| Malnutrition is chronic renal failure(CRF)of the common clinical complications.Although renal replacement therapy was developed,but the mortality rate of end-stage renal disease(ESRD)patients remain very high,Malnutrition plays an important role among factors of death.According to the statistics,in group of patients with ESRD,malnutrition rate is about 20%to 50%.malnutrition can lead to patients immune dysfunction,anemia,frequent infection,even multiple organ failure,which it not only affects the prognosis of patients with CRF,but also is related to its higher case fatality rate.With increasingly the dialysis technology improvement and perfection,maintenance dialysis patients increases,also the incidence of malnutrition in CRF have further increased.At present,on the clinic we commonly used alpha keto-acid(trade name:KT)treatment,but its price is expensive,difficult to universal application.Other treatments such as albumin,energy mixture,water-soluble vitamin given by intravenous drip,Recombinant growth hormone injections,etc,its curative effect is short and limited,and can't improve patients with CRF protein synthesis essentially,so the use is limited.Traditional Chinese herbal compound has the role of improving malnutrition in CRF to varying degrees,but it is not convenient to take,curative effects are not unified and quality difficult to control.But so far,there is no new drugs of Chinese medicine treating malnutrition in CRF domestic.Therefore,developing an effective drug to prevent and treat malnutrition in CRF,is very important to social value and significance.So,its potential market and economic benefits are considerable.The mechanism of malnutrition in CRF,mainly includes the following aspects:1.The nutrients intake reduced;2.Metabolic and endocrine disorders:because of uremic toxins,metabolic acidosis,endocrine is disorder,inflammation and lipid metabolic disorder caused by increased protein decomposition and synthesis to reduce;3.CRF anemia:4.Lost in dialysis,protein and amino acids.In recent years,studies have found that a variety of reasons lead to abnormal protein decomposition is the dominant factor of malnutrition.Especially micro inflammation state,the body start to ubiquitin proteasome pathway(ubiquitin-proteasome pathway,UPP)involved in metabolic pathway of protein and energy for us to further explore the mechanism of malnutrition,and seek for effective intervention measures to provide a new train of thought.Protein ubiquitin ligase E3 is the key enzyme of the pathway.The regulation of this enzyme MuRF-1(muscle RING finger 1),and Atrogin-1(Atrophy gene 1)two important genes,metabolism of the body with specificity in decomposition expression of the various diseases caused by malnutrition and gene expression in skeletal muscle Atrophy is associated with Atrogin-1.But Atrogin-1 and MuRF-1 signaling pathway in the mechanism of malnutrition in CRF in chronic renal failure in is unclear.So the E3 gene pathway is expected to solve the mechanism bottlenecks problems in treatment of malnutrition in CRF.As our motherland medicine thinks,the pathogenesis of malnutrition in CRF belongs to the spleen.and renal failure,qi and blood deficiency,turbid poison resistance.If bennifiting qi and nourishing blood only,it will help evil hinder the spleen,closing the door for the bandit evil of the disadvantages,if we only expel stasis and decrease turbidity toxin,it will weak vital qi and injury spleen,so we must cooperate decreasing turbidity toxin and Detoxifying with benifiting qi and tomifying blood by tonification and purgation in combination,to embody the function of tonifying qi-blood and clearing entrails and decreasing turbidity toxin.Guided by this principle,professor wei according to clinical experience for many years in ginseng tonic soup based on the formula screening,to develop new drugs of Chinese medicine treating malnutrition in CRF a renal failure capsules,nutrition by astragalus,atractylodes,angelica LiuWei etc of traditional Chinese medicine,benefit qi and blood,through inner parts material.The party has obtained national invention patent and two national natural science fund project funding.Preliminary clinical observation and the animal experiments have showed that it could improve malnutrition in CRF very well.But its therapeutic effect on protein metabolism