scholarly journals INDUÇÃO DA EXPRESSÃO DA MOLÉCULA INDOLEAMINA 2,3-DIOXIGENASE (IDO) COMO TERAPIA GÊNICA EM TRANSPLANTE DE ILHOTAS PANCREÁTICAS

2009 ◽  
Vol 12 (4) ◽  
pp. 1176-1185
Author(s):  
Humberto Dellê ◽  
Irene Lourdes Noronha
Keyword(s):  
Sprague Dawley ◽  
Rt Pcr ◽  
Terapia Génica

O bloqueio da rejeição ao enxerto é fundamental para o sucesso do transplante de ilhotas pancreáticas (IP). Uma atraente alternativa surge do paradoxo imunológico durante a gravidez, onde a mãe não rejeita o feto imunologicamente distinto. Esta proteção pode ser devido à produção placentária de IDO. Objetivo: Construir um vetor para induzir a expressão de IDO em IP e analisar se a produção de IDO protege as IP em transplante alogênico experimental. Métodos: O cDNA da IDO foi isolado de placenta de ratas, ligado no vetor pcDNA 3,1 e transfectado em IP através de lipofecção. A expressão da IDO nas IP foi confirmada por RT-PCR, imuno-histoquímica e análise funcional. Ratos Lewis diabéticos induzidos por estreptozotocina (glicemia>300mg/dL) receberam IP sob a cápsula renal e foram divididos em 3 grupos: ISO Tx (isogênico), ratos transplantados com IP de ratos Lewis; ALO Tx (alogênico), transplante com IP de ratos Sprague-Dawley (SD); e ALO+IDO Tx, transplante com IP de ratos SD transfectadas com vetor-IDO. Resultados: O grupo ISO permaneceu normoglicêmico pós-transplante, enquanto que o grupo ALO Tx voltou a apresentar hiperglicemia (>300mg/dL) logo após o transplante (11+1 dias). O grupo ALO+IDO Tx manteve-se com glicemia <300mg/dL. No PO-45, o grupo ISO Tx apresentou níveis normais de insulina sérica (0,55+0,13 ng/mL), enquanto que o ALO Tx apresentou diminuição significativa (0,14+0,02 ng/mL;p<0,05). O grupo ALO+IDO Tx apresentou níveis de insulina sérica significativamente maiores comparado ao ALO Tx (0,33+0,04 ng/mL;p<0,05). Conclusão: A indução da expressão de IDO em IP confere proteção às IP, aumenta a sobrevida e promove um melhor controle metabólico.

scholarly journals Transglutaminases Are Active in Perivascular Adipose Tissue

2021 ◽  
Vol 22 (5) ◽  
pp. 2649
Author(s):  
Alexis N. Orr ◽  
Janice M. Thompson ◽  
Janae M. Lyttle ◽  
Stephanie W. Watts
Keyword(s):  
Adipose Tissue ◽  
Vascular Remodeling ◽  
Coagulation Factor ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Ideal Model ◽  
Perivascular Adipose Tissue ◽  
Tissue Sections ◽  
Insight Into ◽  
Immunohistochemical Analyses

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model to test interactions of amines and TGs. We hypothesized that transglutaminases are active in PVAT. Real time RT-PCR determined that Sprague Dawley rat aortic, superior mesenteric artery (SMA), and mesenteric resistance vessel (MR) PVATs express TG2 and blood coagulation Factor-XIII (FXIII) mRNA. Consistent with this, immunohistochemical analyses support that these PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs when in a catalyzing calcium solution. Both TG2 and FXIII were active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT. Finally, experimentally added NE competitively inhibited incorporation of BAP into MRPVAT adipocytes. Further studies to determine the identity of amidated proteins will give insight into how these enzymes contribute to functions of PVAT and, ultimately, blood pressure.

