| BackgroundDiabetes mellitus (DM) is a kind of metabolic disease of multifactor, characterized by hyperglycaemia. A large number of epidemiological researches suggest that diabetes is an important risk factor for cognitive dysfunction, with the performance of mild cognitive impairment and pathological change in the structure of brain, neural function, mental status and so on. But the mechanism of the cognitive dysfunction is not clear now. By now, the studies suggest that oxidative stress may play an important role in the central nervous system complications of diabetes mellitus. Peroxisome proliferator-activated receptor coactivator1alpha (PGC-1α) is a newly emerging transcription factor,which plays a key role in oxidative stress system. PGC-1α have been shown to induce the expression of antioxidant genes and to be molecular targets of deacetylated by silence regulatory protein1(SIRT1). The SIRT1and PGC-1α mediate the antioxidant and neuroprotective procedure.The pathogenesis of chronic complications of diabetes mellitus is complicated. At present, effect of modern medicine is limited in controlling the complications of diabetes and reducing the mortality. Whereas the traditional Chinese medicine show great advantages in this field, which pay more attentions to the syndrome differentiation. Diabetic cognitive dysfunction, in traditional Chinese medicine, belongs to " Xiaoke " disease, characterized with the symptoms of frequent drinking and urination, with dementia, forgetful etc.pathogenesis is the deficiency of viscera including lung, spleen and kidney. With the extension of course, the viscera function keep declining and the situation become worse and worse. So the therapy should focus on the treatment of Spleen Qi, phlegm and dampness.Jinlida granule, a traditional Chinese medicine patent prescription, contains a variety of ingredients of Chinese Medicine, such as Ginseng, polygonatum, Atractylodes, Radix Sophorae Flavescentis, radix, radix rehmanniae, Radix Polygoni Multiflori, dogwood, Poria, Rhizoma Anemarrhenae, epimedium, Perrin and Salvia miltiorrhiza.With the main function of Supplementing qi,nourishing Yin,and invigorating spleen, Jinlida can be used for the treatment of diabetes. Thus, we speculate that Jinlida particles may improve the cognitive function of diabetic.ObjectiveThe purpose of this study was to detect the expression of SIRT1and PGC-1α in hippocampal tissue of diabetic rats. We tested the expression of SIRT1and PGC-1α and the level of oxidative stress in the hippocampal tissues on the rats of each group, and used TUNEL assay and transmission electron microscopy (TEM) to observe the changes of neuron apoptosis.Based on the results,we can make further efforts on the related mechanism. Then we investigate the protective effect of Jinlida granules on.hippocampus of diabetic, rats, and tested its effects on the expression of SIRT1and PGC-1a and the level of oxidative stress so that we can provide the experimental proof for the clinical application of Jinlida granules.Methods88male SD rats, were provided by the experimental animal center of Second Military Medical University. Two days later, we randomly selected12from them as normal control group, giving common forage.The others were raised by high fat diet.Four weeks later,the model group were given disposable streptozotocin(STZ) intraperitoneal injection with the dosage of30mg/kg.The control group were given solo citrate buffer intraperitoneal injection. Model verification:2weeks later, rats were fasted for8hours.Then oral glucose tolerance test were taken by feeding the rats with20%D-glucose solution according to dosage of2g/kg.The rats whose blood glucose higher than7mmol/L right now and11mmol/L after2hours were considered as the successful model. The72successfully established T2DM rats were divided into model group.Jinlida granule groups(0.75,1.5and3.0g/kg), α-lipoic acid group and insulin group,12of each group.Insulin was given by subcutaneous injection,while the others were given by the way of intragastric administration after dissolved in0.5%sodium carboxymethyl cellulose.The normal control group and model group were given sodium carboxymethyl cellulose only.After8weeks of treatments, rats were tested in Morris water maze and then the hippocampus tissue were taken from each group and were subjected to TUNEL assay and transmission electron microscopy(TEM) observation.The activities of superoxide dismutase(SOD), glutathione (GSH) and the contents of malondialdehyde (MDA) were all examined.Results1. The overall state of the rats in each group.Normal control group rats were generally in good condition, including fine mental status, good exercise capacity, Shiny hair, moderate diet and normal urine and stool, without death; The general condition of rats in model group was poor, with depression, slow movement, messy hair, and the states of polydipsia, polyphagia and polyuria. The different medicine group can change the situation in different extent.2. The results of Morris water maze test in rats of each group. Compared with the normal control group, the diabetic animals spatial learning developed impaired learning and memory8weeks after establishment (P<0.05).Except for those in the low-dose Jinlida group, the impaired learning and memory functions were facilitated in rats of other treatment groups to different extents (P<0.05), especially those in insulin group which has no significant difference with normal control group (P>0.05), followed by alpha-lipoic acid group and high dose group Jinlida.3. TUNEL staining results of rat hippocampus in each groupIn the control group, neurons were compact and neat, with clear cell boundaries. Whereas in the model group, neurons in the hippocampal CA1region were scattered, sparse and appeared an uniform distribution.Compared with normal control group,rats in the model group had more apoptotic neurons in the hippocampal CA1area. Each treatment group can improved the situation in different extent, particularly in the middle and high dose Jinlida, alpha-lipoic acid and insulin group.4. Electron microscopy images of hippocampus in rats of each group.In the control group, neurons had even chromatin, integrity of mitochondrial structure, few mitochondrial crista damage.Whereas in the model group,cells even didn’t have clear structure. Chromatic agglutination could be observed as well as mitochondria cristae damage and incomplete mitochondrial structure.All treatment groups can partially reverse the ultrastructure damage.5. The detection results of free radicals and antioxidant enzymes.Compared with control group, rats in the model group had significantly lower SOD and GSH activity level (P<0.05) and significantly increased MDA level (P<0.05), which was reversed in all the treatment groups, particularly the groups of high dose Jinlida, alpha-lipoic acid and insulin. High dose Jinlida could significantly increase SOD activity level, which is comparable to alpha-lipoic acid (P<0.05),but less effective than insulin.While for the GSH,high dose Jinlida had less effect than alpha-lipoic acid or insulin.And for MDA level, insulin is the best drug followed by alpha-lipoic acid, middle and high dose Jinlida, the effect of which were equal (P<0.05).6. The expression of PGC-1αmRNA, PGC-1α and SIRT1proteinCompared with control group, expression of PGC-1α mRNA, PGC-1α and SIRT1protein(P<0.05) in the model group rats is significantly lower, and is increased to some extent (P<0.05) in all drugs used, especially insulin, alpha-lipoic acid and high dose Jinlida. For PGC-1α protein, expect that alpha-lipoic acid and insulin could increase it, the others didn’t show significantly effect, although middle and high dose Jinlida could increase it if only from the numerical point.But the effect of high dose Jinlida is still better than low or middle group(P<0.05).ConclusionType2diabetes mellitus (T2DM) can lead to cognitive impairment in rats, with obviously destroyed structures and serious neuron apoptosis in the hippocampus. Expression of PGC-1amRNA and protein, SIRT1protein in hippocampus are significantly lowered as well as SOD and GSH activity level while the MDA level is up-regulation. Jinlida granules can protect the hippocampus of T2DM rats and facilitated their cognitive dysfunction, the effect of which is equal to alpha-lipoic acid. The mechanism of the protection maybe partly through regulating the expression of SIRT1and PGC-1α and then regulating the activition of antioxidant enzymes and Mitochondrial biogenesis. |
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