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1.
Life Sci ; 273: 119286, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662429

RESUMO

AIMS: Hepatic ischemia/reperfusion (I/R) injury is a critical factor affecting the prognosis of liver surgery. The aim of this study is to explore the effects of SET8 on hepatic I/R injury and the putative mechanisms. MAIN METHODS: The expression of SET8 and MARK4 in I/R group and sham group were detected both in vivo and in vitro. In addition, mouse and RAW 264.7 cells were transfected with MARK4 siRNA and SET8 siRNA knockdown of MARK4 and SET8, respectively. The expression of SET8, MARK4 and NLRP3-associated proteins were detected after different treatments. The pathology of liver and the serologic detection were detected after different treatments. KEY FINDINGS: Our present study identified SET domain-containing protein 8 (SET8) as an efficient protein, which can negatively regulate hepatic I/R-mediated inflammatory response and ameliorate hepatic I/R injury by suppressing microtubule affinity-regulating kinase 4 (MARK4)/ NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. The data showed that MARK4 deficiency inhibited hypoxia/reoxygenation (H/R)-induced NLRP3 inflammasome activation, while SET8 deficiency showed the opposite effect. We further demonstrated that SET8 restrained NLRP3 inflammasome activation by inhibiting MARK4. Moreover, we verified SET8 made protective effect on hepatic I/R injury. SIGNIFICANCE: SET8 plays an essential role in hepatic ischemia/reperfusion injury in mice by suppressing MARK4/NLRP3 inflammasome pathway. Our results may offer a new strategy to mitigate hepatic I/R injury.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Histona-Lisina N-Metiltransferase/metabolismo , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Células Cultivadas , Histona-Lisina N-Metiltransferase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
2.
Life Sci ; 273: 119292, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33667516

RESUMO

Delta opioids are thought to relieve ischemic injury and have tissue-protective properties. However, the detailed mechanisms of delta opioids have not been well identified. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), have been shown to mediate downstream signals of δ opioid receptor (δOR) activation through the metalloproteinase (MMP)-dependent EGF-like growth factor (HB-EGF) excretion pathway, which is called transactivation. In this study, to investigate the role of EGFR in δOR-induced anti-ischemic effects in the brain, we applied the middle cerebral artery occlusion (MCAO) model followed by reperfusion to mimic ischemic stroke injury in rats. Pre-treatment with the δOR agonist [D-ala2, D-leu5] enkephalin (DADLE) improved the neurologic deficits and the decreased infarct volume caused by cerebral ischemia/reperfusion injury, which were blocked by the EGFR inhibitor AG1478 and the MMP inhibitor GM6001, respectively. Further results indicated that DADLE activated EGFR, Akt and ERK1/2 and upregulated EGFR expression in the hippocampus in a time-dependent manner, which were inhibited by AG1478 and GM6001. The enzyme-linked immunosorbent assay (ELISA) results showed that δOR activation led to an increase in HB-EGF release, but HB-EGF in tissue was downregulated at the mRNA and protein levels. Moreover, this protective action caused by δOR agonists may involve attenuated hippocampal cellular apoptosis. Overall, these results demonstrate that MMP-mediated transactivation of EGFR is essential for δOR agonist-induced MCAO/reperfusion injury relief. These findings provide a potential molecular mechanism for the neuroprotective property of δOR and may add new insight into mitigating or preventing injury.


Assuntos
Isquemia Encefálica/prevenção & controle , Leucina Encefalina-2-Alanina/farmacologia , Receptores ErbB/metabolismo , Infarto da Artéria Cerebral Média/complicações , Receptores Opioides delta/agonistas , Traumatismo por Reperfusão/prevenção & controle , Ativação Transcricional , Animais , Apoptose , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Receptores ErbB/genética , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
3.
Life Sci ; 273: 119293, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33705733

