| Chapter one,Literature ResearchThis chapter systematically summarizes the research progress of diabetes and its chronic complications,and provides scientific basis and theoretical guidance for understanding the pathogenesis of diabetes and its complications.The main active components in Salvia miltiorrhiza Bunge and the stems and leaves of Salvia miltiorrhiza were also summarized.The pharmacological effects of S.miltiorrhiza and the stems and leaves of S.miltiorrhiza on diabetes and its complications was analyzed,in order to providing reference for the comprehensive utilization.It can also provide scientific basis and theoretical guidance for its rational use in clinical practice.Chapter two,Preparation and chemical composition analysis for the effective parts of stems and leaves of Salvia miltiorrhizaIn this experiment,the extraction and purification process of the effective parts from stems and leaves of S.miltiorrhiza was systematically studied and comprehensively evaluated.The total content of phenolic acids and flavonoids in the stems and leaves of S.miltiorrhiza and yield of dry extract were selected as the evaluation indexes.The extraction method for the stems and leaves of S.miltiorrhiza and the factors of orthogonal test were investigated by single factor experiment The effects of extraction solvent,extraction time,ratio of material to liquid and extraction times were investigated by orthogonal test,in order to obtained the optimum condition.Then,the macroporous adsorption resin was used to purify the crude extract of the stems and leaves of Salvia miltiorrhiza.The effect of resin type,sample concentration,the sampling amount,eluent concentration and eluent dosage for the purification of the index composition were further investigated.The purity was further improved by ethyl acetate extraction.The optimum condition for the extraction of the stems and leaves of S.miltiorrhiza is that eight times of 50%ethanol for three times reflux extraction and 1 h for each time and AB-8 macroreticular resin was selected for the purification.Optimum process was as following:The extraction process is as follows:the concentration of sample solution was 1.0 g/mL;the loading quantity of the sample was 0.15 g dried extract of per gram;the resin column chromatography was eluted with 3 BV of 40%ethanol.The extraction process is as follows:the extraction substrate concentration is 0.1 g/mL,the substrate pH value is 2,and the extraction is performed 3 times with 2 volumes of ethyl acetate.After the solvent evaporated,the total phenolic acid in the stems and leaves of S.miltiorrhiza is obtained.The extraction and purification process is stable and feasible.Under these conditions,the total purity of phenolic acids could reach 71.92%.Chapter three,Study on the mechanism of multiple components from the stems and leaves of S.miltiorrhiza on diabetic nephropathySection one.Screening from the stems and leaves of S.miltiorrhiza based on HK-2 and HBZY-1 cellIn this section,high glucose and hydrogen peroxide(H2O2)were used to induce damage of rat mesangial mesenchymal membrane cells(HBZY-1)and human renal tubular epithelial cells(HK-2).The experiment was performed to investigate the protective effects of multiple components from the stems and leaves of S.miltiorrhiza,and to screen the optimal compatibility ratio.As the results shown,salvianolic acid B performed significant protective effects on oxidative damage of HK-2 and HBZY-1 cell.Rutin has protective effect on H2O2-induced HBZY-1 cell injury,and isoquercitrin has certain protection against H2O2-induced HK-2 cell injury.But the effect was no reversal trend in the high glucose induced injury model.Danshensu,astragalin and tanshinones did not show protective activity.In the subsequent monomer compatibility ratio screening experiment,five monomers were selected,including salvianolic acid B,rosmarinic acid,lithospermic acid,Danshensu,and tanshinone ⅡIA.These five monomers will be combined with ratio of root(molar mass ratio,1:0.07:0.04:0.07:0.05),ratio of stem-leaf(molar mass ratio,1:1.16:0.02:0.11:0.05)and ratio of self-selected(molar mass ratio,1:1:1:1:1).The results showed that the compatibility of five different monomers in different proportions had a great influence on the cell viability.Five monomers combined with ratio of root(VG),three monomers combined with ratio of stem-leaf(TJ),three monomers combined with ratio of self-selected(TZ)and four monomers combined with ratio of stem-leaf(FJ)have shown good activity in HBZY-1 and HK-2 cell injury models induced by high glucose and H2O2.These results could provide evidence for subsequent activity experiments in vivoSection two.Study on