Yishentang Combined With Western Medicine To Slow The Progress Of The Role And Mechanism Of Mouse Iga Nephropathy Kidney Damage

Objective: IgA nephropathy (IgAN) has been regarded as the most common primaryglomerular disease in the world and the main pathological type of chronic renal disease. Forthe time being, western medicine usually treat it with glucocorticoid hormone,immunosuppressants, angiotensin converting enzyme inhibitor, angiotensin receptor blockers,fish oil, anticoagulants, anti-platelet aggregation drugs as well as tonsillectomy according to24-hour urinary protein out-put, renal function, the renal pathological change and the age ofpatients. However, according to researches of the evidence-based medicine, shortcomings ofpoor treatment effect or severe adverse reactions exist in the treatment of IgAN with onlywestern medicine. The treatment of IgAN with traditional Chinese medicine (TCM) has had ahistory of nearly 20 years. Furthermore, extensive progress has been made in the clinical andlaboratory study of treatment for IgAN by the combination of western medicine and TCM.TCM treatment for IgAN mainly includes the Supplementing qi and Nourishing yin methodand Nourishing Liver and Kidney method. Based on clinical experiences, the Yishen Decoctioncomes from an experienced modified recipe of the famous ancient Liuwei Dihuang Wan (bolusof Six Drugs Including Rehmannia) described in the pediatric monograph Key to Therapeuticsof Chidren's Diseases. We established the IgAN model in mice and treated it with YishenDecoction combined with the western medicine prednisone and enalapril. Its action andmechanism of alleviating the renal injury in IgAN will then be studied from the view of cellapoptosis and cellular factor and the level of mRNA transcription and protein expression. Indetail, normal control group, model control group, western medicine treatment group, TCMtreatment group and combination of western medicine and TCM group were studiedcomparatively. Their clinical and renal pathological changes, apoptosis of renal mesangial cellsand expression of the modulating genes Bax and Bcl-2, the mRNA and protein expression ofTGF-β1, MMP-9, TIMP-1, PAI-1 were studied thereafter so as to discuss the action andpossible mechanism of alleviation the renal injury by Yishen Decoction combined with western medicine. Meanwhile, whether the combination of Yishen Decoction together with prednisoneand enalapril is better in slowing the developing of renal injury of IgAN than the single use ofwestern medicine or TCM will also be studied. So a theory basis may be provided for thecombination treatment of IgAN by western medicine and TCM in clinical work.Methods1 The establishing and Identification of IgAN Model in Mice105 BALB/c female mice, aged 8 to 10 weeks with body weight of 18 to 22 grams werefeeding for a week for adjusting to the environment. Then 36 mice were chosen randomly to bethe sample. 2 as one group were put in a metabolic cage. The urine within 24 hours wasgathered for the quantitative examination of protein and qualitative test of hematuria. The urineof sample mice showed negative hematuria test. However, the 18 quantitative examinations ofurine protein in 36 tested mice showed that small amount of protein about 5.54±1.46mg wasexcreted in the urine of normal mice. All the 105 mice were divided randomly into 15 ofnormal control group and 90 of model group. The IgAN model was estblished by the methodof oral intake of bovine serum albumin (BSA) together with the injection of staphylococcusenterotoxin B (SEB) through caudal vein provided in the articles' data. By the end of the fifthweek, 3 mice in the model group had been dead. No mice in the normal control group weredead. At the end of the 5th week of the model building, the quantitative examination of proteinand qualitative test of hematuria in the urine of mice of model group and normal control groupwere done the same way as above. 