RESUMO

Experimental studies have shown that ß-caryophyllene (BCP) improved neurological deficits of cerebral ischemia-reperfusion injury (CIRI) rats resulting from Middle Cerebral Artery Occlusion (MCAO). However, research on targets of BCP on CIRI has not been completed. In this study, the mRNA sequencing was used to distinguish various therapeutic multiple targets of BCP on CIRI. Differentially expressed genes (DEGs) were identified from RNA-seq analysis. CIRI induced up-regulated genes (CIRI vs. Sham) and BCP -induced down-regulated genes (BCP vs CIRI) were identified. Significant DEGs were identified only that expressed in each of all samples. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of significant DEGs were determined by cluster Profiler. Protein interactive network (PPI) was analyzed using the String tool and Hub genes was identified by cytoHubba. Transcription factor (TF) regulatory network for the potential Hub genes was constructed. Western blot and ELISA were used to verified hub genes and relative inflammatory cytokines. After mRNA sequencing, a total of 411 DEGs were filtered based on the 2 series (CIRI vs. Sham and CIRI vs. BCP), with Pax1, Cxcl3 and Ccl20 are the most remarkable ones reversed by BCP. GO analysis was represented by DEGs involved in multiple biological process such as extra-cellular matrix organization, leukocyte migration, regulation of angiogenesis, reactive oxygen species metabolic process, etc. KEGG analysis showed that DEGs participated several signaling pathways including MAPK signaling pathway (rno04010), Cytokine-cytokine receptor interaction (rno04060), JAK-STAT signaling pathway (rno04630), and others. The protein-protein interaction (PPI) network consisted of 339 nodes and 1945 connections, and top ten Hub genes were identified by cytoHubba such as TIMP1, MMP-9, and STAT3. Subsequently, a TFs-miRNAs-targets regulatory network was established, involving 6 TFs, 5 miRNAs, and 10 hub genes, consisting of several regulated models such as Brd4 - rno-let-7e - Mmp9, Brd4 - rno-let-7i - Stat3, and Hnf4a- rno-let-7b -Timp1. Finally, western blot demonstrated that BCP could inhibit the increased TIMP1, MMP-9 and STAT3 expression in rat brains after I/R. ELISA represented that BCP could suppress inflammatory cytokines caused by CIRI and present anti-oxidative property. In conclusion, this study shows that the intervention of BCP can significantly reduce neurologic deficit, improve the cerebral ischemia, and a total of ten hub genes were found closely related to the treatment of BCP on CIRI. Prudent experimental validation suggests that the BCP might have the neuro-protective effects in CIRI by decreasing the expression of MMP-9 and TIMP-1, STAT3. In a sense, this study reveals that the MMP-9/TIMP-1 signaling pathway may be involved in the injury after CIRI and thus provides a new treatment strategy as well as a researching method for stroke.


Assuntos
Biomarcadores/análise , Isquemia Encefálica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/complicações , Sesquiterpenos Policíclicos/farmacologia , Traumatismo por Reperfusão/genética , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Biologia Computacional , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia
4.
Methods Mol Biol ; 2269: 139-150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687677

RESUMO

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. The ex vivo NMP hypoxic Rat Liver Perfusion Model represents a feasible approach that allow pharmacological intervention on isolated rat livers by using a combination of NMP and infusion of a number of drugs and/or biological material (cells, microvesicles, etc.). The combination of these two techniques may not only be applied for tissue preservation purposes, but also to investigate the biological effects of molecules and treatment useful in tissue protection. The protocol describes an ex vivo murine model of NMP capable of maintaining liver function despite an ongoing hypoxic injury induced by hemodilution. Furthermore, with this NMP system it is possible to deliver cells treatment or pharmacological intervention to an ex vivo perfused liver and suggests that could represent an innovative approach to recondition organs.


Assuntos
Hepatopatias/metabolismo , Fígado/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Modelos Animais de Doenças , Fígado/patologia , Hepatopatias/patologia , Masculino , Camundongos , Preservação de Órgãos , Perfusão , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia
5.
Life Sci ; 273: 119235, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33607152

RESUMO

Although the central role of Nurr-1/GDNF has been reviewed amply, scarce data are available on their peripheral impact. Carvedilol and morin hydrate have previously conferred their hepatic anti-fibrotic action. AIM: Thus, our aim was to unveil the potential hepatoprotective role of carvedilol (CR) and/or morin hydrate (MH) using a hepatic 70% partial warm ischemia/reperfusion (I/R) rat model. MAIN METHOD: Rats were allocated into sham-operated, hepatic I/R, and I/R preceded by oral administration of CR (10 and 30 mg/kg; CR10/CR30), MH (30 mg/kg), or CR10 + MH for one week. KEY FINDINGS: On the molecular level, pretreatment with CR and/or MH increased the hepatic contents of Nurr-1, GDNF, and the protein expression of active/p-AKT. On the other hand, they inactivated GSK3ß and NF-κB to increase the antioxidant enzymes (GPx, SOD, CAT). All regimens also enhanced the autophagy/lysosomal function and boosted the protein expression of beclin-1, LC3II, and TFEB. Moreover, their antiapoptotic effect was signified by increasing the anti-apoptotic molecule Bcl2 and inhibiting Bax, Bax/Bcl2 ratio, and caspase-3, effects that were confirmed by the TUNEL assay. These improvements were reflected on liver function, as they decreased serum aminotransferases and liver structural alterations induced by I/R. Despite its mild impact, CR10 showed marked improvements when combined with MH; this synergistic interaction overrides the effect of either regimen alone. SIGNIFICANCE: In conclusion, CR, MH, and especially the combination regimen, conferred hepatoprotection against I/R via activating the Nurr-1/GDNF/AKT trajectory to induce autophagy/lysosomal biogenesis, inhibit GSK3ß/NF-кB hub and apoptosis, and amend redox balance.