level of the molecular mechanisms are unclear,needs further research.Therefore,this topic were done in early part of the animal experiment,through the body set up 5/6 nephrectomy malnutrition in CRF rats model and in vitro TNF-a construct malnutrition model and L6 skeletal muscle cells E3 gene silence(MuRF-land Atrogin-1)model,to explore the E3 ubiquitin gene regulation proteosome pathway system(ubiquitin proteasome system,UPS)the role of signaling pathways and serum pharmacology method proved SSYY capsule on ubiquitin proteasome pathway.To study SSYY capsule protein malnutrition in CRF rats in vivo E3 gene pathways.E3 gene signaling pathway in high malnutrition in CRF protein decomposition function and decreasing nutrient capsule renal failure mechanism of malnutrition in CRF protein decomposition.To provide reliable experimental basis for clinical application and development of new drug.Specific as follows:Chapter I Effects of SSYY capsule improving nutritional status on CRF rats with malnutritionObjective:To observe the effect of SSYY capsule in the treatment of malnutrition of CRF rats protection and nutrition status,in order to provide experimental basis for the mechanism of party action of drugs further studyMethods:52 male healthy SD rats of only two months,SPF,weight 180?180g,animal qualified number:SCXK guangdong 2006-0015,provided by experimental animal center,southern medical university.With reference to the literature we established malnutrition model in chronic renal failure by two steps 5/6 nephrectomy(10 for control).fed with containing 4%casein made malnutrition model.Postoperative.If body weight reduced more than 20%,ALB<30 g/L)compared with normal control group rats,it means the models are successfully established,then calculating the rate of them,The Success models were randomly divided into 3 groups according to the weigh:blank model control group;KT(KT)positive control group;Traditional Chinese medicine group(SSYY).Give the dose according to the 200 g of rats,as 70 g adult and body surface area ratio is 1/56.SSYY medicine for adult dose of 15g/d(about 85 g containing crude drugs).Into rats dose of medicine 1.4 g/kg/d(about 8 g crude drugs).KT take a dose of 11.34 g/d.Converted to rats dose of 1g/kg/d.before lavaging evryday With KT?SSYY group were in after grinding the prepared with distilled water to dissolve fresh,SSYY capsule 0.28 g/ml(1.6 g)containing crude drugs,KT 0.21 g/ml,1 ml per 200 g rats lavage,1/d,for 4 weeks continuously,continue to be low protein feed.Blank control group,model control group to the distilled water to fill the stomach.Normal control group does not impose any processing,forage standard in rats.Every group rats were free drinking water.administration intervention for 4 weeks.Respectively on the morning,before the first administration and before 1 day at the end of treatment,put the rat into metabolism cages,fast water,collecting 24-hour urine,measuring amount of urine by measuring cylinder,to the test 24 hours urine protein(24hUpro)amount.by returning a moderate amount of urine specimens,Prior to the first delivery took 24 h urine of rats,then cut its tails to take blood,separaed serum,automatic determination of blood biochemical analyzer propagated,Scr and BUN level.And fasting 12 hours after the last Drug delivery,3%pentobarbital sodium each group anesthesiaed by abdominal cavity of rats(45 mg/kg).Abdominal aorta puncture blood,to return 2 ml to EDTA blood vessels,blood routine test;The rest of the blood serum separation,automatic biochemical analyzer test ALB?Scr?BUN,etc,enzyme-linked immunosorbent assay(ELISA)method to determine the serum PA?TF?IGF-1 concentration;select a certain amount of kidney,skeletal muscle are generally observed.Fixed in 4%paraformaldehyde,block,conventional paraffin embedding,sectioning,HE staining to observe histopathological changes.under light microscopy.Results:At the end of 16 weeks of establishing models,33 rats conform to the malnutrition models caused by CRF amony 42 rats.The rate of successful models was78.57%(33/42),after models established.The fatality rate was 6.06%(2/33).Before treating,All model groups' ALB were<30 g/L,or less weight Comparison d with normal group weight reduced by more than 20%on average.The weight of each successful modules rat,have no significant difference(P>0.05)at