To Explore Mechanism Of Traditional Chinese Medicine In Treating β Thalassemia Based On Proliferation Of Hematopoietic Stem Cell And Hematopoietic Niche

β thalassaemia is a kind of autosomal single gene, recessive inherited hemoglobin disease. Thalassemia have a wide distribution in tropical or subtropical regions, mainly in Mediterranean region and southeast Asia(India,Thailand, Indonesia). In China thalassemia is mainly distributed in Guangxi and Guangdong provinces. Pathogenesis of thalassemia is clear, and there is no fundamental treatment at home and abroad.Thalassemia is a disease with high mobility, hazard and high fatality rate in the worldwide. Birth of heavy and middle type thalassemia children is a public problem recognized by the world, and how to deal with these newborn, and improve the quality of survival of this group are unsolved problem.Professor Wu Zhikui have treated thalassemia patients based on "kidney born marrow, marrow generate blood" and gained a definite clinical effect. Accumulated more than 30 years of clinical practice, for the first time, professor Wu put forward with the core pathogenesis, therapeutic principle and the possible mechanism and fill the blank of traditional Chinese medicine in treating thalassemia.The main content of this paper is, for the first time, to develop a randomized, double blinded, placebo controlled clinical trial to evaluate effect of Yisuishengxue granules based on previous work, and put forward the research hypothesis about the effect mechanism:"Improving hematopoietic niche, regulating differentiation of bone marrow CD34+ cells, influencing DNA methylation modifications, thus promoting bone marrow hematopoietic effectively may play a role". We separate bone marrow MSC and HSC, using RT-PCR, Western blot, DNA methylation, transmission electron microscopy(TEM) and other modern technologies, combining experiments in vivo and vitro, to test the hypotheses.PurposeWe want to reveal the connotation of "kidney born marrow, marrow generate blood" from improving hematopoietic niche, regulating the differentiation of HSC and DNA methylation. This paper develop from two parts:clinical and experimental research.1 Clinical research1.1 TCM syndrome distributionInvestigation of kidney deficiency syndrome of traditional Chinese medicine about thalassemia patients(kidney essence deficiency, kidney Yin deficiency, kidney Yang deficiency).1.2 Clinical RCT trialUsing randomized controlled trial(randomized, double blind, placebo controlled), international registration before clinical trial, to quantify the TCM symptoms in patients with grade, observe the dynamic changes of Hb、RBC、Ret、 HbF、WBC、PLT. Results are analyzed by a third party.2 Mechanism researchTo study the mechanism of Yisuishengxue granules from improving bone marrow hematopoietic niche, regulating differentiation of bone marrow HSC, DNA methylation modifications, thus promoting bone marrow hematopoiesis effectively.2.1 Study on proliferation of endogenous bone marrow HSC, differentiation of HSC treated with medicine serum and medicine monomer, influence of globin gene expression.2.2 To observe the influence of marrow hematopoietic niche with tonifying kidney by detecting bone marrow hematopoietic cytokine activity, erythrocyte immune enzyme activity, proliferation of BMSC, cytokine secretion and gene expression of BMSC.2.3 Finding key functional genes associated with hematopoietic through DNA methylation.Methods and Results1 Clinical methods and results1.1 TCM syndrome distributionTo investigate TCM syndrome distribution of 299 thalassemia patients by collecting clinical basic data and establishing epidemiological survey, classify syndromes(kidney essence deficiency, kidney Yin deficiency, kidney Yang deficiency syndrome) and score TCM symptoms.According to this survey, kidney Yin deficiency syndrome is the most common with 195 cases, followed with kidney essence deficiency syndrome 89 cases, and less proportion of kidney Yang deficiency syndrome. Frequencies of kidney associated symptoms and signs showed that sallow complexion、night sweats、oral pale white、fatigue、dry throat、dry mouth are most common. Score of kidney deficiency syndrome with homozygous and heterozygous genotype in β thalassemia patients is with no statistically significant difference.1.2 RCT of Yisuishengxue granules in treating thalassemiaThis study used randomized, double blind, and placebo controlled method, and registrated before clinical trials.40 patients was selected and randomly divided into experimental group and control group. Patients in experimental group is given Yisuishengxue granules, and placebo in control group. The whole course are 12 weeks. Peripheral blood Hb、RBC、Ret、HbF、WBC and PLT were detected before and after 4、8 and 12 weeks treatment. Patient whose Hb>5g/L is considered a clinical effective case.According to the results of RCT, effective rate of experimental group is 47.06%、47.06% and 58.82% respectively after 4、8 and 12 weeks treatment, and 11.11%、5.56%、5.56% in control group. Comparison of effective rate between two groups showed effective rate in experimental and control group had a significant difference at 4 weeks(p<0.05),8 weeks(p<0.01) and 12 weeks(p<0.01).Comparison within group, Hb and RBC in experimental group started to rise since 4 weeks treatment, and there is statistical significance compared with before treatment; HbF started to rise after 8 weeks treatment, with a statistical significance compared with before.After 12 weeks treatment, Hb in experimental group is 71.35g/L higher than 68.50g/L in control group with no statistical significance. Yisuishengxue granules can improve Hb、RBC、HbF content, but with no obvious function on WBC and PLT.2 Experiment Methods and Results2.1 Bone marrow hematopoietic stem cell proliferationBefore and after 12 weeks treatment, bone marrow blood nucleated cells from 5 thalassemia