| Purpose:1.Based on the activating blood know luo of traditional Chinese medicine and traditional Chinese medicine compound Qi Ji Shenkang Jiawei decoction’s mechanism of human glomerular mesangial cells.This research adopts the angiotensin Ⅱ as a stimulating factor,induction of in vitro cultured human glomerular mesangial cell proliferation and extracellular matrix proliferation,by using the method of serum pharmacology,Choose the different point in time,By observing Qi Ji Shenkang Jiawei decoction pharmacology of in vitro cultured human glomerular mesangial cell FN,Ⅳ and TGF-beta/Smad TGF-beta,Smad2 in signal transduction pathways,the influence of Smad3 and Smad7 targets,to explore Qi Ji Shenkang Jiawei decoction treatment of the mechanism of action of glomerular disease;2.Based on the method of activating blood know luo,Select the prince of Qi Ji Shenkang Jiawei decoction which is the medicine of salvia miltiorrhiza,on behalf of the role of blood,interference induced by AngⅡ human glomerular mesangial cells,to observe the cell proliferation at different time points,choosing the cell activity of stability observation time point of the TGF-beta/Smad TGF-beta,Smad2 in signal transduction pathways,the influence of Smad3 and Smad7 targets,to explore the mechanism of action of salvia miltiorrhiza treatment of glomerular disease;3.Based on the method of activating blood know luo,selection of the maker of Qi Ji Shenkang Jiawei decoction medicine pepper stem,for tongluo by representatives of the medicine,interference induced by angiotensin Ⅱ human glomerular mesangial cells,to observe the cell proliferation at different time points,choose cell activity of stability time points observed TGF-beta/Smad TGF-beta,Smad2 in signal transduction pathways,the influence of Smad3 and Smad7 targets,to study the effect of wind cane treatment of glomerular disease mechanism.Material and method:1 Material1.1 Experimental animals SPF SD rats,liaoning immortality biological technology co.,LTD.,license: SCXK(liao),30,2015-0001,only body quality of 180-220 g,male and female half and half.1.2 The cells :cell lines are glomerular mesangial cell lines,guangzhou jeannie biological technology co.,LTD.,the Catalog website article number # 4200.1.3 Chinese traditional medicine: astragalus,field thistle,salvia miltiorrhiza,spreading hedyotis herb etc.Nine Chinese medicine yinpian provided by liaoning university of traditional Chinese medicine affiliated hospital pharmacy department.1.4 Main instruments and reagents: FC enzyme standard instrument(Thermo)angiotensin Ⅱ(Ang Ⅱ),Wako,batch number 014-18211;The calf serum(FBS),GIBICO company,batch number,1108862;RPMI-1640 culture,Hy Clone company,batch number ABB212915;Prednisone piece,its group rongsheng pharmaceutical co.,LTD.,batch number,16032631;ELISA kit: all by AMEKO company purchases,TGF-beta ELISA kit(AE98029Hu)96 t,96 t ELISA kit(AE90205Hu)FN,Col Ⅵ 96 t ELISA kit(AE90348Hu).Rabbit polyclonal antibody against Smad2 Abclonal company,batch number,respectively: A7699.Rabbit polyclonal antibody against Smad3 Abclonal company,batch number,respectively: A7536.Rat polyclonal antibody against beta actin,Proteintech company,batch number: 60008-1-1 g.Injection with salvia miltiorrhiza polyphenols acid salt,Shanghai green valley pharmaceutical co.,LTD.,batch number: 16070123;Pepper stem granules,jiangyin pharmaceutical co.,LTD.,batch number: 1604053.2 method2.1 Cell culture: Human glomerular mesangial cells,training in 1 to 2 days replacement RPMI-1640 culture medium containing 10% FBS(completely culture),the cell vaccination in 75 cm2 culture flasks.Stay in logarithmic growth phase cells,cell culture bottle cover the bottom 80%,suck out the original culture,with PBS buffer twice,add 2 to 3 m L of trypsin digestion,placed in 37 ℃ 0.5% CO 2incubator culture digest 4-5 min,the microscopic observation cells,to narrow cell edge,basic from the bottom of the bottle,add completely culture 14 m L termination of digestion.Through a straw repeated gently blowing bottle wall cells absorb the culture medium,to ensure that the bottle full blown.When the cells from cell suspension liquid is formed after bottle,collected in 50 m L centrifuge tube,1800 r/min after centrifuge for 10 min,suck out the supernatant,join culture completely,made the density is 1.0 x 105 / m L cell suspension liquid,set aside.2.2 Pharmacological serum preparation:30 SD rats,the male and female half,body quality of 180 ~ 220 g in weight were randomly divided into 5 groups,each group of 6,respectively,the normal group,prednisone group,stilbene thistle renal flavored decoction high,medium and low concentration group.Rats(mass)1 m L / 100 g/day to fill the stomach,lavage for five days in a row,the last delivery 1 h after abdominal aortic blood collection and separation of serum,the same set of medicated serum of rats together,set aside.2.2.1 Specific drug delivery method(experimental group)as follows: Normal group given saline 1 ml / 100 g(mass)lavage once a day.Stilbene thistle renal flavored decoction high concentration group According to the content of 47.2 g/kg/d(4 times the dose of rats with equivalent)to fill the stomach,1 times a day;Stilbene thistle renal flavored concentration groups according to the content of 23.6 g/kg/d(2 times the equivalent dose in rats lavage,1 times a day;Stilbene thistle renal flavored low concentration group According to the content of 11.8 g/kg/d(rat and equivalent dose)to fill the stomach,1 times a day.