| Purpose：This experiment use macroporous purification Qiji shenkang granule extract flavonesadsorption resin method, and to observe the effects of Qiji shenkang granule flavonoidsextracts on urine red blood cell count、24hour urinary protein quantitative、pathologicalchanges in kidney and renal immunofluorescence examination in rats with IgA nephropathy,proving the Qiji shenkang granule can treat the rats with IgA nephropathy.The angiotensin IIas a stimulating factor, rat glomerular mesangial cells cultured in vitro, induced excessivesecretion of fibronectin and collagen type IV, leading to the extracellular matrix accumulationoccurs. Choose a different time point, by observing Qiji shenkang granule effect on thesecretion of fibronectin and collagen type IV, to investigate the role of the target by Qijishenkang granule of total flavonoids extract on glomerular disease. The signal transductionpathway through glomerulus mesangial cell in vitro, to detect the expression of activatedmitogen activated protein kinase and nuclear factor kappa b, and to explore the role ofprotecting kidney and the mechanism of prevention and treatment of glomerular diseases.And to provide reliable experimental data for the treatment of diseases of glomerular by Qijishenkang granule, in order to guidance clinical provide new ideas and methods in future.Material and method：1Using purification technology of total flavonoids from Qiji shenkang granule bymacroporous resinThe sample of Qi ji shen kang granule was passed through the column with flow rate of1BV·h-1，eluted by2BV water，3BV30%ethanol，4BV50%ethanol respectively，elutionflow rate was2BV·h-1.Combined30%and50%ethanol elutes were concentrated to yield thepurification of total flavonoids. The purity of total flavonoids was up to76%.2. The use of oral bovine serum albumin replication of experimental IgA nephropathy modelrats and the treatment groupThe dose of oral immunogen bovine serum albumin (BSA) increased one times (400mg·kg-1every other day), the method of injecting carbon tetrachloride CCl4was subcutaneousinjection instead of intraperitoneal injection, which dose was one third of content to induce hepatic fibrosis (benne oil0.5ml once a week and CCl40.10ml once a week, continue9weeks). Combined with0.05mg lipopolysaccharide once a everyday, at the sixth and ninthweek once.forty SD rats (specific pathogen free), in which the half was male and the other half wasfemale and200±20g body weight. The rats were randomly divided into five groups, namely:the control group, model group, the Qiji shenkang granule total flavonoids high does group,the Qiji shenkang granule total flavonoids low does group.the telmisartan group. N=8.Thenormal group, model group were given normal saline1ml/100g (weight) once daily. the Qijishenkang granule total flavonoids high does group was treated by60g·kg-1of Qiji shenkanggranule total flavonoids everyday and Qiji shenkang granule total flavonoids low does groupby30g·kg-1of Qiji shenkang granule total flavonoids everyday. The telmisartan group wastreated by8mg·kg-1of telmisartan everyday. In the twelfth week, after the last administration，collection of samples, determination all kinds of indexs.3. Through the pharmacology of serum method,we got the control group，the Qiji shenkanggranule total flavonoids high does group, the Qiji shenkang granule total flavonoids low doesgroups’ pharmacological serum．The Qiji shenkang granule total flavonoids high does group was treated by30g·kg-1·d ofQiji shenkang granule total flavonoids and Qiji shenkang granule total flavonoids low doesgroup by15g·kg-1·d of Qiji shenkang granule total flavonoids. The normal group was givennormal saline1ml/100g (weight) once daily for five days.1hour after the last administration,blood was taken from the abdominal aorta. Blood using a centrifuge, condition is1500rpmcentrifugation for5minutes, taking supernatant. After56℃water bath,30min inactivation.By filter sterilization of0.22μ m,-20℃preservation reserve.4. The angiotensin II as a stimulating factor, rat glomerular mesangial cells in vitro, make itsproduce extracellular matrix accumulation.The glomerular mesangial cells were cultured in25cm2culture flask, with mediumcontained10%FBS DMEM. The cells were incubated at37℃in humidified5%CO2-95%air. The cell medium was left untouched for2-3days and changed every other day untilconfluence. Glomerulus mesangial cell were grown to80%-90%confluence, washed oncewith serum-free DMEM.According to the concentration of2x104cells/hole, MCs cells was seeded in24well plates, each set of5hole. Cells were made arrested in0.5%FBS DMEM for24h to synchronize the cell growth. After this time period, the media was changed to DMEMcontaining10%pharmacological serum for24h、48h、72h, Cells were collected at differenttime points to be measured。