| Objective:Diabetic nephropathy(DN)is the main cause of end-stage renal disease,which have a high incidence rate,and its prevention and control is great significance.Autophagy is the self-protection mechanism of cell,and also the stabilizing mechanism of apoptosis and necrosis.In the early DN,Autophagy is renal cell protecting mechanism against damage by self,and the regulation of autophagy activity is a new target for the prevention and treatment of early DN.But there are few researches progresses at home and abroad.TCM believes that DN is Qi deficiency and blood stasis,and kidney-energy's Yin and Yang state of estrangement.To cure the disease of Qi and blood,which is beneficial to use the method(Danggui Buxue Decoction)as the foundation.There were three parts including theory,clinical,and experiment research about my study.On the basis of theoretical research,the clinical prescriptions for the treatment of early DN in medical institutions were analyzed,and it was found that the composition of Danggui Buxue Decoction including astragalus and angelica drugs have some application rules,and Pharmacological mechanism of Danggui Buxue Decoction would be researched by the animal experiments,whether it can regulate autophagy active when it treatment early DN.Comparing the prevention and control of the early DN is given priority to with tonifying qi,that make kidney essence of solid and blance the Yin and Yang,and whether intervention in the kidney tissues miR-21 target Akt/mTOR control autophagy protection mechanism of kidney are consistent.Thus revealing its effectiveness mechanism.To explore whether autophagy is the Yin and Yang and the theory of micro basis,provide ideas for TCM prevention and treatment of DN.Theoretical Discussion:1.Based on the theory of Qi and blood in traditional Chinese medicine.The method of invigorating Qi and generating blood can supplement Yang Qi and generate Yin blood,that play the role of harmonizing Yin and Yang,and promoting the self-harmony of Yin and Yang.2.Angelica nourishing blood decoction:it is the representative of nourishing Qi and giving birth to blood.The original prescription mainly treats blood deficiency and fever.3.Autophagy activity:it is a self-stabilizing mechanism to maintain the stability of the intracellular environment,which is similar to the core idea of yin-yang self-harmony theory in TCM theory.4.Based on the thinking of system biology:from clinical to experimental research is an important method of TCM disease prevention and treatment research,and an effective way to explain the scientific connotation of TCM theory.Methods:Clinical research:1.Taking the cases of diabetic nephropathy in the Department of endocrinology of Hubei Traditional Chinese Medicine Hospital in 2017 as samples,and based on the research method of the real world.Screening of prescription,the choice of diabetes as the main diagnosis,and screening merger cases of early diabetic nephropathy,which is an effective prescription.Prescription of entry and check will be carried out in accordance with the above requirement,and by the professionals of Chinese medicine into"heritage of traditional Chinese medicine auxiliary systems(V2.5)" drugs,frequency analysis,characteristics analysis and the analysis of new prescription,etc.2.Experiment research:1)Cells in vitro experiment:the preparation of normal medicated serum(I),high dose Danggui Buxue Decoction medicated serum(7g/Kg d)(II),low doses of Danggui Buxue Decoction medicated serum(1.75g/Kg d)(III),gliclazide medicated serum(2mg/Kg·d)(IV),in vitro culture of rat kidney cells,can be divided into:normal control group(group A),the high sugar group(group B),Danggui Buxue Decoction high dose group(group C),Danggui Buxue Decoction low dose group(group D)and gliclazide group(group E).Group A was treated with I drug serum and 5mmol/L glucose.Group B was treated with I drug serum and 30mmol/L glucose.Group C was treated with II drug serum and 30mmol/L glucose.Group D was treated with III drug serum 30mmol/L glucose.Group E was supplemented with IVdrug serum+30mmol/L glucose.The cells were cultured for 12h,24h,48h and 72h,respectively.MTT assay was used to detect the proliferation of podocytes in different time periods.The apoptosis of podocytes in each group was detected by flow cytometry.The morphology of autophagy was observed by transmission electron microscopy.Application Western-Bolt testing cells,Belcinl,LC3-land LC3-IIprotein expression,RT-PCR detection,Belcinl,LC3-I and LC3-? mRNA expression level.2)In vivo animal experiment:the alcohol extract of Danggui Buxue Decoction for enriching blood was prepared.After the successful modeling of early diabetic nephropathy,SD rats were randomly divided into group A:blank group;Group B:model group,group C:Danggui Buxue Decoction high-dose