Study The Dynamic Influence Of Nourishing Yin Clearing Heat Formula On Gene And Protein Expression Of PBMC Of SLE With Internal Heat Due To Yin Deficiency

BackgroundSystemic lupus erythematosus (SLE) as a serious autoimmune disease, theetiology, pathogenesis of this disease had not been fully explained and thetreatment are still a world's problems. Currently, corticosteroids andimmunosuppressants are the main treatments, but the application will bringlong-term significant toxicities. Clinical Practice has proved thatintegrated traditional and western medicine methods can significantly reducedrug toxicity and improve life quality of SLE patients. Internal Heat due toYin Dificiency(IHDYD) is the basic types of SLE, Nourish yin, tonify thekidney, clear away heat and suppress fire are the basic rules for the treatmentof SLE. Many studies showed that many genes take part in the onset of SLE.Study the dynamic changes of gene regulation networks and protein interactionnetwork of SLE will help us to understand the pathogenesis of SLE deeply.ObjectiveObserved the Treatment Effects of Nourishing Yin Clearing Heat formula(NYCHF) on patients of SLE with Internal Heat due to Yin Oificiency(IHDYD);Studied the dynamic changes of gene and protein expression of the peripheralblood mononuclear cell (PBMC) according to three different stages: Before cure,six weeks and 12 weeks after treatment among 3 months. Try to learn thesimilarities and differences of gene and protein expression patterns betweenpatients and normal persons, explore treatment mechanism of NYCHF and prepareto establish methods and platform for Chinese medical research for SLE at thelevel of gene and protein expression.Methods1. ObjectsFifteen IHDYD female SLE patients were 18 to 50 years old, outpatient ofthe TCM hospital of GuangDong province of China from November 2005 to November 2006, with diagnosis as SLE according to the American Rheumatism Association1997 revised diagnostic criteria, in line with IHDYD certification standards,and in the mild or moderate activity with the score between 5 to 25 accordingto SLEDAI system of the United States and Canada SLE center. If had been usingglucocorticoids, then must maintain the dosage at 10-25 mg/day within the nextthree months study. Must withdrawal non-steroidal anti-inflammatory drug overa week and hadn't used immunosuppressant over six months. All of the casesmust finish 3 months courses of treatment and record detailed according toCase Report Form. Fifteen normal control were healthy voluntaries fromGuangzhou University of Traditional Chinese Medicine with a mean age: 28.4±6.50. Treatment group had included 18 cases, two cases exodus and one lost,15 cases were effective, with a mean age:31.60±9.37 years. The age varianceof two groups were homogeneity (P=0.098), and there were nonsignificantstatistically difference between two groups acording to two sample t test(P=0.289).2. Treatment and observationPatients were given five NYCHFs three times per day during three months,and score of IHDYDwere got every two weeks. C₃, C₄, anti-nuclear antibody (ANA),anti-dsDNA, blood routine, urine routine, and other indicators were detectedbefore, 6 weeks and 12 weeks after cured with NYCHF, and PBMC were extractedand stored in refrigerator at -70℃. Only samples of those cases that hadfinished three months observation were sended to gene or protein microarraychips detect. Case information was collected detailed acording to "Case ReportForm."3. DNA microarray chip experimentcDNA were directly pointed on chips to made the human cDNA microarray chipsby the Chipscreen Biosciences, with total genes as 8,064, probes were markedwith Cy3 single fluorescent, data were revised standardizd, Calculated theratio of gene expression in the experimental group and control group. If Ratiowas greater than 2 or less than 0.5, there were significant differences betweenthem. GeneCluster 2 software was use to analysis the data with self-organizingmaps, functional classification and clustering analysis methods.4. Protein microarray experimentUsing Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI) methods established the PBMC protein expression data ofSLE patients before treatment, six