5.24hour urinary protein quantitative was determined by end-point method。After the last administration, rats were placed in metabolic cages with single cagefeeding (fasting, but the water), collected24hours urine of rats in metabolic cage.24hourslater, accurate records of each rat urinary total24hours of content, and collected the urine5ml, for determination of urine red blood cell count and24hour urinary protein quantitative.Experimental conditions: Temperature:37°C, wavelength:600nm, optical diameter:1.0cmthe absorbance range to:0-2A, reaction time:5min Sample: Reagents=1:604.6. Pathological evaluation of renal tissueThe renal lesions using20%formalin-fixed, paraffin embedded sections, and throughHE, to object pathological changes of IgA nephropathy.7. Immunofluorescence stainingA direct method for dyeing, and renal cortex of rats with routine frozen sections of3μ m,with a hair dryer drying, sections by acetone fixed5min, PBS liquid rinse3times, each time5min, drop of sheep anti-mouse immune fluorescence IgA, IgM and C31:10dilutionfluorescence labeling (labeled with fluorescein isothiocyanate.) antibody in tissue section, thetemperature of37℃incubating45min, remove the slices in PBS solution and wash3times,each time5min, remove the slices with buffer glycerol.8. The secretion of fibronectin、collagen type Ⅳand the expression of mitogen activatedprotein kinase、nuclear factor-kappa b were 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 respectively controlled holewithout samples, affinity,and standard reagents, the rest of the enzyme each step for the sameoperation）, sample hole. The enzymes standard bag was board to be added sample and then40μl add meat10μl Sample billogical. And when the Sample billogical and in a sample ofenzyme panels at the bottom of the hole, try not to touch the hole wall, gently shaking blending. Incubate: After closing plate with Closure plate membrane, incubate for30min at37℃.（2）Configurate liquid:30-fold wash solution diluted30-fold with distilled water andreserve. washing: Uncover Closure plate membrane dry by swing, add washing buffer toevery well, still30s then drain, repeat5times, dry by pat.（3）add enzyme: Add ELISAreagents50μl to each well, except the blank well.（4）Uncover Closure plate membrane dryby swing, add washing buffer to every well, still30s then drain, repeat5times, dry by pat（.5）color: add color reagent A50μl and color B50μl to each well. Gently mix, incubate for15min at37℃.（6）Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the blue color change to yellow color Immediately）.（7）assay: take blank well as zero,measure the optical densit（OD）at450nm after Adding Stop Solution and within15min.（8）To calculate the sample concentration according to the standard concentration and thecorresponding OD.Results：1Compared with the normal group,(the urine red blood cell count:26.30±10.64/μ L,24hour urine protein:168.38±55.71mg/24hours), the urine red blood cell count of modelgroup rats (113.57±32.5/μ L) and24hour urine protein excretion (593.51±114.01mg/24hours) was significantly higher than that in normal group, p＜0.05; The Qiji shenkang granuletotal flavonoids high, low dose and telmisartan treatment after5weeks, urine red blood cellcount of rats in all treatment groups (the high dose group48.42±22.27/μ L, the low dosegroup51.38±25.94/μ L, the telmisartan group59.15±11.01/μ L) and24hour urine proteinexcretion (the high dose group was286.46±134.33mg/24hours, the low dosage group was358.43±167.79mg/24hours, the telmisartan group263.91±126.42mg/24hours) decreasedsignificantly, which has statistical difference compared with model group (p <0.05), thetreatment group showed no statistical difference. The results suggest that, Qiji Shenkanggranule total flavonoids can effectively improve the rat IgA nephropathy hematuria andproteinuria symptoms.2. Pathological observation: normal group kidney glomerular structure normal, model groupshowed glomerular swelling, enlargement and severe diffuse mesangial proliferation,glomerular staining showed some lobulated, mesangial cells, mesangial matrix increased. Total flavonoids of