group[7g/(kg d)];Group D:Danggui Buxue Decoction low-dose group[1.75g/(kg d)];Group E:glitter group[2mg/(Kg·d)].Ten mice in each group were sampled after continuous gavage for 4 weeks.Blood urea nitrogen(BUN),creatinine(Cr),urinary albumin excretion rate(uAE)were detected,and the general condition and renal histopathology(PAS staining)were observed.Western Blotting was used to analyze the expression of Akt protein kinase B and total mTOR(mammalian target of rapamycin),and RT-PCR was used to detect the mRNA expression of mir-21 and Akt and mTOR.Results:1.Clinical study:Early DN clinical symptoms:fatigue,dry mouth,edema,low back acid,chills and so on are common symptoms of diabetic nephropathy.Common drugs used to treat early DN include astragalus root,angelica root,salvia miltiorrhiza root,Chinese yam,poria root,rehmannia rehmannii,etc.Angelica blood-enriching decoction is the first drug for"astragalus root-angelica root",the four Qi characteristics of drug use indicate that the temperature and cold are the main ones,the five tastes are sweet and bitter in order,and the frequency of drug use indicates spleen,liver,kidney,heart and lung in order.2,Experiment research:1)Lab 1(cells in vitro experiment):(1)The Danggui Buxue Decoction can restrain high sugar stimulate cell proliferation,compared with group B(P<0.05),while angelica blood tonic soup group C is the most significant effect of high dose group,with significant,compared with D and E group(P<0.01),and found that 48 hours proliferation situation is the most significant;(2)The cell under high sugar induced apoptosis obviously,comparing different drug intervention that angelica blood tonic soup high dose group can obviously reduce apoptosis,due to the high sugar stimulation after renal cell damage,cell apoptosis,given after incubation 48h after drug intervention,the patients renal sertoli cell apoptosis rate statistical analysis results:apoptosis rate of group A was 4.79%and 39.28%in group B;Group C was 13.66%,group D was 19.43%,and group E was 12.82%.(3)Compared with group A and group B,the glomerular balloon adhesion,renal interstitial fibrosis and the decrease of autophagosomes were observed under electron microscope.The pathological changes in group C,D and E were all higher than those in the model group,and the increase of autophagosomes was found.2)Lab 2(cells in vitro experiment):After sugar intervention of kidney podocytes autophagy standard protein expression decreased(P<0.05),including high dose group angelica blood tonic soup on the control results suggest autophagy protein markers,Belcinl,LC3-?,LC3-? increased significantly(P<0.05),which is most evident effect of group C(P<0.01).3)Lab 3(in vivo animal experiment):(1)Generally,SD rats in group A were agile and flexible,with normal diet,fur and defecation;SD rats in groups B,C,D and E started to lose activity and weight after about 7 days,gradually losing weight and losing luster,and some of them showed impaired renal function such as polydipsia,polyphagia and polyuria,which were very prominent after 3 weeks.The situation of group C,D and E was better after administration than that of group b.after administration for 1 week,animals in group C and D showed active reaction,obvious increase in food intake,shiny hair,improvement in polydipsia,and gradual weight gain.(2)In terms of biochemical indexes,the comparison between group B and group A was also statistically significant(P<0.05).There were significant differences between group C,D and E with drug intervention and group B(P<0.01).Blood urea nitrogen and creatinine showed statistically significant differences between group B and group A(P<0.05),and between group B and group A(P<0.05).(3)Renal PAS staining showed that the kidney of group A was basically normal without obvious mesangial hyperplasia or capillary degeneration.In group B,obvious mesangial stromal hyperplasia was observed in the kidney,and balloon fusion was observed locally.Compared with group A,renal lesions were obvious and all had lesions,indicating successful modeling.Obvious renal lesions were observed in groups C,D and E,presenting as glomerular tubular lesions of different severity and mild proliferation of mesangial matrix.PAS-positive area of mesangial matrix indicated that compared with group A,mesangial hyperplasia was obvious in group B(P<0.05).Compared with group B,mesangial hyperplasia was improved in groups C,D and E(P<0.05),with group C having the best effect(P<0.01).(4)According to electron microscope observation,compared with group A and group B,glomerular balloon adhesion,renal interstitial fibrosis,and decreased autophagosome were observed.The lesions in group C,D,and E were all higher than those in the model group,and the autophagosome was found to be increased,with no significant difference among the three groups.