Assuntos
Carvedilol/farmacologia , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Isquemia/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Antioxidantes/farmacologia , Apoptose , Autofagia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
6.
J Vis Exp ; (168)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33616119

RESUMO

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.


Assuntos
Lesão Renal Aguda/etiologia , Lesão Renal Aguda/patologia , Arteriopatias Oclusivas/complicações , Artéria Renal/patologia , Lesão Renal Aguda/fisiopatologia , Animais , Arteriopatias Oclusivas/fisiopatologia , Modelos Animais de Doenças , Rim/irrigação sanguínea , Rim/patologia , Rim/fisiopatologia , Testes de Função Renal , Masculino , Artéria Renal/fisiopatologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Suínos
7.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440708

RESUMO

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood-brain barrier, which triggers gliosis after ischemic insults.


Assuntos
Calbindina 1/genética , Jejum , Expressão Gênica , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Animais , Calbindina 1/imunologia , Morte Celular/genética , Morte Celular/imunologia , Gerbillinae , Gliose/etiologia , Imunoglobulina G/imunologia , Masculino , Neurônios/metabolismo , Neurônios/patologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia
8.
Nutr Metab Cardiovasc Dis ; 31(1): 333-343, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33500109

RESUMO

BACKGROUND AND AIMS: Diabetes is one of the most important risk factors and comorbidities of ischemic stroke. Endoplasmic reticulum stress (ERS) is considered to be the major injury mechanism of ischemic stroke with diabetes. Studies have found that incretin can inhibit ERS in ischemia-reperfusion injury of the liver and heart. We aimed to explore the effects of GLP-1/GIP double agonist DA3-CH and GLP-1 single agonist liraglutide on ERS and apoptosis in diabetic rats with cerebral ischemia-reperfusion injury. METHODS AND RESULTS: 72 Sprague-Dawley (SD) male rats were randomly divided into 4 groups: ① blank group (Sham group, n = 18); model group (Saline group, n = 18); DA3 treatment group (DA3 group, n = 18); liraglutide treatment group (Lir group, n = 18). The Sham group was not given any treatment and was only raised in the same environment as the other groups. The remaining 3 groups used STZ-induced diabetes models. After the successful membrane formation of diabetes, DA3-CH and liraglutide (10 mmol/kg, once-daily for 14 days) were injected intraperitoneally. Thereafter, rats were subjected to middle cerebral artery occlusion followed by 24-h reperfusion. Animals were evaluated for neurologic deficit score, infarct volume, and biomarker analyses of the brain after ischemia. The DA3-CH-treated and liraglutide-treated groups showed significantly reduced scores of neurological dysfunction and cerebral infarction size, and reduced the expression of ERS markers GRP78, CHOP and Caspase-12, and the expression of apoptosis marker bax. Anti-apoptotic markers bcl-2 and neuronal numbers increased significantly. CONCLUSIONS: DA3-CH and liraglutide have obvious neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury with diabetes, which can reduce the infarct size and the neurological deficit score. Their exert neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury with diabetes by inhibiting endoplasmic reticulum stress and thereby reducing apoptosis. DA3 is better than liraglutide.


