| ObjectiveTo study the effect and mechanism of kidney essence deficiency on bone marrow hematopoiesis by observing the apoptosis of bone marrow hematopoietic stem cells on kidney essence deficiency male SD rat model.To observe the therapeutic effect of Gui Lu Erxian Glue on myelosuppressive rats with kidney essence deficiency,and to analyze its mechanisms of "treatment from the kidney" in the treatment of kidney essence deficiencyMethods1)Theoretical research: By systematically reviewing the ancient and modern literatures,explaining the TCM theoretical research and modern medical research of kidney essence,marrow and blood,and clarifying the relationship among them,so as to provide TCM theoretical basis for anemia caused by deficiency of kidney essence.To analyze the prescription of Gui Lu Erxian Glue and the clinical application of tonifying kidney and essence in modern medicine.2)Animal experiments: 30 SPF grade male SD rats in 8 weeks old weighing 270 ±20g were randomly divided into control group(n = 10),model group(n = 10)and kidney-tonifying group(n = 10).The rat model of kidney essence deficiency was established by simulated seismic vibration,photoelectric stimulation,stress intimidation,long-term low-dose benzene subcutaneous injection and short-term high-dose cyclophosphamide intraperitoneal injection.It took 49 days to establish the model.In the first 4weeks,the rats in the model group and kidney-tonifying group were subcutaneously injected with benzene(1m L/kg)every other day according to their weigh.And the rats were injected intraperitoneally with cyclophosphamide(25mg/kg)for 3 days from the 25 th day.At the same time,the rats were simulated by earthquake platform(twice a day,a total of 15-20minutes)and the plantar were shocked by the photoelectric stimulation box(1-3 times a day at random time).The control group was fed normally.On the last day of the fourth week,one rat in the model group and one in the kidney-tonifying group were randomly selected to examine the peripheral hemogram and serum testosterone to make it clear whether the model was successful or not.The kidney-tonifying group was treated with Gui Lu Erxian Glue with a dose 11g/kg each day(divided into two times a day)from the 29 th day according to the ratio of rat surface area to human body surface area.The weight was tested every other day during the administration period in order to adjust dose for 21 consecutive days.The rats in the model group and the control group were given the same amount of deionized water by intragastric for 21 days.The general conditions such as fur,food intake and activity of rats in each group were observed every day in the process of model establishing.The peripheral venous blood red blood cell(RBC)and hemoglobin(HGB)were measured to observe the degree of anemia.Serum testosterone(T)was detected by ELISA and epididymal sperm quality was detected by sperm analyzer.Testicular tissue embedded sections were taken and stained with HE to observe the morphological changes.Bone marrow smear and Wright-Giemsa staining were used to observe the morphological changes of bone marrow cells.HE staining was used to observe bone marrow hyperplasia.Fresh bone marrow was embedded into ultra-thin section to observe its ultrapathological changes.The content of erythropoietin(EPO)was detected by ELISA.The apoptosis of CD34+ cells in bone marrow tissue was detected by flow cytometry and TUNEL method.Western Blot(WB)were used to detect the protein expression of Bcl-2,Bax,caspase-3,Fas and Fasl in rat tibial bone marrow fresh tissue were detected by using.RT-PCR was used to detect the m RNA expression of Bcl-2,Bax,caspase-3,Fas and Faslin fresh tissue of rat tibia bone marrow.3)Statistical analyze: the data were compared among each group,and the data were expressed by mean ±standard deviation(sx ?).The data were analyzed by SPSS 17.0.One-way ANOVA was used to compare between groups.P < 0.05 indicates that the difference is statistically significant.ResultsThe main results were as follows:1)Compared with the control group,the rats in model group showed dry and uneven standing hair,decreased food intake,reduced range of activity and so on.In the kidney-tonifying group,the hair regained luster,food intake and activity increased,and the symptoms of kidney deficiency were improved.2)Compared with the control group,the number of RBC and the content of HGB in the model group decreased from the 14 th day(P<0.01).Compared with the model group,the number of RBC and the content