| Objective:Application of B ultrasound method for monitoring acute on chronic liver failure(ACLF)model making,To investigate the feasibility of the method for screening of ACLF model.Selected animal model successfully by this method,further study of SHYCD high,low dose of ACLF rat survival rate,liver function,blood coagulation function,the effect of serum inflammation related factors;Effects of SHYCD for APE1/Ref 1 expression in liver cellular different location,and effects on p53 and apoptosis related proteins in the ACLF;Clarifying SHYCD inhibit the P53 regulation of apoptosis related protein in prevention and treatment of ACLF by APE1/Ref-1.Methods:1.CCL4 combine with D-GaIN and LPS establish ACLF model and Screening successful model by B ultrasound.Specific pathogen free of SD rats,200?240g(certificate No.4402102032),were purchased from Experimental Animal Center of Southern Medical University.Rats were allowed to adapt to the new environment for 1 week before experiment,Except the normal control group,the other rats inject 40%CCL4(2ml/kg.bw)peanut oil solution by abdominal cavity,2 times a week,for 10 weeks.Rats were observed activity state and recorded the death every day.From the beginning of the sixth week,Application of B ultrasonic monitoring of abdomen of rats one times a week,observed size of liver and ascites,Screening successful model by B ultrasound.Before modeling the fasted for 12 hours,Except control group,the other rats were injected 400 mg/kg D-GalN combined with 10mg/ml LPS in acute attack,copy of ACLF model,the control group were injected with equal volume of physiological saline.ad libitum access to rodent chow and water.Observe the activity state of the rat and record the circumstances of death,survival analysis.Abdominal aortic blood after 48 hour,Detection of serum AST,ALT,TBIL level,Take liver tissue pathological slices,Comprehensive analysis of ACLF model.2.preparation of the traditional Chinese medicine and grouped to treatment.Sanhuangyinchi decoction consists of rhubarb 15 g,turmeric 15 g,astragalus membranaceus 30 g,artemisia capillaris 30 g,radix paeoniae rubra 30 g(the dose for adults 1),and bought in TCM pharmacy of nanfang hospital of southern medical university,Eath dose of TCM add water 960 ml(according to the method of modern pharmacology,tisanes is 8 times the quality of traditional Chinese medicine),Soaked 30 min,High heat to a boil,slow fire decoction 40 min again,twice in a row,to slag merged,60 ? concentrated into crude drugs containing 2 g/mL concentrate,4 ?refrigerator.The paeoniflorin,emodin,chlorogenic acid,curcumin as the standard to control the quality of the SHYCD by high performance liquid chromatography(HPLC),ensure the quality of drug.-20 ? storage for later use.According to the above method Screened successful animal model,the successful models were randomly divided into 5 groups:model group,SHYCD high,middle,low dose group and Angong Niuhuang Pill group,Except the control group.SHYCD with 30g/kg crude drug concentration after intragastric administration,continuous administration for7days after the copy ACLF model,before modeling the fasted for 12 hours.Except control group,other 5 group swere injected 400 mg/kg D-GalN combined with 10mg/ml LPS in acute attac k,copy of ACLF model,the control group were injected with equal volume of physiological saline,continue to give medicine intervention model after 4 h ours,ad libitum access to rodent chow and water.Observe the activity state of therat and record the circumstances of death,survival analysis.48 hours after sacrificed all the rats,collect all kinds of specimen.3.Detection of liver function,blood ammonia,blood coagulation index.The serum level of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL)levels of rats in each groups were detected by automatic biochemical detector while the prothrombin time(PT),blood coagulation time international standard ratio(INR),plasma fibrinogen(Fbg)were detected by automatic blood coagulation detector.blood ammonia(NH3)was detected by enzyme method.4.Histological and Transmission Electron Microscopy(TEM)Examination.HE stain:The big liver lobe of the tissue was fixed in 10%formalin,embedded in paraffin,cut into 4 ?m sections,and mounted on slides,Dewaxing,hydration.The samples were stained with hematoxylin and eosin(HE)for histopathological examination.Each sample of rats to make two slices,randomly select one section,applying the Nikon Eoipse Ti-s inverted microscope at high magnification to determine five vision observation liver tissue pathology and analysis image.TEM examination,samples containing a 2 mm portion from the edge of the liver tissue were immediately fixed in 0.1 M phosphate buffer containing 2.5%glutaraldehyde and 2%paraformaldehyde for 4h.The samples were then fixed with 1%osmium tetroxide for 2 h,dehydrated through a graded ethanol series,and embedded in epoxy resin.Resin-embedded blocks were cut into 60 nm to 80 nm ultrathin