The Protective Function And Cell Metabolomics Study Of Erzhiwan On Ageing Kidney Cells

Er-Zhi-Wan(EZW),named privet Dan,is composed of only two Chinese herbs,Fructus Ligustri Lucidi(FLL)and Herba Ecliptae(HE).EZW,firstly described in"FuShouJingFang" by Wu Min during Ming Dynasty(AD 1530),has the function of invigorating liver and kidney,nourishing Yin and stopping bleeding.It is mainly used for symptoms caused by Yin deficiency of liver and kidney,vertigo tinnitus,dry throat,knee pain,and hyper-menorrhea.The book recorded,it could increase elderly physical strength in a short period of time,and turn the white hair into the blackness.It also can nourish elderly Yin to make bones strong and improve sleep quality.Some scholars have proposed that "the aging of people is due to the Yin deficiency of liver and kidney" in recent years.Based on this,nourishing Yin of liver and kidney is the best way for antiaging.Although many articles reported that EZW has anti-aging effect,there are no EZW anti-aging studies in vitro.And the mechanism of the anti-aging of EZW is not clear,so that its clinical application is limited.In this paper,the quality of EZW and the extract was evaluated comprehensively.Based on the NRK senescence model,EZW made of different extraction methods of FLL was studied on the anti-aging effect in vitro.The theory of serum drug chemistry of traditional Chinese medicine is used to study and quantify the absorbed constituents in rat serum after administered EZW.According to the method of serum pharmacology,the protective effect of EZW-containing serum on aging NRK cells was studied based on the technique of cell metabolomics,and we explored the possi-ble mechanism of EZW anti-aging.The research content of the dissertation mainly includes the following five parts:Part one:Literature research.The Chinese and foreign literatures are consulted to compare the similarities and differences of various cell aging modeling methods,and the advantages and disadvantages of various methods for cell senescence detection are summarized,and practical detection methods are selected to pave the way for further establishment of cell senescence model and anti-aging research in vitro.Part two:A comprehensive quality evaluation method based on C30-HPLC and analytic hierarchy process for Chinese herbal formula,ErzhiwanThe quality evaluation method based on quantitative analysis of multiple indexes has always been one of the important contents of the quality study of traditional Chinese medicine(TCM)herbal formula.Chinese pharmacopoeia 2015 only stipulates the content of specnuezhenide as a single component in EZW from the FFL powder and water decoction of EH.This could not generalize the intrinsic quality of the TCM herbal formula.A novel C30-HPLC method with good precision,accuracy and reproducibility,was developed for simultaneous determination of six compounds for quality control of EZW.This method was applied to quantitative evaluation of the eight samples among which the four were prepared by different methods and another four samples were extracted by different solvent systems.The quantitative results of eight samples were then analyzed comprehensively by the Analytic Hierarchy Process analysis,and the results of analysis were in agreement with the results of the antioxidant tests(2,2-dibhenyl-1-picrylhydrazyl,DPPH method)in vitro.The comprehensive strategy could provide references and suggestions for the improvement of quality evaluation method of EZW and other TCM herbal formula.Part three:Qualitative and quantitative study of the absorbed constituents in rat serum after administering of EZW1.Qualitative analysis of the absorbed constituents in rat serum after administered EZW on UFLC-Q-TOF-MS technologyAn 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 the absorbed constituents in rat serum after administered EZW.As a result,a total of 27 compounds were identified in the absorbed constituents in rat serum after administered EZW,including flavonoids,phenyl alcohol,cyclic ether terpenes,triterpenoids,phenols,etc al.Among these compounds,18 compounds were origined from LLF while 7 from EH,and the luteolin and apigenin were found to be in both LLF and EH.This study could provide valuable information for the quality control and further investigation of the absorbed constituents in rat serum after administered EZW.2.Quantitative analysis of the absorbed constituents in rat serum after administered EZWThe salidroside and specnuezhenide from the absorbed constituents in rat serum after administered EZW,was quantified by UPLC-MS/MS.UPLC was conducted using an ACQUITY UPLC(?)BEH C18 column(4.6 mm×250 mm,5 ?m),the mobile phase consisted of(A)acetonitrile-(B)0.1%formic acid water.The flow rate