Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Hypoglycaemic effect of Berberis vulgaris L.in normal and streptozotocin-induced diabetic rats

Objective:To achieve a primary pharmacological screening contained in the aqueous extract of Berberis vulgaris(B.vulgaris) and to examine the hypoglycaemic effect and biochemical parameters of aqueous and saponins extract on groups of rats rendered diabetic by injection of streptozotocin.Methods:The phytochemicol tests to detect the presence of different compounds were based on the visual observation of color change or formation of precipitate after the addition of specific reagents.Diabetes was induced in rats by intraperitoneal(i.p.) injection of streptozotocin(STZ) at a dose of 65 mg/kg bw.The fasting blood glucose levels were estimated by glucose oxidase-peroxidase reactive strips(Dextrostix,Bayer Diagnostics).Blood samples were taken by cutting the tip of the tail.Serum cholesterol and serum triglycerides were estimated by enzymatic DHBS colorimetric method.Results:Administration of 62.5 and 2S.0 mg/kg of saponins and aqueous extract respectively in normal rats group shows a significant hypoglycemic activity(32.33%and 40.17%respectively) during the first week.However,diabetic group treated with saponin extract produced a maximum fall of 73.1%and 76.03%at day 1 and day 21 compared to the diabetics control.Also,blood glucose levels of the diabetic rats treated with aqueous extract showed decrease of 78.79%on the first day and the effect remains roughly constant during 3 week. Both extracts also declined significantly biochemical parameters(20.77%-49.00%).The control in the loss of body weight was observed in treated diabetic rats as compared to diabetic controls. Conclusions:These results demonstrated significant antidiabetic effects and showed that serum cholesterol and serum triglycerides levels were decreased,significantly,consequently this plant might be of value in diabetes treatment.

Hypoglycemic and anti-hyperglycemic study of Ocimum tenuiflorum L.leaves extract in normal and streptozotocin-induced diabetic rats

Objective:To investigate the antidiabetic activity of Ocimum tenuiflorum L.(O.tenuiflorum) leaves used in the traditional medicine management of diabetes in Malaysia.Methods:O.tenuiflorum leaves were extracted sequentially with hexane,chloroform,ethyl acetate,methanol,and water.The extracts were evaluated in terms of antidiabetic activity by using acute,subcutaneous glucose tolerance,and sub-chronic tests in streptozotocininduced diabetic rats.The extracts were also subjected to phytochemical analyses.Results:With an acute dose(1 g/kg),the methanol extracts showed significant reduction(31%) in fasting blood glucose(FBG) of the streptozotocin-induced diabetic rats.The FBG-decreasing effect of ethyl acetate extract was more rapid than that of the other extracts;the decreasing rates were 20%after 2 h,21%after 3 h,and 8%after 5 and 7 h.After 7 h(31%),the effect of methanol extract on FBG was significantly lower than that of metformin.In the subcutaneous glucose tolerance test,only methanol and hexane extracts showed the similarity of metformin in diabetic rats.After 14 days,the effects of these extracts were similar to those of metformin(63.33%).The total flavonoid and phenolic contents of extracts decreased as the polarity of the extraction solvent increased.Conclusions:The results obtained provide support for a possible use of O.tenuiflorum leaves in managing hyperglycemia and preventing the complications associated with it in type 2 diabetic.

Effect of insulin in combination with selenium on blood glucose and GLUT4 expression in skeletal muscle of streptozotocin-induced diabetic rats

Objective To evaluate the effect of low-dose insulin [1 U/(kg·d)] in combination with selenium [180 g/(kg·d)] on general physiological parameters and glucose transporter (GLUT4) level in skeletal muscle of streptozotocin (STZ)-induced diabetic rats. Methods Diabetic rats were treated with insulin,selenium,and insulin and selenium in combination for four weeks. The level of blood glucose was determined using One Touch SureStep Blood Glucose meter and the level of GLUT4 in skeletal muscle was examined by immunoblotting and immunohistochemistry. Results Our data showed that insulin in combination with selenium could significantly lower blood glucose level and restore the disturbance in GLUT4 level in skeletal muscle. Treatment with insulin was only partially effective in restoring diabetic alterations. Conclusion It can be concluded that there is a synergistic action between insulin and selenium,and that treatment of diabetic rats with combined doses of insulin and selenium is effective in the normalization of blood glucose level and correction of altered GLUT4 distribution in skeletal muscle of diabetic rats.

