| Objective:To select the activity of effective parts and the compatibility combination from radix rehmanniae and Cornus officinalis by glomerular mesangial cells(GMC)experiment.To observe the therapeutic effect of the compatibility combination from radix rehmanniae and Cornus officinalis effective parts in whole animal experiments and in vitro experiments.In order to explore its mechanisms of improving diabetic nephropathy.Method:1 The preparation and content determination of the effective parts 1)Take some Radix Rehmanniae smashed,and extract through 3 times.First time is adding 10 times volume of water,extract about 2 h,filtrate to get the extraction liquid I;the second time is adding 6 times volume of water into the herbal residue,extract about 1h,and get the extraction liquid II;the third time is adding 4 times volume of water into the herbal residue,extract about 1 h,and get the extraction liquid III.Combine each time of the extarction liquid,concentrate until 1g crude drug/ml,and add alcohol to concentration 70%.Place four 24h and filtrate it.Oligosaccharides parts extracted:The supernatant was waving to be up to no alcohol taste,than concentrated and loaded D101 resin.And wash untile the molish reaction becoming negative with distilled water.Polysaccharide parts extracted:After ethanol precipitation the resulting residue was dissolved with distilled water,centrifuged to remove insoluble material,the supernatant was subject to no alcohol volatile flavor,freeze-dried to obtain a polysaccharide powder.Iridoid glycosides parts extracted:Concentratedang and loaded D101 resin,eluted with water,collecting water eluent until catalpol peak disappeared.The eluate was collected and then concentrated under reduced pressure r,loaded H103 resin,eluted with water.With 20%ethanol eluent is collected,concentrated and freeze-dried to obtain habitat iridoid glycosides powder.Determine the purity of iridiod glycoside using the catalpol as an indicator by HPLC;Cornus officinalis total iridoid glycosides parts extracted:Weigh some Cornus officinalis,extracted several times with water,combined water extracts,concentrated adding ethanol,filtration.Loaded macroporous resin column,washed with distilled water,30%aqueous ethanol elution,collected effluent,evaporated,freeze-dried,then dissolved in methanol,adding silica mixed sample,vacuum recovery of methanol to dryness to give a reddish brown powder.Column chromatography on silica using chloroform-methanol isocratic solvent system,effluent was collected and lyophilized to give the iridoid glycosides powder.Cornus officinalis total triterpene acid parts extraction:After draining the water extraction residue,the Soxhlet extraction with ethyl acetate,the solvent recovered by rotary evaporation to dryness,washed with 40%ethanol,60 ? heated water bath,filtration,residues 50 ? drying to obtain a triterpene acids powder.The loganin and morroniside,used as the indicators of iridoid glycoside purity determined by HPLC.The ursolic acid and oleanolic acid,used as the indicators of triterpenic acid purity determined by HPLC.In vitro experiments:Cells were cultured in vitro and stimulated by 200 mg/L AGEs.Than intervened with compatibility combinations from radix rehmanniae and Cornus officinalis effective parts,after 24h,the cells were observed under a microscope state by MTT assay and cell viability;Cells were cultured in vitro and stimulated by 30 mmol/L glucose.Than intervened with compatibility combinations from radix rehmanniae and Cornus officinalis effective parts,the cells were observed under a microscope state by MTT assay and cell viability after 24h;Cells were cultured in vitro and stimulated by 200 mg/L AGEs.Than intervened with compatibility combinations containing serum from radix rehmanniae and Cornus officinalis effective parts,the cells were observed under a microscope state by MTT assay and cell viability after 24h;Cells were cultured in vitro and stimulated by 30 mmol/L glucose.Than intervened with compatibility combinations containing serum from radix rehmanniae and Cornus officinalis effective parts,the cells were observed under a microscope state by MTT assay and cell viability after 24h;The whole animal experiment:Take 6 weeks old male C57BL/6J mice,adaptive feeding,respectively,on the first day,the first four days in accordance with a dose of 100mg/kg body weight by intraperitoneal injection of STZ solution,and the remaining two groups injected with equivalent citric acid-citric acid sodium citrate buffer,the first eight days of fasting blood glucose measurements mouse,select the glucose values greater than 15mmol/L mice as a successful model of diabetes in mice.The successful modeling according to blood glucose in mice divided into model group,aminoguanidine group,metformin group,captopril group,A2B1 low-dose group,A2B1 medium-dose group,A2B1 high-dose group,separate the normal group,n = 10,in addition to the normal group than in mice fed with normal diet the rest of the group are outside the high AGEs diet.Each group were administered doses of the drug for 8 weeks,in mice of the model group and blank control group distilled water.In 8 weeks,the mice eyeballs blood,the pancreas,kidney was separated,weighed to 10%formalin solution for fixation,part of the slice to 2.5%glutaraldehyde fixed,and the remaining kidney to-80 ? save the determination indicators are as follows:(1)every four weeks Determination of blood glucose,body weight,food intake,water intake,urine output.(2)renal indicators:urinary protein,urine creatinine,serum urea nitrogen(3),pancreas,kidney pathological examination(including optical microscopy,electron microscopy).(4)RT-PCR detection the renal cortex RAGEmRNA of expression,Western Blot renal cortical RAGE protein expression.ResuIts:1)radix rehmanniae iridoid glycosides catalpol content 32.03%;Cornus officinalis loganin content 33.82%.Cornus officinalis iridoid glycosides morroniside content 21.95%.2)With 200 mg/L of AGEs stimulation,after adding drug intervention,at a concentration of 25 ?g/mL,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B2 = A1B1>A1>B1;At a concentration of 50 pg/mL,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B2>A1B1? A1? B1;At a concentration of 100 ?g/mL,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B1>A1B2>A1>B1.With 30 mmol/L glucose stimulation,after adding drug intervention,at a concentration of 25 ?g/mL,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B2=A1B1>A1=B1;At a concentration of 50 ?g/mL,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B1? A1B2>A1?B1;At a concentration of 100 ?g/mL,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1? A1B1>A1B2>A1>B1;With 200 mg/L of AGEs stimulation,after adding drug serum intervention,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B2>A1B1>A1>B1.With 30 mmol/L glucose stimulation,after adding drug serum intervention,The intensity of radix rehmanniae and Cornus officinalis effective parts compatibility to inhibite GMC proliferation were:A2B1>A1B2= A1B1>A1>B1.Model group showed a significant hyperglycemia,polydipsia,polyphagia,polyuria,proteinuria and kidney damage and other symptoms.A2B1 group can significantly reduce blood glucose in diabetic nephropathy(P<0.01),improvement of "a little" symptoms(P<0.01),increased insulin secretion(P<0.01),lower urinary protein,creatinine,serum urea nitrogen(P<0.01),to reduce the pancreas,kidney pathological changes,decreased serum,kidney AGEs levels(P<0.05,P<0.01),reduced renal cortical RAGE,NF-?B TGF-? protein in protein expression.Conclusion:experimental results show:radix rehmanniae and Cornus officinalis effective parts of the best compatibility combination is A2B1;Radix rehmanniae and Cornus officinalis effective parts of the best compatibility combination can significantly improve the compatibility of diabetic nephropathy in mice "a little" symptoms,lowering blood sugar levels,increase insulin secretion,decreased serum creatinine,blood urea nitrogen,serum AGEs levels,reduce the discharge of urine protein to improve kidney function and renal pathological changes,reduced renal cortical parts of the expression of RAGE,NF-?B,TGF-?,to delay the process of glomerulosclerosis. |
PDF Full Text Request