and column temperature were set to 0.3 mL·min-1,30 ?,respectively.Mass spectrometry conditions:Electrospray ionization(ESI)ion source was used for multiple reaction monitoring(MRM)mode detection of each compound in negative ion mode.The salidroside and specnuezhenide were not detectable in blank serum,and were separated well.The content of salidroside and specnuezhenide from the Group Erzhiwan were 3.19,0.10 ?g·mL-1,and the Group EZW-3 were 22.29,0.14?g·mL-1.The method can rapidly and accurately detect the contents of salidroside and specnuezhenide in the absorbed constituents in rat serum after administered EZW.A quality control of EZW-containing serum was established in the further pharmacodynamic experiments.Part four:The protective function of EZW on D-galactose-induced senescence of normal rat kidney cellsTo investigate the protective function of EZW on aged Normal Rat Kidney(NRK)Cells induced by D-galactose,different concentrations of D-galactose(1,5,10,20,40 g·L-1)were added into the culture medium for different processing time(24,48 and 72 h)to induce NRK cells senescence.Cellular senescence was described by senescence-associated ?-galactosidase staining and cell viability was evaluated by MTT assay.Fluorescein di-?-D-galactopyranoside(FDG)method was used to detect the quantity of ?-galactosidase.The levels of SOD,MDA and GSH-PX in the supernatant were measured to show the changes of oxidative stress.The establishment of the NRK cell aging model was determined according to the outcome of these detections.After that,different concentrations of EZW(10,100,500 and 1000 mg·L-1)were added into the culture medium,and the effect of EZW on the aged NRK cell was observed with FDG detection.The activity of SOD,GSH-PX and MDA were measured.Compared with control group,the number of?-galactosidase-positive cells in aging group(20 g·L-1 D-galactose exposure for 48 h)were apparently increased.In aging group,the quantity of ?-galactosidase detected by FDG was markedly increased,and the activity of SOD,GSH-PX was down-regulated while the content of MDA in the cells was increased.No significant change of the cell viability between the 2 groups was observed.Compared with aging group,the quantity of ?-galactosidase in EZW treatment group was decreased.EZW also up-regulated the activity of SOD,GSH-PX and decreased the content of MDA in the cells.Up-regulation of oxidative stress level exists in D-galactose-induced aged NRK cells.EZW attenuates D-galactose-induced senescence in NRK cells.Part five:Study on the protective effect of EZW on aging NRK cells based on cell metabolomics1.The protective effect of EZW-containing serum on aging NRK cellsThe concentration of drug-containing serum(5%;10%,20%),administration time(24,48,72 h)and drug-containing serum type(EZW drug-containing serum,EZW-3 drug-containing serum)were used as influencing factors,and the intracellular ?-galactosidase activity was used as an evaluation index to design a mixed orthogonal experiment.The preferred condition for EZW drug-containing serum is that 20%of EZW-3 containing serum act on ageing NRK cells for 48 h.Compared with blank serum group,drug-containing serum can significantly inhi-bit the aging of NRK cells.2.The protective function of EZW on ageing NRK cells based on cell metabolomicsThe NRK cell ageing model was replicated by D-galactose and the cell samples were determined by GC-MS.The PCA,PLS-DA and other methods for pattern recognition,Kruskal-Wallis nonparametric test and Dunn's multiple comparison were used for univariate statistical analysis.After the PLS-DA pattern recognition analysis,the cell samples of the control group,the model group,and the administration group were well distinguished.Based on the variable importance projection(VIP)of the PLS-DA model and the P value of the univariate analysis,metabolites that significantly contributed to the classification were screened.Metabolites were identified by mass spectrometry matching with the reference spectra in the NIST 2014 standard database,and a total of 33 potential biomarkers were identified.The intracellular levels of endogenous metabolites in the senescent NRK cells of the administration group recovered to varying degrees.EZW could restore the intracellular abnormal metabolism of D-galactose-induced aging NRK cells.The therapeutic effects maybe related to the regulation of the following 10 pathways:(A)Phenylalanine,tyrosine and tryptophan biosynthesis,(B)Glycine,serine and threonine metabolism,(C)Phenylalanine metabolism,(D)Glyoxylate and dicarboxylate metabolism,(E)Arachidonic acid metabolism,(F)Glycerolipid metabolism,(G)Alanine,aspartate and glutamate metabolism,(H)Inositol phosphate metabolism,(I)Cysteine and methionine metabolism,(J)Citrate cycle.