Improvement Effect And Mechanism Of Mulberry Leaf Active Components On Diabetes And Liver And Kidney Injury Complicated

This dissertation was divided into four chapters.Chapter one,Literature StudySection one,This paper traced the historical origin of mulberry leaf as medicinal materials and the main application directions in ancient times through textual research of herbal medicine;collected the growth sites and environmental information of 15 species and 4 varneties of mulberry plants in China to show the distribution of mulberry leaf resources.The main chemical constituents and pharmacological activities of mulberry leaf and the application of mulberry leaf in traditional Chinese medicine were summarized.The research progress of mulberry leaf medicinal resources was discussed comprehensively,which could provide scientific reference for further research and efficient comprehensive utilization of mulberry leaf medicinal plant resources.Section two,The pathogenesis of diabetes mainly includes insulin resistance,decreased transmembrane transport capacity of glucose,abnormal glucose receptor function and inflammatory factors.Common complications of diabetes include diabetic nephropathy,cardiovascular and cerebrovascular diseases,diabetic liver injury,retinopathy and neuropathy.Western medicine’s common treatments for diabetes include methods to promote insulin synthesis and secretion,increase insulin sensitivity,use insulin and its analogues,and slow down carbohydrate absorption.Traditional Chinese medicine treatment of diabetes often involves single Chinese medicine,Chinese medicine monomer and Chinese herbal compound preparation.This section summarizes the pathogenesis and treatment of diabetes,in order to provide evidence and reference for the treatment of diabetes and the application of traditional Chinese medicine to improve diabetes.Chapter two,Preparation and analysis of active components in mulberry leafSection one,According to the previous results of our research group,the mulberry leaf flavonoids and phenolic acids were extracted by 70%ethanol reflux and purified by AB-8 macroporous resin;the mulberry leaf alkaloids were prepared by water extraction and alcohol precipitation,and the supernatant was partially concentrated by cation exchange resin;the mulberry leaf polysaccharides were prepared by water extraction and alcohol precipitation and purified by AB-8 macroporous resin.Section two,The contents of 6 components of mulberry leaf flavonoids and phenolic acids were determined by HPLC,including neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,rutin,isoquercitrin and astragaloside.The content of neutral polysaccharides in the purified mulberry leaf polysaccharides were determined by phenol-sulfuric acid colorimetry with detection wavelength of 490 nm,and the content of acidic polysaccharides was determined by sulfuric acid-carbazole colorimetry and the detection wavelength was 512 nm.The contents of DNJ and fagomine in the purified mulberry leaf alkaloids were determined by UPLC-TQ/MS.The contents of flavonoids and phenolic acids were 3.85%,3.43%,4.38%,3.56%,1.58%and 1.02%,respectively,total flavonoids were 17.82%.The contents of neutral and acidic polysaccharides were 18.03%,0.83%,total polysaccharides were 18.86%,and the contents of DNJ and fagomine in mulberry leaf alkaloids were 27.7%and 3.78%,total alkaloids were 31.48%.These methods are simple and easy to operate,with good repeatability,precision and stability,which provide a basis for subsequent experimental research.Chapter three,Effect and mechanism of mulberry leaf multi-component on diabetes and liver and kidney injury in db/db miceSection one,In this section,self-diabetic diabetes db/db mice were used as experimental model,and mulberry leaf flavonoids and phenolic acids,alkaloids,polysaccharide components were used for interventional treatment.Biochemical indicators were detected,and liver and kidney pathological sections were analyzed.Western blot was used to analysis the insulin signal transduction pathway and TGF-β/smad signaling pathway.The results of biochemical indicators showed that fasting blood glucose,trnglyceride,total cholesterol,microalbuminuria,alanine aminotransferase,aspartate aminotransferase and insulin resistance index increased significantly in model group;liver and kidney pathological sections also showed significant pathological changes in the model group.After the drug intervention,the fasting blood glucose level and the urine microalbumin content in the mulberry leaf polysaccharide group and the alkaloid group decreased significantly,while the flavonoids and phenolic acids group and alkaloid group played a significant role in reducing the levels of alanine aminotransferase and aspartate aminotransferase.Compared with the model group,the blood lipid levels(TG,T-CHO)of the mulberry leaf treatment groups decreased,which tend to be in the normal group direction;the pathological changes of each drug-administered group were also improved.Western blot results showed that the levels of InsRβ and PI3K protein in liver tissue were significantly decreased,and the expression of IRS-1 and NF-κB protein was increased,suggesting that insulin resistance and inflammatory reaction occur in db/db mice,leading to disorder of insulin receptor signaling pathway.The drug-regulated groups had different degrees of callback,and the alkaloid group and the polysaccharide group had obvious effects,but the administration groups had no significant regulation on IRS-1 protein.The level of TGF-β1,CTGF,Smad2,Smad3 and Smad4 protein in the kidney was significantly increased.It was speculated that the TGF-β1 signaling pathway was activated and renal fibrosis occurred.Flavonoids and phenolic acids group and polysaccharides group were particularly effective in the expression of these proteins,which was superior to the