Abstract 333: Existence of Functional Norepinephrine Transport in Perivascular Adipose Tissue

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Nadia Ayala-Lopez ◽  
Robert Burnett ◽  
Janice M Thompson ◽  
Stephanie W Watts
Keyword(s):  
Blood Vessel ◽  
Contractile Function ◽  
Organic Cation Transporter ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Perivascular Adipose Tissue ◽  
Healthy State ◽  
Health And Disease ◽  
Organic Cation Transporter 3 ◽  
Beta 2 Microglobulin

Perivascular Tissue (PVAT) is a recent focus of studies for its regulation of blood vessel tone. Contraction to norepinephrine (NE) is reduced by PVAT through mechanisms not entirely clear. Loss of this anti-contractile function of PVAT occurs in obesity-related hypertension. We hypothesized that PVAT can remove NE from the blood vessel environment through transporters and reduce NE induced contraction in health. RT-PCR of mesenteric PVAT was positive for norepinephrine transporter (NET; Ct=33.2±1.4) and serotonin transporter (SERT; Ct=31.9±0.9) relative to beta-2-microglobulin. To study NE uptake in the healthy state, mesenteric and aortic PVAT from the male Sprague Dawley rat was incubated with 10 μM NE after pre-incubation with an inhibitor of transport (10 μM desipramine to inhibit NET and SERT, 100 μM corticosterone to inhibit organic cation transporter 3) or vehicle and quantified by HPLC for NE. Desipramine plus corticosterone inhibited NE uptake in mesenteric PVAT (figure) but desipramine (410.5±80.8 ng/g vs. 414.4±67.0 ng/g NE only; n=8) or corticosterone (225.0±26.1 ng/g vs. 319.2±35.9 ng/g NE only; n=5) alone did not, indicating a robust uptake capacity. In aortic PVAT, NE uptake was not inhibited by desipramine plus corticosterone (figure), desipramine (1763.5±460.8 ng/g vs. 1702.7±298.4 ng/g NE only; n=6), or corticosterone (1085.8±205.3 ng/g vs. 2245.8±506.8 ng/g NE only; n=4). Protein staining revealed bands positive for NET in mesenteric PVAT by Western blot. Taken together, we found that PVAT is a reservoir of NE that can remove NE from the blood vessel environment, an important finding to understand vasculature-PVAT interactions in health and disease.

scholarly journals Cardioprotective Effect of Danhong Injection against Myocardial Infarction in Rats Is Critically Contributed by MicroRNAs

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Jingrui Chen ◽  
Jing wei ◽  
John Orgah ◽  
Yan Zhu ◽  
Jingyu Ni ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiac Function ◽  
Ventricular Remodeling ◽  
Nuclear Translocation ◽  
Inflammatory Factors ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Danhong Injection ◽  
Sprague Dawley Rats ◽  
Masson Staining

Background. Danhong injection (DHI) has been mainly used for the treatment of myocardial infarction, atherosclerosis, and coronary heart disease in clinical practice. Our previous studies have shown that DHI improves ventricular remodeling and preserves cardiac function in rats with myocardial infarction (MI). In this study, we focused on the potential mechanism of DHI in protecting cardiac function in MI rats. Methods. Sprague-Dawley rats were subjected to ligation of the left anterior descending coronary artery (LAD) to prepare a myocardial infarction (MI) model. After 14 day DHI intervention, cardiac function was measured by echocardiography and myocardial fibrosis was assessed by Masson staining. Differentiated miRNAs were screened using rat immunopathology miScript miRNA PCR arrays, and their results were verified by RT-PCR, immunofluorescence, and immunoblotting. Results. DHI treatment significantly reduced infarct size and improved cardiac function and hemodynamics in MI rats by echocardiography and morphology. miRNA PCR array results showed that DHI reversed 25 miRNAs known to be associated with inflammation and apoptosis. Moreover, the expression of inflammatory factors TNF-α, IL-1β, and IL-6 was significantly reduced in the treated DHI group. Mechanistically, DHI downregulated the inflammatory transcription factor NF-κB (as reflected by inhibition of NF-κB p65 nuclear translocation and phosphorylation of the IκBα). Conclusions. DHI is effective in mitigating inflammation associated with MI by preventing NF-κB nuclear translocation and regulating miRNAs, thereby improving cardiac function in myocardial infarction rats.

scholarly journals Triptolide Attenuates Podocyte Injury by Regulating Expression of miRNA-344b-3p and miRNA-30b-3p in Rats with Adriamycin-Induced Nephropathy

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Chun-Bo Jiang ◽  
Ming-Gang Wei ◽  
Yue Tu ◽  
Hao Zhu ◽  
Chun-Qing Li ◽  
...  
Keyword(s):  
Renal Cortex ◽  
Group Model ◽  
Biological Parameters ◽  
Healthy Male ◽  
Model Group ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Podocyte Injury ◽  
Sprague Dawley Rats

