Effect Of Vanadium-enriched Chickpea Sprout On Glycolipids Metabolism And Learning-memory In Diabetic Rats

Diabetes mellitus(DM) is clinic syndromes caused by absolute or relative deficient of insulin,with the main manifestation of glycometabolism disorders. Chronic diabetes mellitus can result in structural and functional lesions of multiple organs in human.Even more,it will cause diabetic neuropathy,including peripheral and central neuropathy.And these complications may become the primary causes of mutilation or case fatality in diabetic patients.In present,the therapeutics of DM,such as alimentary control,insulin and oral antidiabetic drug,cannot achieve expected goals in tiuman—controlling blood glucose in normal level,preventing glycometabolism disorders and chronic complications,shieldingβ-cell function,and,in the final,delaying the progression of DM.For these reasons,it is significantly necessary to develop new therapeutic path and to investigate the corresponding mechanism of action.Vanadium compounds have demonstrated insulin-mimetic effects with in vitro and in vivo studies.Vanadium compounds improve glucose homeostasis in animal models of both Type 1 and Type 2 diabetes mellitus,but the acceptance of vanadium compounds as antidiabetic agents has been slowed due to the concern for short-term gastrointestinal stress and potential long-term toxicity with vanadium accumulation. Recently,some studies of vanadium permeability and toxicity suggested that some kinds of vanadium organic compounds,for example,the combination of vanadium with some botanic antioxidants,might be benefit in reducing vanadium toxicity and increasing vanadium hypoglycemic effects.Chickpea has been traditional hypoglycemic foodstuff,what more,its seeds and its sprout contain variety of antioxidants such as,isoflavone,biochanin A,C and Formoononetin B,and have been investigated their potential hypoglycemic effects in diabetic animal models by several groups.Diabetic cognitive disorder is one of the common complications in DM. Nevertheless,as the nerve center of learning -memory,hippocampus is also the structural foundation.In the present study,with streptozotocin-induced diabetes in Wistar rats as objects,the effects and side effects of vanadium-enriched chickpea sprout(VCS),a new possible vanadium organic compounds,on impaired glycolipids metabolism and learning-memory ability of diabetes rats in morphological,functional and molecular biology aspects have been investigated.Objectives1.To incubate chickpea sprouts(CS) and vanadium-enriched chickpea sprout (VCS).2.To determine the most effective and safe dosage VCS as the therapeutic animal food.3.To observe the changes of some parameters in experimental rats after 8 weeks VCS- treatment,the parameters including:diabetic symptoms,blood glucose and lipid metabolism,space learning-memory ability,and morphology and molecular biology aspects.Methods1.Based on the method of Burguillo PD,the CS was performed with tap water, and the VCS were incubated with 8 dosages of sodium orthovanadate(SOV) solution as following:50μg/mL,100μg/mL,200μg/mL,400μg/mL,600μg/mL,800μg/mL, 1600μg/mL and 2000μg/mL(VCS 50～2000),respectively.2(1).The vanadium concentrations of vanadium-enriched chickpea sprout powder,of blood specimen and tissues in diabetic,were determined using a biological specimen analysis method(GB/T5009—2003) by use of a plasma emission spectroscopy(IRIS IntrepidⅡ).Spearman analysis was used to correlate the serumⅤconcentration with clinical response.2(2).Male Wistar rats weighting between 188 and 220g were obtained from the Institute of Experimental Animals,Tongji Medical School,Huazhong Technology University(SCXK2004-0007),and housed in air-conditioned room(temperature: 23±10℃;relative humidity:55±5%;12-h/day photoperiod:) in the animal facility of Taishan Medical University.Diabetes was induced by a single intravenous tail vein injection of streptozotocin(STZ) 45 mg/kg.On day 14 following STZ administration, animals with a blood glucose level≧15mM were considered diabetic.2(3).Then,the rat food containing vanadium-enriched chickpea sprout (VCS-food) was prepared by adding 5%(w/w) the VCS powder in the standard rat food pellet.After 4-week's intervention to diabetic rats with VCS-food as animal feeds,the most effective dosages of VCS-food(VCS100) was determined according to the changes