Assuntos
Encéfalo/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Incretinas/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Liraglutida/farmacologia , Peptídeos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos Sprague-Dawley , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Estreptozocina
9.
Am J Physiol Renal Physiol ; 320(3): F359-F374, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33427061

RESUMO

Lysophosphatidic acid (LPA) increases platelet-derived growth factor-B (PDGFB) and connective tissue growth factor (CTGF) production and secretion by proximal tubule (PT) cells through LPA2 receptor-Gqα-αvß6-integrin-mediated activation of transforming growth factor-ß1 (TGFB1). LPA2, ß6-integrin, PDGFB, and CTGF increase in kidneys after ischemia-reperfusion injury (IRI), coinciding with fibrosis. The TGFB1 receptor antagonist SD-208 prevents increases of ß6-integrin, TGFB1-SMAD signaling, and PDGFB/CTGF expression after IRI and ameliorates fibrosis (Geng H, Lan R, Singha PK, Gilchrist A, Weinreb PH, Violette SM, Weinberg JM, Saikumar P, Venkatachalam MA. Am J Pathol 181: 1236-1249, 2012; Geng H, Lan R, Wang G, Siddiqi AR, Naski MC, Brooks AI, Barnes JL, Saikumar P, Weinberg JM, Venkatachalam MA. Am J Pathol 174: 1291-1308, 2009). We report now that LPA1 receptor signaling through epidermal growth factor receptor (EGFR)-ERK1/2-activator protein-1 cooperates with LPA2-dependent TGFB1 signaling to additively increase PDGFB/CTGF production and secretion by PT cells. Conversely, inhibition of both pathways results in greater suppression of PDGFB/CTGF production and secretion and promotes greater PT cellular differentiation than inhibiting one pathway alone. Antagonism of the LPA-generating enzyme autotaxin suppressed signaling through both pathways. After IRI, kidneys showed not only more LPA2, nuclear SMAD2/3, and PDGFB/CTGF but also increased LPA1 and autotaxin proteins, together with enhanced EGFR/ERK1/2 activation. Remarkably, the TGFB1 receptor antagonist SD-208 prevented all of these abnormalities excepting increased LPA2. SD-208 inhibits only one arm of LPA signaling: LPA2-Gqα-αvß6-integrin-dependent production of active TGFB1 and its receptor-bound downstream effects. Consequently, far-reaching protection by SD-208 against IRI-induced signaling alterations and tubule-interstitial pathology is not fully explained by our data. TGFB1-dependent feedforward modulation of LPA1 signaling is one possibility. SD-208 effects may also involve mitigation of injury caused by IRI-induced TGFB1 signaling in endothelial cells and monocytes. Our results have translational implications for using TGFB1 receptor antagonists, LPA1 and LPA2 inhibitors concurrently, and autotaxin inhibitors in acute kidney injury to prevent the development of chronic kidney disease.


Assuntos
Lesão Renal Aguda/metabolismo , Citocinas/metabolismo , Túbulos Renais Proximais/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Linhagem Celular , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Modelos Animais de Doenças , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Túbulos Renais Proximais/patologia , Linfocinas/metabolismo , Masculino , Camundongos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos Sprague-Dawley , Receptores de Ácidos Lisofosfatídicos/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
10.
Am J Physiol Heart Circ Physiol ; 320(3): H1185-H1198, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416452

RESUMO

Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.


Assuntos
Caveolina 2/deficiência , Adesão Celular , Células Endoteliais/metabolismo , Doenças do Jejuno/metabolismo , Jejuno/irrigação sanguínea , Migração e Rolagem de Leucócitos , Leucócitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Migração Transendotelial e Transepitelial , Vênulas/metabolismo , Animais , Caveolina 2/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Doenças do Jejuno/genética , Doenças do Jejuno/patologia , Leucócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Vênulas/patologia
11.
Am J Physiol Renal Physiol ; 320(3): F308-F321, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33427060

RESUMO

Renal ischemia-reperfusion (I/R) injury is associated with markedly reduced protein expression of aquaporins (AQPs). Membrane G protein-coupled bile acid receptor-1 (TGR5) has shown protective roles in some kidney diseases. The purpose of the current study was to investigate whether activation of TGR5 prevented the decreased protein expression of AQPs in rodents with renal I/R injury and potential mechanisms. TGR5 agonist lithocholic acid (LCA) treatment reduced polyuria after renal I/R injury in rats. LCA prevented the decreased abundance of AQP2 protein and upregulated hypoxia-inducible factor (HIF)-1α protein expression, which were associated with decreased protein abundance of NF-κB p65 and IL-1ß. After renal I/R, mice with tgr5 gene deficiency exhibited further decreases in AQP2 and HIF-1α protein abundance and increases of IL-1ß and NF-κB p65 protein expression compared with wild-type mice. In primary cultured inner medullary collecting duct cells with hypoxia/reoxygenation, LCA induced markedly increased protein expression of AQP2 and HIF-1α, which were partially prevented by the PKA inhibitor H89. FG4592, a prolyl-4-hydroxylase domain-containing protein inhibitor, increased HIF-1α and AQP2 protein abundance in association with decreased NF-κB p65 protein expression in inner medullary collecting duct cells with hypoxia/reoxygenation. In conclusion, TGR5 stimulation by LCA prevented downregulation of renal AQPs in kidney with I/R injury, likely through activating HIF-1α signaling and suppressing inflammatory responses.NEW & NOTEWORTHY Stimulation of the membrane G protein-coupled bile acid receptor TGR5 by lithocholic acid (LCA) reduced polyuria in rats with renal ischemia-reperfusion (I/R) injury. LCA increased abundance of aquaporin-2 (AQP2) protein and upregulated hypoxia-inducible factor (HIF)-1α protein expression in association with decreased NF-κB p65 and IL-1ß. After I/R, mice with tgr5 gene deficiency exhibited more severe decreases in AQP2 and HIF-1α protein abundance and inflammatory responses. TGR5 activation exhibits a protective role in acute renal injury induced by I/R.