(stilbene thistle renal flavored by liaoning university hospital of traditional Chinese medicine decoction tisanes room preparation)prednisone group According to the adult 1.5 mg/kg/d,up to 60 mg/d,or large amount of 6.25 mg/kg/d rat poison to fill the stomach,once a day.2.2.2 Qi Ji Shenkang Jiawei decoction: Decocting method with reference to the state administration of traditional Chinese medicine decoction related regulations.Tisanes meet the national hygienic standards for drinking water,should be used for frying drugs should precede soak 30 min,general with decoction to water soaked in powder 2-5 cm is advisable.Using tisanes machine tisanes,boil medicine decoction after 20-30 min,boiled decoction again after 15 to 20 min.Tisanes temperature does not exceed 100 ℃ in general.2.3 determined by MTT method: On the basis of preliminary experiment results with 15% concentration of drug-containing serum and the concentration of 10-8 mol/L Ang Ⅱ experiment,1.0 x 105 / m L cell suspension liquid vaccination in 96-well culture plate,divided into normal group,model group,prednisone group,stilbene thistle renal flavored high,medium and low concentration group,each group have 3 holes.Cultivate suck out a day after the original culture,each hole to join 150 mu L RPMI-1640 culture medium containing 0.5% FBS,the synchronous hunger cells after 24 h,epispastic supernatant,each hole with 200 mu L drug-containing serum,37 ℃ 5% CO2 incubator synchronous culture(24 h,48 h,72 h)after each hole add 20 u L determined by MTT(5 mg/m L),continue to develop 4 h,termination,suction hole broth,add 150 mu L DMSO/hole,shaking table shock for 10 min at low speed,make the crystal dissolved in full,by enzyme-linked immune detector at 590 nm wavelength measure its light absorption value(OD value).2.3.1 experimental group(1)Experiment 2 groups: Normal group(completely culture),model group(completely culture + 10-8 mol/L Ang Ⅱ)and salvia miltiorrhiza phenolic acid salt high dose(completely culture + 10-8 mol/LAng Ⅱ + 200umol/L salvia miltiorrhiza polyphenols acid salt),middle dose group(completely culture + 10-8 mol/LAng Ⅱ + 20umol/L salvia miltiorrhiza polyphenols acid salt),low dose group(completely culture + 10-8 mol/LAng Ⅱ + 2umol/L salvia miltiorrhiza polyphenols acid salt).(2)Experiment 3 groups:Normal group(completely culture),model group(completely culture + 10-8 mol/L Ang Ⅱ),wind cane high dose(completely culture + 10-8 mol/L Ang Ⅱ + 10 mg/m L wind cane solution),middle dose group(completely culture + 10-8 mol/L Ang Ⅱ + 1 mg/m L wind cane solution),low dose group(completely culture + 10-8 mol/L Ang Ⅱ + 0.1 mg/m L wind cane solution).2.4 ELISA method to detect TGF-beta,FN,Col Ⅳ contains quantity:Respectively in 24 h,48h,72 h after collecting cells that,supernatant fluid by using enzyme-linked immunosorbent(ELISA),in accordance with the kit attached manual operation,each group set up 10 hole,detection of TGF-beta,FN,Col Ⅳ protein concentration.Detect the OD value,based on the standard curve to calculate the corresponding concentration.2.5 Weatern Blot:Collect 48 h point cultured glomerular mesangial cells and extract groups of total protein,using coomassie brilliant blue method determination of protein content in each sample,according to the result of quantitative protein,to join the corresponding volume of total protein sample with 5 x on the protein gel electrophoresis buffer,100 ℃ heat 5 min the protein denaturation.10% to 12% according to conventional methods of electrophoresis sds-page denaturation,plus 10 ul sample,every lane 80 v electrophoresis 30 min,90 min,100 v electrophoresis transferred to PVDF membrane,membrane membrane in the buffer 60 ma turns 90 min,TBST closed 1 h,respectively with rabbit anti Smad2,Smad3 polyclonal antibody(the latter)4 ℃ for the night,after the oscillation,TBST washing,respectively,to join the sheep fight rabbit 2 fight(1:50 00),DAB chromogenic,taking pictures.The Scion Image software analysis of grey value analysis.2.5 Real-Time-PCR: Collect treated cells,48 h time points by trizol one-step extraction cell total RNA,and determination of A260/280,purity identification of RNA.The above each cell clones