3 mice in the normal control group and model grouprespectively were chosen randomly as a sample. Blood was taken through eye orbit aftereyeball extirpation to test the urea nitrogen and creatinine. Then, the kidney was dissected todo the following tests:Light Microscope Test: The kidney tissue of mice was fixed with paraformaldehyde andembedded in paraffin and sectioned by 3μm. The sections were washed in 3 jars of xylene, thenwashed by absolute alcohol, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol and distilled water successively. Finally the sections were stained by HE, MASSON, PAS,argentum hexamethy lenamine respectively.Immunofluorescence Test: Fresh kidney tissues were embedded in OCT, then frozenly slicedby 4 to 6μm. The sections were washed in PBS for 5 minutes and 3 times then dried slightlyunder room temperature. Rabbit anti mouse antibody IgA of 1:400 labeled by FITC weredropped onto the sections. The sections were put overnight in the dark box under 4℃. Theywere then washed in PBS for 5 minutes, 3 times. After that, they were dried and mounted undertemperature and observed under fluorescence microscope and the pictures were taken. Theimmunofluorescent semi-quantitative standard was formulated by the common method of 5grades (0 to ++++) used inside the country and overseas.The Image Pattern Analysis of Morphological Change of Kidney Tissues:The PAS stained sections were examined under light microscope. The microscope field wasamplified by 400 times. 8 to 10 glomeruli were chosen randomly in a section. By the use of allautomatic image analysis system of German KONTRON IBAS 2.5, the mesangial regions ofrenal glomeruli were labeled by mouse of the computer and the areas of the regions werecalculated. The areas of capillary plexuses were also measured and the percentages of themesangial region in the whole capillary plexuses area were calculated.2 Animal grouping, experimental intervention and materials:After it was certain that the model was successful, the 84 IgAN mice still alive were dividedrandomly into 6 groups, 14 mice in each group including the normal control group. There werestill 12 mice, which got stomach lavage with 1 milliliter of distilled water, once daily. So therewere altogether 7 groups of mice. Model control group: stomach lavage by 1ml of distilledwater, once daily.2.1 Prednisone plus enalapril group: prednisone 5mg per kg of body weight per day andenalapril 10mg per kg of body weight per day by stomach lavage mixed with 1ml of distilledwater, once a day. 2.2 Prednisone plus cnalapril plus low concentration Yishen Decoction group:prednisone 5mg per kg of body weight per day and enalapril 10mg per kg of body weight perday dissolved in 1 ml of low concentration Yishen Decoction by stomach lavage, once daily.2.3 Low concentration Yishen Decoction group: 1ml of low concentration YishenDecoction by stomach lavage, once daily.2.4 Prednisone plus enalapril plus high concentration Yishen Decoction group: prednisone 5mgper kg of body weight per day and enalapril 10mg per kg of body weight per day dissolved in1 ml of high concentration Yishen Decoction by stomach lavage, once daily.2.5 High concentration Yishen Decoction group: 1 ml of high concentration Yishen Decoctionby stomach lavage, once daily.From the sixthth week, all groups except the normal control were all injected BSA and SEB bythe means introduced in articles. All mice drank and ate freely except that the aboveexperimental conditions for the associated mice were different. The urine of the mice in the 7groups was collected to get the 24-hour protein quantitative examination and hematuriaqualitative test at the end of observation for 12 weeks when the trial was ended. Blood wastaken from the eye orbit after the eyeball extirpation. Then the mice were injected 50mg/kg of2% pentobarbital sodium into abdominal cavity for anesthesia. Their bodies were incised openalong the median line of the abdominal cavity. The kidneys were taken out then sliced openhalf by half along the coronary line. The kidneys were kept sliced along the coronary section.The outer cortical part was taken for frozen section for immunofluroscence IgA test. The otherpart was taken for paraffin section and fixed with 10% neutral formaldehyde, embedded andsectioned in paraffin and stained by HE, MASSON, PAS and argentum hexamethy lenaminerespectively for pathological examination. The following detections were accomplishedaccording to the experimental design.3 Influence of Experimental Intervention on Clinical Treatment Effect and PathologicalChanges of Kidney in Mice The urine quantitative protein examination, hematuria qualitative test, blood urea nitrogen andcreatinine as well as the area of mesangial membrane region, capillary plexuses of glomeruliand the percentage of the former in the latter were all detected among each of the 7 groups ofthe mice. The pathological