of HGB in the kidney-tonifying group increased significantly from the 42 nd day(P<0.05).3)Compared with the control group,the content of serum EPO in the model group decreased significantly(P<0.01).Compared with the model group,the content of serum EPO in the kidney-tonifying group increased significantly(P<0.01).4)The pathological section of testis showed that in the control group,the capsule of testicular convoluted seminiferous tubule was intact,the morphology of Sertoli cell was at normal,and a large number of sperm could be seen in the lumen of convoluted seminiferous tubule.In the model group,the seminiferous epithelium became thinner,the cell level was disordered,and the morphology of Sertoli cell was broken.In the kidney-tonifying group,the seminiferous tubule capsule was intact,the cell arrangement was tight,the number of spermatogenic cells decreased slightly,and a small number of spermatogenic cells could be seen in some areas,but the structure was clear,and there were more spermatozoa in the lumen.5)Compared with the control group,the epididymal sperm concentration and sperm motility in the model group decreased(P<0.05),and the serum T content in the model group decreased significantly(P<0.01).The epididymal sperm concentration,sperm motility and the serum T content in the kidney-tonifying group were significantly higher than those in the model group(P<0.01).6)The bone marrow detection showed that the number of bone marrow nucleated cells in model group was lower than that in control group(P<0.05).The number of bone marrow nucleated cells in the kidney tonifying group was significantly higher than that in the model group(P<0.05).Bone marrow smears showed that the bone marrow hematopoietic structure of the control group was intact and the tissue was rich,while in the model group,the bone marrow hyperplasia and megakaryocytes decreased;although the hematopoietic reticulocyte increased in the kidney-tonifying group,it still did not return to the normal level.The pathological sections of bone marrow showed that in the control group,there were much hematopoietic cells and uniform distribution.The scaffold structure of bone marrow hematopoietic tissue was compact,and with less adipocytes.While in the model group,the hematopoietic tissue area decreased,megakaryocytes were rare,and the number of hematopoietic cells decreased.In the kidney-tonifying group,the hematopoietic structure was basically repaired,the number of megakaryocytes and hematopoietic cells increased,and non-hematopoietic cells such as adipocytes could still be seen.7)Ultrastructural pathomorphological examination of bone marrow showed that the hematopoietic stem cells in the control group were round,mononuclear,large nucleus,less cytoplasm,mitochondria,and the inner membrane of mitochondria folded into a deep crest.In the model group,the number of mitochondria decreased,the crest became shallow or disappeared,the mitochondria swelled and apoptotic bodies could be seen in the hematopoietic stem cells.In the kidney-tonifying group,the number of nucleated cells of hematopoietic stem cells increased,apoptotic bodies were rare,the number of mitochondria increased and the mitochondrial crest recovered.8)Compared with the control group,more green fluorescent spots of apoptotic CD34+ cells were found in the model group tested by TUNEL,the number of apoptotic CD34+ cells in the model group was significantly increased(P<0.05).Compared with the model group,the green fluorescent spots of bone marrow tissue and the number of apoptotic CD34+ cells in the kidney-tonifying group were lower than those in the model group(P<0.05).9)Bcl-2,Bax,caspase-3,Fas and Fasl protein expression in rat tibial bone marrow fresh tissue were detected using WB.Compared with the control group,the expression of Bcl-2 protein in the model group decreased(P<0.05).The protein expression of Bax,caspase-3,Fas and Fasl increased(P<0.05).Compared with the model group,the protein expression of Bcl-2 in kidney-tonifying group increased(P<0.05).The protein expression of Bax,caspase-3,Fas and Fasl decreased(P<0.05).10)RT-PCR was used to detect the m RNA expression of Bcl-2,Bax,caspase-3,Fas and Faslin in the fresh tissue of rat tibia bone marrow.Compared with the control group,the m RNA exprssion of Bcl-2 in the model group decreased(P<0.05),while the m RNA exprssion of Bax,caspase-3,Fas and Fasl increased(P<0.05).Compared with the model group,the m RNA exprssion of Bcl-2 in kidney-tonifying group increased(P<0.05),the m RNA