Assuntos
Aquaporina 2/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos Wistar , Receptores Acoplados a Proteínas-G/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fator de Transcrição RelA/metabolismo
12.
J Stroke Cerebrovasc Dis ; 30(3): 105595, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33450605

RESUMO

BACKGROUND: Endovascular thrombectomy (EVT) is highly effective but may also lead to hemorrhagic transformation (HT) and edema, which may be more pronounced in severe ischemia. We sought to determine whether glibenclamide can attenuate HT and edema in a severe ischemia-reperfusion model that reflects EVT. METHODS: Using a transient middle cerebral artery occlusion (tMCAo) rodent model of stroke, we studied two rat cohorts, one without rt-PA and a second cohort treated with rt-PA. Glibenclamide or vehicle control was administered as an intravenous bolus at reperfusion, followed by continuous subcutaneous administration with an osmotic pump. RESULTS: Compared to vehicle control, glibenclamide improved neurological outcome (median 7, interquartile range [IQR 6-8] vs. control median 6 [IQR 0-6], p = 0.025), reduced stroke volume (323 ± 42 vs. 484 ± 60 mm3, p < 0.01), swelling volume (10 ± 4 vs. 28 ± 7%, p < 0.01) and water content (84 ± 1 vs. 85 ± 1%, p < 0.05). Glibenclamide administration also reduced HT based on ECASS criteria, densitometry (0.94 ± 0.1 vs. 1.15 ± 0.2, p < 0.01), and quantitative hemoglobin concentration (2.7 ± 1.5 vs. 6.2 ± 4.6 uL, p = 0.011). In the second cohort with rt-PA coadministration, concordant effects on HT were observed with glibenclamide. CONCLUSIONS: Taken together, these studies demonstrated that glibenclamide reduced the amount of edema and HT after severe ischemia. This study suggests that co-administration of glibenclamide may be worth further study in severe stroke patients treated with EVT with or without IV rt-PA.


Assuntos
Edema Encefálico/prevenção & controle , Glibureto/administração & dosagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Hemorragias Intracranianas/prevenção & controle , Fármacos Neuroprotetores/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Edema Encefálico/diagnóstico por imagem , Edema Encefálico/patologia , Modelos Animais de Doenças , Fibrinolíticos/farmacologia , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/patologia , Infusões Subcutâneas , Injeções Intravenosas , Hemorragias Intracranianas/diagnóstico por imagem , Hemorragias Intracranianas/patologia , Masculino , Ratos Wistar , Traumatismo por Reperfusão/diagnóstico por imagem , Traumatismo por Reperfusão/patologia , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/farmacologia
13.
Life Sci ; 266: 118879, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310030

RESUMO

Acute renal injury (AKI) is a risk factor for the development of hypertension, which involves oxidative stress, changes in Na+ handling, and the intrarenal renin-angiotensin-aldosterone system (RAAS) as underlying mechanisms. We investigated in rats whether renal ischemia-reperfusion (IR) leads to changes in the proximal tubule ATP-dependent Na+ transport and the intrarenal content of RAAS components, as well as the role of NADPH oxidase. Rats weighing 300-350 g were submitted to AKI by bilateral IR (n = 25). After IR injury, the animals were followed up for 4 weeks. One part (n = 7) received daily treatment with the NADPH oxidase inhibitor apocynin (100 mg/kg, drinking water), while another part (n = 9) received apocynin 24 h before and after IR. One group was submitted to sham surgery (n = 8). Four weeks after IR, the rats presented elevated systolic blood pressure, as well as increased lipid peroxidation, NADPH oxidase activity, (Na++K+)ATPase activity, and upregulation of type 1 angiotensin II receptor in the renal cortex. On the other hand, there was a decrease in Na+-ATPase activity and downregulation of the isoforms 1 and 2 of the angiotensin-converting enzyme, type 2 angiotensin II receptor, and of the α and ε isoforms of protein kinase C. Most of these alterations was prevented by both apocynin treatment protocols. Thus, we conclude that AKI-induced by IR may induce changes in proximal tubule ATPases and RAAS components compatible with renal Na+ retention and hypertension. These data also indicate that the NADPH oxidase represents a key factor in the origin of these alterations.