Objectives. We investigated the action of triptolide in rats with adriamycin-induced nephropathy and evaluated the possible mechanisms underlying its protective effect against podocyte injury.Methods. In total, 30 healthy male Sprague-Dawley rats were randomized into three groups (normal group, model group, and triptolide group). On days 7, 28, 42, and 56, 24 h urine samples were collected. All rats were sacrificed on day 56, and their blood and renal tissues were collected for determination of biochemical and molecular biological parameters. Expression of miRNAs in the renal cortex was analyzed by a biochip assay and RT-PCR was used to confirm observed differences in miRNA levels.Results. Triptolide decreased proteinuria, improved renal function without apparent adverse effects on the liver, and alleviated renal pathological lesions. Triptolide also elevated the nephrin protein level. Furthermore, levels of miR-344b-3p and miR-30b-3p were elevated in rats with adriamycin-induced nephropathy, while triptolide treatment reversed the increase in the expression of these two miRNAs.Conclusions. These results suggest that triptolide may attenuate podocyte injury in rats with adriamycin-induced nephropathy by regulating expression of miRNA-344b-3p and miRNA-30b-3p.

scholarly journals Posttraumatic therapeutic hypothermia alters microglial and macrophage polarization toward a beneficial phenotype

2016 ◽  
Vol 37 (8) ◽  
pp. 2952-2962 ◽  
Author(s):  
Jessie S Truettner ◽  
Helen M Bramlett ◽  
W Dalton Dietrich
Keyword(s):  
Brain Injury ◽  
Therapeutic Hypothermia ◽  
Macrophage Polarization ◽  
Temperature Sensitive ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
M2 Microglia ◽  
Sprague Dawley Rats ◽  
Inflammatory Processes ◽  
Clinical Models

Posttraumatic inflammatory processes contribute to pathological and reparative processes observed after traumatic brain injury (TBI). Recent findings have emphasized that these divergent effects result from subsets of proinflammatory (M1) or anti-inflammatory (M2) microglia and macrophages. Therapeutic hypothermia has been tested in preclinical and clinical models of TBI to limit secondary injury mechanisms including proinflammatory processes. This study evaluated the effects of posttraumatic hypothermia (PTH) on phenotype patterns of microglia/macrophages. Sprague-Dawley rats underwent moderate fluid percussion brain injury with normothermia (37℃) or hypothermia (33℃). Cortical and hippocampal regions were analyzed using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) at several periods after injury. Compared to normothermia, PTH attenuated infiltrating cortical macrophages positive for CD11b+ and CD45high. At 24 h, the ratio of iNOS+ (M1) to arginase+ (M2) cells after hypothermia showed a decrease compared to normothermia. RT-PCR of M1-associated genes including iNOS and IL-1β was significantly reduced with hypothermia while M2-associated genes including arginase and CD163 were significantly increased compared to normothermic conditions. The injury-induced increased expression of the chemokine Ccl2 was also reduced with PTH. These studies provide a link between temperature-sensitive alterations in macrophage/microglia activation and polarization toward a M2 phenotype that could be permissive for cell survival and repair.

The P2Y12 Receptor Antagonist Ticagrelor Ameliorates Pulmonary Hypertension

2021 ◽  
Author(s):  
nannan li ◽  
jie yin ◽  
yugen shi ◽  
li sun ◽  
qingshan zhang ◽  
...  
Keyword(s):  
Vascular Remodeling ◽  
Adenosine Diphosphate ◽  
P2y12 Receptor ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Sprague Dawley Rats ◽  
Haemodynamic Changes ◽  
P2y12 Receptor Antagonist ◽  
Pulmonary Arterial ◽  
Adp Receptor