exprssion of Bax,caspase-3,Fas and Fasl decreased(P<0.05).ConclusionIt is said that "Kidney stores essence,produces marrow,transforms blood",kidney stores congenital essence and kidney qi promotes spleen and stomach to turn water grain into essence,while "essence and blood is homologous",kidney essence is an important material basis for metaplasia of blood.Deficiency of kidney essence leads to blood deficiency due to passivity of blood metaplasia.Kidney dominates not only human body development,but also bone marrow growth and development.While kidney essence is sufficient,then marrow is full.On the contrary,kidney essence is insufficient leading to marrow empty.Bone marrow could transform into blood and depend on the function of kidney,deficiency of kidney essence will lead to low function of bone marrow thus lead to blood deficiency.This project is based on the argument of "kidney storing essence,producing marrow and transforming blood" and deficiency of kidney essence leading to blood deficiency.In this project we used hematopoietic stem cells to study the effect of kidney essence deficiency on hematopoiesis and its mechanism.The traditional etiological model of "fear of injuring the kidney" combined with drug intervention was used to guide the rat model establishing of kidney essence deficiency.Simulated seismic platform,photoelectric stimulation,long-term low-dose benzene and short-term high-dose cyclophosphamide were used to establish the rat model.The rats showed symptoms of kidney deficiency: the number of red blood cells and hemoglobin decreased,the content of serum EPO,serum testosterone and the number of bone marrow nucleated cells decreased,the structure of bone marrow hematopoietic tissue was loose,the hematopoietic area decreased and hematopoietic cells decreased,the mitochondria of bone marrow hematopoietic stem cells swelled,the mitochondrial crest became shallower,and the pathological and ultrapathological changes of apoptotic bodies appeared.While the rats were treated by Gui Lu Erxian Glue by intragastric,the symptoms of kidney essence deficiency could be improved: the number of red blood cells and hemoglobin increased,the content of serum EPO,serum testosterone as well as the number of bone marrow nucleated cells increased,the bone marrow hematopoietic tissue could be repaired.The number of mitochondria of bone marrow hematopoietic stem cells increased,the number of apoptotic bodies decreased,and the apoptosis of bone marrow CD34+ cells decreased.The conclusions of this experiment are summarized as follows:1)The kidney essence deficiency model of male rats was successfully established by using "imitation earthquake platform and photoelectric stimulation,benzene and cyclophosphamide".2)The content of serum EPO in rats with kidney essence deficiency decreased.The expression of anti-apoptotic protein Bcl-2 protein and Bcl-2m RNA in bone marrow tissue of rats with kidney essence deficiency decreased,while the expression of pro-apoptotic protein Bax protein and Bax m RNA increased,which activated mitochondrial / cytochrome C pathway,activated Caspase-3 protein and induced apoptosis of bone marrow CD34+cells.The increased protein expression and m RNA expression of Fas and Fasl in bone marrow activated the death receptor pathway,Caspase-3 protein,and induced apoptosis of CD34+ cells.Excessive apoptosis of CD34+ cells and decrease of bone marrow hematopoietic stem cell activity may be one of the mechanisms of myelosuppression affecting bone marrow hematopoietic function caused by deficiency of kidney essence.3)Gui Lu Erxian Glue was used to treat kidney essence deficiency for the first time.Gui Lu Erxian Glue can effectively relieve the symptoms of rats with kidney essence deficiency,increase the content of serum EPO,promote the expression of anti-apoptotic protein Bcl-2,inhibit the expression of pro-apoptotic protein Bax,Fas and Fasl,reduce the expression of Caspase-3protein,and inhibit the apoptosis of CD34+ cells,so as to protect hematopoietic stem cells.Stimulating the proliferation of bone marrow hematopoietic stem cells and avoiding excessive apoptosis may be the therapeutic mechanisms of Gui Lu Erxian Glue.4)EPO may be one of the material bases of kidney essence in the view of source formation and biological function.5)Hematopoietic stem cells are the expression of kidney essence at the cellular level and they are the executor of the function of "kidney essence transforms blood". |
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