| BackgroundDiabetes Mellitus (DM), a common endocrine-metabolic disease caused by defects ofinsulin secretion and/or insulin resistance, is chacterized by increased blood glucose. Ithas been the third major disease following tumor and cardio-cerebrovascular disease.Diabetic nephropathy (DN) is the most frequent and serious complications of DM andbecomes the main cause of deaths in DM. Approximately20to30persents of patientswith Type1diabetes mellitus (T1DM) or Type2diabetes mellitus (T2DM) have DN,most of which are further developed to end-stage renal disease (ESRD). Pathogenesy ofDN has not been clarified so far. It is believed to be influenced by multiple determintswith the high glucose level as the initiation factor. Strong oxidative stress is observed inDM and plays an important role in occurance and development of DM. Therefore,oxidative stress is regarded as the key reason of DM complications. As to the treatmentand prevention of DN, it is lack of ideal strategies and drugs. Thus, it would be of of greatsignificant to seek for effective methods for DN control.In Traditional Chinese Medicine (TCM), it is considered that DM belongs to consumptivethirst and oedema and characterised with asthenia in origin, asthenia in superficiality andasthenia and sthenia. It treatment must focus on strengthening body resistance andeliminating pathogen. In clinical medication of TCM, supplementing qi and nourishingyin and disperse blood stasis and dredge collateral are suggested to be the predominanttreatments.Astragalus membranaceus (AS) is one of the most commonly used herbal medicines in TCM, which possesses "Buqishengyang, Yiweigubiao, Tudushengji, Lishuituizhong"function. It can be used in deficiency of vital energy and calor internus. AS is a principaldrug to control diseases in heart, liver and kidney. Single AS can decrease urine protein innephritic syndrome and is one of the commonest traditional medicine to control nephriticsyndrome. Astragalosides (AST) is the effective fraction extracted from astragalus.Through many years of research by our team, AST is found to have many biologicaleffects, including anti-stress, anti-oxidation and immunological regulation. There havebeen lots of clinical and experimantal researches on DM treatment with astragalus orextract of astragalus, but the systematic study about the effect of AST on DM and itspotential mechanisms is still limited. In this thesis, we take the pharmacologic action ofastragalus effective component as the particular target and aim to understand the effect ofAST on DM and its potential mechanisms with both in vivo experiment and ex vivoexperiment. Findings generated from the exproments are expected to provide elementaryinformation on DM treatment and drug design for DN.ObjectivesTo understand effect of AST on DM and its potential mechanisms by treating diabetic ratsand diabetic mice induced by streptozotocin (STZ) and the molecular mechanismsunderline the effect is investigated using high-glucose-incubated human mesangial cell(HMC) as the targeted cell.Methods1The diabetic mouse model was established by intraperitoneal injection with STZ (100mg·kg-1) and the model mice were randomly divided into Model group, Tempol group (90mg·kg-1), AST-L group (30mg·kg-1), AST-M group (60mg·kg-1), and AST-H group (120mg·kg-1). The animals were given the drugs with successive intragastric administration for4-6weeks, the same volume of distilled water were admistered to Control group and Model group, respectively. After4weeks and6weeks of administration, half of the mice(10) in each group were randomly selected and blood sampkes were drawn to assessfasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), glycosylated serumprotein (GSP). The mice were sacrificed with cervical dislocation after blood sampleswere taken. And then, livers, spleens and kidneys were removed immediately, douchedwith ice physiological saline, dried with filter papers, weighed and organ index werecalculated. Liver tissue bomogenate (10%) was made by liver tissue of0.5g. Activities ofT-SOD and MDA and contents of GSH-Px were evaluated