Effects Of Total Flavonoids Of Qijiang And Kidney On Signal Transduction Pathway Of MC Proliferation And ECM Accumulation In Rat Glomeruli

Purpose：To investigate the mechanism which Qi ji shen kang total flavonoidsserum with medicines influences the proliferation of mesagil cells and abnormalaccumulation of extracellular matrix through ERK signal pathway、NF-κB signalpathway and R-Smad signal pathway.Material and method：1The prepatation of Qi ji shen kang total flavonoids:Extracting Huang Qi and Dan Shen with95%ethanol two times, taking one hourwhen extracting the Huang Qi and Dan Shen each time. Then to filter the ethanolwith Huang Qi and Dan Shen, getting the ethanol with Huang Qi and Dan Shen andpreparing. Gathering the rest of Huang Qi and Dan Shen which had been extratedand Xiao Ji, Bai hua she she cao and Xian He cao, then adding the water whichwas16times than the mixture which gathered the rest of Huang Qi and Dan Shenwhich had been extrated and Xiao Ji, Bai hua she she cao and Xian He cao. Thento extracr the mixure with16times water two times, taking one hour whenextracted the mixture with16times water each time and filtering. Gatheringthe mixture with16times water and the ethanol with Huang Qi and Dan Shen,concentrated the mixture with16times water and the ethanol with Huang Qi andDan Shen, as result we could get the extractum of Qi ji shen kang(desity:1.12).The sample of Qi ji shen kang was passed through the column with flow rateof1BV·h-1, eluted by2BV water3BV30%ethanol,4BV50%ethanol respectively,elution flow rate was2BV·h-1.Combined30%and50%ethanol elutes wereconcentrated to yield the purification of total flavonoids. The purity of totalflavnoids was up to76%.2The preparation of Qi ji shen kang total flavonoids serum with medicines:The rats which in Qi ji shen kang total flavonoids high dose group were takenmedicine by30g/kg·d of Qi ji shen kang, The rats which in Qi ji shen kang total flavonoids low dose group were taken medicine by15g/kg·d of Qi ji shen kang,one time each day. The rats which in normal group were taken equal dose normalsaline one time each day. The rats were taken the different medicines5days.In the last time to take medicines, getting the blood from the abdominal aortaafter one hour taking the medicines. Blood was centrifuging with TDL-5-Acentrifugal machine, condition is1500rpm centrifugation for5minutes, suckingthe supernatant. After56oC water bath, half one hour inactivation. To filterand sterilize with the filtrator which the diameter is0.22μm, reserved thesupermatant in-20oC preservation.3Dividing into groups and culturing of rats glomerular mesangial cellsThe glomerular mesangial cells were cultured in25cm2culture flask, puttingthe10%FBS DMEM in the culture flask. The glomerular mesangial cells werecultured at37oC and5%carbon dioxide incubator. The glomerular mesangial cellsculture flasks were left untouched for2-3days and changed every other day untilconfluence. After the glomerulus mesangial cells were grown to80%-90%confluence, washed once with serum-free DMEM. According to the concentrationof2x104cells/hole, MCs cells were seeded in24well plates, each group wereset into5holes. The glomerular mesangial cells were cultured in0.5%FBS DMEMfor24h to synchronize the cell growth. After this time period, the media waschanged to DMEM containing10%pharmacological serum for48h. The glomerularmesangial cells were collected in order to be tested. To divide the glomerularmesangial cells into five groups: normal group which the glomerular mesangialcells were fed10%the normal rat serum; model group which the glomerularmesangial cells were fed10-7mol/LAngII and10%the normal rat serum; qi ji shenkang total flavonoids high dose group which the glomerular mesangial cells werefed10-7mol/LAngII and10%qi ji shen kang total flavonoids high dose serum;qiji shen kang total flavonoids low dose group which the glomerular mesangial cellswere fed10-7mol/LAngII and10%qi ji shen kang total flavonoids low dose serum;losartan group which the glomerular mesangial cells were fed10-7mol/LAngII and 10%the normal rat serum and10-2mol/L losartan.4The secretion of fibronectin、collagen type IV and the expression of ERK，NF-κB，TAK1，TRAF6,Smad2，Smad3，Smad4were tested by enzyme-linked immunosorbentassay (ELISA)(1) add the standard: according to the order on board hole concentration in50μlstandard product configured. Add samples: a blank hole (blank respectivelycontrolled hole without samples, affinity, and standard reagents, the rest ofthe enzyme each step for the same operation),sample hole. The enzymes bag wasboard to be added sample and then40μl add meat10μl Sample billogical. Andwhen the Sample billogical and a sample of enzyme panels at the bottom of thehole, try not to touch the hole wall, gently shaking blending. Incubate: Afterclosing plate with Closure plate membrance, incubate for30minutes at37oC.