weeks, and 12 weeks after treatment andthe normal control group. ZUCIPDAS package designed by Jie-Kai Yu of CancerInstitute of Zhejiang University were used to identify the protein peaksdiscriminant model. The name and the preliminary function of the protein peakswere identified by Crosslinked compareing between Gene microarray expressiondatabase and the Expasy protein database.Results1. Glucocorticoid with NYCHF can relieve pathogenetic condition of IHDYDSLE quickly, soft self discomfortable sense, decrese scores of SLEDAI and IHDYDsignificantly, and incerese the level of C3 and C4. Scores between IHDYD andSLEDAI was positively correlated which suggested that Scores of IHDYD canreflect the condition of the patient fairly integratly and objectively.2. 2,946 gene expression data were acquired by using normalized dataprocess methods. 167 genes which has marked differences expression at leastone time in three independent experiments were categoried to 25 differentpatterns and analysis the difference among three stages used the repeatmeasurements analysis of variance, the differences were statisticallysignificant (P=0.000). 20 different genes had statistically significantdifferent (P＜0.05) expression at three different test times: IGKC expressiondecreased after cure 12 weeks and lower than the expression of normal control,the expression of another 19 genes (Arp2, ARCN1, CRY2, DAF, DPYSL2, FMR1. FYN,GRO3, LFNG, IVNS1ABP, P125, PBEF, PRKCB1. PSCDBP, PTPRE, Carrier, TMEM2, TNF,ZFP103) were increased, decreased or normal in SLE than in the normal control,but compared with the expression of themself before cure, they all increasedafter treatment.3. Genes with similar changes in the expression of three samples (before,during and after the cure) were analyzed with cluster method, and 87 geneswere clustered: 21 genes expressed decreased and 66 genes expression increasedafter cure than before cure. down expressed Gene mainly related to the immuneresponse genes (DXS1357E, LY117, IGHG3, IGKC, IGL@, PTPRCAP), inflammationrelated genes (LY117, ADORA3, MIF, EDG6, CTSH), and some cell apoptosis andproliferation, signal pathway and metabolic associate gene (H2AFO, RPL28, FEZ1, EMP3, CAPN4, MIF, DXS1357E, APRT, COXBA1, PLEC1, RPS5, DGKA, NMBR,PTPRCAP). Up-regulated expression genes mainly related to the signaltransduction genes (SHOC2, SEL1L, JAK2, NGB, BIG2, DPYSL2, RGS19, PTGS2, BAI3),immune inflammation related genes (ILF3, B4-2, PTPRC, KLRC1, CSF2RB, PBEF,GRO3, DAF, SDCBP, TNF, TRA1, SSFA2, HSPA9B, JAK2), the transcriptionalregulation of gene-modified (B4-2, ZNF9, RYBP, TFEC, ZNF217, SSX2, ATF3,HUMHOXY1, MAF, SMARCA5, LFNG, PTGS2, NS1-BP, EGR2, SF3B1, prpf4, GRSF1), cellproliferation, apoptosis and differentiation-related genes (Aug, BTG3, E6,RYBP, TNF, CCNG2, ERCC5, IDN3, CLK1, CDC23), and the biosynthesis and membraneprotein related genes (TBCC, DLEU2, ZNF217, PLSCR1, TMEM2, SCP2, CD47, CGGBP1,TACC1, DNM1, EBI2, PIGA, SLCTA6, KIAA1382, PTPRC, IPLA2, FOSB).Conclusions1. Score of IHDYD symptom decreased significantly along with the time oftreatment, suggesting NYCHF can improve the symptoms of IHDYD SLE patients.2. Apoptosis, immune-related gene expression disorder were found in IHDYDSLE patients before or during treatment, Gene expression patterns changesignificantly in the three months study. NYCHF may adjust YIN and YANG in arelative equilibrium through regulates the gene expression disorder patientsand achieves treatment effect. Further study on SLE and other immune-relateddiseases use large sample gene clustering analysis may be able to establishearly diagnosis, differential diagnosis and build different gene expressionpatterns of Chinese medicine syndromes.3. Different protein expression patterns of PBMC were observed betweenIHDYD SLE Patients and normal samples, Suggesting protein chip can be usedto build the method for early diagnosis of SLE, pending further study.4. Many differences of protein expression observed before, during and afterthe treatment, suggesting NYCHF may cure IHDYD SLE patients by regulatingprotein expression of PBMC. Further study on the discovered protein-markermay lead to find treatment-related protein target. It is worth to depthstudying.