parameters including the semi-quantitative immunofluroscent testof the kidney tissues were calculated. Comparison between one group and two groups wereanalyzed.4 The Study of Apoptosis and Its Modulating Genes Bax and Bcl-2 of Mesangial Cells of MiceThe TUNEL method was adopted to detect the cell apoptosis that was finished under theinstruction of the reagent kit's manual. Apoptosis was determined by the morphological changeof cell nucleus, that is, the cell nucleus was condensed, fragmented and became brown granules.100 mesangial cells were counted in each section under light microscope and the apoptoticcells within them were also counted. The apoptotic rate was defined as the percentage ofapoptotic cell amount in the total cell amount. The Bax and Bcl-2 proteins were detected byimmunohistochemistry method. The staining was finished by routine procedures. The stainingof the proteins in mesangial cells was observed under light microscope. Five high powerfields (×400) whose cells dispersed evenly were observed and the cells under various stageswere calculated. The average amount in the five fields was calculated and the positive cellswere counted. The positive ones were expressed by percentage. The determination of theresults were as follows: the color of the background was observed under low power field andthe staining degree of glomerular mesangial region was observed under high power field andscored according to the routine standard, that is, the protein expression of Bcl-2 and Bax wasclassified as 4 grades according to the staining depth of the positive products and the amount ofpositive cells: (-): no positive cells, positive cells were less than 10% or the backgroundshowed the same color with normal control; (+):only less than 25% of cells showedobviously positive or most of the cells showed slightly positive; (++): 25 to 50% of cellsshowed obviously positive; (+++): 50% of cells showed obviously positive. 5 The mRNA Expression of TGF-β1, MMP-9, TIMP-1, PAI-1 in the Kidney Tissue of MiceDetected by FQ-RT-PCR:5.1 Extraction of RNA in kidney tissue:5.1.1 The tissue slice was put in homogenizer and mixed evenly with 1ml of Trizol liquid,then frozenly homogenized.5.1.2 The mixture was put into a 1.5ml Eppendorf tube. Then add 0.2ml of choroform into it.The tube was sealed tightly and shaken strongly for 15 seconds. It then was incubatedunder 15 to 30℃for 2 to 3 minutes.5.1.3 The tube was centrifuged for 15 minutes under 4℃by 12000r/min. The supernate wasthen put into a new 1.5ml Eppendorf tube and mixed by the same volume ofisopropanol.5.1.4 Incubation under 15 to 30℃for 10 minutes.5.1.5 Centrifuge for 10 minutes under 4℃by 12000r/min. The supernate was discarded.5.1.6 The remnants were washed and precipitated by 75% alcohol with DEPC water in it,which was centrifuged for 5 minutes under 4℃by 7500r/min. The alcohol wasdiscarded.5.1.7 The remnants were dried in air or vacuum for 5 to 10 minutes. However, it was notfully dried. Then DEPC water was added into the remnants so that RNA was dissolvedin it.5.1.8 Stored under -80℃for further use. In case of store for long period, alcohol 2.5 times ofits volume shall be added.5.2 The Design of Primer Probe: The forward and reverse primers and TaqMan probe ofTGF-β1, MMP-9, TIMP-1, PAI-1 and GAPDH was designed and synthesized by Primerexpress2.0 software on the Model ABI 3900 Table High Flux DNA Synthesizer.5.3 Reverse Transcriptional Reaction: 4μl of RNA templates were taken to do thetranscriptional reaction. The device was the PE9600 PCR device and the reaction system was: 4μl of 5×reverse transcriptional buffer, 0.4μl of forward primers and reverse primersof target gene, 0.5μl of dNTPs, 1μl of MMLV, 9.7μl of DECP water, 4μl of RNAtemplates, the total volume was 20μl. The reaction condition was under 37℃for 1 hourthen 95℃for 3 minutes.5.4 Fluorescence Quantitative PCR: The reaction system: 10μl of 5×quantitative PCR buffer,1μl of forward primers F1 and 1μl of reverse primers of target gene R1, 0.5μl of dNTPs,1μl of fluorescent primer probe of target gene, 1.5μl of Taq enzyme, 5μl of cDNA, 30μlof ddH₂O, the total volume was 50μl. The reaction condition was under 93℃for2minutes, then 93℃for 45 seconds, and 55℃for 45 seconds, and totally 40 cycles.5.5 Contents of TGF-β1, MMP-9, TIMP-1, PAI-1 in Kidney Tissue of Mice Detected byImmunohistochemistry:5.5.1 Staining Procedures:5.5.1.1 Section slicing, application and