(2) Sonfigurate liquid:60-fold wash solution diluted30-fold with distilledwater and reserve. Washing:Uncover Closure plate membrance dry by swing, addwashing buffer to every well, still30s then drain, repeat5times, dry by pat.(3) add enzyme:add ELISA reagents50μl to each well, except the blank well.(4)Uncover Closure plate membrance dry by swing, add washing buffer to everywell, still30s then drain, repeat5times, dry by pat.(5) color: add color reagent A50μl and color B50μl to each well. Gently mix,incubate for15min at37oC.(6) Stop the reacton: add stop solution50μl to each well, stop the reaction(the blue color change to yellow immediately).(7)assay: take blank well as zero, measure the optical densit (OD) at451nmafter adding stop solution and within15min.(8)To calculate the sample concentration according to the standard concentrationand the cooresponding OD.5. The secretion of fibronectin、collagen type IV and the expression of ERK，NF-κB， Smad2，Smad4were tested by Real Time RT-PCR(1) liquid formulation: putting one gram DEPC in the distilled water in order to make the1‰DEPC water (in the high-pressure environment). And then put the1‰DEPC water in the1000ml container for4hours.75%ethanol: using ethanolwithout water and DEPC water, then, put the mixture water in the negative20℃to reserve.(2) culture rats glomerular mesangial cells: the glomerular mesangial cells werecultured in25cm2culture flask, putting the10%FBS DMEM in the culture flask.The glomerular mesangial cells were cultured at37oC and5%carbon dioxideincubator. The glomerular mesangial cells culture flasks were left untouchedfor2-3days and changed every other day until confluence. After the glomerulusmesangial cells were grown to80%-90%confluence, washed once with serum-freeDMEM. According to the concentration of2x104cells/hole, MCs cells were seededin24well plates, each group were set into5holes. The glomerular mesangialcells were cultured in0.5%FBS DMEM for24h to synchronize the cell growth.After this time period, the media was changed to DMEM containing10%pharmacological serum for48h. The glomerular mesangial cells were collectedin order to be tested.(3) to extract the total RNA: to put the chloroform in the tube with cellsupernatant, then shocking it in the vortex for15seconds, after that, puttingit in the room temperature for5minutes, at last, centrifuging in the4℃for15minutes. To put the samples in the ice, sucking0.5ml supernatant into1.5mlEP tube and adding0.5ml promethazine, shocking it in the vortex for15seconds,then putting the mixture in the negative20℃for30minutes, centrifuging inthe4℃for10minutes. To put the samples on the ice, discarding the supernatant,putting1ml75%ethanol (using ethanol without water and DEPC water) to washthe sediment, centrifuging in the4℃for10minutes. Repeating the process fortwo times. Discard the supernatant, air drying the sediment in the roomtemperature for10minutes. To add20μlDEPC water to dissolve and precipitatethe RNA and test the density of RNA, at last, putting the mixture in the negative20℃to reserve momently. The density and purity of RNA: to get1μl RNA sample, putting the79μl DEPC water to test the OD260and OD280, the ratio of the OD260and the OD280is1.8-2.0, it means that the purity of the sample is up to standard.The density of RNA(μg/μl) equals OD260×40×80/1000.(4) to reverse transcription: to put1μg total RNA and0.5μg/μl O1igo(dT)primer1μl, adding the DEPC water to12μl total volume. To put the sampleon the PCR machine and making the incubation in the42℃for5minutes, and thencooling to4℃. Reversing transcription: the condition is37℃for15minutesand85℃for5minutes. To put the sample on the ice, adding the reagents, puttingthe sample on the PCR machine and making the incubation in the42℃for60minutes,and then making the incubation in the70℃for10minutes, cooling to4℃. Toreserve the cDNA in negtive20℃.