of blood glucose,clinical manifestation,mental status and praxiology of the diabetic.3(1).On day 14 following STZ administration,diabetic animals were randomly divided into five groups.The control rats(C) and diabetic rats(D) received only standard pellet food and were given tap water ad libitum.Four treated groups(V group,treated with 0.5mg/mL SOV solution;CS group,treated with pellet food containing 5%of chickpea sprout powder;V+CS group,treated with 0.2mg/mL SOV solution combined with pellet food containing 5%of chickpea sprout powder;and VCS group,treated with pellet food containing 5%of vanadium-enriched chickpea sprout powder;) were received different therapeutics for eight weeks.At the end of 8-weeks,some diabetic parameters were measured as following: Plasma insulin(ins),and leptin(LEP) levels were measured using radioimmunoassay kits(Boster biological company,Wuhan,China) using the manufacturer protocol. Plasma total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol(LDL-C),and function of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen (BUN),and creatinine clearance rate(Cr) were measured using the diagnostic kits (Ortho-Clinical Diagnostic,Inc.USA) with manufacturer protocol.The plasma GHbA1 was measured by HPLC.The OGTT studies were then carried out in the morning after an overnight-fasting.The 0-hour(baseline) blood samples were drawn for glucose, insulin and lipid profile analysis.Then a glucose solution(1g/ml) was immediately given to the animal at a dose of 2g/kg body weight via gavages,and then the glucose levels were checked at the intervals of 30,60,90 and 120 min,respectively.And plasm specimens were prepared for OGRIT test.What's more,before sacrifice,a part of experimental rats were trained to search for the escape platform under the pool water by using Morris water maze for four days to observe their space learning-memory ability.The testes were carried out in the fifth day,escape latencies and swimming paths were recorded using the video tracking system for each rat.3(2).In order to investigate the changes ofhippocampal neurons and the effect of VCS,electronmicroscope and immunohistochemistry were used.Then the hippocampus mRNA was then extracted,and corresponding cDNA was synthesized. Real Time-PCR was used to detect mRNA expressions of amyloid protein precursor (App),and Caspase3.At the same time,the brain was sliced,and immunoblotting (western-blotting) was used to detect the protein expressions of Caspase3,bax,bcl-2, IGF-1,and IGF-1R.4.Statistical analysisOne-way analysis of variance(ANOVA) was used to compared the Means±SEM.Correlation analysis was made using Spearman analysis.The significance levels were tested at P＜0.05.Results2(1).After 9d from the administration with STZ(45mg/kg,i.p.),the diabetic rats had diabetic symptoms such as polydipsia,polyuria,and hyperglycemia.And after 14d,the rats with blood glucose≧15mM/L were determined to be diabetic ones.This showed that the administration with STZ at dose of 45mg/kg,i.p.to induce diabetic model was a successful method,with achievement ratio:92%,death rate:4%.1.The growth of CS and VCS were well with little rotten sprout.But observation of sprout growth manifested that SOV might slightly inhibited the growth of chickpea sprout,but there were no significant difference among 8 different dosages.2(3).After 4-week preliminary experiment,the results showed that VCS 100 was the best select for the diabetic therapeutic,according to the changes of blood glucose, diabetic manifestation,mental status,and praxiology of the diabetic rats.2(1).The vanadium concentrations were related with the doses of the different SOV,and the vanadium concentrations in blood specimen and organs differentiated from intervention in different diabetes groups.3(1).Compared with the C group,the D group rats hadn't only typically diabetic symptoms such as polydipsia,polyuria,and hyperglycemia,but also increased levels of plasma lipid and GHbA1,decreased levels of serum insulin(Ins),and leptin(LEP), impaired function ofALT,AST,BUN,and Cr,and,abnormal levels ofOGTT,OGIRT, and their AUCs.Otherwise,After 8-weeks therapeutics for diabetes,much expected changes of above parameters had appeared in four treated groups,and the most rational results happened in VCS group as following:the typical diabetic symptoms of polydipsia,polyuria,and emaciation disappeared,blood