alkaloid group.Section two,UPLC/QTOF-MS was used to analyze the changes of endogenous metabolites in mouse serum,combined with the evaluation of the efficacy described above to study the effect and mechanism of the three effective components in the treatment of diabetes and complications.Through metabolomics analysis,13 endogenous potential biomarkers were identified in serum,including PGH3,lysophosphatidylcholine,phospholipid choline,palmitic acid,cis-aconitic acid and other biomarkers related to diabetes.Its metabolic pathway involves ether lipid metabolism(influence value 0.36),dicarboxylic acid metabolism(influence value 0.13)and arachidonic acid metabolism(effect value 0.1).The effective components of mulberry leaf can be clearly distinguished from the model group and close to the normal group,which is more effective than the positive drug metformin.The flavonoids and phenolic acids group and alkaloids group show better improvement effects,suggested that the multiple effects multi-target action mode of mulberry leaf is different from the traditional hypoglycemic agents,which provides a scientific basis for further revealing the mechanism and advantages of mulberry leaf.Section there,The db/db mice were used to explore the mechanism of multi-component intervention of mulberry leaf from the perspective of intestinal microenvironment by 16S rDNA sequencing.The results showed that the intestinal microflora of db/db mice changed significantly from phylum level to genus level.The proportion of Firmicutes and Proteobacteria in model group decreased significantly,and the proportion of Bacteroidetes increased.The difference in species abundance distribution between model group and other groups was significant,which indicated that the community distribution was disordered in model group.After administration,the Bacteroidetes,Lachnospiraceae,Roseburia and Desulfovibrio were effectively regulated,especially in the alkaloid group.Intestinal flora function prediction shows that membrane transport,carbohydrate metabolism,amino acid metabolism,energy metabolism,nucleotide metabolism,cell processes and signaling,lipid metabolism,sugar biosynthesis and metabolic pathways are abnormal,indicating that the imbalance of the intestinal environment in db/db mice leads to the disorder of the above-mentioned related pathways in the body,which directly or indirectly promotes the development of diabetes,suggesting that the effective components of mulberry leaf have the effect of relieving the dysregulation of intestinal flora in db/db mice to improve diabetes.Chapter four,Study on biological effect and mechanism of mulberry leaf active components based on cell modelSection one,Human umbilical vein endothelial cells(HUVEC)were cultured to investigate the protective effect and mechanism of mulberry leaf on high glucose-induced model.The model was established in a culture medium containing 40 mg·ml-1 glucose.Different doses of mulberry leaf flavonoids and phenolic acids,mulberry leaf polysaccharide,rutin,chlorogenic acid and isoquercitrin were given.The positive drug was aminoguanidine.The cell viability was observed by MTT assay.The content of ET-1,VCAM-1 and MAD in supernatant was detected.The expression of phosphorylated p38,Erk1/2,c-jun and AKT was detected by Western blot.Compared with the blank group,the cell injury rate of the model group was about 50%,the content of ET-1,VCAM-1 and MAD increased,and the p38,Erkl/2 and c-jun increased,the expression of AKT is reduced.After the intervention of effective components of mulberry leaf,the survival rate of each group was significantly increased,and the concentration was correlated.The expression of each factor in the cell supernatant and the expression of each protein in the cells were also significantly regulated.The protective effect of mulberry on injured HUVEC is related to the regulation of oxidative stress,inflammatory reaction and inhibition of intercellular adhesion factor expression,which provides a reference for revealing the mechanism of mulberry leaf treatment of diabetes.Section two,Human hepatocellular carcinoma HepG2 cells were selected and insulin resistance model was established by in vitro induction of insulin.The protective effect and synergistic mechanism of mulberry leaf flavonoids and phenolic acids with mulberry leaf alkaloids on insulin resistance in HepG2 cells were investigated.The ratio of flavonoids and phenolic acids to alkaloids,chlorogenic acid to DNJ were studied successively.The ratios were set to 1:2,1:1,2:1,3:1,4:1,5:1,6:1,10:1,the results showed that the best ratio of the extract and the monomer was the 5:1 group.The dose-response relationship was studied for the ratios selected,and the equivalent line method and isoradiation analysis method were used to evaluate the effect of the combination of the two extract.The results showed that the ratio of 5:1 has a synergistic effect to insulin resistance.The content of hexokinase(HK),pyruvate kinase(PK),glucose-6-phosphate(G-6-P),triglyceride(TG),acetyl-CoA carboxylase(ACC),lipoprotein fat The enzyme(LPL)and sterol regulatory element binding protein 1C(SREBP-1C)was measured.The content of HK,PK and LPL in model group significantly lower than in blank group,and the content of G-6-P,TG,ACC and SREBP-1C higher than that.After drug intervention,the expression of related factors in cells of each group tended to control group.Overall,DNJ have a more obvious effect on the regulation of HK,G-6-P,chlorogenic acid effect is slightly better regulation effet of ACC,SREBP-1C than DNJ;and the ratio of the two is better than the effect of the single use.It is speculated that the improvement of insulin resistance by flavonoids and phenolic acids focus on the regulation of lipid metabolism,while alkaloids tend to regulate the abnormalities associated with glucose metabolism to produce an effect.