sections with an ultramicrotome.The ultrathin sections were placed on carbon-coated nickel grids and examined with an H-7500 transmission electron microscope operating at 80 kV.Analyzing the change of the liver subcellular structure.5.Luminex detect serum inflammation related factor of TNF-?,IL-1,IFN-?,IL-6,IL-10.Synthesis consists of TNF-?,IL-1,IFN-?,IL-6,IL-10 factors of kit by millipore companies.Experiments according to the instructions,A:Preparation of Serum Samples;B.Preparation of Quality Controls;C.Preparation of Wash Buffer D.Preparation of Standard.1.Add 200?L of Wash Buffer into each well of the plate.Seal and mix on a plate shaker for 10 minutes at room temperature(20-25?).2.Decant Wash Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.3.Add 25 ?L of each Standard or Control into the appropriate wells.Assay Buffer should be used for 0 pg/mL standard(Background).4.Add 25 ?L of Assay Buffer to the sample wells.5.Add 25 ?L of serum Sample into the appropriate wells.6.Vortex Mixing Bottle and add 25 ?L of the Mixed or Premixed Beads to each well.(Note:During addition of Beads,shake bead bottle intermittently to avoid settling.)7.Seal the plate with a plate sealer.Wrap the plate with foil and incubate with agitation on a plate shaker overnight at 4?.8.Gently remove well contents and wash plate 2 times following instructions listed in the PLATE WASHING section.9.Add 25 ?L of Detection Antibodies into each well,Seal,cover with foil and incubate with agitation on a plate shaker for 1 hour at room temperature(20-25?).10.Add 25 ?L StreptavidinPhycoerythrin to each well containing the 25 ?L of Detection Antibodies,Seal,cover with foil and incubate with agitation on a plate shaker for 30 minutes at room temperature(20-25?).11.Gently remove well contents and wash plate 2 times following instructions listed in the PLATE WASHING section.12.Add 150 ?L of Sheath Fluid to all wells.Resuspend the beads on a plate shaker for 5 minutes,13.Run plate on Luminex 200TM,with xPONENT software.14.Save and analyze the Median Fluorescent Intensity(MFI)data using a 5-parameter logistic or spline curve-fitting method for calculating cytokine/chemokines concentrations in samples.6.Apoptosis Assay by TUNEL.Tissue sections placed 60 ? bake 1 hour,Xylene dewaxing,gradient alcohol dehydration,PBS washed 5min × 3 times.Conduct citrate microwave repair,natural cooling at room temperature.1%Triton-100 penetration 5min.3%H2O2 endogenous blocking 10min at room temperature,5%normal goat serum blocking exogenous 30min,Add the TdT enzyme reaction drops 37? avoid light incubation 60 min in a humidified chamber.PBS washed 5min × 3 times,add the working fluid DAPI stained nuclei were incubated at room temperature for 10min.PBS washed 5min x 3 times,Add 5-10 ul fluorescence decay resistance sealing seal tablets,applying the Nikon Eoipse Ti-s inverted microscope at high magnification to determine five vision observation of the apoptotic index of liver tissue.7.Western bolt detected APE1/Ref-1,p53,caspase3,bcl-2,bax protein expression50 mg liver tissue were homogenized in liquid nitrogen and dissolved in lysis buffer.Protein concentrations were determined by BCA protein quantitative assay.The protein lysates were loaded onto 10%SDS-polyacrylamide gel for separation,electrotransferred to PVDF membranes,and blocked in 5%non-fat milk in Tris-buffered saline,Membranes were incubated overnight using primary antibodies,APE1/Ref-1(1:1000),p53(1:500),caspase3(1:1000),bcl-2(1:1000),bax(1:1000)at 4?.This step was followed by secondary antibody,We performed enhanced chemiluminescence(Merck-Millipore,Germany)detection.The images were captured and documented using a CCD system by Kodak 2000MM.The expression of objective protein was optical density of objective protein ratio actin.8.qPCR detected APE1/Ref-1 mRNA expression.30mg liver tissue were homogenized in liquid nitrogen,Each sample with 1 mlTrizol cracking 1h on the ice,Trizol method of extracting total RNA,Rev erse transcriptioncDNA.QPCR reference TaqMan universal PCR Master Mix manual for operation.Based on PCR denaturing,annealing,extend the three principles,grope for good reaction conditions.According to the reaction system and samples,each sample do after three holes,then put the samples on the f luorescence quantitative instrument,according to the set procedures,using Strat egen Mx3000p software for real-time data collection and quantitative analysis.9.Immunofluorescence detected APE1/Ref-1,p53,caspase3,bcl-2,bax protein expression in situ.Tissue sections placed 60 ? bake 1 hour,Xylene dewaxing,gradient alcohol dehydration,PBS washed 5min × 3 times.Conduct citrate microwave repair,natural cooling at room temperature.1%Triton-100 penetration 5min.3%H2O2 endogenous blocking 10min at room temperature,5%normal goat serum blocking exogenous 30min,a solution of primary antibodies APEl/Ref-1(1:40),p53(1:100),caspase3(1:200),bcl-2(1:200),bax(1:200),the sections were placed in