Effects of Bofutsushosan and Gardeniae Fructus on Diabetic Serum Parameters in Streptozotocin-Induced Diabetic Mice

Streptozotocin (STZ)-induced diabetic mice increased levels of serum glucose, triglyceride and cholesterol, and decreased level of serum insulin. Effects of Bofutsushosan (BOF: Pulvis ledebouriellae compositae: 防風通聖散) and its composed crude drug, gardeniae fructus (GF: 山梔子) were investigated on levels of these diabetic parameters (serum glucose, insulin, triglyceride and cholesterol) in STZ-diabetic mice. BOF and GF were extracted in 10 volumes of distilled water with an automatic extractor “Torobi”. STZ-induced diabetic mice with serum glucose level of over 600 mg/dl at 3 - 4 weeks after intravenous injection of 150 mg/kg STZ were used for experiments. BOF extract, GF extract, geniposide (a main constituent of GF), and glibenclamide were administered intraperitoneally into 3-hour-fasted STZ-diabetic mice. At 6 hours after administration, BOF extract (100 - 300 mg/kg) decreased high levels of serum glucose, triglyceride and cholesterol, and also increased low level of serum insulin in STZ-diabetic mice in a dose-dependent manner, respectively. Anti-diabetic drug glibenclamide (0.3 - 1 mg/kg) as positive control significantly decreased serum glucose and cholesterol levels, and increased serum insulin level in the diabetic mice. GF extract (30 - 300 mg/kg) decreased serum glucose, triglyceride and cholesterol levels but did not affect serum insulin level in the diabetic mice. Geniposide (10 - 100 mg/kg), decreased serum glucose level but did not affect serum insulin and triglyceride levels in the diabetic mice. These results demonstrated that intraperitoneally administrated BOF extract improved abnormal levels of serum glucose, insulin, triglyceride and cholesterol in the STZ-diabetic mice as being similar to glibenclamide. GF extract has an important role in a part of improving actions of BOF in the diabetic mice. The action of GF extract on serum glucose was parallel with the action of geniposide in the diabetic mice, supporting roles of geniposide in anti-hyperglycemic action of GF.

Garlic (<i>allium sativum</i>) modulates the expression of angiotensin II AT₂receptor in adrenal and renal tissues of streptozotocin-induced diabetic rats

The loss of balance between the antagonistic activities of angiotensin II AT1/AT2 receptors has been impli-cated as a major mediator in the development of hypertension and progressive nephropathy in expe-rimental diabetes. The present study was designed to investigate the potential of garlic to modulate the level of expression of the AT2 receptor in the adrenal and renal tissues of diabetic rats. Three groups of rats were studied after 8 weeks following diabetes induction: normal, streptozotocin-induced diabetic (control diabetic), and garlic-treated diabetic rats. A polyclonal antibody of proven specificity to the AT2 receptor, as verified by Western blotting and emplo- yed in immunohistochemical assays, indicated that compared to normal rats, the highest adrenocortical AT2 receptor expression was significantly shifted from the zona glomerulosa to the zona fasciculate/ reticularis, and was significantly reduced in adrenomedul- lary chromaffin cells of control diabetic rats. In the kidney, STZ treatments were associated with a signi- ficant decrease in AT2 receptor expression throughout glomeruli and all cortical and medullary tubular segments. Compared to control diabetic rats, the labeling of the AT2 receptor in the garlic-treated diabetic group was restored among adrenocortical zona glomerulosa cells and adrenomedullary chromaffin cells and significantly reduced in the zona fasiculata, and was also restored in glomeruli and throughout renal cortical and medullary tubular segments, to le- vels comparable to those observed in normal rats. The capacity of garlic to modulate diabetes-induced AT2 receptor down-regulation may be implicated in restoring the recuperative processes mediated by AT2 receptors, which interfere with the development of hypertension and nephropathy.