were taken of total RNA,reverse transcription into c DNA,according to the reagent instructions step line of Real-time-PCR detection,with GADPH primers to contrast analysis.TGF-beta-F: 5 ’-TAC TAC GCA AGG AGG TCA-3’,TGF-beta-R: 5 ’-AGC AAC ACG GGT TCA GGT-3’.Smad7-F: 5 ’-CCA AAG GTC ATC CC-3’ ACC ACC,Smad7-R: 5 ’-CTT the CAG TTT CTT GAG CAC CGA GT-3’);GADPH-f: 5 ’-the GAA ACG GCT ACC ACA TCC-3’,GADPH-R: 5 ’ACC AGA CTT GCC CTC CA-3’.Primers by the Beijing country prosperous biological technology co.,LTD.Amplification conditions: 95 ℃ for 2 min.15 s 95 ℃,60 ℃,30 s,40 cycles;Take its cycle threshold(Ct),USES the comparative 2-△△Ctmethod TGF-beta,Smad7 gene relative contained in the cell.2.6 Statistical methods: Our data using(x±s)said,using SPSS17.0 statistical software for statistical analysis.Statistics using single factor analysis of variance between groups multiple comparison analysis(P < 0.05)for the difference was statistically significant.Results:1.MTT results:1.1 Compared with normal group,model group OD values for each time point of the model group were statistically significant(P < 0.05),and increased with the extension of time;Compared with model group,24 h Qi Ji Shenkang Jiawei decoction group and prednisone group,high and low concentration of 48 h Qi Ji Shenkang Jiawei decoction decoction high,medium and low concentration group and prednisone group were statistically significant(P < 0.05).1.2 Compared with normal group,model group time point of 24 h,48 h,72 h were statistically significant(P < 0.01),and increased with the extension of time.Salvia miltiorrhiza polyphenols acid salt group compared with model group,24 h,48 h and 72 h time point were statistically significant(P < 0.01)and salvia miltiorrhiza polyphenols acid salt each dose group,24 h,48 h,72 h had no statistical significance(P < 0.01).1.3 Compared with normal group,model group each time point of 24 h,48 h,72 h statistically significant(P < 0.01),and increased with the extension of time.Wind cane group compared with model group,24 h and 48 h time points were statistically significant(P < 0.01),72 h point only wind cane high dose group and model group with statistical significance(P < 0.01).2 ELISA results:2.1 ELISA method to detect TGF-beta content:Compared with normal group,model group each point in time(24 h,48 h,72 h)of the model group TGF-beta statistically significant(P < 0.01);Compared with model group of each point in time the drug-containing serum dosage groups were statistically significant(P < 0.01);Compared with prednisone group,24 h Qi Ji Shenkang Jiawei decoction each concentration groups were statistically significant(P < 0.05);48 h Qi Ji Shenkang Jiawei decoction low concentration group has statistical significance(P < 0.05);72 h high Qi Ji Shenkang Jiawei decoction and concentration in the group have statistical significance;High concentrations and Qi Ji Shenkang Jiawei decoction group,low concentration at each time point were statistically significant(P < 0.01).2.2 ELISA method to detect FN content: Compared with normal group,model group each point in time(24 h,48 h,72 h)of the model group FN statistically significant(P < 0.01);Compared with model group of each point in time the drug-containing serum dosage groups were statistically significant(P < 0.01);Compared with prednisone group,24 h,Qi Ji Shenkang Jiawei low concentrations in the decoction group were statistically significant(P < 0.05);48 h Qi Ji Shenkang Jiawei decoction each concentration groups were statistically significant(P < 0.05);72 h Qi Ji Shenkang Jiawei decoction high and low concentration groups have statistical significance;High concentrations and stilbene thistle renal flavored decoction group,low concentration at each time point group were statistically significant(P < 0.01).2.3 ELISA method to detect ColⅣcontent: Compared with normal group,model group each point in time(24 h,48 h,72 h)of the model group Col Ⅳ statistically significant(P < 0.01);Compared with model group of each point in time the drug-containing serum dosage groups were statistically significant(P < 0.01);Compared with prednisone group,24 h Qi Ji Shenkang Jiawei decoction each concentration groups were statistically significant(P < 0.05);48 h high Qi Ji Shenkang Jiawei decoction and concentration in the group were statistically significant(P < 0.05);72 h Qi Ji Shenkang Jiawei decoction low concentration groups have statistical significance;High concentrations and stilbene thistle renal flavored decoction group,low concentration at each time point group were statistically significant(P < 0.01).3.Western Blot results3.1 The experiment 1:Collect 48 h point cell experiments,model group compared with normal group,model group Smad2 and Smad3 statistically significant(P < 0.01);Compared with the model group the drug-containing serum