after isolating supernatants.One side of kidney was put into4%formaldehyde solution and made to paraffin sectionfor histomorphological examination. This side of kidney was also used to measure thelevel of TRPC6expression by immunohistochemistry as well as the apoptosis ofglomerular cells in glomeruli by TUNEL. The other side of kidney, however, was undercryopreservation at-80℃for the assessment of TGF-β1mRNA, Col Ⅳ mRNAexpression.2The diabetic rat model was established by intraperitoneal injection with STZ (60mg·kg-1) and the model rats were randomly divided into Model group, Tempol group (60mg·kg-1) and AST group (40mg·kg-1). The animals were given the drugs with successiveintragastric administration for4-6weeks, the same volume of distilled water wererespectively admistered to Control group and Model group. Survival status and activitiesof the animals were observed every day. Via metabolic cage, body weight, daily foodintake, daily water intake and daily urine output (UO) were recorded respectivelypre-therapy,0W,1W,4W and6W after administration. After urion prepared in standing,uric osmotic pressure was assessed by osmotic pressure molar concentration determinator.Proper amount of urion was centrifugated and prepared for measurement of biochemicalindicators under cryopreservation at-80℃, such as urine creatinine (Ucr),urinaryalbumin excretion (UAE) β2-microglobulin (β2-MG), tumor necrosis factor (TNF-α), transforming growth factor (TGF-β1). After4weeks and6weeks of administration, halfof the rats (10) in each group were randomly selected and blood samples were drawn fromabdominal aorta after anesthesia to assess FBG, GSP, fasting serum insulin (FINS), insulinsensitivity index (ISI), TC, TG, high density lipoprotein (HDL), low density lipoprotein(LDL), as well as activities of T-SOD and GSH-Px and contents of MDA, blood ureanitrogen (BUN), serum creatinine (Sc). The rats were sacrificed with cervical dislocationafter blood samples were taken. And then, livers, spleens and kidneys were removedimmediately, douched with ice physiological saline, dried with filter papers, weighed andorgan index were calculated. Liver tissue bomogenate (10%) was made by liver tissue of0.5g. One side of kidney was put into4%formaldehyde solution and made to paraffinsection for histomorphological examination. This side of kidney was also used to measurethe level of TRPC6expression by immunohistochemistry. The other side of kidney,however, was under cryopreservation at-80℃for the assessment of TRPC6mRNA,TGF-β1mRNA and Col ⅣmRNA expression.3Human mesangial cells (HMC) was taken as target cells and divided into Normal group(NG), Mannitol (MA), high glucose group (HG), Tempol (100μmol·L-1), AST-L(50mg L-1), AST-M (100mg L-1) and AST-H (200mg L-1). NG group were incubated withlow glucose DMEM culture solution (5.5mmol L-1D-glocose), MA group wereincubated with low glucose culture solution (24.5mmol L-1Mannitol) and HG groupwere with high glucose DMEM culture solution (30mmol L-1D-glocose). Similarly,Tempol group were incubated with high glucose culture solution (100μmol L-1Tempol),and high glucose culture solution (50mg L-1AST) in AST-L group, high glucose culturesolution (100mg L-1AST) in AST-M group and high glucose culture solution (200mg L-1AST) in AST-H group. The duration of administration was all defined as24hours. Then,different methods were adopted to assess various indicators. For instance, MTT Assay forthe effect of AST on proliferation and viability of high-glucose-incubated HMC, flow cytometry for ROS contents in HMC, fluorescent quantitative real time PCR for TRPC6mRNA,TGF-β1mRNA,NOX4mRNA expression in HMC, Western blot for TRPC6,TGF-β1,NOX4expression in HMC. In addition, enzyme-linked immunosorbent assaym(ELISA) was introduced to assess contents of SOD,MDA,GSH-Px,TGF-β1,FN,Col Ⅳ in HMC supernatant in each group.Results1Protective effects of AST on experimental diabetic mice1.1AST significantly improved the typical symptoms (polyuria, polydipsia, polyphagia,weight loss) and enhanced the anti-oxidative ability in liver of STZ diabetic mice. Indetail, it