Assuntos
Lesão Renal Aguda/complicações , Hipertensão/patologia , Túbulos Renais Proximais/patologia , NADPH Oxidases/metabolismo , Sistema Renina-Angiotensina , Traumatismo por Reperfusão/complicações , Sódio/metabolismo , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Aldosterona/metabolismo , Animais , Hipertensão/enzimologia , Hipertensão/etiologia , Túbulos Renais Proximais/metabolismo , Masculino , Estresse Oxidativo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
14.
Biomed Pharmacother ; 133: 111086, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378987

RESUMO

Testicular torsion is an acute urological emergency condition that occurs due to obstruction of blood flow to the testicles which may result in ischemia and loss of testicular functions. This study examined the protective effects of Proxeed Plus (PP), a dietary supplement on testicular ischemia/reperfusion (I/R) injured rats using oxidative stress markers, hormonal levels, apoptotic parameters, histological and immunohistochemistry analysis at 4 h and after 7 days of reperfusion. The protective treatment of the I/R injured rats with PP at 1000 and 5000 mg/kg body weight (bw) resulted in significant increases in the serum and tissue antioxidative defense capacities (superoxide dismutase, reduced glutathione, catalase, glutathione-s-transferase, and glutathione peroxidase), sex hormones (luteinizing hormone, follicle-stimulating hormone, and testosterone), also reduce pro-oxidative markers (malondialdehyde and hydrogen peroxide), serum iNOS and apoptotic parameters (Caspase -3 and Caspase -9) in comparison to the results detected in the I/R untreated rats. It was also observed that PP ameliorated histological changes of I/R injured rats; increased spermatogenetic activity, seminiferous tubular diameter, Leydig cell mass, and reduced expressions of testicular inducible nitric oxide synthase (iNOS). Therefore, the therapeutic use of Proxeed Plus could be considered a promising approach in averting testicular damage against I/R injury.


Assuntos
Antioxidantes/farmacologia , Suplementos Nutricionais , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Torção do Cordão Espermático/tratamento farmacológico , Testículo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Modelos Animais de Doenças , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Torção do Cordão Espermático/enzimologia , Torção do Cordão Espermático/patologia , Espermatogênese/efeitos dos fármacos , Testículo/enzimologia , Testículo/patologia
15.
Biomed Pharmacother ; 134: 111130, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33348309

RESUMO

OBJECTIVE: Dimethyl fumarate (DMFU), a known Nrf2 activator, has proven its positive effect in different organs against ischemia/reperfusion (Is/Re) injury. Nevertheless, its possible impact to modulate intestinal Is/Re-induced injury has not been previously demonstrated before. Hence, this study aimed to investigate DMFU mechanistic maneuver against intestinal Is/Re. METHODS: To accomplish this goal, Wistar rats were allocated into four groups; Sham-operated (SOP), intestinal Is/Re (1 h/6 h), and 14 days pre-treated DMFU (15 and 25 mg/kg/day, p.o). RESULTS: The mechanistic maneuver divulged that DMFU safeguarded the intestine partly via amplifying the expression/content of Nrf2 along with enhancing its downstream, HO-1 expression/content. In addition, DMFU lessened GSK-3ß expression/content accompanied by enriching ß-catenin expression/content. The antioxidant action was affirmed by enhancing total antioxidant capacity, besides reducing MDA, iNOS, and its by-product, NOx. The DMFU action entailed anti-inflammatory character manifested by down-regulation of expression/content NF-κB with subsequent rebating the contents of TNF-α, IL-1ß, and P-selectin, as well as MPO activity. Moreover, DMFU had anti-apoptotic nature demonstrated through enriching Bcl-2 level and diminishing that of caspase-3. CONCLUSION: DMFU purveyed tenable novel protective mechanisms and mitigated events associated with intestinal Is/Re mischief either in the lower or the high dose partly by amending of oxidative stress and inflammation through the modulation of Nrf2/HO-1, GSK-3ß, and Wnt/ß-catenin pathways.