(5) Real-time Quantitative PCR Detecting: using the cycler thermal cyciermachine and fluorescent quantitation PCR reagent to amplificate. Using10μ SYBRPremix Ex Taq (2X),6.8μl dH2O,0.4μl control fluorescent dyes, the upstreamprimer and downstream primer2μl respectively. The condition of amplificationis to be pre degenerated in95℃for3minutes and degeneration for5seconds.To anneal in60℃for one minute, collecting the fluorescence signal andrepeating the process for40times. To test the curve of dissolution: after thecirculation, the condition is95℃for15seconds,55℃for60seconds, and95℃for30seconds. During the process of55℃to95℃, collecting thefluorescence signal one time every up to5℃.Results：1Qi ji shen kang total flavonoids inhibit ERK activation causing AngII bystimulating glomerulus mesangial cell: Glomerulus mesangial cell could secretea little bit ERK. After AngII stimulating, comparing with normal group, theexpression of ERK of model group was increasing （p＜0.01）, it means which ERKcould be activated by AngII. Given Qi ji shen kang total flavonoids and losartan,after48hours, comparing with the model group, the expressions of ERK from Qiji shen kang total flavonoids group and losartan group had decreased （p＜0.01）, it means total flavonoids total flavonoids could inhibit the expression of ERKcausing by AngII. There is no difference between the groups of Qi ji shen kangtotal flavonoids high dose and Qi ji shen kang total flavonoids low dose（p>0.05）,it means that there is not dose-effect relationship of Qi ji shen kang totalflavonoids. The expression of ERK of Qi ji shen kang total flavonoids group andlosartan group is different （p＜0.01）, the result shows that the effect oflosartan is a little bit better than Qi ji shen kang total flavonoids2Qi jishen kang total flavonoids inhibits NF-κB activation causing AngII bystimulating glomerulus mesangial cell: Glomerulus mesangial cell could secretea little bit TAK1,TRAF6and NF-κB. After AngII stimulating, comparing withnormal group, the expression of TAK1、TRAF6and NF-κB from model group wereincreasing （p＜0.01），it means that TAK1、TRAF6and NF-κB could be activatedby AngII. Given Qi ji shen kang total flavonoids and losartan, after48hours,comparing with model group, the expressions of TAK1、TRAF6and NF-κB from Qiji shen kang total flavonoids group and losartan group had decreased （p＜0.01）,it means Qi ji shen kang total flavonoids could inhibit the expression of TAK1、TRAF6and NF-κB causing by AngII. There is no difference between the groupsof Qi ji shen kang total flavonoids high dose and Qi ji shen kang total flavonoidslow dose（p>0.05）, it means that there is not dose-effect relationship of Qiji shen kang total flavonoids. The expression of TAK1、TRAF6and NF-κB of Qiji shen kang total flavonoids group and losartan group is different （p＜0.01）,the result shows that the effect of losartan is a little bit better than Qi jishen kang total flavonoids3Qi ji shen kang total flavonoids influenced the expression of FN: Glomerulusmesangial cell could secrete a little bit FN. AngII as stimulus, the expressionof FN had increased through ERK signal pathway and NF-κB signal pathway,comparing with normal group, the expression of FN from model group was increasing（p＜0.01）. Comparing with model group, the expressions of FN from Qi ji shenkang total flavonoids group and losartan group had decreased （p＜0.01）, it means Qi ji shen kang total flavonoids could inhibit the expression of FN causingby AngII through ERK signal pathway and NF-κB signal pathway. There is nodifference between the groups of Qi ji shen kang total flavonoids high dose andQi ji shen kang total flavonoids low dose（p>0.05）, it means that there is notdose-effect relationship of Qi ji shen kang total flavonoids. The expressionof FN of Qi ji shen kang total flavonoids group and losartan group is different（p＜0.01）, the result shows that the effect of losartan is a little bit betterthan Qi ji shen kang total flavonoids4. Qi ji shen kang total flavonoids inhibit Smad2、Smad3activation causing byAngII stimulating glomerulus mesangial cell: Glomerulus mesangial cell couldsecrete a