(5)Nephrin immunofluorescence staining,A group of podocytes in protein nephrin along in the glomerular capillary loops homogeneously continuous linear distribution,group B compared with group A nephrin group protein expression decreased,C,D,E group of nephrin eggs begin to increase the white expression,by continuous line into A difuse granular distribution,prompt between groups in group B compared with group A nephrin protein significantly(P<0.05),compared with group B,C,D,E group can improve the mesangium hyperplasia(P<0.05),which works best group C(P<0.01).4)Lab 4(in vivo animal experiment):(1)The expressions of Akt and mTOR were decreased,and the differences were statistically significant between group B(model group)and group D(P<0.05).Compared with group D,the expressions of Akt and mTOR in renal tissues of group C were statistically significant(P<0.05).(2)mRNA expression of mima-21,Akt and mTOR showed statistically significant differences between group B and group A(P<0.05),and significant differences between groups C,D and E(P<0.01).In terms of down-regulating the expression of AktmRNA and mTORmRNA,the difference between group B and group A was found to be statistically significant(P<0.05),and that among groups C,D and E,there were significant differences compared with group B(P<0.01).Conclusion:1,Using data analysis software,the formula in the clinical treatment of DN prescription medication rule is analyzed,and the treatment of early DN lack basic pathogenesis of deficiency of Qi stasis,"Reinforcing Qi to Promote Blood" treatment of the comprehensive advantages,especially in Danggui Buxue Decoction for Chinese traditional medicine astragalus and angelica characteristics of the drug,and it can reduce or avoid the side effects of western medicine,which is the basis for the further experiment research.2.Experimental research:1)Cells in vitro experiments suggest the sugar induced renal cell apoptosis,and renal cell damage is the pathologic basis of diabetic nephropathy,protecting renal function can start from protecting kidney podocytes,Reinforcing Qi to Promote Blood method(Danggui Buxue Decoction)the effect of medicated serum is intervention of the autophagy body to promote the formation of the renal cells of self-healing,which is also the foundation of maintain cell function.So use Danggui Buxue Decoction of sertoli cell protection mechanism in addition to the influence cell apoptosis,also associated with autophagy,high sugar stimulation Beclin-1,LC3-?expression significantly lower than normal group,prompt autophagy activity is restrained,Under normal conditions,the ability of autophagy to maintain the stability of the intracellular environment was decreased,and the increase of autophagy marker protein was found after the intervention of angelica buxue decoction,suggesting enhanced autophagy activity.To sum up,autophagy plays an important role in kidney disease,and may be involved in the occurrence and development of DN,while angelica blood-enriching decoction may prevent the decline of autophagy activity caused by high concentration of glucose,suggesting that autophagy can delay DN kidney damage.2)In vivo animal experiments suggest podocyte damage and autophagy activity decline,angelica blood tonic soup treatment after the kidney was delayed by the extent of the damage and hardening,biochemical index improved significantly,the pathologic structure changes and nephrin protein hints about intervention autophagy,based on this,the study on the biochemical indicators and pathological morphology description:tonifying qi huoxue method(Danggui Buxue Decoction)of different extracts group can obviously improve the early DN rat renal function,proteinuria and drop on angelica blood tonic soup is better than that of gliclazide;Also found thatChinese angelica blood tonic soup can reverse the inhibition of nephrin,give play to the role of reducing proteinuria,at the same time "angelica blood tonic soup" suppress kidney miR-21 expression,then control the pathological state of imbalance of Akt/mTOR signaling pathways and keep the kidney cell autophagy activity,suppress the high sugar stimulation caused by renal mesangial proliferation,have the effect of delayed renal damage,the above shows that tonifying Qi huoxue method(angelica blood tonic soup)inhibit the expression of miR-21,target Akt/mTOR signaling pathway enhances renal cell autophagy activity,this could be one of the important mechanism for the treatment of early treatment of DN,It also indicates that the method of invigorating qi and invigorating blood(angelica nourishing blood decoction)can prevent DN by regulating autophagy,and the autophagy activity may be one of the microcosmic bases of the theory of "Yin and Yang self-balance". |
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