pharmacodynamic effects of multiple components from the stems and leaves of S.miltiorrhiza on diabetic nephropathy in db/db miceIn this section,spontaneous diabetic db/db mice were used as experimental animal models,and multiple components from the stems and leaves of S.miltiorrhiza were used for intervention treatment.The biochemical indicators and pathological changes of renal tissue and liver were analyzed.The experimental results showed that multiple groups can change the pathological state.Some drug-administered groups can significantly adjust the pathological changes of biochemical indicators in vivo.Comprehensively evaluated the biochemical indicators and pathological section results,and combined with previous cell-level activity screening results,we can initially determine the optimal compatibility ratio among five monomers:three monomers combined with ratio of stem-leaf(TJ)and five monomers combined with ratio of root(VG).The high-dose group of TJ and VG can significantly regulate the biochemical indicators,blood glucose and plasma lipid levels.At the same time,TJ and VG group could prevent the damage of HBZY-1 and HK-2 cell injury models induced by high glucose and H2O2.The results indicated that TJ and VG group had good therapeutic effect both in vivo and in vitro.Section three.Regulatory effect of multiple components from the stems and leaves of S.miltiorrhiza on intestinal flora diversity in db/db miceIn this section,16Sr DNA technology was used to analyze the diversity of intestinal flora in feces and cecal contents samples from db/db mice,which could show the intervention effect of multiple components from the stems and leaves of S.miltiorrhiza.The experimental results in this section showed that compared with the cotrol group,intestinal flora of the model group had significant differences from phylum units to the genus units.The relative abundance of Lachnoclostridium,Rikenellaceae_RC9_gut_group,Eubacterium nodatum group,Firmicutes,Christensenellaceae,Lachnospiraceae UCG-001,Coriobacteriaceae,Bacteroidales S24-7 group,Lactobacillus,Ruminococcaceae_UCG-013 and Eubacterium_coprostanoligenes group in the model group were significantly changed.This result indicated that a certain degree of intestinal flora imbalance occurred in the mice with diabetic nephropathy,and the multiple components from the stems and leaves of S.miltiorrhiza could adjust the imbalance of intestinal flora.Three monomers combined with ratio of stem-leaf(TJ)group and five monomers combined with ratio of root(VG)group showed good therapeutic effect,which can further prove that TJ and VG might be the optimal ratio of multiple componentsSection four.Regulation mechanism of multiple components from the stems and leaves of S.miltiorrhiza of diabetic nephropathy based on TGF-β/Smad signaling pathwayIn this section,high glucose was used to induce damage of human renal tubular epithelial cells(HK-2)in vitro as the model of oxidative stress,and multiple components from the stems and leaves of S.miltiorrhiza were operated for intervention.The expression of cytokines in supernatant was determined by ELISA.Western blotting and real-time PCR was used to detect the expression of related proteins and mRNAs in the cell.Also,the content of related proteins in kidney tissues was detected by Waitern blotting.The results showed that the multi-components can improve the level of oxidative stress,the content of related proteins from TGF-β/smad signaling pathway and the expression of mRNA from TGF-β/smad signaling pathway,thereby preventing the damage of HK-2 cell injury models induced by high glucose and improving renal tissue damageChapter four,Study on the effect of multiple components from the stems and leaves of S.miltiorrhiza in diabetic nephropathy based on metabolomics,transcriptomics and proteomicsSection one.Study on the effect of multiple components from the stems and leaves of S.miltiorrhiza in diabetic nephropathy based on metabolomicsIn this section,we used metabolomics technique to detect endogenous metabolites in serum and urine samples from diabetic nephropathy mice.The multiple components from the stems and leaves of S.miltiorrhiza were evaluated by the metabolism of molecular substances in vivo.The experimental results showed that 14 potential metabolites could be identified in the serum of diabetic mice,68 potential metabolites could be identified in urine.Endogenous metabolites in serum are mainly involved in ether lipid metabolism,biotin metabolism,steroid hormone biosynthesis and sphingolipid metabolism.Endogenous metabolites in urine are mainly involved in ubiquinone and other terpenoid-quinone biosynthesis and D-glutamine and D-glutamate