1,25-Dihydroxyvitamin D_(3) effects on the regulation of the insulin receptor gene in the hind limb muscle and heart of streptozotocin-induced diabetic rats

In the present study, we examine the effects of the treatment with 1,25-dihydroxyvitamin D3 [150 IU/Kg (3.75 μg/Kg) once a day, for 15 days] to non-diabetic and streptozotocin-induced diabetic rats. The results indicate that treatment with 1,25-dihydroxyvitamin D3 had minor effects in non-diabetic rats. The same treatment in streptozotocin-induced diabetic rats, although it did not correct the hyperglycemia and hypoinsulinemia induced by the diabetes, caused other actions that could mean beneficial effects on the amelioration of diabetes e.g., it avoided body weight loss, increased calcium and phosphorus plasma levels, and corrected the over-expression of the insulin receptor mRNA species of 9.5 and 7.5 Kb present in the hind limb muscle and heart of these animals. These genomic 1,25-dihydroxyvitamin D3 effects could involve transcriptional mechanisms of repression mediated by vitamin D response elements in the rat insulin receptor gene promoter. Using computer analysis of this promoter, we propose the -249/-235 bp VDRE (5’GGGTGACCCGGGGTT3’) with a pyrimidine (T) in the (+7) position of the3’half-site as the best candidate for negative control by 1,25-dihydroxy-vitamin D3. In addition, posttranscriptional mechanisms of regulation could also be implicated. Thus, computer inspection of the5’untranslated region of the rat insulin receptor pre-mRNA indicated the presence of a virtual internal ribosome entry segment whereas the computer inspection of the3’untranslated region localized various destabilizing sequences, including various AU-rich elements. We propose that through these virtual cis-regulatory sequences, 1,25-dihydroxyvitamin D3 could control the translation and stability of insulin receptor mRNA species in the hind limb muscle and heart of diabetic rats.

Protective Effects of Leukemia Inhibitory Factor on Retinal Vasculature and Cells in Streptozotocin-induced Diabetic Mice

Background： Leukemia inhibitory factor （LIF） has been reported to possess various pharmacological effects, including displaying vascular and neuroprotective properties, during retinal disease. The aim of this study was to investigate the vascular and structural changes in the retina of diabetic mice and to explore whether LIF prevents experimental diabetes-induced retinal injury in the early stages. Methods： Diabetes was induced in C57BI/6J mice with streptozotocin （STZ） injections. Successful diabetic animal models were randomly separated into two groups： the diabetic group （n = 15） and the LIF-treated group （n = 15）. Normal C57BL/6 mice served as the normal control group （n = 14）. Recombinant human LIF was intravitreally injected 8 weeks after the diabetic model was successfully established. Retinas were collected and evaluated using histological and immunohistochemical techniques, and flat-mounted retinas and Western blotting were performed at 18 weeks after the induction of diabetes and 2 days after the intravitreal injection of LIF. The analysis of variance test were used. Results： Histological analysis showed that there were fewer retinal ganglion cells （RGCs） and the inner nuclear layer （INL） became thinner in the diabetic model group （RGC 21.8 ± 4.0 and INL 120.2 ± 4.6 μm） compared with the normal control group （RGC 29.0 ± 6.7, t = -3.02, P = 0.007; INL 150.7 ±10.6 lain, t = -8.88, P 〈 0.001, respectively）. After LIF treatment, the number of RGCs （26.9 ± 5.3） was significantly increased （t = 3.39, P = 0.030） and the INL （ 134.5± 14.2 lain） was thicker compared to the diabetic group （t - 2.75, P = 0.013）. In the anti-Brn-3a-labeled retinas, the number of RGCs in the LIF-treated group （3926.0 ± 143.9） was obviously increased compared to the diabetic group （3507.7 ± 286.1, t = 2.38, P = 0.030）, while no significance was found between the LIF-treated group and the control group （4188.3 ± 114.7, t= -2.47, P- 0.069）. Flat-mounted retinas demonstrated that a disorganized, dense distribution of the vessel was prominent in the diabetic model group. Vessel distribution in the LIF-treated mouse group was typical and the thickness was uniform. The levels of phosphosignal transducer and activator of transcription 3 activation were obviously higher in the LIF-injected retinas than those in the diabetic control group （t = 3.85, P = 0.019） and the normal control （t = -3.20, P - 0.019）. Conclusion： The present study provides evidence that LIF treatment protects the integrity of the vasculature and prevents retinal injury in the early stages of diabetic retinopathy in STZ-induced diabetic models.