drying (in incubator of 60℃for 1 to 2 hours).5.5.1.2 Degrease the sections by dimethylbenzene for 5 minutes and 3 times5.5.1.3 Gradient washing by alcohol (absolute alcohol for 10 minutes, twice; 95% alcohol for10 minutes, twice; 75% alcohol for 5 minutes).5.5.1.4 Incubate in 3% hydrogen peroxide under room temperature for 5 to 10 minutes so as todemolish the activity of endogenous peroxidase.5.5.1.5 Sections were washed by distilled water and soaked in PBS for 5 minutes and twice.5.5.1.6 Sections were put in blocking 1.5% serum for half an hour.5.5.1.7 The TGF-β1 antibody (1:400) was added to the sample and left alone overnight under4℃.5.5.1.8 The next day sections were washed by PBS for 5 minutes, 3 times, then incubated inantibody for 30 minutes.5.5.1.9 Sections were washed by PBS for 5 minutes, 3 times5.5.1.10 Incubate in second antibody for 30 minutes. 5.5.1.11 Sections were washed by PBS for 5 minutes, 3 times5.5.1.12 DAB coloring.5.5.1.13 Irrigated by tap water, counter stain, hyalinization, dehydration, and mounting.5.5.2 Calculation of PU (positive unit) of TGF-β1,MMP-9,TIMP-1,PAI-1:The immunohistochemistry sections of kidney tissues were observed under a light microscope.10 renal glomeruli were counted and amplified by 400 times. The pictures of the fields werethen taken into fully automatic German KONTRON IBAS 2.5 image analysis system. Anequation was adopted to calculate the value of PU, that is, PU=100×(G_a-GA)/[(1-A_a)×G_max].The PU values of each group were compared. Notes: Ga, the average gray scale of positivereactions; GA, the average gray scale of whole field; A_a, the area density of positive reactions;G_max, the highest grade of gray scale (256). All the data were calculated automatically thesystem.Results1 IgAN Model: At the end of the fifth week of model building, the results of urine proteinquantitative examination and hematuria qualitative test of mice in model group were obviouslyhigher than those of mice in normal control group (P＜0.05), while the blood urea nitrogen andereatinine between groups showed no statistical significance (P＞0.05). Under light microscope,the renal glomeruli of mice in model group were slightly enlarged, the mesangial cells andmatrix were moderately or severely proliferated. Part of Bowman's capsules became adherent,the cavities of glomerular capillaries were compressed and narrowed. Nevertheless, no obviousabnormalities were shown in the renal glomeruli of mice in normal control group.Immunofluroscence showed that the mesangial region of mice in model group showed strongpositive, the IgA was strongly positive. No IgA sediments were found in the mesangial regionof mice in normal control group, which showed obvious significant difference (P＜0.01). Thearea of mesangial region, renal glomeruli and the ratio of area of the former to the latter ofmice in model control group were all higher than that of mice in normal control group (P＜0.05).2 The Change ofApoptosis of Mesangial Cells and Its Modulating Genes Bax and Bcl-2 inIgAN Mice:When the trial ended, a little cell apoptosis was found in the mesangial region of mice innormal control group, while no cell apoptosis was found in that of mice in model group, whichshowed significant difference (P＜0.05). A little expression of Bax was found in mice of twogroups, which showed no statistical significance (P＞0.05). But the expression of Bcl-2 in miceof model group was higher than that in normal group, which showed significant difference(P＜0.05).3 Influence of Experimental Intervention on the Expression of Bax and Bcl-2 and MesangialCell Apoptosis in IgAN Mice:Expression of Bcl-2 in renal tissue of mice in either the low or high concentration YishenDecoction group were obviously lower than that of the model group (P＜0.01). However, theapoptosis rate of mesangial cell and Bax expression of the Yishen Decoction groups wereobviously higher than that of the model group (P＜0.01). Among the 5 intervention group, theBcl-2 expression in mesangial region of mice in prednisone plus enalapril plus Yishen TangDecoction group were obviously lower than that of other groups (P＜0.01), while its apoptosisrate of mesangial cells and expression of Bax were obviously higher than that of other groups(P＜0.01).4 Influence of Experimental Intervention on the Urine Protein Quantitative Examination,Hematuria Qualitative Test and Renal Function Test of IgAN Mice:The results of urine protein