metabolism.They could both involved in steroid hormone biosynthesis and sphingolipid metabolism.Section two.Study on the effect of multiple components from the stems and leaves of S.miltiorrhiza in diabetic nephropathy based on transcriptomicsAccording to the results of previous activity studies in vivo and in vitro,in this section,we selected five groups of experimental samples:blank group,model group,positive drug(metformin)group,three monomers combined with ratio of stem-leaf(TJ)group and five monomers combined with ratio of root(VG)group,to perform transcriptomics experiments,in order to elucidate the effect of multiple components on diabetic nephropathy from and to explore its molecular mechanism.After combining the results of WGCNA analysis,we ultimately selected eleven candidate genes:Egr1,Foxo3,Pik3r3,Fgfl,Sost,Wnt10a,Tgif2,Akt2,Meplb,Collal and Apoe These candidate genes can involve multiple signaling pathways in diabetic nephropathySection three.Study on the effect of multiple components from the stems and leaves of S.miltiorrhiza in diabetic nephropathy based on proteomicsIn this section,we selected five groups of experimental samples:blank group,model group,positive drug(metformin)group,three monomers combined with ratio of stem-leaf(TJ)group and five monomers combined with ratio of root(VG)group,to perform proteomics experiments.The samples were subjected to high-throughput proteomic assay to elucidate the effect of multiple components from the stems and leaves of S.miltiorrhiza on diabetic nephropathy.The experimental results showed that 539 significant differential proteins could be screened after comparison between the control group and the model group.After the biochemical analysis(GO enrichment analysis,KEGG enrichment analysis,PPI analysis)of the differential proteins,they were found to be mainly involved in oxidative phosphorylation and fatty acid degradation.At the same time,differential proteins were identified based on the analysis of differentially expressed genes in pre-transcriptome and WGCNA.We ultimately selected seven candidate proteins:P61148,Q80XU3,Q91W43,Q61847,P19324,P24472 and P52480Section four.Study on the multi-omics joint analysis and mechanism of multiple components from the stems and leaves of S.miltiorrhiza on diabetic nephropathyIn this section,we combined the data of metabolism,transcriptomics and proteomics to explore the mechanisms of multiple components in diabetic nephropathy.The results showed that 12 genes were both up-regulated in the proteome and transcriptome;163 genes were up-regulated only in the proteome;20 genes were up-regulated only in the transcriptome;49 genes were both down-regulated in the proteome and transcriptome;310 genes were down-regulated only in the proteome;and 16 genes were down-regulated only in the genome.When the data of metabolomics,transcriptomics,and proteomics were combined,the mainly involved biological pathways were steroid metabolism,lipid metabolism,cellular response to glucose stimulus,steroid binding,linoleic acid metabolism and arachidonic acid metabolism.In coalition analysis,the mainly involved biological pathways included steroid metabolism,ABC transporters and choline metabolism in cancer.The results of previous experiments could show that the multiple components can improve the endogenous metabolites and the genes that associated with the biological pathway.Section five.Molecular mechanism verification of multi-components of the stems and leaves of S.miltiorrhiza to improve diabetic nephropathyIn this chapter,the candidate genes selected in the previous transcriptomics were further verified by real-time PCR and the possible molecular mechanisms were speculated.The experimental results showed that,compared with the model group,the difference folds of other candidate genes except Akt2 gene were significant;the P-Value of Foxo3,Pik3r3,Fgfl,WntlOa and Meplb genes was significant.After being regulated by multi-component drugs from stems and leaves of S.miltiorrhiza,the differential candidate genes in the model mice have changed.The differential multiple value and P-value were used as screening criteria.Metformin and multi-component drugs from stems and leaves of S.miltiorrhiza may regulate different molecular pathways to treat diabetic nephropathy.The positive drug group could significantly change the Foxo3,Fgfl,Sost,Akt2 and Meplb genes.Three monomers combined with ratio of stem-leaf(TJ)group and five monomers combined with ratio of root(VG)could significantly regulate the Egrl,Pik3r3 and Collal genes.Its regulatory gene is the key gene in the PI3K/Akt/FoxO signaling pathway. |
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