GLUCOSE TRANSPORTER 2: PREVENTION OF STREPTOZOTOCIN-INDUCED REDUCTION OF 5-THIO-D-GLUCOSE

Decreased expression of glucose transporter 2 (GLUT2) has been found to correlate with impaired secreting β-cell function in diabetes. Here, the aim was to study if the GLUT2 of pancreatic islets is a target structure for streptozotocin (STZ) in vitro. GLLT2 expression was determined by Western blot analysis, using lysates prepared from islets of C57BL / 6mice. A dose-dependent decrement of GLUT2 expression was found after incubation of islets with streptozotocin for 30 min. Compared with solvent-incubated islets, a subtoxic dose (4mM) of STZ resulted in a reduction of approximate 58% of GLUT2 expression and a toxic dose

Hypoglycemic and anti-hyperglycemic study of Gynura procumbens leaf extracts

Objective:To study the antidiabetic activity of Gynura procumbens(G.procumbens)used in the traditional management of diabetes in Southern Asia.Methods:G.procumbens leaves were extracted sequentially with graded percentage of ethanol in water(95%,75%,50%,25%and 0%),and the extracts were tested for antidiabetic activity using acute(7 h),subcutaneous glucose tolerance test and sub-chronic(14 d)test in non-diabetic and streptozotocin-induced diabetic rats.The extracts were further subjected to phytochemical studies.Results:In acute dose(1 g/kg),the extracts significantly lowered fasting blood glucose(FBG)in streptozotocin-induced diabetic rats(P<0.05).However,the FBG-lowering effect of the 25%extract compared to the other extracts,was rapid(47%after 2 h)and the highest:53%,53%and 60%in the 3rd,5th,and 7th h,respectively(P<0.05),comparable only to the effect of metformin.Furthermore,the extracts suppressed peak FBG in subcutaneous glucose tolerance test,but only the 0%and 25%extracts,and metformin sustained the decrease until the 90th min(P<0.05).Moreover,in the 14 days study,the 25%extract exerted the highest FBG-lowering effect,namely 49.38%and 65.43%on days 7 and 14,respectively(P<0.05),similar to the effect of metformin(46.26%and 65.42%).Total flavanoid and phenolic contents in the extracts were found to decrease with increase in polarity of extraction solvents.The composition of reference compounds(chlorogenic acid,rutin,astragalin and kaempferol-3-O-nrtinoside)followed a similar trend.Conclusions:G.procumbens contains antidiabetic principles,most extracted in 25%ethanol.Interaction among active components appears to determine the antidiabetic efficacy,achieved likely by a metformin-like mechanism.

Antidiabetic and antioxidant activities of Nypa fruticans Wurmb. vinegar sample from Malaysia

