Study On The Prepartion,Characterization Of Acidic Polysaccharide From Buddleja Officinalis And Its Effect And Mechanism On Diabetic Retinopathy

Part 1 Preparation and characterization of acid polysaccharide from Buddleja officinalisObjective:Using Buddleja officinalis Maxim as raw material,applying extraction,deproteinization,and fractional purification technology to optimize the best preparation process of acidic polysaccharide from Buddleja officinalis Maxim(APBOM)and provide a basis for the preparation of APBOM.The application of analytical instrument technology to characterize the chemical composition and primary structure of APBOM provides a basis for the quality control of APBOM.Method:The ultrasonic-assisted hot water extraction method with short extraction time,which had high extraction rate and low solvent consumption,was used to extract polysaccharide from Buddleja officinalis Maxim(PBOM).And BOX-Behnken Design(BBD)response surface optimization was used to optimize the conditions of the ultrasonicassisted hot water extraction method.Then,the classic and easy-to-operate Sevag method was used to remove the protein in PBOM.The combined application of DEAE-52 cellulose anion exchange column chromatography and Sephadex G100 gel column chromatography were used to fractionate and purify PBOM.By measuring the potential and calculating the yield,the purified polysaccharide(APBOM)with relatively uniform ionic conductivity and molecular weight was finally obtained.By using the phenol-sulfuric acid method,the Coomassie brilliant blue method,the barium chloride-gelatin method,and the sulfuric acidmet-hydroxy biphenyl method to determine the total sugar,protein,sulfate group,uronic acid content,and chemical components of the purified polysaccharide(APBOM).Ultraviolet-visible spectrophotometry,Fourier infrared spectroscopy,molecular exclusion high performance liquid chromatography,and 1-phenyl-3-methyl-5-pyrazolone(PMP)column derivatization method were used to characterize the primary structure of APBOM.Results:The process conditions and results of the ultrasonic-assisted hot water extraction method optimized by BBD were as follows:the liquid-to-material ratio(mL:g)was 26:1,the ultrasonic power was 240 W,the ultrasonic time was 45 min,the ultrasonic temperature was 62℃,the extraction was repeated three times,and finally the extraction rate of PBOM was 5.39±0.16%.The DEAE-52 cellulose anion column chromatography was used to fractionate and purify PBOM.Considering the factors of electrical properties and yield,a purified component with relatively uniform ionic electrical properties was finally obtained,which was PBOM-2.Sephadex G100 gel column chromatography was used to fractionate and purify PBOM-2.Considering the yield factor,a purified component with a relatively uniform molecular weight was finally obtained,which was APBOM.The total sugar content of APBOM is 94.37±1.14%,the uronic acid content is as high as 17.41±0.35%,the sulfate group content is 1.68±0.17%,and APBOM does not contain protein.APBOM is an acidic polysaccharide,which is a heteropolysaccharide composed of glucose,galactose,fucose,glucuronic acid and galacturonic acid.The molar ratio of the above composition is 6.75:3.33:1.79:1.42:1.00.APBOM is a homogeneous polysaccharide with a relative molecular weight of 165.4 kDa.In the APBOM structure,characteristic absorption peaks were as follows:OH bond stretching vibration,CH bond stretching vibration and deformation vibration,asymmetric and symmetric vibration,C=O bond,S-O asymmetric vibration,α-glucosidic bond,S-O-S symmetric stretching vibration,etc.Conclusion:Using the BBD response surface optimization method to optimize the ultrasonic-assisted hot water extraction method,the extraction rate of PBOM was high and the extraction time was short.The combined application of DEAE-52 cellulose and Sephadex G100 column chromatography was used to obtain a purified component(APBOM)with relatively uniform ionic conductivity and molecular weight.The chemical composition and primary structure of APBOM were determined by modern technology.Part 2 The alleviation effect of acid polysaccharide from Buddleja officinalis on diabetic retinopathyObjective:To explore whether APBOM can relieve diabetic retinopathy(DR).Methods:Transgenic db/db mice were used to construct a DR model to explore the intervention effect of APBOM on DR.Randomly divided into 5 groups:normal(N,db/m mice)group,model group(DM,high-fat and high-sugar feed),positive group(P,high-fat and high-sugar feed,5 mg/mL metformin),APBOM high Dose(H,high-fat and high-sugar feed,400 mg/kg)group,APBOM low-dose(L,high-fat and high-sugar feed,100 mg/kg)group.To evaluate the effect of drugs on blood lipids,total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),and high-density lipoprotein(HDL)in serum were measured.The determination of serum fasting blood glucose and fasting insulin(FINS)content to evaluate the disease process of diabetes.Hematoxylin-eosin(H&E)and retinal vascular spread Periodic Acid-Schiff staining were used to observe the pathological changes of retinal structure and retinal vascular