Assuntos
Anti-Inflamatórios/farmacologia , Fumarato de Dimetilo/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Enteropatias/prevenção & controle , Intestinos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/genética , Heme Oxigenase (Desciclizante)/genética , Enteropatias/enzimologia , Enteropatias/genética , Enteropatias/patologia , Intestinos/enzimologia , Intestinos/patologia , Masculino , Fator 2 Relacionado a NF-E2/genética , Estresse Nitrosativo/efeitos dos fármacos , Ratos Wistar , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia
16.
J Stroke Cerebrovasc Dis ; 30(2): 105516, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33310074

RESUMO

OBJECTIVE: To investigate the effects of different degrees of carotid artery stenosis (CAS) on the expression of XIAP and Smac in ischemic penumbra of rats with cerebral ischemia-reperfusion (I/R). MATERIALS AND METHODS: Samples were collected at 12 h and 24 h after reperfusion, and then the treated groups were divided into the NC-12 group, NC-24 group, MIS-12 group, MIS-24 group, MOS-12 group, MOS-24 group, SES-12 group and SES-24 group. HE staining was used to observe the pathological changes of the brain tissue. TUNEL assay was used to detect the apoptosis in the ischemic penumbra. IHC and RT-qPCR were used to detect the expression of XIAP and Smac in the brain tissue. RESULTS: By observing the pathological sections of brain tissue, the rats in MIS, MOS and SES groups showed loose brain tissue on the infarcted side and neuronal pyknosis in the ischemic penumbra. And with the aggravation and prolongation of the degree of stenosis, the degree of brain injury deepened. It was further found that the TUNEL positive rate was significantly increased in the ischemic penumbra in the SES and MOS groups compared with that in the normal control (NC) group. The results of IHC and RT-qPCR showed that the mRNA expression of XIAP and Smac in the ischemic penumbra was significantly up-regulated in the MIS, MOS and SES groups compared with that in the NC group. CONCLUSIONS: CAS may activate XIAP/Smac signaling pathway to induce neuronal apoptosis and promote the injury in the ischemic penumbra caused by cerebral I/R.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Isquemia Encefálica/etiologia , Encéfalo/metabolismo , Estenose das Carótidas/complicações , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Traumatismo por Reperfusão/etiologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Encéfalo/patologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Proteínas Inibidoras de Apoptose/genética , Masculino , Proteínas Mitocondriais/genética , Neurônios/patologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Índice de Gravidade de Doença , Transdução de Sinais , Fatores de Tempo
17.
Methods Mol Biol ; 2225: 275-292, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108669

RESUMO

Ischemia-reperfusion injury (IRI) drives early and long-term damage to organs as well as compounding damage from acute transplant rejection and surgical trauma. IRI initiates an aggressive and prolonged inflammation leading to tissue injury, organ failure, and death. However, there are few effective therapeutic interventions for IRI. The destructive inflammatory cell activity in IRI is part of an aberrant innate immune response that triggers multiple pathways. Hence, immune-modulating treatments to control pathways triggered by IRI hold great therapeutic potential. Viruses, especially large DNA viruses, have evolved highly effective immune-modulating proteins for the purpose of immune evasion and to protect the virus from the host immune defenses. A number of these immune-modulating proteins have proven therapeutically effective in preclinical models, many with function targeting pathways known to be involved in IRI. The use of virus-derived immune-modulating proteins thus represents a promising source for new treatments to target ischemia-reperfusion injury. Laboratory small animal models of IRI are well established and are able to reproduce many aspects of ischemia-reperfusion injury seen in humans. This chapter will discuss the methods used to perform the IRI procedure in mice, as well as clinically relevant diagnostic tests to evaluate liver injury and approaches for assessing histological damage while testing novel immune modulating protein treatments.


Assuntos
Anti-Inflamatórios/farmacologia , Hepatite/prevenção & controle , Fatores Imunológicos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Proteínas Virais/farmacologia , Isquemia Quente/métodos , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Biomarcadores/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Hepatite/genética , Hepatite/imunologia , Hepatite/patologia , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/biossíntese , Fatores Imunológicos/imunologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/imunologia , Fígado/patologia , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Coloração e Rotulagem/métodos , Proteínas Virais/biossíntese , Proteínas Virais/imunologia
18.
Vasc Endovascular Surg ; 55(1): 11-17, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32878581