glucose level lowered significantly,the levels of plasma lipid and GHbA1 decreased,serum Ins,and LEP recovered,at the same time,the impaired function of hepar and kidney were ameliorated,and,the levels of OGTT,OGIRT,and their AUCs normalized,as well, without any side effects(gastrointestinal stress) during the experimental period caused by vanadium inorganic compounds as seen before.The effect of VCS food on spatial learning and memory was measured using a water maze test.After 8 weeks of diabetes,D group rats could swim for more than 3h, which means that the movements of diabetic rats were not harmed.Compared with the C group rats,the D ones exhibited significantly delayed latencies to locate the platform and decreased score of platform-searching in all four quadrants(P＜0.05). After treatment,the time for locating the platform and the score of platform-searching were improved significantly.The best effect was observed for VCS group.3(2).After 8 weeks of diabetes,morphological analysis of the hippocampal neurons by HE staining revealed that the neuronal cells were in the state ofraritas and disorder,accompanied with reduced neuronal density.The ultra structural analysis of the hippocampal pyramidal neurons by electronmicroscope revealed that the cell body and cell process were swelling obviously,endochylema was vacuolated,the cytoplasmic organoids of neurons were ambiguous,rough endoplasmic reticulum and free ribosome were significantly decreased,chondriosome was swelling,lyzing and demolishing,and cellular membrane was disaggregated.The ultrastructural analysis also showed that the nerve fiber and nerve plexus were ambiguous,synaptic cleft was widened.While,VCS food fed group had the improved morphological changes, obviously diminished synapse cleft and increased neuron density.Apoptosis was demonstrated after 8 weeks of diabetes by electronmicroscope observation.Compared with C group,increased expressions of positive cells were shown with increased Caspase3 and ratio of bax/bcl-2 in the D group hippocampus. And the same changes of protein expressions and mRNA expression with Caspase3 and bax/bcl-2 were shown by Western-blotting and Real Time-PCR in D group,as well.However,VCS food could ameliorate the changes above.The result of Real Time-PCR analysis was that,the mRNA expressions of App in D group were higher than that in C.But the treatment results showed that VCS could improve the impaired learning- memory ability of the diabetic rats,and inhibit the mRNA expressions of App.In this experiment,immunchistochemistry and western-blotting were used to detect the expressions of insulin-like growth factorl(IGF-1) and insulin-like growth factor 1 receptor(IGF-1R).Compared with the C group,the results of protein expression in IGF-1 were significantly decreased in D group.And IGF-1R protein expression was obviously up-regulated compensatedly in the diabetic rats with deficiency of learning and memory.This indicated that the deficiency of IGF-1 and up-regulation of IGF-1R might account for the impaired learning memory ability in diabetic rats.While VCS food could reverse the changes of IGF-1 and IGF-1R in diabetic rats,and produce its neuroprotective effect,by improving the deficiency of learning-memory in the diabetic rats.4.After 8w treatment,the results of relationship analysis showed that,the VCS antidiabetic effect significantly related with the vanadium intakes of VCS in diabetic rats,and the vanadium in blood,but not with the vanadium concentrations in vital organs.Conclusions1.It is the first time to enrich the vanadium into the chickpea sprout through the sprout process of seeds by biological method,so that to form vanadium-enriched chickpea sprout(VCS)-a new possible biological actived vanadium,which may have some new expected characteristics such as:not only enhancing the antidiabetic effect of both vanadium and chickpeas,but attenuating the adverse reaction resulted from the use of inorganic vanadium compound alone.2.VCS can significantly ameliorated the impaired glycolipids metabolism and learning-memory in diabetic rats.3.The possible mechanism of VCS in protecting the hippocampus injury from diabetes,maybe relate with the effects of VCS on lessening the apoptosis of hippocampal neurons,inhibiting the expression of App and IGF-1R,and increasing the expression of IGF-1 in diabetic hippocampus.