RESUMO
CONTEXT: Calenduloside E (CE), one of the primary natural products found in Aralia elata (Miq.) Seem. (Araliaceae), possesses prominent anti-apoptotic potential. A previous study found that one of the anti-apoptotic CE targets is heat shock protein 90 AB1 (Hsp90AB1) by probe CE-P, while the other targets of CE still need to be identified with more efficient probes. OBJECTIVE: This study investigates CE analogue (CEA) as one clickable activity-based probe for use in exploring anti-apoptotic CE targets. MATERIALS AND METHODS: Pretreatment of HUVECs with CEA (1.25 µM) for 8 hr, followed by ox-LDL stimulation for 24 h. Flow cytometry analysis and JC-1 staining assays were performed The kinetic constant measurements were tested by the Biacore T200, CM5 Sensor Chip which was activated by using sulpho-NHS/EDC. Ligands were dissolved and injected with a concentration of 12.5, 6.25, 3.125, 1.56, 0.78 and 0 µM. RESULTS: CEA was confirmed to possess an anti-apoptotic effect. The probable targets of CE/CEA were calculated, and as one of the higher scores proteins (Fit values: 0.88/0.86), Hsp90 properly got our attention. Molecular modelling study showed that both CE and CEA could bind to Hsp90 with the similar interaction, and the docking scores (S value) were -7.61 and -7.33. SPR assay provided more evidence to prove that CEA can interact with Hsp90 with the KD value 11.7 µM. DISCUSSION AND CONCLUSIONS: Our results suggest that clickable probe CEA could alleviate ox-LDL induced apoptosis by a similar mechanism of anti-apoptotic CE, and afforded the possibility of identifying additional anti-apoptotic targets of CE.
Assuntos
Apoptose/efeitos dos fármacos , Química Click , Modelos Moleculares , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas LDL/metabolismo , Simulação de Acoplamento Molecular , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/farmacologia , Saponinas/administração & dosagemRESUMO
We investigated whether Xiao-Xu-Ming decoction reduced mitophagy activation and kept mitochondrial function in cerebral ischemia-reperfusion injury. Rats were randomly divided into 5 groups: sham, ischemia and reperfusion (IR), IR plus XXMD (60 g/kg/day) (XXMD60), IR plus cyclosporin A (10 mg/kg/day) (CsA), and IR plus vehicle (Vehicle). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion (MCAO). Cerebral infarct areas were measured by triphenyl tetrazolium chloride staining. Cerebral ischemic injury was evaluated by hematoxylin and eosin staining (HE) and Nissl staining. Ultrastructural features of mitochondria and mitophagy in the penumbra of the ischemic cortex were observed by transmission electron microscopy. Mitophagy was detected by immunofluorescence labeled with LC3B and VDAC1. Autophagy lysosome formation was observed by immunofluorescence labeled with LC3B and Lamp1. The expression of LC3B, Beclin1, and Lamp1 was analyzed by Western blot. The rats subjected to MCAO showed worsened neurological score and cell ischemic damage. These were all significantly reversed by XXMD or CsA. Moreover, XXMD/CsA notably downregulated mitophagy and reduced the increase in LC3, Beclin1, and Lamp1 expression induced by cerebral ischemia and reperfusion. The findings demonstrated that XXMD exerted neuroprotective effect via downregulating LC3, Beclin1, Lamp1, and mitochondrial p62 expression level, thus leading to the inhibition of mitophagy.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Medicina Tradicional Chinesa , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
This study examined the course of mitophagy following cerebral ischemia with reperfusion and the role of the PTEN-induced kinase 1 (PINK1)/Parkin/p62 signalling pathway. The middle cerebral artery of male Sprague-Dawley rats was occluded for 90â¯min and was followed by different time-points of reperfusion. Cerebral infarct areas were detected by 2,3,5-triphenyl tetrazolium chloride staining, while brain damage was observed by haematoxylin and eosin staining. Levels of LC3, Beclin1 and LAMP-1 were estimated by western blots. LC3B location was observed in various cells in the neurovascular unit. In addition, PINK1 accumulation in damaged mitochondria and Parkin/p62 mitochondrial translocation were investigated by double immunofluorescence staining. Finally, the levels of PINK1, Parkin and p62 expression in mitochondrial