RESUMO

INTRODUCTION: Naftidrofuryl and cilostazol are drugs with proven efficacy in the treatment of claudication in peripheral vascular disease. In this experimental study, we evaluated the effects of naftidrofuryl and cilostazol in ischemia-reperfusion (IR) injury on various tissues. MATERIALS AND METHODS: 40 male albino Wistar rats (8-12 weeks old, 250-350 g.) are randomly divided into 4 groups: Control (Group 1), sham (group 2), cilostazol pre-treatment (group 3), naftidrofuryl pre-treatment (group 4). During 21 days placebo is given to group 2, 12 mg/kg/day cilostazol is given to group 3, 50 mg/kg/day naftidrofuryl is given to group 4 orally. Ischemia and reperfusion are induced at the lower hind limb in Groups 2, 3 and 4. Ischemic muscle, kidney, liver, heart, brain and blood samples are obtained. The total antioxidant capacity, oxidant levels and oxidative stress index are studied for each group. RESULTS: Both drugs have protective effects of remote organ injury following IR. Systemic effects are similar to each other, both have protective effects of IR injury. It showed no statistical significance in the total antioxidant capacity. Total oxidant levels are significantly affected by cilostazol in the heart (p < 0.01) and by naftidrofuryl in the liver (p < 0.01). The effect on oxidative stress was only significant with cilostazol on the heart (p < 0.01). CONCLUSION: Cilostazol and naftidrofuryl had beneficial effects in all tissues against tissue damage caused by IR injury. In ischemic muscle, kidney and heart cilostazol had improved outcomes comparing to naftidrofuryl. Naftidrofuryl had benefits over cilostazol in liver tissue.


Assuntos
Antioxidantes/farmacologia , Encéfalo/irrigação sanguínea , Cilostazol/farmacologia , Rim/irrigação sanguínea , Fígado/irrigação sanguínea , Músculo Esquelético/irrigação sanguínea , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Nafronil/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
19.
J Ethnopharmacol ; 265: 113410, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32980487

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The Tibetan turnip (Brassica rapa L.) has a wide array of medicine properties including heat-clearing, detoxifying and anti-hypoxia as listed in the famous centuries-old Tibetan medicine classic "The Four Medical Tantras". Evidence-based medicine also indicated the anti-hypoxic effect of turnips, suggesting a potential link to neuroprotective effect on ischemic stroke. This thereby enables turnips to serve as a novel nontoxic agent in related treatment. AIM OF THE STUDY: This study aimed to investigate the neuroprotective effect and elucidate the mechanism of aqueous extract of turnip (AET) on cerebral ischemia/reperfusion. MATERIALS AND METHODS: The experimental models of cerebral ischemia included transient middle cerebral artery occlusion/reperfusion (MCAO) in C57BL/6J mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in HT-22 cells. Long-term effect of AET on infarct volume was evaluated by microtubule-associated protein 2 (MAP2) immunofluorescence 28 days after MCAO, and on neurofunctional outcomes determined by rotarod, grid walking, and cylinder tests in the meantime. Efficacy of AET was determined by the cell viability, the release of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) in neurons. The underlying mechanism of AET rescued OGD/R cells were characterized by PI3K, Akt and mTOR expressions, which were further used to validate AET's role in the pathway. RESULTS: AET can reduce cerebral infarct volume and ameliorate behavioral deficits of MCAO/R mice dose-dependently. In vitro experiment further demonstrated that suitable concentrations of AET inhibited ROS, LDH production and restored mitochondrial expression induced by OGD/R. AET pretreatment can reverse the OGD/R-induced decreased level of phosphorylation of PI3K, Akt, mTOR, whereas this effect was blocked in the LY294002 (PI3K inhibitor) treatment group. CONCLUSIONS: AET improved the survival of OGD/R-injured HT-22 cells by activating the PI3K/Akt/mTOR pathway. Based on the results above, aqueous extract of turnip has a protective effect on focal cerebral ischemic injury.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Brassica rapa/química , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Animais , Isquemia Encefálica/patologia , Linhagem Celular , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/isolamento & purificação , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Serina-Treonina Quinases TOR/metabolismo , Tibet
20.
J Vis Exp ; (166)2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33369603

RESUMO

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.


Assuntos
Traumatismo por Reperfusão/patologia , Traumatismos da Medula Espinal/patologia , Isquemia do Cordão Espinal/patologia , Medula Espinal/patologia , Animais , Aorta/cirurgia , Modelos Animais de Doenças , Feminino , Hemodinâmica , Masculino , Microcirculação , Medula Espinal/irrigação sanguínea , Suínos
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