the 4 weekend of intervention,groups of rats body weight had significant difference(F=51.065,P=000,<0.001);Various mental health treatment group rats,reaction,the colour,than before delivery as well.And model group weight did not increase,instead of a downward trend.After the treatment,the treatment group rats compared with model group,the weight gain were significantly higher than model group(P<0.05);Com-pared with KT group,SSYY rats weight gain than KT group(p<0.05);Compared with model group SSYY group of Scr and BUN were significantly reduced(P<0.05);Scr SSYY compared with before treatment group were significantly reduced(P<0.01);SSYY group and the KT group 24h Upr were significantly reduced,compared with before treatment with significant difference(P<0.05),compared with the model group had significant difference(P<0.001);SSYY?KT group,Alb?PA?TF?Hb level increased significantly,compared with the model group had significant difference(P<0.01,P<0.001),compared with before treatment with significant difference(P<0.05),SSYY and KT group IGF-1 levels increased significantly,compared with before treatment with significant difference(P<0.01,P<0.05),compared with the model group had significant difference(P<0.001).SSYY group,KT,there was no significant difference between groups(P>0.05);SSYY group,KT kidney pathological changes compared with model group all have varying degrees of ease,SSYY group is more significant.Conclusions:SSYY capsule can reduce serum BUN,Scr and 24 h Upr,increase ALB?PA?TF?Hb improve renal histopathological change in malnutrition rats of CRF,to a certain extent,alleviate the kidney pathological damage,has the treatment of malnutrition in CRF rats,protect the function of rats,improved experimental malnutrition in CRF rats nutritional status.Chapter ? Effects of SSYY capsule on CRF rats with malnutrition in E3 gene pathwaysObjective:To study the effect of high protein decomposition through E3 gene signaling pathway and the mechanism of SSYY capsule which decreased protein decomposition with malnutrition in CRFMethods:With PBS,fresh skeletal muscles in vitro were rinsed,4%paraformald-hyde fixed in neutral buffer for 24 h,apply Streptomyces avidin peroxidase links the immunohisto chemical staining(SP)method.Don't sustain rat polyclonal antibody as a resistance,Ub detection rats skeletal muscle protein expression.Take the first part of each right kidney tissue of rats,PBS is rinsed clean,after-80?freezer,around 30 mg of skeletal muscle tissue homogenate in ice bath after dissolved in 300ul pyrolysis liquid RIPA,according to the kit instruction to extract total RNA.Take the M-1.5 the total RNA MLV transcribed into cDNA,using SYBR Premix TaqTM in FTC-2000-a real-time fluorescent quantitative PCR on the real-time fluorescence quantitative polymerase chain reaction(RT-PCR),in reference to using GAPDH,detectiing the expression of Atrogin-1?MuRF-1 in skeletal muscle tissues.Results:Compared with normal group,model group Ub in the cytoplasm of skeletal muscle protein expression quantity increased significantly;Compared with model group,SSYY capsule with treatment can reduce the Ub protein expression in cell plasma,with statistical significance(P<0.01),including SSYY group the effect is more obvious.Ub protein expression in normal skeletal muscle fiber cells scattered;Model group in the nucleus,cell membrane expression increased;SSYY capsule,open group Ub nucleus,the membrane expression quantity less than model group(P<0.01).Compared with normal group,model group rats skeletal muscle,Atrogin-1?MuRF-1 mRNA expression quantity increases;Compared with model group,the group of SSYY and KT can reduce Atrogin-1?MuRF-1 mRNA expression.Conclusions:Malnutrition in CRF rats in Ub high expressed,raised Atrogin-1?MuRF-1,exacerbate malnutrition;SSYY capsule can effectively inhibit the expression of E3 gene pathway,thereby delaying malnutrition in CRF progress.Chapter ? The model construction of L6 cells malnutrition and E3 of gene silencingObjective:To construct the model of L6 cells malnutrition and E3 gene silence,study the silencie E3 gene effect on L6 cells of malnutrition,to establish the ideal of E3 gene is malnutrition in CRF treatment targets.Methods:TNF-a(0?3?7.5?15?30?60?100 ng/ml)stimulate 96h differentiated L6 cells to construct malnutrition cell model for 48 