Effect Of Compound Shenlu Granules On Apoptosis Of Bone Marrow CD34~+ Cells In Lower-risk MDS With Kidney-Yang Deficiency By Inhibiting P38MAPK Phosphorylation

Object1.To study the effect of Compound Shenlu Granules(CSG)on peripheral hemogram and apoptosis rate of bone marrow CD34~+cells in lower-risk MDS with kidney-yang deficiency.To explore the mechanism of nhibiting p38MAPK phosphorylation to improve hematopoietic function of bone marrow.2.In vitro culture of bone marrow CD34~+cells from patients with low risk MDS of kidney-yang deficiency.To study the effect of CSG containing serum on apoptosis rate,p38MAPK signaling pathway and normal hematopoiesis recovery.Methods1.The lower-risk MDS with kidney-yang deficiency were randomly divided into two groups:CSG group and Andriol treatment group.All patients were treated for 3months.The changes of peripheral blood cell count were observed.Acquisition of bone marrow mononuclear cells(BMMNC)from the control group and two groups of MDS patients.All the cases from control group were age-matched patients with non-MDS cytopenia.The apoptosis rates of bone marrow CD34~+cells were detected by Annexin V/PI double staining flow cytometry(FCM).The expressions of p38 and p-p38 protein were tested using Western Blot assay(WB).The correlation between the expression of p-p38 protein and the apoptosis rates of CD34~+cells was analyzed.2.The CSG containing serum and blank serum were prepared.Bone marrow CD34~+cells were cultured in vitro and divided into four groups:blank serum group,CSG containing serum group,SB202474 group and SB203580 group.After obtaining the informed consent of the patients,BMMNC from lower-risk MDS patients with kidney-yang deficiency,co-cultured with 10%CSG containing serum or p38 inhibitor in vitro.The apoptosis rate of CD34~+cells was detected by Annexin V/PI double staining FCM.The expression of p38 and p-p38 protein were detected by WB.BMMNC CD34~+cells were isolated by magnetic activated cell sorting,and then co-cultured with 10%CSG containing serum or p38 inhibitor,in Human Methylcellulose Complete Medium Containing Cytokines.To observe the formation of colony forming unit-hematopoietic stem/progenitor cell colony(CFU-HSPC).The percentages of aberrant clones in CFU-HSPC were detected by fluorescence in situ hybridization(FISH).ResultsIn the clinical study,the peripheral blood cell count of CSG group and Andriol group increased after treatment compared with those before treatment,and the red blood cell and hemoglobin count of CSG group had significant difference(P<0.01).There was a significant difference in the platelet count(P<0.05),but no significant difference in the white blood cell count(P>0.05).These results suggest that CSG has a significant effect on the ineffective hematopoiesis of erythroid of bone marrow.In this study,we demonstrated that compared to cytopenia caused by diseases other than MDS,lower-risk MDS with kidney-yang deficiency is featured by high apoptosis rate of bone marrow CD34~+cells and p38MAPK overphosphorylation.After the intervention of CSG,the apoptosis rate of bone marrow CD34~+cells decreased,whereby the apoptosis rate is proportional to the expression of p-p38 protein(P<0.01).The results of cell culture in vitro showed that the CSG containing serum affected the apoptosis rate of bone marrow CD34~+cells and the formation of hematopoietic stem/progenitor cell colony in lower-risk MDS with kidney-yang deficiency.Compared with the blank serum group and SB202474 group,the apoptosis rates,the expressions of p-p38 protein and the proportions of abnormal clone cells of CSG containing serum group were lower(P<0.05),the numbers of CFU-HSPC in CSG containing serum group were higher(P<0.01),the percentages of aberrant clones in CFU-HSPC were lower(P<0.05).The correlation analysis found that,p-p38 protein expressions and the proportions of abnormal clone cells of CSG containing serum group and SB203580group were positively correlated(P<0.01).ConclusionThis study has preliminarily verified that CSG is a TCM compound which can effectively restore bone marrow hematopoiesis in lower-risk MDS,especially in the treatment of ineffective erythropoiesis.These results suggest that may be related to the inhibition of p38 hyperphosphorylation,the reduction of apoptosis in bone marrow CD34~+cells and the restoration of normal hematopoietic stem/progenitor in bone marrow.Furthermore,it provides the scientific evidence for the clinical treatment of lower risk MDS with"boosting marrow by warming kidney".But the mechanism of the CSG regulating the abnormal inflammatory signal of low-risk MDS with the kidney-yang deficiency still needs further research.