Study On The Therapeutic Effect Of Er Zhi Wan On Yin-deficiency Rat Based On Metabolomics

Er Zhi Wan(EZW)also named Nv Zhen Dan,a famous TCM formulation,which contains only two herbs,Ligustri Lucidi Fructus(LLF,the fruits of Ligustrum lucidum Ait.)and Ecliptae Herba(EH,the herbs of Ecliptaprostrata L.).EZW was firstly introduced in an ancient book titled Fu Shou Jing Fang which was compiled by Wu-Min during Ming Dynasty(AD 1530).In Chinese clinical practice,EZW is mainly used for diseases caused by liver and kidney yin deficiency,which are characterized by a series of symptoms,such as vertigo,tinnitus,dry nose and throat,lumbago,knee pain and menorrhagia.Liver and kidney yin deficiency is a TCM terminology,which is involved in yin deficiency pattern and has a great deal to do with the occurrence and development of many modern diseases.According to Chinese medicinal theory,EZW is claimed to have the effects of nourishing yin and supplementing liver and kidney,while its specific therapeutic effects and mechanism have not been fully understood or studied in depth,which has limited the clinical application of EZW.In this paper,the multiple chemical components in EZW were unambiguously identified or tentatively characterized.Moreover,the traditional pharmacological method and the powerful metabolomics were combined to investigate the pathological mechanism of yin deficiency and the therapeutic effects of EZW base on the rat model induced with thyroxine and reserpine.The main content of this paper includes the following five parts:Part one:Literature SearchBased on the related literature at both ancient and modern time,the origin and history of EZW were investigated.In addition,the pharmacology and clinical application of EZW were summarized,which provided a theoretical basis for the further development and research on EZW.Part two:Identification of multiple constituents in EZW based on UFLC-Q-TOF-MSAn ultra-fast liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry(UFLC-ESI-Q-TOF-MS)method was established for rapid separation and identification of multiple constituents in EZW,LLF and EH.As a result,a total of 84 compounds were identified in EZW,including phenylethanoids,phenylethanoid glycosides,flavonoids,iridoids,triterpenoids,coumarins,phenols and phenolic acids.Among these compounds,43 compounds were origin from LLF while 32 from EH,and other 9 compounds were found to be in both LLF and EH.This study enriched the chemical profiling of EZW and could provide valuable information for the quality control and further investigation of EZW.Part three:Study on the therapeutic effect of EZW on the yin deficiency rats induced by thyroxine and reserpineThyroid tablets and reserpine injection were oral administrated to female SD rats for 10 days to establish the yin deficiency rat model.As many as 13 substances in the serum were detected to evaluate the yin deficiency rat model and the therapeutic effect of EZW,including free triiodothyronine(FT3),free thyroxine(FT4),estradiol(E2),testosterone(T),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),aspertate aminotransferase(AST),superoxide dismutase(SOD),malondialdehyde(MDA),creatinine(Cr)and blood urea nitrogen(BUN).In addition,the pathological morphological changes of liver and adrenal were also observed.Comparing with control group,yin deficiency group showed a significant increase in the level of FT3,FT4,E2,T,TG,LDL-C,ALT,AST and MDA(P<0.0l or 0.05)and a decrease in the level of HDL-C and BUN(P<0.01 or 0.05),while the level of SOD and Cr didn’t exhibited significant changes.Moreover,the liver and adrenal both showed pathological injury.Above all,the yin-deficiency rat model was successfully established.Based on the above rat model,the therapeutic effect of EZW was investigated.Contrast to model group,EZW-high dose group could significantly reduce FT3(P<0.05),EZW-high,middle and low dose group could significantly reduce FT4 and E2(P<0.01 or 0.05),EZW-middle dose group could significantly reduce LDL-C(P<0.01)and increase HDLC(P<0.05),and EZW-middle and high dose group could significantly reduce TG(P<0.01),EZWhigh,middle and low group could significantly reduce MDA,ALT and AST(P<0.01),EZWmiddle and low dose group could significantly increase BUN(P<0.01).EZW also showed protective effect on the injury liver and adrenal tissue.Part four:Study on the serum metabolomics of thyroxine and reserpine-induced yin deficiency rats and the therapeutic effect of EZWBased on the yin deficiency rat model induced by thyroxine and reserpine,changes of metabolic profile in rat serum and the therapeutic effect of EZW towards abnormal metabolism of yin deficiency rats were investigated by UFLC-Q-TOF-MS.Principal Component Analysis(PCA)and Partial Least Squares-Discriminant Analysis(PLS-DA)were used for multivariate data analysis.For metabolite data,all comparisons were made using Kruskal-Wallis non-parametric analysis followed by Dunn’s multiple comparison test.The VIP value form PLS-DA model and the P value from non-parametric analysis and multiple comparison test were taken into account for selecting the significant variables which contribute a lot to the classification.As a result,a total of 16 potential biomarkers were identified in the serum,which were associated with yin deficiency syndrome.And these metabolites were recovered in EZW groups at different levels,among which 5 metabolites showed significant differences.Pathway analysis showed that these biomarkers were involved in 7 metabolic pathways,among which there were 4 pathways showing more potential,namely arachidonic acid metabolism,steroid hormone biosynthesis,primary bile acid biosynthesis and glycerophospholipid metabolism.EZW may achieve the protective effect on yin deficiency rats by acting on 3 pathways including arachidonic acid metabolism,sphingolipid metabolism and glycerophospholipid metabolism.Part five:Study on the urine metabolomics of thyroxine and reserpine-induced yin deficiency rats and the therapeutic effect of EZWBased on the yin deficiency rat model induced by thyroxine and reserpine,changes of metabolic profile in rat urine and the therapeutic effect of EZW towards abnormal metabolism of yin deficiency rats were investigated by UFLC-Q-TOF-MS.And the method was same as the part four.As a result,a total of 16 potential biomarkers were identified in the urine,which were associated with yin deficiency syndrome.And these metabolites were recovered in EZW groups at different levels,among which 6 metabolites showed significant differences.Pathway analysis showed that these metabolites were involved in 10 metabolic pathways,among which there were 6 pathways showing more potential,namely taurine and hypotaurine metabolism,steroid hormone biosynthesis,primary bile acid biosynthesis,TCA cycle,tyrosine metabolism and alanine,aspartate and glutamate metabolism.EZW may achieve the protective effect on yin deficiency rats by acting on 5 pathways including taurine and hypotaurine metabolism,primary bile acid biosynthesis,steroid hormone biosynthesis,lysine degradation,arginine and proline metabolism.