Abstract Background: Pulmonary arterial hypertension (PAH) is a disease that the pulmonary artery is abnormally elevated. P2Y12 is an adenosine diphosphate (ADP) receptor and it act as the target of thienopyridine antiplatelet drugs by controlling vascular remodeling. Inhibition of P2Y12 receptor in the process of PAH was explored in this study.Methods: The PAH model was established in Sprague-Dawley rats by single subcutaneous injection of 60 mg/kg monocrotaline (MCT). The ticagrelor solution (a selective P2Y12R inhibitor) was intraperitoneally injected into rats at a dose of 14 mg/kg from the time of MCT injection to day 28.Results: In the lung tissues of PAH rats, the marked P2Y12R was detected. Treatment with ticagrelor greatly decreased P2Y12R level and efficiently abolished the upregulation of α-SMA as demonstrated by Western blot and RT-PCR. The wall thickness and occlusion score of the pulmonary arterioles showed that blockade of P2Y12R could relieve lung remodeling caused by PAH. The haemodynamic changes at 4 weeks determined that P2Y12R inhibition affected RV pressure and right heart hypertrophy.Conclusions: P2Y12R might be involved in the pathogenesis of PAH. Blockade of P2Y12R has potential in treating PAH.

Gold Nanoparticles with Dexmedetomidine Regulate GSK-3β to Reduce Neurocognitive Effects in Anesthetized Rats

2021 ◽  
Vol 21 (12) ◽  
pp. 6205-6211
Author(s):  
Xiaoxia Zhang ◽  
Zumin Xing ◽  
Jiyuan Li ◽  
Shuyi Tang ◽  
Yiwen Zhang
Keyword(s):  
Gold Nanoparticles ◽  
Mrna Expression ◽  
Intraperitoneal Injection ◽  
Control Group ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Anesthetized Rats ◽  
Sprague Dawley Rats ◽  
Gsk 3Β ◽  
Neurocognitive Effect

The aim of this study was to explore the neurocognitive effects of dexmedetomidine-loaded gold nanoparticles (AuNPs-dexmedetomidine) on anesthetized rats. Sixty Sprague Dawley rats (age, 2–3 weeks; weight, 250–280 g) were randomly divided into three groups (n = 20): the control group and two groups that received intraperitoneal injection of AuNPs-dexmedetomidine at 50 and 100 μg/kg each. Western blotting and RT-PCR were used to determine the protein and mRNA expression of GSK-3β, respectively. Compared with that in the control group, GSK-3β expression in AuNP-dexmedetomidine groups increased (P < 0.05). The protein expression of GSK-3β was higher and mRNA expression was significantly lower in the 100 μg/kg AuNP-dexmedetomidine group (P < 0.05). AuNPs-dexmedetomidine reduced the neurocognitive effect on anesthetized rats through the regulation of the GSK-3β signaling pathway.

Impact of proestrous milieu on expression of orexin receptors and prepro-orexin in rat hypothalamus and hypophysis: actions of Cetrorelix and Nembutal

2007 ◽  
Vol 292 (3) ◽  
pp. E820-E828 ◽  
Author(s):  
Patricia Silveyra ◽  
Paolo N. Catalano ◽  
Victoria Lux-Lantos ◽  
Carlos Libertun
Keyword(s):  
Energy Balance ◽  
Real Time ◽  
Anterior Pituitary ◽  
Hormonal Status ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Adult Rats ◽  
Orexin Receptors ◽  
Brain Expression ◽  
Frontoparietal Cortex

Orexins and their receptors OX1 and OX2 regulate energy balance and the sleep-wake cycle. We studied the expression of prepro-orexin (PPO), OX1, and OX2 in brain and pituitary under the influence of the hormonal status in adult rats. Primarily, PPO, OX1, and OX2 expression was determined in Sprague-Dawley female cycling rats during proestrus and in males. Animals were killed at 2-h intervals. Anterior (AH) and mediobasal (MBH) hypothalamus, anterior pituitary (P), and frontoparietal cortex (CC) were homogenized in TRIzol, and mRNAs were obtained for screening of PPO, OX1, OX2 expression by semiquantitative RT-PCR. Main findings were confirmed and extended to all days of the cycle by quantitative real-time RT-PCR. Hormones and food consumption were determined. Finally, OX1, OX2, and PPO were measured by real-time RT-PCR in tissues collected at 1900 of proestrus after treatments at 1400 with ovulation-blocking agents Cetrorelix or pentobarbital. OX1 and OX2 expression increased at least threefold in AH, MBH, and P, but not in CC, between 1700 and 2300 of proestrus, without variations in estrus, diestrus, or in males. PPO in AH and MBH showed a fourfold or higher increase only during proestrus afternoon. Cetrorelix or pentobarbital prevented increases of OX1 and OX2 only in the pituitary and blunted gonadotropin surges, but left OX1, OX2, and PPO brain expression unchanged. Reproduction, energy balance, and sleep-wake cycle are integrated. Here, we demonstrate that, in the physiological neuroendocrine condition leading to ovulation, information to the orexinergic system acts in hypothalamus and pituitary by different mechanisms.