dosage groups were statistically significant(P < 0.01);Compared with prednisone group,high stilbene thistle renal flavored decoction and concentration in the group were statistically significant(P < 0.01);High concentrations and stilbene thistle renal flavored decoction group,medium and low concentration group has statistical significance(P < 0.01).Compared with concentration in the group,the low concentration group has statistical significance(P < 0.01).3.2 The experiment 2:Collect 48 h point cell experiment,compared with normal group,model group Smad2 and Smad3 expression increased(P < 0.01);Salvia miltiorrhiza polyphenols acid salt group compared with model group,Smad2 and Smad3 protein expression decreased(P < 0.01);Salvia miltiorrhiza polyphenols acid salt group comparison,Smad2 protein: high,middle dose group of protein expression levels,low dose group with statistical significance(P < 0.01);Smad3 protein: each dose group is no statistical significance.3.2 The experiment 3:Collect 48 h time points of each cell experiment,compared with normal group,model group Smad2 and Smad3 expression increased(P < 0.01);Wind cane group compared with model group,Smad2 and Smad3 protein expression reduced statistically significant(P < 0.01);Wind cane between groups,Smad2 protein: wind cane high,middle dose group of protein expression levels lower in the low dose group(P < 0.01),Smad3 protein: pepper stem high dose group of the middle dose group,low dose group lowered expression levels(P < 0.01).4.Real-Time-PCR results4.1 The experiment 1:Collect 48 h time points of each cell experiment,compared with normal group,model group TGF-beta m RNA statistically significant(P < 0.01);Compared with the model group the drug-containing serum dosage groups were statistically significant(P < 0.01);Compared with prednisone group,stilbene thistle renal flavored decoction each concentration groups were statistically significant(P < 0.01);And Qi Ji Shenkang Jiawei decoction decoction high concentration group,medium and low concentration has statistical significance(P < 0.01);Compared with concentration in the group,the low concentration group has statistical significance(P < 0.01).Compared with normal group,model group Smad7 m RNA statistically significant(P < 0.01);Compared with the model group the drug-containing serum dosage groups were statistically significant(P < 0.01);Compared with prednisone group,high Qi Ji Shenkang Jiawei decoction decoction and concentration in the group were statistically significant(P < 0.01);High concentrations and Qi Ji Shenkang Jiawei decoction group,medium and low concentration were statistically significant(P < 0.01);Compared with concentration in the group,the low concentration group has statistical significance(P < 0.01).4.2 The experiment 2:Choose 48 h point in time the cells were collected for experiment,TGF-beta m RNA expression in relatively: compared with normal group,model group with statistical significance(P < 0.01);Salvia miltiorrhiza polyphenols acid salt group compared with model group,with statistical significance(P < 0.01);Smad7 m RNA expression in relatively: compared with normal group,model group with statistical significance(P < 0.01);Salvia miltiorrhiza dove acid salt group compared with model group,with statistical significance(P < 0.01)4.3 The experiment 3:Gather 48 h point groups of cells,TGF-beta m RNA expression in relatively: compared with normal group,model group with statistical significance(P < 0.01);Wind cane group compared with model group,with statistical significance(P < 0.01);Smad7 m RNA expression in relatively: compared with normal group,model group with statistical significance(P < 0.01);Wind cane group compared with model group,with statistical significance(P < 0.01)Conclusion:1.Angiotensin Ⅱ can induce human glomerular mesangial cell proliferation and extracellular matrix.2.Prednisone can effectively restrain human glomerular mesangial cell proliferation and extracellular matrix,and inhibit the TGF-beta/Smad signaling pathway conduction.3.Qi Ji Shenkang Jiawei decoction can inhibit proliferation of glomerular mesangial cells and extracellular,have kind of hormone kind function,may inhibit TGF-beta/Smad signaling pathways in various targets,therefore the glomerular sclerosis.4.Qi Ji Shenkang Jiawei decoction has the hormone kind function of flavored decoction showed a trend of strengthening as the dose increases.5.Salvia miltiorrhiza polyphenols acid salt can inhibit glomerular mesangial cell proliferation,through the role in the TGF-beta/Smad each target signal transduction pathway,thereby inhibit glomerular sclerosis.6.Wind cane can inhibit glomerular mesangial cell proliferation,through the role in the TGF-beta/Smad each target signal transduction pathway,thereby inhibit glomerular sclerosis. |
PDF Full Text Request