Objective: To study the antidiabetic and antioxidant activities of nipa palm vinegar(NPV) used in traditional Malay medicine for treating diabetes. Methods: NPV was extracted using liquid-liquid extraction method and the obtained samples were subjected to antidiabetic studies using normal and streptozotocin-induced diabetic rat models whereas antidoxidant activities were investigated via in vitro antioxidant tests namely 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid free radicals scavenging activities and the reducing power assay. Results: Single administration of NPV and its extracts were not effective in both normal and diabetic rats. In intraperitoneal glucose tolerance test, NPV and its aqueous extract showed significant blood glucose lowering effect. In the sub-acute study, compared with the diabetic control, aqueous extract of NPV showed the most notable blood glucose lowering effect(56.6%) and a significant improvement in serum insulin levels(79.8%, P<0.05). To assess NPV's antioxidant activity, three in vitro antioxidant tests were employed: 2,2-diphenyl-1-picryhydrazyl and 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid free radical-scavenging assays, and the reducing power assay. Ethyl acetate extract had the greatest antioxidant potential and content of phenolic and flavonoid compounds. A linear positive correlation between the antioxidant parameters was observed. Chemical profiling analysis of aqueous extract of NPV revealed the presence of acetic acid(35.25%), the main active constituent which significantly contributed to the observed antidiabetic activity. Conclusions: Aqueous extract of NPV possesses antihyperglycaemic activities comparable to the metformin, while the ethyl acetate extract precipitated significant antioxidant effects attributable to its high phenolic content. These findings suggest that antioxidant compounds of NPV do not contribute much towards the overall observed antidiabetic effect.

DIABETOGENIC T CELLS INDUCE AUTOIMMUNE DIABETES IN BALB/c MICE

Objective To investigate the role of T cell and its subsets in the induction of insulifis and type 1 diabetes mellitus （T1DM） in BALB/c mice. Methods Autoimmune diabetes mellitus was developed by intraperitoneal injection of 40 mg/kg streptozotocin （STZ） daily for 5 consecutive days in BALB/c mice as sources of donor cells. Spleen cells from diabetic mice were then cultured for 7 days in the stimulation of interleukin-2 （IL-2） to harvest diabetogenic T cells, which were subse-quently transferred into normal BALB/c mice recipients. MTT, ELISA, and HE staining were used to analyze the lym- phocyte proliferation, cytokine （ IL-2, interferon-γ, IL-4, and IL-10） levels, and pathological changes in pancreatic is- lets. Results As few as 3×10^6 diabetogenic T cells successfully induced diabetes mellitus in recipients pretreated with STZ twice, whereas transfer of equal amount of normal splenocytes, T cell-depleted diabetogenic splenocytes, or diabe-togenic CD4^＋ T cells alone in recipients receiving STZ twice pretreatment was proved not to induce diabetes mellitus either. A markedly increased lymphocyte proliferation, high levels of interferon-γ/and IL-2 in the supernatants of diabeto-genic T cells were observed. In addition, a markedly enhanced lymphocyte proliferation, a high level of interferon-γ/secretion in serum, and numerous lymphocytes infiltration in pancreatic islets were detected in the diabetic mice induced by diabetogenic T cells transfer. Conclusions A novel T1DM murine model is established in STZ-pretreated BALB/c mice by adoptive transfer of diabetogenic T cells. CD4^＋ T cells with interferon-γ/may promote the onset of diabetes mellitus.

Combinatory effect of hesperetin and mesenchymal stem cells on the deteriorated lipid profile,heart and kidney functions and antioxidant activity in STZ-induced diabetic rats