network structure,and to observe important pathological changes in DR-the condition of new blood vessels.Immunofluorescence technique was used to stain Vascular endothelial growth factor(VEGF)and Hematopoietic progenitor cell antigen(CD34)to observe the expression of neovascular factors CD34 and VEGF.Results:The blood glucose,blood lipids,and insulin levels of the mice in N group were at normal levels.The retinal structure was clear,the cells and vascular network of each layer were arranged in an orderly manner,and no new blood vessels were seen.The fluorescence intensity of CD34 and VEGF was the weakest.Compared with N group,the levels of blood glucose,blood lipids,and insulin in DM group were significantly higher.H&E staining showed that the structure of the mouse retina was obviously loose,vacuolated,and the ganglion cell layer was arranged disorderly.The retinal vascular network was disorderly,and new blood vessels increased significantly.The fluorescence of CD34 and VEGF were both strongly positive.After the intervention of APBOM,the blood glucose,blood lipids,and insulin indicators were improved.The pathological state of the mouse retinal tissue structure and the structural state of the vascular network were improved,and the number of new blood vessels was significantly reduced compared with the DM group.The fluorescence of CD34 and VEGF were weakened.And the improvement effect of H group was better than L group.Conclusion:Through the establishment of DR model,it was found that APBOM could improve the pathological changes of DR and delay the development process of DR by inhibiting new blood vessels.Part 3 The alleviation mechanism of acid polysaccharide from Buddleja officinalis on diabetic retinopathyObjective:To clarify the mechanism of APBOM to relieve DR.Methods:Using a combination of in vivo and in vitro mechanisms,immunohistochemistry,immunofluorescence,PCR,Western Blot and other techniques were used to determine the expression of angiogenesis factors,autophagy and oxidative stress pathway related proteins in mouse retina and Human umbilical vein endothelial cell(HUVEC).In the in vitro mechanism study,the MTT method was used to determine the survival rate of the cells,the cell scratch and Transwell migration test were used to investigate the migration ability of the cells,the angiogenesis test was used to investigate the effect of APBOM on the formation of the lumen,and the shape and changes of cells were observed by phalloidin immunostaining.Results:In the in vivo mechanism study,the expression of Keapl/Nrf2/ARE signaling pathway in the model group was inhibited,LC3B expression increased,p62 expression increased,Beclin-1 expression decreased,and autophagy flow was inhibited.After administration of APBOM,the expression of Keapl/Nrf2/ARE signal pathway was promoted,LC3B expression increased,p62 expression decreased,Beclin-1 expression increased,and autophagy flow was activated.In the study of in vitro mechanism,APBOM could significantly inhibit the migration of HUVEC and the formation of the lumen of HUVEC.After high glucose induction,the morphology of endothelial cells was not significantly changed,and the expression of CD34,CD31,VEGF was enhanced,and a large amount of ROS was generated.Besides,P62 protein expression increased,Beclin-1 protein expression decreased,LC3II/LC3I ratio increased significantly,autophagy function was inhibited,mRNA and protein expression levels of HO-1,NQO1,SOD,Nrf2 pathway related factors decreased significantly.After APBOM intervention,the expression of CD34,CD31,VEGF was reduced,the generation of ROS was inhibited,the expression of P62 protein was reduced,the expression of LC3II/LC3I and Beclin-1 was increased,the autophagy flux was activated,mRNA and the protein level of HO-1,NQO1,SOD,Nrf2 had increased on average,and the effect of 160 mg/mL APBOM was the most obvious.Conclusion:The induction of high glucose could cause the increase of ROS,promote the effect of oxidative stress,reduce the ability of mitochondrial autophagy,and promote the production of vascular endothelial growth factor VEGF.APBOM could inhibit the generation of ROS,inhibit the effect of oxidative stress,activate the Keap1/Nrf2/ARE signaling pathway,promote autophagy,and inhibit the expression of VEGF,CD34 and other angiogenesis factors,thereby intervening in anglogenesis and improving DR.In vivo and in vitro mechanism studies have jointly verified that APBOM could alleviate diabetic retinopathy by activating the Keap1/Nrf2/ARE signaling pathway,promoting autophagy,and inhibiting neovascularization.This research also lays the foundation for the controllability of the samples of Buddleja officinalis Maxim,provides a theoretical basis for the preparation and quality control of acidic polysaccharides from Buddleja officinalis Maxim,provides a practical basis for the comprehensive development and application of Buddleja officinalis Maxim,provides a new idea for the prevention and treatment of diabetic retinopathy,and clarifies the mechanism of action of the acidic polysaccharides from Buddleja officinalis Maxim in the interfering diabetic retina.