little bit Smad2、Smad3. After AngII stimulating, comparing with normalgroup, the expression of Smad2、Smad3of model groups were increasing （p＜0.01）, it means which Smad2、Smad3could be activated by AngII. Given Qi jishen kang total flavonoids and losartan, after48hours, comparing with modelgroup, the expressions of Smad2、Smad3from Qi ji shen kang total flavonoidsand losartan group had decreased （p＜0.01）, it means Qi ji shen kang totalflavonoids could inhibit the expression of Smad2、Smad3causing by AngII. Thereis no difference between the groups of Qi ji shen kang total flavonoids highdose and Qi ji shen kang total flavonoids low dose（p>0.05）, it means that thereis not dose-effect relationship of Qi ji shen kang total flavonoids. Theexpression of Smad2、Smad3of Qi ji shen kang total flavonoids group and losartangroup is different （p＜0.01）, the result shows that the effect of losartanis a little bit better than Qi ji shen kang total flavonoids5. Qi ji shen kang total flavonoids influenced the extral expression of Smad4:Glomerulus mesangial cell could secrete a little bit Smad4. R-Smad signal pathwayhad been activated, comparing with normal group, the expression of Smad4frommodel group was increasing （p＜0.01）, it means Smad4had been strengthenedafter R-Smad signal pathway activated. comparing with model group, theexpressions of Smad4from Qi ji shen kang total flavonoids and losartan group had decreased （p＜0.01）, it means Qi ji shen kang total flavonoids could inhibitthe expression of Smad4. There is no difference between the groups of Qi ji shenkang total flavonoids high dose and Qi ji shen kang total flavonoids low dose（p>0.05）, it means that there is not dose-effect relationship of Qi ji shenkang total flavonoids. The expression of Smad4of Qi ji shen kang total flavonoidsgroup and losartan group is different （p＜0.01）, the result shows that theeffect of losartan is a little bit better than Qi ji shen kang total flavonoids6. Qi ji shen kang total flavonoids influenced the expression of ColIV:Glomerulus mesangial cell could secrete a little bit ColIV. AngII as stimulus,the expression of ColIV had increased through R-Smad signal pathway, comparingwith normal group, the expression of ColIV from model group was increasing （p＜0.01）. Comparing with model group, the expressions of ColIV from Qi ji shenkang total flavonoids group and losartan group had decreased （p＜0.01）, itmeans Qi ji shen kang total flavonoids could inhibit the expression of ColIVcausing by AngII through R-Smad signal pathway. There is no difference betweenthe groups of Qi ji shen kang total flavonoids high dose and Qi ji shen kangtotal flavonoids low dose（p>0.05）, it means that there is not dose-effectrelationship of Qi ji shen kang total flavonoids. The expression of ColIV ofQi ji shen kang total flavonoids group and losartan group is not different（p>0.05）, the result shows that the effect of Qi ji shen kang total flavonoidsand losartan is similar.Conclusion：1The mechanism of Qiji shenkang total flavonoids slowing down glomerulussclerosis is to inhibit the ERK and NF-κB signal pathway with the result thatthe extral expression of FN could decrease in the earier period.2Qiji shenkang total flavonoids could inhibit the activation of Smad signalpathway, as a result which it could decrease the expression of ColIV. Thereby,this effect of Qijij shenkang total flavonoids could reduce the hyperplasia ofGlomerulus mesangial cell, mitigate accumulation of ECM, relieve the glomerulus damnification and slow down glomerulus sclerosis.3Both Qiji shenkang total flavonoids and losartan could lower the expressionof ColIV and FN, it shows that Qiji shenkang total flavonoids has the same effectas losartan.4The Qiji shenkang total flavonoids could reduce the hyperplasia of Glomerulusmesangial cell, mitigate accumulation of ECM. It means that total flavonoidsis the pharmacological effect basis for Qiji shenkang5The Qiji shenkang total flavonoids could reduce the hyperplasia of Glomerulusmesangial cell, mitigate accumulation of ECM through three signal pathways (ERK、NF-κB and R-Smad) and nine targets (ERK、FN、NF-κB、TAK1、TRAF6、Smad2、Smad3、Smad4and ColIV). The result shows that the mechanism of Qiji shenkang totalflavonoids treating kidney disease is multi-approach and multiple target points.