quantitative examination, hematuria qualitative test and renalfunction test of mice in high concentration Yishen Decoction group were respectively lowerthan that of low concentration Yishen Decoction group (P＜0.05). The parameters ofprednisone plus enalapril group were respectively lower than that of low concentration andhigh concentration Yishen Decoction group (P＜0.05), while the 3 groups were lower than that of model control group (P＜0.05). No statistical significant difference was shown between theprednisone plus enalapril plus low concentration Yishen Decoction group and prednisone pluscnalapril plus high concentration Yishen Decoction group (P＞0.05). But parameters of eitherof them were respectively lower than single western group (prednisone plus enalapril) or singleTCM (Yishen Decoction) groups (P＜0.05).5 Influence of Experimental Intervention on the Pathological Parameters (area of mesangialregion, area of renal glomruli and the ratio of the former to the latter, IgA immunofluorescencein mesangial region) of IgAN Mice:No statistical significance of the parameters was shown between the low and highconcentration Yishen Decoction group (P＞0.05). The parameters of prednisone plus enalaprilgroup were respectively lower than that of either the low or high concentration YishenDecoction group (P＜0.0). The parameters of prednisone plus enalapril group and YishenDecoction group were respectively lower than that of model control group (P＜0.05). Nostatistical significant difference was shown between the prednisone plus enalapril plus lowconcentration Yishen Decoction group and prednisone plus enalapril plus high concentrationYishen Decoction group (P＞0.05). But parameters of either of them were respectively lowerthan single western group (prednisone plus enalapril) or single TCM (Yishen Decoction) group(P＜0.05).6 Influence of Experimental Intervention on the mRNA and Protein Expression of TGF-β1,MMP-9, TIMP-1, PAI-1 in Kidney Tissues:No significant differences of mRNA and protein expression of TGF-β1, MMP-9, TIMP-1,PAI-1 were found between the low concentration and high concentration Yishen Decoctiongroup. But the secretion and mRNA expression of TGF-β1, TIMP-1, PAI-1 in the low and highconcentration Yishen Decoction group were decreased than the IgAN model group, while thatof MMP-9 were stronger than the model control group. Among the five treatment groups, theprednisone plus enalapril plus Yishen Decoction group showed the weakest mRNA and protein expression of TGF-β1, PAI-1, IMP-1 and the strongest expression of MMP-9.Conclusions:1 The results of urine protein quantitative examination, hematuria degree, area of mesangialregion, area of renal glomruli, the ratio of area of mesangial region area to renal glomruli area,pathological findings including the light microscopy and immunofluorescent test shows thatthe IgAN mice model built through oral intake of bovine serum albumin (BSA) together withthe injection of staphylococcus enterotoxin B was successful.2 Mice in IgAN model group show obvious poor apoptosis of mesangial cells, meanwhile, Alittle expression of Bax was found in mice of two groups, which showed no statisticalsignificance. But the expression of Bcl-2 in mice of model group was higher than that innormal group, which showed significant difference.That indicates that the modulating gene Bax and Bel-2 for apoptosis play important roles in theonset and development of IgAN.3 The abnormal expression of TGF-β1, MMP-9, TIMP-1, PAI-1 mRNA and protein playimportant roles in the onset and development of IgAN.4 The TCM Yishen Decoction can alleviate the hematuria and proteinuria, stabilize renalfunction, modulate renal mesangial cell apoptosis and the expression of Bax and Bcl-2, reducethe abnormal expression of the mRNA and protein of TGF-β1, TIMP-1 and PAI-1, strengthenthe mRNA and protein expression of MMP-9 so as to slow down the renal injury that was lessobvious than the combination ofprednisone with enalapril.5 The combination of prednisone plus enalapril plus Yishen Decoction has better treatmenteffect than single use of prednisone or combination of prednisone and enalapril or the singleuse of Yishen Decoction to reduce urine protein and stabilize the renal function. It has alsosome advantages of alleviating pathological injury of IgAN, modulating apoptosis ofmesangial cell as well as the abnormal expression of TGF-β1, MMP-9 TIMP-1, PAI-1 mRNAand protein.