h.Using RNA interference(RNAi)technology to design 4 respectively Atrogin-1-siRNA,Murf-1-target sequence and control siRNA sequences,gradually cloning to Lenti virus(LV)core carrier FG12 in restructuring FG12 with three kinds of packaging plasmid(pRSVREV,Pmdlg/pRRE,pHCMV-G)common transfection cells to packaging,virus infection L6 cells,and differentiate into muscle tubes.Photoed by Inverted fluorescence microscope,through the qRT-PCR,and Western Blot method to detect E3 gene expression.Results:15 ng/1 TNF-a is to construct a rat L6 skeletal cells most effective concentrations of malnutrition,malnutrition in the muscle cell model was constructed successfully..Microscopic cells shrink significantly.Recombinant lentivirus vector containing the correct size,sequence fragment,can be successfully infected L6 cells of E3 gene(MuRP-1,Atrogin-1)RNAi Atrogin group of cells-ImRNA,MuRF-1 mma,Atrogin-1?MuRF-1 protein expression,cell has no obvious atrophy.RNAi L6 cells of E3 gene expression and normal L6 cells and malnutrition L6 cells have significant difference(P<0.01).Conclusions:Apply TNF-a can successfully construct L6 cells malnutrition mode.Use of RNAi can turn stabilization technology to E3 gene L6 cells(MuRF-1?Atrogin--1)gene silencing,obtain stable transfection of L6 cell lines.The E3 gene silencing can avoid malnutrition in muscle cells,E3 is malnutrition in CRF treatment ideal targets for intervention.Chapter ? Effectsof SSYY capsule on E3 gene pathway in L6 cellsObjective:To study effect of SSYY capsuleon E3 gene pathways in L6 cellsMethods:15 male SD rats,SPF,were randomly divided into 3 groups,SSYY,KT group and normal serum group,each group 5 only.After 7 days,each filling and take the corresponding drug drug serum.In vitro L6 cells in rats.The in vitro cultured L6 is divided into 5 groups:model control group,the group of normal rat serum,KT and SSYY capsule high,low dose group.In silence and Atrogin-1?MuRF-1 set.After 24 h,ELISA to detect the contents of each L6 cells cultivate nucleoprotein the NF-?B,Using Westen blot method to detect the L6 cells the Ub,and Atrogin 1?MuRF-1?FoXO3a protein expression,by RT-PCR method,observe Atrogin-1,MuRF-1 and NF-? B expression,in L6 cells each group,Results:Compared with normal serum group,the model group NF-?B content increased(P<0.01)in the L6 cell nucleus,Ub,and Atrogin-1?MuRF-1 protein expression increased(P<0.01).Compared with model group,drug-containing serum after the intervention,the NF-?B were decreased(p<0.01),and Atrogin-1?MuRF-1 protein expression decreased(p<0.01).In one of the SSYY dose group is obvious.Compared with normal serum group,the TNF-? intervention,Atrogin-1?MuRF-1 L6 cells the NF-?BmRNA gene expression,after the intervention,SSYY and KT group can reduce the TNF-? induction,Atrogin-1?MuRF-1,the NF-BmRNA gene expression.SSYY capsule medium dosage group effect is significant.Conclusions:After inducing malnutrition of L6 cells,the activity of Atrogin-1?MuRF-1 mediated pathway increases,Suggested,TNF-? can activate the pathways,and induction of protein decomposition pathways start UPS,SSYY capsule can inhibit the E3 gene mediated pathway activation in L6 cells,reduce the amount of protein catabolism,delay the malnutrition in CRF process.Conclusion of the whole paper:This study through experimental research on malnutrition modelsS in CRF rats,proved that SSYY capsule has treat malnutrition in CRF and improve skeletal muscle malnutrition,protect the renal function of rats;By observing the malnutrition in CRF rats and in vitro to construct L6 cells malnutrition and E3 model of gene silencing,gene E3 promote muscle protein catabolism,and inhibition of protein anabolism to cause muscle atrophy;E3 gene silencing can improve TNF-a caused muscle atrophy.E3 gene is effective targets which can reverse muscle wasting,the expression of gene E3(MuRF-1?Atrogin-1)mediated pathway and SSYY capsule intervention effect,It suggested that the mechanism of SSYY capsule inhibiting reduce the skeletal muscle protein decomposition,promote the synthesis,delay the metabolism of protein.may be related to the pathway activity,For the application of SSYY capsule which treat malnutrition in CRF in clinical treatment of,provided a more comprehensive and deeply molecular mechanism of the theoretical and experimental basis. |
PDF Full Text Request