a humidified chamber and incubated overnight at 4 ?.PBS washed 5min × 3 times,add second antibody of immunofluorescence(dark),37 ? incubate 1 hour.PBS washed 5min ×3 times,dropping the working fluid DAPI stained nuclei were incubated at room temperature for 10min.PBS washed 5min x 3 times,Add 5-10 ul fluorescence decay resistance sealing seal tablets,confocal microscope observation taking pictures.10.Statistical analysisThe data were statistically analyzed by using SPSS 13.0 software,Use(x±s)for measurement data.Compared with One-Way ANOVA analysis of variance of the mean diversity,multiple comparisons between groups if they meet the homogeneity of variance test by LSD method,if the missing variance with Tamhane T2 method.Count data and rate compared with chi-square test.Correlation between the two indexes,using bivariate correlation analysis.The value of P<0.05 would mean significant difference.Result:1.The first time,B Ultrasound screening model success rate was 86.7%.Compared with control group,ALT,AST,TBIL level in serum was significantly increased(P<0.05).Liver pathological damage to 100%,which reaches 12 cases of liver failure,B Ultrasound positive and pathological morphological damage reached 11 cases of liver failure.B Ultrasound results and pathology,serology results of the correlation coefficientr r1=0.786,r2=12.The second time,B Ultrasound screening model success rate was 83.3%,control group,model group,SHYCD-H,SHYCT-M,SHYCD-L group,AGNHW group rat survival rate,respectively:100%,60%,80%,80%,70%,80%;The liver is smooth,bright.lobe is complete in the control group.liver,spleen are swelling and into nodular,Ascites is obvious,lobe is incomplete in the liver failure model.Compared with control group,the ratios which are liver,spleen weights vs body weights increased significantly3.SHYCD to slow and the prevention and control of acute liver failure dose dependent,Compared with model group,SHYCD dose dependence,high,medium and low dose could reduce the serum content of ALT,AST,TBIL,NH3,shorten the PT and INR time in whole blood,increase the content of FIB(P<0.05),Reduce TNF-?,IL-1,IFN-?,IL-6,IL-10 of the serumthe expression level(P<0.05).4.Pathological morphology observation,HE staining under normal contrast hepatic lobule structure clear,liver cells arranged orderly rules;Model group have chunk of necrosis,liver disorders,lobular structure fuzzy in liver tissue.hepatic sinus expansion and bleeding,lobules and portal area in inflammatory cells infiltration,remnant liver cell edema,degeneration,reached the standard of liver failure.SHYCD high,medium and low dose could improve liver pathological.Electron microscopy(sem),show to the results of model group liver cell line is not clear,the visible apoptotic body.Mitochondria swelling,membrane damage,crest fracture or disappear;The nucleus pycnosis is obvious.High,medium and low dose of SHYCD has different extent improve the ultrastructure of liver cell.Reduce or disappear of apoptosis body;Mitochondria swelling subsided,crest increased,The nucleus structure is complete.5.Normal control group of APE1/Ref-1 mainly expressed in the nuclei,But its expressed the nucleus and plasma total expression in the model group.Compared with model group,high,medium and low dose of SHYCD can make APE1/Refl increase in total protein andnucleus protein,decrease in cytoplasmic protein(P<0.05).Shows the results of fluorescencequantitative PCR,High,medium and low dose SHYCD can increase the expression of APE1/Ref1 mRNA(P<0.05).6.Compared with model,SHYCD can Down-regulated the p53,bax,caspase3,Cyt-C upregulated the Bcl-2 protein expression levels(P<0.05).Reduce the a poptosis index.Conclusion:1.B ultrasound examination results close correlation with pathology and serological.B ultrasound examination can be used as a living donor screening of liver cirrhosis,liver failure model is one of the diagnostic index of rats2.SHYCD high,medium and low doses has improved ACLF rat liver function,blood coagulation function,reduce serum inflammatory factors related,improve the survival rate in rats.3.High,medium and low dose of SHYCD can make APE1/Refl increase in total protein and nucleus protein,decrease in cytoplasmic protein and Up-regulated mRNA level.This may be the APE1/Ref-1 of biological macro molecular involved in liver cell DNA damage repair,and participate in the process of liver cell apoptosis.4.SHYCD can reduce p53,bax,caspase3,enhance the Bcl-2 protein,Reduce the apoptosis index.Possible mechanism is SHYCD inhibits p5 3 of apoptosis induced protein,thus suppresses p53 transcription of target molecules Bax,regulate apoptosis pathway proteins,eventually reduce liver cell apoptosis.5.SHYCD have prevention and treatment of ACLF,its possible mechanism which promote APE1/Ref 1 inhibit the expression of p53,to regulate apoptosis related proteins,finally,reach the role of prevention and treatment of ACLF. |
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