood-derived hematopoietic stem cells and maintain the proportion of CD34+ cells during the expansion process.2.Whether caffeic acid was added to the expansion process of human umbilical cord blood-derived hematopoietic stem cells had no significant effect on the subsequent colony formation ability and the induction of differentiation into various cell lines,but could advance the expression of surface specific markers of megakaryocytes.3.Whether caffeic acid is added to the process of colony formation or induction of differentiation into different lines has no significant effect on the differentiation of human umbilical cord blood-derived hematopoietic stem cells.4.After 14 days of oral administration of caffeic acid in mice,there was no significant change in peripheral blood routine,but DNA synthesis of bone marrow hematopoietic stem cells was vigorous and tissue-specific.No similar phenomenon was observed in Lgr5+ intestinal crypt stem cells and Sca-1+ pulmonary epithelial stem cells.Part Ⅱ Reproductive and developmental toxicity of caffeic acid in miceBackground:The incidence of thrombocytopenia during pregnancy is 6%-10%in pregnant women,which is about four times that of non-pregnant women of the same age,mainly including thrombocytopenia during pregnancy and immune thrombocytopenia.Considering the known or unknown risk of thrombocytopenia in pregnancy to the fetus,the treatment of thrombocytopenia in pregnancy has been paid more attention in recent 10 years at home and abroad.The main therapeutic drugs are glucocorticoid,thrombopoietin receptor agonist and intravenous immunoglobulin.However,the main treatment options have more or less adverse effects on the mother and fetus.Considering the placental effect,prednisone is better than dexamethasone,but long-term prednisone can increase the risk of fetal cleft palate and maternal gestational diabetes mellitus;intravenous immunoglobulin can bind to the surface of fetal red blood cells through the placenta,only for hormone-ineffective patients;there is no report on the reproductive toxicity of thrombopoietin receptor agonists and recombinant human thrombopoietin.Therefore,it is urgent to find a kind of platelet-elevating drug that can be used in pregnancy.Caffeic acid,as a platelet-elevating drug independently developed in China,may be helpful to elevate platelet levels in patients with thrombocytopenia during pregnancy.Caffeic acid is a commonly used hemostatic and hemostatic drug in clinic,but its reproductive and developmental toxicity has not been systematically studied.We systematically studied the reproductive toxicity of caffeic acid in pre-pregnant,pregnant and lactating female mice and the developmental toxicity in embryonic and lactating offspring of mice.Purpose:To explore the effects of caffeic acid on maternal,embryonic and fetal development in mice,and to systematically evaluate the reproductive and developmental toxicity of caffeic acid in order to provide a preliminary basis for clinical application.Methods:1.All the experimental procedures conform to the quality management standards for non-clinical drug research promulgated by the State Food and Drug Administration,the technical guidelines for non-clinical drug safety research and the technical guidelines for drug reproductive toxicity research.2.Caffeic acid suspension was prepared with 0.5%sodium carboxymethyl cellulose(CMC-Na)with granularity less than 100 mesh.Suspensions were prepared daily before use and were administered to mice via gavage once a day.The doses were set as follows:1.control group:0.5%CMC-Na;2.low-dose group:CFA 0.15 mg/kg/d;3.mid-dose group:CFA 5 mg/kg/d;4.high-dose group:CFA 150 mg/kg/d.3.Section I Fertility and Early Embryo Development Toxicity Test.This experiment included the period from pre-mating to conception and from conception to implantation.Female mice of F0 generation were administered with caffeic acid or vehicle from 2 weeks before mating to mating until embryo implantation(day 5,G5).The effects of the tested materials on animal fertility were evaluated by mating rate,luteal number and implantation number.4.Section II Embryo-foetal developmental toxicity test.This experiment included embryo implantation to termination of pregnancy and from birth to weaning of offspring.CFA was administered to female mice from implantation(gestation day 6,G6)to day 15 of gestation(G15).The effect of caffeic acid intervention on embryos and fetuses was evaluated by Staple method.5.In the whole course reproductive toxicity test,the female mice were exposed to test materials throughout the reproductive process in order to observe the long-term effects of caffeic acid on reproductive toxicity.F0 female mice were given caffeic acid or vehicle from 2 weeks before mating to weaning of offspring(post-parturition day 21,P21).To evaluate the effect of caffeic acid intervention on reproduction and development of mice,the survival rate and development indexes of fetuses were observed.Results:1.In Segment I fertility and early embryo development toxicity test,the number of implanted embryos,implantation rate and viable fetus in mid-and high-dose groups decreased significantly,and the pre-implantation loss increased significantly.No maternal toxicity was observed in F0 females in each dose group.Maternal weight gain,mating rate,corpus luteum number,dead fetus number,post-implantation loss and live fetal weight were not significantly different from those in control group.2.In Segment Ⅱ embryo-fetal developmental toxicity test,the fetal weight of high-dose group was significantly lower than that of control group,but this phenomenon was not observed in mid-and low-dose group.No maternal toxicity was observed in F0 females of each dose group.No abnormalities or variations in fetal appearance,viscera and skeleton were observed.There were no significant differences in the number of live fetuses,dead fetuses and teratogenic fetuses between dose groups and control group.3.In the whole course reproductive toxicity test,the birth weight of F1 offspring in high-dose group was significantly lower than that of control group,and the weight at weaning showed no significant difference between groups.No maternal toxicity was observed in FO females of each dose group.There were no significant differences in the number of live births,stillbirths,4-day survival rate.21-day survival rate and neurodevelopmental indicators between groups.Conclusions:In this study,when caffeic acid was administered at doses of 5 mg/kg/d and 150 mg/kg/d,significant anti-implantation effects were observed in early pregnant mice.No adverse events such as abortion or teratogenesis were observed after caffeic acid administration started after embryo implantation.The effect of whole course administration of high-dose caffeic acid on fetal weight gain at birth can be restored over time.No observed adverse effect level(NOAEL)of caffeic acid in maternal was 0.15 mg/kg/d under the conditions of this study.