| Chapter one,Literature ResearchDiabetic Kidney Disease(DKD)is a severe complication of chronic microangiopathy,which is common in patients with advanced diabetes.It is difficult to cure and makes people helpless.It is also one of the main inducing factors leading to end-stage renal disease(ESRD).In the next decade,the number of people suffering from diabetes worldwide will increase to 439 million.More than 30%of these diabetic patients will develop into DKD,which is a major cause of severe disability and death in diabetic patients.Therefore,the research on the mechanism of diabetic nephropathy and the development of new therapeutic drugs has become a hot research in the pharmaceutical industry at home and abroad.This paper systematically summarizes the pathogenesis of diabetic nephropathy at home and abroad,including hyperglycemia,hypertension,dyslipidemia,activation of renin-angiotensin-aldosterone system,insulin resistance and oxidative stress,and describes in detail the mechanism of Salvianolic acid and tanshinone in alleviating the pathological process of diabetic nephropathy,which is expected to provide theoretical basis for further research and new drug development.Chapter two,Preparation and composition analysis of Salvianolic acid and tanshinone.(a),Based on the research on the separation and purification process of salvianolic acids,the extraction,separation and purification process previously studied by the research group was followed,and the crude extract of salvianolic acid was obtained from Salvia miltiorrhiza by the method of water extraction and alcohol precipitation;The high purity salvianolic acids were further purified by ethyl acetate extraction and macroporous adsorption resin column chromatography.The purity of salvianolic acid(Protocatechualdehyde,Affeic acid,Rosmarinic acid,lithospermic acid,Salvianolic acid,Salvianolic acid B,Salvianolic acid C)was 81.6%.(b),The purification process of tanshinone class composition has carried on the research and evaluation system,with four kinds of ingredients like tanshinone in salvia miltiorrhiza(Dihydrotanshinone,Cryptotanshinone,Tanshinone Ⅰ,Tanshinone ⅡA)of the total content of evaluation indexes,through the macroporous resin purification to determine the best purification process.The purity of tanshinone was further improved by ethyl acetate extraction,and the optimal separation and purification process was finally determined.The purity of the total tanshinone after purification was 67.33%.Chapter three,Study on compatible effect and dose-effect characteristics of Salvianolic Acid and Tanshinone based on Cell Model.(a),In this study,high glucose(GLU)was used to model human renal tubular epithelial cells(HK-2),causing cell damage.It was found that 60mg·mL-1 high glucose was the best concentration for modeling after screening.After high glucose model administration,it was found that salvianolic acid,tanshinone and their combination groups had protective effect on cells,and the medium and low doses had better protective effect on cells.Compared with the single drug group,the combination group had a stronger protective effect on GLU induced HK-2 cell injury,and the higher protective rates were mainly concentrated in the range of 10:1 and 9:1 with high compatibility ratios.In the high glucose model,the levels of inflammatory cytokines in the model group were significantly higher than those in the normal group.The abnormal inflammatory cytokines induced by high glucose in each compatibility group had a tendency to return to the normal group.In the inflammatory cytokines IL-6,TNF-α and MCP-1,the ratio of compatibility group with obvious down-regulation effect was mainly 4:1;in the IL-1β,the ratio of compatibility group was 10:1 and 9:1,indicating the best regulation effect.(b)In this study,hydrogen peroxide was used to model human renal tubular epithelial cells(HK-2),causing oxidative damage.After the screening of modeling concentration,the optimal concentration of hydrogen oxide was 2.5 mmol·L-1.After hydrogen oxide modeling,it was found that salvianolic acid,tanshinone and their combination groups had protective effects on cells.The higher the dose of salvianolic acid,the better the protection rate of HK-2 cells.Tanshinone also has a good protective effect on it.In the compatibility group,the compatibility ratio was 4:1,3:1,2:1,and the protective effect was more obvious.In the hydrogen peroxide model,the levels of inflammatory cytokines in the model group were significantly higher than those in the normal group.In the combination administration group,the down-regulation effect of inflammatory cytokines IL-1β,IL-6,TNF-α and MCP-1 was better,mainly when the compatibility ratio was 4:1,and the effect of other ratios was not good.