This study aimed to assess the effect of hesperetin and/or bone marrow-derived mesenchymal stem cells(BM-MSCs)on disturbed lipid profile,heart and kidney functions,oxidative stress and antioxidant defense system in streptozotocin(STZ)-induced diabetic rats.Type 1 diabetes mellitus(T1DM)was induced in male Wistar rats by injecting 40 mg/kg body weight(b.w.)STZ dissolved in citrate buffer(pH 4.5).The diabetic rats were treated with hesperetin orally administered at dose 20 mg/kg b.w.,BM-MSCs intravenously injected at a dose of 1 x 106 cells/rat/week and their combination for 6 weeks.The diabetic rats exhibited lipid abnormalities manifested by elevated serum levels of total cholesterol,triglycerides,LDL-cholesterol and VLDL-cholesterol and lowered HDL-cholesterol as well as elevated liver cholesterol and triglycerides content in association with the resultant fasting and postprandial hyperglycemia and insulin deficiency.The heart function biomarkers including CK-MB,AST and LDH activities as well as levels of kidney function parameters,creatinine,and urea,were significantly raised in the serum of diabetic rats.These changes were concomitant with abnormal redox balance represented by elevated lipid peroxidation,decreased glutathione content,and suppressed antioxidant enzyme activities in both heart and kidney of diabetic rats.The previous deleterious alterations were significantly ameliorated after the treatment of diabetic rats with hesperetin and BM-MSCs singly or in combination;the treatment with hesperetin together with BM-MSCs was the most potent.Based on these findings,it can be concluded that the use of hesperetin with BM-MSCs may have more additive therapeutic value than their uses singly in T1DM.In addition,the ameliorative effects of hesperetin and BM-MSCs on lipid profile and heart and kidney functions in diabetic rats may be mediated,at least in part,via their suppressive effects on oxidative stress and ameliorative effects on the antioxidant defense system secondary to improvement in the hyperglycemia and insulin secretory response.

Effect of <i>Pleurotus ostreatus</i>on hyperglycemia, DNA damage and chromosomes aberrations

One of the main health problems with high and markedly increased complications is diabetes. Despite several projects with preventative strategies and armories of medication, the arrangement of diabetes remains grossly unsatisfactory. Thus, it is vital to identify unfamiliar drugs or novel nutraceuticals for treating and preventing diabetes without side effects. The present study deals with scientific information on mushrooms with regards to its potential use as anti-diabetic active food. In addition to the anti-hyperglycemic action of mushrooms, the present study presents its effect on DNA damage, chromosome aberrations and sperm alternations in streptozotocin-induced diabetic rats. These animals have been treated, for 30 days, with amaryl (as control treatment) (0.03 mg/kg·b·wt/dl), low-dose mushroom (100 mg/kg·b·wt/dl) and high-dose mushroom (200 mg/ kg·b·wt/dl). The glucose level GL of streptozotocin-induced diabetic animals has been markedly improved by mushroom treatment;for example GL has decreases from 167.6 mg/dl down to 116.0 mg/dl for treatment with high-dose mushroom and 128.9 mg/dl for treatment with low-dose mushroom, comparing with amaryl treatment that decreases GL down to 92.6 mg/dl. But, the experimental results show that treatment with mushroom is better than treatment with amaryl in case of genetic changes (DNA fragmentation, disappear of some base pairs and chromosome aberrations. So, it is proposed that more close scientific attention be paid to precede more research of functional mushrooms for preventive and curative treatments for diabetes.

The in Vitro Antioxidant Capacities of Hydroalcoholic Extracts from Roots and Leaves of Smallanthus sonchifolius (Yacon) Do Not Correlate with Their in Vivo Antioxidant Action in Diabetic Rats

Leaf and root extracts of Smallanthus sonchifolius (yacon), have antihyper-glycemic activity and antioxidant properties. The present study aims to compare the in vivo hepatic antioxidant activity of hydroalcoholic extracts of yacon leaves and roots in rats with streptozotocin-induced diabetes in terms of their in vitro antioxidant capacity. Rats were treated during 14 days with 1060 mg·Kg-1 root extract or 400 mg·Kg-1 leaf extract. The latter was richer in phenolics and possessed a much higher in vitro antioxidant activity. Both extracts prevented hyperglycemia in diabetic rats. The liver of diabetic rats presented increased levels of protein carbonyls and ROS and decreased activities of antioxidant enzymes. Treatment with both root and leaf extracts restored the protein carbonyl levels to normality. The root extract also restored the ROS levels to normality, but the leaf extract was not effective. The root extract was also more effective in restoring the activity of at least two important antioxidant enzymes (glucose 6-phosphate dehydrogenase and glutathione peroxidase). In terms of the antioxidant load (which was 17 times lower in the root extract treatment), the in vivo action of the root extract was more effective than the leaf extract in reducing the hepatic oxidative stress that accompanies diabetes.