Qiji Shenkang granula high dose group mesangial proliferative lighter.Total flavonoids of Qiji Shenkang granule low dose group glomerular swelling, moderatemesangial proliferation. Telmisartan group glomerular swelling and lighter, with moderate,mild mesangial proliferation.3. Immunofluorescence examination showed: normal group glomerular mesangial areas IgA,IgG, IgM and C3were negative. The model group rats were all glomerular IgA deposition, itsstrength is+++, mainly in the mesangium granular deposition, the individual also affectedthe capillary wall and associated with IgG, IgM and C3deposition. Each IgA depositiondecreased fluorescence intensity of treatment in different degree, especially the totalflavonoids of Qiji Shenkang granule effect of high dose group was more significant, part ofthe IgA fluorescence intensity in±~-.4. The normal rat glomerular mesangial cells can secrete a small amount of collagen type IVand fibronectin, show that under normal circumstances, glomerular mesangial cells in vitrocan secrete collagen type IV and fibronectin. After the angiotensin II stimulation, the ratglomerular mesangial cells secretion of collagen IV and fibronectin increased significantly,about2times the normal group, there was significant difference compared with the normalgroup (p <0.01), suggesting that angiotensin II can obviously promote mesangial cellssecrete collagen type IV and fibronectin. At different time points, the Qiji Shenkang granuletotal flavone high, low dose group the content of collagen type IV and fibronectin weresignificantly reduced in24hours,48hours,72hours, compared with angiotensin II group，there were significant differences between them (p <0.01), and showing a certaindose-effect relationship. But the treatment group showed no statistical difference. Inhibitionof extracellular matrix accumulation effect by Qiji shenkang granule total flavonoids hasemerged from the24hours, and continued to72hours.5. Ang II activates mitogen-activated protein kinase and nuclear nuclear factor kappa b signaltransduction pathway. The contents of mitogen-activated protein kinase (3.365±0.265)andnuclear factor kappa b(3.355±0.265)compared with the normal group(mitogen-activatedprotein kinase1.805±0.065, nuclear factor kappa b1.660±0.100), there were significantdifferences (p <0.01). Given the Qiji shenkang granule total flavonoids and losartan intervention for48hours, two signal transduction pathways were inhibited in glomerulusmesangial cell in vitro. The content of mitogen-activated protein kinase(the high group2.590±0.150, the low group2.465±0.285, the losartan group2.215±0.015) and nuclearnuclear factor kappa b(the high group2.435±0.165, the low group2.450±0.160, the losartangroup1.995±0.095) were significantly decreased, at the same time content of fibronectin andcollagen type IV was significantly lower. Compare with the angiotensin II group, all of themwere statistically significant, p <0.01. Although every treatment group showed no statisticaldifference between them, but the effect of losartan group is better than the Qiji Shenkanggranule total flavonoids high and low dose group.Conclusion：1. Qiji shenkang granule total flavonoids can effectively reduce urinary red blood cell countin and the excretion of urine protein in24hours in rats with IgA nephropathy. Reduce therenal pathological changes, reduce the deposition of immune factors. Confirm the QijiShenkang granule total flavonoids have a certain therapeutic effect on experimental IgAnephropathy.2. Qiji Shenkang granule total flavonoids can inhibit Ang II induced glomerular mesangialcell extracellular matrix–fibronectin and collagen type IV expression. Through inhibitexcessive expression of extracellular matrix to delay the occurrence and development of renaldisease. The target was clear about Qiji Shenkang granule total flavonoids. Also providingexperimental data, the aim is useing Qiji Shenkang granule in clinical treatment of glomerulardisease.3. Both of the mitogen-activated protein kinase and nuclear factor kappa b two signaltransduction pathways were effectively inhibited by Qiji Shenkang granule total flavonoids,and it prevent the excessive accumulation of extracellular matrix, meanwhile reduce theglomerular injury. Play a role in renal protection. Qiji Shenkang granule total flavonoidsprevent the occurrence of glomerular disease and further development of glomerular disease. |
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