(c)In this experiment,HBZY-1 cells in the mesangial of rat glomerulus were modulated by high glucose,causing cell damage.It was found that high glucose concentration of 50mg-mL-1 was the best model concentration in HBZY-1 cells.After high glucose model administration,it was found that salvianolic acid,tanshinone and their combination groups had protective effects on cells.The protective effect of tanshinone was better than that of salvianolic acid,and the combination group had a significant protective effect on Glu induced damage of HBZY-1 cells.The results showed that the higher the content of salvianolic acid in the compatibility group,the better the protective effect.In the high glucose model,the levels of inflammatory cytokines in the model group were significantly higher than those in the normal group.All the compatible groups showed a tendency to regulate the inflammatory cytokines,and IL-1β,TNF-α and MCP-1 were significantly down-regulated,and the effect was obvious when the ratio was 9:1 and 4:1.However,there was no trend of IL-6 callback in all the compatibility groups.(d)In this study,hydrogen peroxide was used to model rat mesangial HBZY-1 cells,causing oxidative damage.It was found that 1.63mmol L-1 hydrogen peroxide was the best concentration for modeling.After hydrogen oxide modeling and administration,it was found that salvianolic acid,tanshinone and the combination group had protective effect on HBZY-1 cells.The protective effect of salvianolic acid on oxidative damage of HBZY-1 was enhanced with the increase of dosage.Tanshinone also has a good protective effect on mesangial cells of the glomerulus.Compatibility group can also effectively prevent oxidative damage of cells,and the compatibility ratio of 10:1,9:1,6:1 and 4:1 is more obvious.In the hydrogen peroxide model,the comparison of the levels of inflammatory factors can be found that the model group was significantly higher than the normal group.The proportions of significant down-regulation effects of inflammatory cytokines IL-1β,IL-6,TNF-α and MCP-1 in each combination group were mainly 10:1,9:1,5:1,4:1 and 3:1,while the other proportions were generally regulated.This result is similar to the trend of cell protection rate.Chapter four,Study on the compatible effect and dose-effect characteristics of Salvianolic acid and tanshinone based on animal model.(a),Rats with chronic kidney injury model were induced by low dose STZ combined with diet,and irberartan(IRB),metformin(MEL),salvianolic acid(SAL),tanshinone(TAN),and different compatibility ratios of salvianolic acid and tanshinone were 4:1(COM1)and 9:1(COM2)for drug intervention.The results of pathological section showed that the pathological conditions of renal injury in diabetic nephropathy rats were improved to a certain extent in each administration group,and the pathological conditions of renal injury in MEL group,COM1 group and COM2 group were significantly improved.24h urine protein test results showed that IRB,COMI,COM2 had obvious callback effect;As for biochemical indexes,such as serum creatinine SCR,urea nitrogen BUN,triglyceride TG,total cholesterol TC,glutamic-alanine transaminase AST,glutamic-oxalacetic transaminase ALT,etc.,it was found that each administration group had a downward trend,among which MEL and COM1 had significant therapeutic effects.The results of TNF-α,IL-1β,IL-6 and MCP-1 in renal tissue showed that IRB,Sal and Com2 had significant down-regulation effect.Based on the pathological section,biochemical indexes,24h urine protein results and inflammatory factors,as well as the activity screening results at the previous cell level,the optimal compatibility ratio of the two components was determined to be 4:1,and the combined use of salvianolic acid and tanshinone was better than that of salvianolic acid and tanshinone alone.(b),In this study,the metabolic profile characteristics of urine and plasma of rats with diabetic nephropathy were explored through comprehensive analysis and evaluation of various variables using UPLC-Q-TOF-MS and statistical methods.The biological characteristics and action mechanism of salvianolic acid,tanshinone and their compatibility groups were also studied.The results showed that 23 potential biomarkers were identified in serum and 22 potential biomarkers were identified in urine,including metabolism of taurine and taurine,phenylalanine,linoleic acid,arachidonic acid and other metabolic pathways.The PLS-DA diagram of each group showed great differences,and the normal group,model group,positive drug group,salvianolic acid,tanshinone and its compatibility group could be clearly distinguished.All the drug administration groups returned to normal state.(c),The intestinal flora diversity in the colon contents of diabetic nephropathy rats was analyzed by 16SR DNA technique.The results showed that compared with the blank group,the intestinal flora in the model group was significantly different at the phylum level.At the phylum level,the abundance of Firmicutes and Bacteroidetes in model group decreased.Abundance increased in Actinobacteria,Proteobacteria,and Microflora verrucosa.Among the families classified,Bifidobacteriaceae,Enterobacteriaceae and Erysipelotrichaceae showed an upward trend in the diabetic nephropathy model group,while Lactobacillaceae,Lachnospiraceae and Prevotellaceae showed a downward trend in the model group.The results showed that intestinal flora of diabetic nephropathy mice was dysregulated to a certain extent. |
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