Elevated hypothalamic orexin signaling, sensitivity to orexin A, and spontaneous physical activity in obesity-resistant rats

2006 ◽  
Vol 291 (4) ◽  
pp. R889-R899 ◽  
Author(s):  
J. A. Teske ◽  
A. S. Levine ◽  
M. Kuskowski ◽  
J. A. Levine ◽  
C. M. Kotz
Keyword(s):  
Gene Expression ◽  
Physical Activity ◽  
Caloric Intake ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Orexin A ◽  
Daily Caloric Intake ◽  
Behavioral Studies ◽  
Sprague Dawley Rats ◽  
Spontaneous Physical Activity

Selectively-bred obesity-resistant [diet resistant (DR)] rats weigh less than obesity-prone [diet-induced obese (DIO)] rats, despite comparable daily caloric intake, suggesting phenotypic energy expenditure differences. Human data suggest that obesity is maintained by reduced ambulatory or spontaneous physical activity (SPA). The neuropeptide orexin A robustly stimulates SPA. We hypothesized that DR rats have greater: 1) basal SPA, 2) orexin A-induced SPA, and 3) preproorexin, orexin 1 and 2 receptor (OX1R and OX2R) mRNA, compared with DIO rats. A group of age-matched out-bred Sprague-Dawley rats were used as additional controls for the behavioral studies. DIO, DR, and Sprague-Dawley rats with dorsal-rostral lateral hypothalamic (rLHa) cannulas were injected with orexin A (0, 31.25, 62.5, 125, 250, and 500 pmol/0.5 μl). SPA and food intake were measured for 2 h after injection. Preproorexin, OX1R and OX2R mRNA in the rLHa, and whole hypothalamus were measured by real-time RT-PCR. Orexin A significantly stimulated feeding in all rats. Orexin A-induced SPA was significantly greater in DR and Sprague-Dawley rats than in DIO rats. Two-mo-old DR rats had significantly greater rLHa OX1R and OX2R mRNA than DIO rats but comparable preproorexin levels. Eight-mo-old DR rats had elevated OX1R and OX2R mRNA compared with DIO rats, although this increase was significant for OX2R only at this age. Thus DR rats show elevated basal and orexin A-induced SPA associated with increased OX1R and OX2R gene expression, suggesting that differences in orexin A signaling through OX1R and OX2R may mediate DIO and DR phenotypes.

scholarly journals Increased Activity of the Intracardiac Oxytocinergic System in the Development of Postinfarction Heart Failure

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Agnieszka Wsol ◽  
Kaja Kasarello ◽  
Marek Kuch ◽  
Kamila Gala ◽  
Agnieszka Cudnoch-Jedrzejewska
Keyword(s):  
Heart Failure ◽  
Mrna Expression ◽  
Protein Level ◽  
Oxytocin Receptor ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Sham Surgery ◽  
Sprague Dawley Rats ◽  
The Right ◽  
Increased Activity

Aim.The present study was designed to test the hypothesis that the development of postinfarction heart failure is associated with a change of activity of the intracardiac oxytocinergic system.Methods.Experiments were performed on male Sprague-Dawley rats subjected to myocardial infarction or sham surgery. Four weeks after the surgery, blood samples were collected and the samples of the left ventricle (LV) and right ventricle (RV) were harvested for evaluation of the mRNA expression (RT-PCR) of oxytocin (OT), oxytocin receptor (OTR), natriuretic peptides, and the level of OT and OTR protein (ELISA). The concentration of N-terminal B-type natriuretic peptide was measured to determine the presence of heart failure.Results.Plasma NT-proBNP concentration was higher in the infarcted rats. In the infarcted rats, the expression of OT mRNA and the OT protein level were higher in the RV. There were no significant differences between infarcted and noninfarcted rats in the expression of OT mRNA and in the OT protein level in the fragments of the LV. In both the left and the right ventricles, OTR mRNA expression was lower but the level of OTR protein was higher in the infarcted rats.Conclusions.In the present study, we indicate that postinfarction heart failure is associated with an increased activity of the intracardiac oxytocinergic system.

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