increased activities of T-SOD and GSH-Px and decreased MDA level. Althoughthe levels of serum FBG, GSP, TC and TG decreased, the reduction could not return tonormal level.1.2AST decreased kidney index to some extent. The effects of alleviating glomerulusdefects and apoptosis were shown dose-dependent and this effect was most significant atthe dosage of60mg·kg-1.1.3AST inhibited the high expression of collagen Type Ⅳ mRNA and TGF-β1mRNA inkidney. AST raised the TRPC6expression in glomerulus in STZ diabetic mice.2Protective effects of AST on experimental diabetic rats2.1AST significantly improved the typical symptoms (polyuria, polydipsia, polyphagia,weight loss) in STZ diabetic rats. The body weights of rats didn't continuously decrease,while daily food intake, daily water intake and daily urine output decreased gradually.2.2AST decreased the levels of FBG and GSP and increase ISI. But the content of FBGstill kept at a relatively high level. 2.3AST decreased the level of serum TC, TG and LDL while enhance the level of HDL.This kind of effect could rectify lipid metabolic disorders in STZ diabetic rats.2.4AST enhanced the anti-oxidative ability in STZ diabetic rats. It increased activities ofT-SOD and GSH-Px and decreased MDA level.2.5AST decreased daily amount of urine protein excretion as well as daily amount ofurine β2-MG, TNF-α and TGF-β1excretion. AST improved kidney function of STZdiabetic rats. It decreased serum levels of creatinine, urea nitrogen and enhanced thecreatinine clearance rate. It decreased kidney index and alleviate the defects of glomerulus.It also decreased expression of collagen Type Ⅳ mRNA and TGF-β1mRNA, enhancedexpression of TRPC6mRNA and increased expression of TRPC6in glomerulus in STZdiabetic rats.3Protective effect of AST on high glucose incubated glomerular mesangial cells3.1At the range of0-400mg L-1, the cell proliferation inhibiting rate caused by ASTshowd dose-dependent in high glucose incubated incubated glomerular mesangial cells.3.2Activities of T-SOD and GSH-Px in HMC supernatant decreased and increasedcontent of MDA. AST increased activities of T-SOD and GSH-Px in HMC and decreasedthe level of MDA.3.3The level of TGF-β1, FN and Col Ⅳ increased in high glucose incubated HMCsupernatant. AST with high, median and low dosage all decreased the contents of TGF-β1,FN and Col Ⅳ. The three indicators returned to normal level in groups with high andmedian dosage.3.4The level of ROS significantly increased in high glucose incubated HMC. AST withhigh, median and low dosage all decreased the contents of ROS in HMC. And ROSdecreased to normal level in group with high AST dosage. 3.5The expression of TRPC6mRNA decreased in high glucose incubated HMC while theexpression of TGF-β1mRNA and NOX4mRNA significantly increased. AST with highand median dosage both increased the expression of TRPC6mRNA and decreased theexpression of TGF-β1mRNA and NOX4mRNA.3.6The expression of TRPC6protein decreased in high glucose incubated HMC while theexpression of TGF-β1and NOX4protein significantly increased. AST with high, medianand low dosage all decreased the the expression of TRPC6protein in HMC and decreasedthe expression of TGF-β1protein. The expression of NOX4protein significantlydecreased in group with high and median dosage.Conclusions1There is protective effect of AST on high glucose incubated glomerular mesangial cells.The potential mechanisms may include the enhancement of anti-oxidative ability of HMC,reduction of the levels of TGF-β1, FN and Col Ⅳ, inhibitation in the high expression ofTGF-β1and NOX4and promotion of the TRPC6expression by AST.2There are some protective effects of AST on kidney of experimental diabetic rats andmice. The potential mechanisms may include improvement of anti-oxidative ability inserum and liver, regulation of lipid metabolism, protection of kidney function, inhibitationin apoptosis in glomerulus, inhibitation in the high expression of Col ⅣmRNA,TGF-β1mRNA in kidney and enhancement of the expression of TRPC6mRNA andTRPC6protein in glomerulus by AST. |
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