fractions were estimated by western blots. Cerebral ischemia with different time-points of reperfusion resulted in infarct in the territory of the middle cerebral artery accompanied by overall brain damage. In addition, we found up-regulation of LC3B, Beclin1, and LAMP-1, as well as mitophagy activation after reperfusion, with peak expression of these proteins at 24â¯h after reperfusion. Electron microscopy and immunofluorescence indicated that LC3B was primarily located in neurons, although lower levels of expression were found in astrocytes and even less in vascular endothelial cells. Moreover, significant increases in PINK1 accumulation in the outer membrane of mitochondria and increased Parkin/p62 mitochondrial translocation were shown at 24â¯h after reperfusion. These ï¬ndings suggest that the PINK1/Parkin/p62 signalling pathway was involved in the pathophysiological processes following ischemia and reperfusion.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Mitofagia/fisiologia , Traumatismo por Reperfusão/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Autofagia/fisiologia , Encéfalo/patologia , Isquemia Encefálica/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Expressão Gênica , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Quinases/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Modulation of apoptosis is therapeutically effective in cardiomyocytes damage. Calenduloside E (CE), a naturally occurring triterpenoid saponin, is a potent anti-apoptotic agent. However, little is known about its synthetic analogues on the protective effects in apoptosis of cardiomyocytes. The present research was performed to investigate the potential protective effect of CE analogues against H2O2-induced apoptosis in H9c2 cardiomyocytes and the underlying mechanisms. Sixteen novel CE anologues have been designed, synthesized and biological evaluation. Among the 16 CE anologues, as well as the positive control CE tested, compound 5d was the most effective in improving cardiomyocytes viability. Pretreatment with anologue 5d inhibited ROS generation, maintained the mitochondrial membrane potential and reduced apoptotic cardiomyocytes. Moreover, exposure to H2O2 significantly increased the levels of Bax, cleaved caspase-3, and cleaved PARP, and decreased the level of Bcl-2, resulting in cell apoptosis. Pretreatment with anologue 5d (0.02-0.5 µg/mL) dose-dependently upregulated antiapoptotic proteins and downregulated proapoptotic proteins mentioned above during H2O2-induced apoptosis. These results suggested that CE analogues provide protection to H9c2 cardiomyocytes against H2O2-induced oxidative stress and apoptosis, most likely via anti-apoptotic mechanism, and provided the basis for the further optimization of the CE analogues.
RESUMO
The aging processes of copper and zinc in single and combined metal-contaminated typical soils of China, red soil, paddy soil and cinnamon soil, were studied. The results showed that available copper and zinc (metals extracted by 0.01 mol x L(-1) CaCl2 ) decreased rapidly at initial stages, and then reduced slowly, where the turning point occurred at about 90 d. The difference in aging of copper and zinc was insignificant whether in single metal-contaminated soils or in combined metal-contaminated soils, suggested there were similar chemical behaviors between copper and zinc. The aging processes of copper and zinc in red soil, paddy soil and cinnamon soil fitted best a second-order equation (R2 = 0.9940-0.9999, p < 0.0001), whereas parabolic diffusion equation has less goodness of fit. It indicated that the transformation from availability to unavailability of metals, i.e. aging, was not completely controlled by diffusion, but controlled by the interactions from surface nucleation/precipitation, occlusion by organic matter, and diffusion, etc. The aging of copper and zinc in soils was significantly affected by pH. In soils with low pH such as red soil, the ratio of available metals was higher and the aging rate was slower [constant of rate, k2 4.36x10(-3)-7.05x10(-3) kg x (mg x d)(-1)]; whereas in soils with high pH, for example in cinnamon soil, the ratio of available metals was lower and the aging rate was faster [k2 1.095x10(-2)-1.377x10(-2) kg x (mg x d)(-1)]. That is, the aging rate of metals in soils increased obviously with increasing pH.