Study On Mechanism Of Hematopoietic Function Of Maoji Jiu By Using Plasma Metabonomics

Background and objective: Maoji Jiu(MJ)is a Chinese traditional health-care wine commonly used in Chinese folk to enrich and nourish the blood.However,its effect and mechanism on hematopoiesis has not been reported.The aim of this study is to investigate the hematopoietic function of MJ on blood deficiency model rats induced by acetylphenylhydrazine(APH)and cyclophosphamide(CTX)and to explore the mechanism of hematopoietic function by analyzing the metabolites of plasma samples in blood deficiency model rats by using metabonomics,which would lay the foundation for further studies and clinical application of MJ.Methods: 36 Female Sprague-Dawley rats(SPF)were randomly divided into 6 groups with 6 rats in each: control group,model group,Fufang E'jiao Jiang group(FEJ,6.0 mL/kg),and high-dose of MJ group(MJ-H,10.0 mL/kg),medium-dose of MJ group(MJ-M,5.0 mL/kg)and low-dose of MJ group(MJ-L,2.5 mL/kg).Except control group,all rats in the other groups werehypodermically injected with APH and intraperitoneally injected with CTX.After 10 days of administration,all rats were killed,and their blood samples,livers,thymuses,spleens and lungs were collected.The white blood cell(WBC),red blood cell(RBC),Hemoglobin(HGB),Hematocrit(HCT)and Platelet(PLT)levels in peripheral blood samples were detected by automatic hematological analysis.The liver index,thymus index,spleen index and lung index were calculated with gravity method.The activity of superoxide dismutase(SOD)and total antioxidant capacity(T-AOC)and the content of malondialdehyde(MDA)in liver tissues were tested by using commercially available kits.The pathologic changes of spleen tissues were observed by hematoxylin and eosin(HE)staining.The expressions of Bcl-2 and Bax proteins in spleen tissues were detected by immunohistochemistry.The metabolites of plasma in the rats of control group,model group,MJ-H,MJ-M and MJ-L were detected by using ultra high performance liquid tandem chromatography quadrupole time of flight mass spectrometry(UHPLC-Q/TOF-MS).The principal component analysis(PCA),orthogonal partial least-squares discriminant analysis(OPLS-DA)and t-test were used to analyze the variation of metabolites in the plasma samples and look for potential biomarkers.The mechanism of hematopoietic function of MJ could be illuminated by metabolic pathways related to biomarkers.Results:(1)Effects of MJ on peripheral blood routine of the blood deficiency model rats: Compared with control group,the WBC,RBC,HGB,HCT and PLT levels of peripheral blood routine in model group were decreased significantly(P<0.01).Compared with model group,the WBC,RBC,HGB and HCT levels in FEJ,MJ-H and MJ-M were increased significantly(P<0.05),and the WBC,RBC,HGB and HCT levels in MJ-L shown an increasing trend,but there was statistical difference(P<0.05)only in the RBC increase.There was nostatistical difference in the PLT levels among all doses groups of MJ compared to model group.(2)Effects of MJ on visceral index of the blood deficiency model rats: The liver index and spleen index in model group were increased significantly(P<0.01),while thymus index in model group was decreased significantly(P<0.01)compared to model group.The liver index and spleen index in FEJ and all doses groups of MJ shown a decreasing trend,and the thymus index in FEJ and all doses groups of MJ shown an increasing trend compared to model group.There was no statistical difference in the lung index among control group,FEJ and all doses groups of MJ compared to model group.(3)Effects of MJ on SOD,T-AOC and MDA in livers of the blood deficiency model rats: Compared with control group,the T-AOC in model group was decreased significantly(P<0.01)and the levels of MDA in model group were increased significantly(P<0.01).Compared with model group,the T-AOC in FEJ,MJ-H,MJ-M and MJ-L was increased significantly(P<0.05),and the levels of MDA in FEJ,MJ-H and MJ-M were decreased significantly(P<0.05).There was no statistical difference in the activity of SOD among control group,FEJ and all doses groups of MJ compared to model group.(4)Effects of MJ on expressions of Bcl-2 and Bax in spleen tissues of blood deficiency model rats:The expressions of Bcl-2 and Bax of spleen tissues in model group were reduced significantly(P<0.05)in comparison with control group.The expression of Bcl-2 and the value of Bcl-2/Bax in FEJ,MJ-H and MJ-M were increased significantly(P<0.05),and the expression of Bax in FEJ and MJ-H was increased significantly in comparison with model group(P<0.05).(5)Metabonomics analysis of plasma samples of blood deficiency model rats:Sample points of control group were separated completely from sample points of control group in the PCA score plot and OPLS-DA score plot,whichsuggested that the metabolites of plasma samples of blood deficiency model rats had been changed,and the changes of metabolites shown that blood deficiency model made a success.According to variable importance in the projection(VIP)of metabolites in OPLS-DA and t-test,15 potential biomarkers were selected from plasma samples of rats.Compared with control group,the relative intensity of Uracil,5-L-Glutamyl-L-alanine acid,Cytidine,L-Glutamic acid,L-Arginine,Orotic acid,Uridine Bilirubin,Protoporphyrin IX and Linoleic acid in model group was increased significantly(P<0.05),while the relative intensity of Oxidized glutathione,Picolinic acid,L-Tryptophan,5-Hydroxyindoleacetic acid and Formylanthranilic acid was decreased significantly(P<0.05).Compared with model group,the relative intensity of Uracil,5-L-Glutamyl-L-alanine acid,Cytidine,L-Glutamic acid,L-Arginine,Orotic acid,Uridine Bilirubin,Protoporphyrin IX and Linoleic acid in MJ-treatment groups shown a decreasing trend,and the relative intensity of Oxidized glutathione,Picolinic acid,L-Tryptophan,5-Hydroxyindoleacetic acid and Formylanthranilic acid shown an increasing trend.By analyzing metabolic pathways,the results shown that the potential biomarkers were correlated to the metabolic pathways of linoleic acid metabolism,D-glutamine and D-glutamate metabolism,alanine,aspartate and glutamate metabolism,tryptophan metabolism,arginine and proline metabolism,glutathione metabolism,porphyrin and chlorophyll metabolism,and pyrimidine metabolism.Conclusion: The improvement of the peripheral blood routine,liver index,thymus index,spleen index,oxidative injury and parenchyma injury of spleen suggests that MJ has a therapeutic effect on blood deficiency model rats induced by APH and CTX.The metabonomics proved that the hematopoietic function of MJ was corresponding to the regulation of abnormal status of linoleic acidmetabolism,D-glutamine and D-glutamate metabolism,alanine,aspartate and glutamate metabolism,tryptophan metabolism,arginine and proline metabolism,glutathione metabolism,porphyrin and chlorophyll metabolism,and pyrimidine metabolism.This survey indicates that metabonomics technique is a promising method to investigate the efficacy and mechanism of traditional Chinese medicine.