patients were obtained, and bone marrow CD34+ cells was separated using density gradient centrifugation and immune positive magnetic bead method. Bone marrow CD34+ cells, before and after treatment, were cultured in vitro. CCK8 is used to detect OD value after two、four、six and seven day. Results found that OD value of CD34+ cells after treatment is higher(p<0.01) compared with that before treatment. Results suggest that Yisuishengxue granules significantly promote endogenous bone marrow hematopoietic stem cell proliferation.2.2 Erythroid differentiation by medicated serum of Yisuishengxue granulesUsing semisolid colony culture method, to observe colonies differentiation of HSC and y globin gene expression of K562 cells with medicated serum of Yisuishengxue granules. Benzidine staining and RT-PCR technique are used to observe erythroid differentiation of K562 cells. Results show that after 13 days, colonies in high dose group is higher than that in control group (p<0.001), that in low dose group(p<0.001),and that in the medium dose group(p<0.001).2.3 Hematopoietic stem cell colony differentiation with medicine monomerDiphenylethylene glycosides and emodin are two monomer from radix polygoni multiflori. Colonies of bone marrow HSC are observed and globin gene (α, β,γ,ε) expression as well as protein contend of colonies are detected using RT-PCR and Western blot technologies.Results showed that CFU-E in 9.25uM emodin group and in 3.70uM group are higher compared with control group. After 13 days, emodin with 9.25uM can promote γ globin gene expression of colony cells(p<0.05). Western blot results show that emodin can promote y globin content of colonies.2.4 Cytokine activity from bone marrow bloodBefore and after treatment, bone marrow blood was centrifugated by density gradient to collect plasma and RBCs. Hematopoietic cytokine activity was detected using enzyme-linked immune experiment. Results show that after 12 weeks treatment SCF、G-CSF、IL-6 and EPO in bone marrow blood have a rising tendency; SOD and GSH-PX within RBC increased, MDA decreased; Yisuishengxue granule can reduced the number of RBC inclusions, promoting antioxidant capacity.2.5 Proliferation of mesenchymal stem cellsBone marrow MSC are collected with adherent culture. Proliferation of bone marrow MSC treated with medicine monomers was detected with CCK8 method. Results showed that certain concentration of emodin can promote bone marrow mesenchymal stem cell proliferation.2.6 Cytokines secreted by mesenchymal stem cellsSCF and IL-6 activity secreted by MSC was detected using ELISA and RT-PCR technologies. Results showed that emodin can improve the activity of SCF, little influence on IL-6; Emodin can promote SCF gene expression(p<0.05), Diphenylethylene glycoside has no effect on SCF and IL-6 gene expression.2.7 DNA methylation before and after treatmentPeripheral blood cell from 5 thalassemia patients was collected to observe the genome wide DNA methylation using DNA methylation chip technology.Results showed LGR4 and PP11159H22.2 on chromosomell, CCDC101, NUPR1, RP1166602.1 on chromosome 16 were in a low methylation gene functions after treatment. Role of these genes are related with protein coding. JAK3 and MAPK10 are two key genes existing in bone marrow and lymphatic system, and JAK3 is considered to have a relationship with hematopoietic cytokines in the process of early hematopoietic.ConclusionThis article have investigated kidney deficiency syndrome of thalassemia patients. Based on TCM syndrome investigation, for the first time we carry out a randomized、double blind、placebo controlled clinical trial. Based on clinical efficacy, we studied the mechanism from proliferation and differentiation of bone marrow HSC, hematopoietic niche, and DNA methylation level.1.Kidney Yin deficiency is the most common syndrome among thalassemia patients.2.Results of clinical RCT show that effective rate in experimental group after 4、8 and 12 weeks treatment are respective 47.06%、47.06% and 58.82%. Yisuishengxue granules can increase Hb content and improve clinical symptoms.3.Yisuishengxue granule can improve patients with bone marrow hematopoietic stem cell proliferation. Emodin can promote y globin gene expression of colonies. High dose of medicated serum can induce mRNA expression of y globin in K562 cells.4.Yisuishengxue granules can promote SCF、IL-6、G-CSF、EPO activity, improve SOD in RBC. Emodin can promote bone marrow MSC proliferation, and SCF activity secreted by MSC.5.Genome wide DNA methylation found that JAK3 and MAPK10 may play a role in the process in hematopoietic.6.Hematopoietic niche can regulate proliferation of bone marrow HSC, reduce oxidative damage of colonies, promote y globin gene expression of differentiation colonies, promote effective hematopoiesis. "Kidney born marrow, marrow generate blood" can be embodied proliferation and differentiation of bone marrow HSC under the regulating of bone marrow niche.Innovation1.This study for the first time, using a randomized、double blind、placebo controlled clinical trial and international registration(NCT01549080) to study the clinical effect of Yisuishengxue granules. With an international recognized standard, to evaluate the clinical effect and promote the international influence of Chinese medicine in treating thalassemia.2.Yisuishengxue granules can promote the proliferation of endogenous bone marrow HSC in vitro. Bone marrow CD34+ cells after treatment increased significantly than CD34+ cells before. Genetic background and curative effect have a relationship with proliferation of bone marrow CD34+ cells.3.Theoretical innovation, this paper based on obvious curative effect, from promoting differentiation and proliferation of HSC, improving hematopoietic niche and DNA methylation epigenetic modifications level, to interpret "kidney born marrow, marrow generate blood" theory scientific connotation.