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1.
Invest Ophthalmol Vis Sci ; 62(7): 6, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34086044

RESUMO

Purpose: To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. Methods: Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. Results: We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. Conclusions: Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Retinopatia Diabética/enzimologia , Receptores Virais/metabolismo , Retina/enzimologia , SARS-CoV-2/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sítios de Ligação , Western Blotting , Células Cultivadas , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/virologia , Retinopatia Diabética/patologia , Retinopatia Diabética/virologia , Endotélio Vascular/enzimologia , Endotélio Vascular/virologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Pericitos/enzimologia , Pericitos/virologia , Vasos Retinianos/enzimologia , Vasos Retinianos/patologia , Vasos Retinianos/virologia , Serina Endopeptidases/metabolismo
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(6): 551-556, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34060450

RESUMO

Objective To prepare the fusion protein MVF-ErbB3II composed of measles virus fusion (MVF) protein 288 to 302 amino acid peptide and human epidermal growth factor receptor 3 (ErbB3) 236 to 308 amino acid (ErbB3II) peptide, then prepare and characterize the anti-MVF-ErbB3II polyclonal antibody (pcAb). Methods The MVF-ErbB3II gene was synthesized artificially and subcloned into pET-21b plasmid using DNA ligase. After transformation, the recombinant MVF-ErbB3II protein was expressed in E. coli BL21 (DE3) and purified using nickel ion affinity chromatography. Subsequently, the purified MVF-ErbB3II protein was used as antigen to immunize rats subcutaneously for induction of anti-MVF-ErbB3IIpcAb. The titer of anti-MVF-ErbB3II pcAb was analyzed by ELISA. The ErbB3 specificity and targeting ability of pcAb were evaluated by Western blotting, immunoprecipitation (IP) and flow cytometry (FCM). Results SDS-PAGE confirmed that MVF-ErbB3II protein was successfully expressed and purified. ELISA showed that the titer of pcAb was 1 024 000. Western blotting, IP and FCM assays showed that the anti-MVF-ErbB3II pcAb not only had good antigen specificity against purified MVF-ErbB3II and native ErbB3 but targeted the ErbB3 dimerization interface. Conclusion The prokaryotic expression and purification of MVF-ErbB3II is successfully achieved, rat anti-MVF-ErbB3II pcAb is prepared and characterized successfully.


Assuntos
Escherichia coli , Receptor ErbB-3 , Animais , Especificidade de Anticorpos , Western Blotting , Dimerização , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Ratos , Receptor ErbB-3/genética
3.
Methods Mol Biol ; 2255: 1-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033089

RESUMO

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.


Assuntos
Apoptose , Western Blotting/métodos , Caspase 8/metabolismo , Caspase 9/metabolismo , Neoplasias Ovarianas/patologia , Ativação Enzimática , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
4.
Methods Mol Biol ; 2255: 21-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033091

RESUMO

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.


Assuntos
Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Western Blotting , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
5.
Methods Mol Biol ; 2255: 119-134, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033099

RESUMO

The study of necroptosis is a rapidly growing field in current research of cell death mechanisms and cancer treatment strategies. While apoptotic cells can be reliably identified via annexin V assay, necroptosis is not associated with exposure of easily detectable markers. The most reliable way to identify necroptotic events is immunochemical detection of active phosphorylated RIPK1, RIPK3, and MLKL proteins facilitating necroptosis execution. This chapter describes a detailed protocol on necroptosis induction in human colon adenocarcinoma HT-29 cells, preparation of various positive and negative controls, detection of necroptosis mediator proteins via Western Blot analysis, and interpretation of results. This protocol allows reliable and specific detection of necroptosis in cell culture or tissue samples, and it provides a well-established model suitable for detailed studies of necroptosis molecular mechanisms in vitro.


Assuntos
Adenocarcinoma/patologia , Western Blotting/métodos , Neoplasias do Colo/patologia , Necroptose , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Humanos , Indóis/farmacologia , Fosforilação , Células Tumorais Cultivadas
6.
Anticancer Res ; 41(5): 2297-2306, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33952455

RESUMO

BACKGROUND/AIM: Phosphodiesterase 5 (PDE5) holds clinical relevance in several pathological states, including lung, breast, and prostate cancer. In this study, we examined PDE5 expression in oral squamous cell carcinoma (OSCC)-derived cell lines and tissues, and the anti-tumour effect of PDE5 inhibitor, sildenafil citrate (SC). MATERIALS AND METHODS: Cell proliferation, cell invasion, and gap closure assays were performed in six OSCC-derived cell lines upon treatment with varying concentrations of SC. PDE5 expression was determined in primary OSCC tissues by western blotting and immunohistochemistry. RESULTS: Elevated PDE5 expression was observed in all cell lines. A concentration-dependent decrease in cell viability, invasion rate, and migration was observed after SC treatment. A significant correlation (p=0.05) was observed between elevated PDE5 expression and lymphatic infiltration in OSCC tissues. CONCLUSION: PDE5 plays an important role in carcinogenesis of OSCC, and the specific inhibition of PDE5 may be an effective chemotherapeutic strategy.


Assuntos
Carcinoma de Células Escamosas/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Neoplasias Bucais/enzimologia , Citrato de Sildenafila/farmacologia , Western Blotting , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Neoplasias Bucais/patologia , Inibidores da Fosfodiesterase 5/farmacologia
7.
Nat Commun ; 12(1): 2736, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980844

RESUMO

Endothelial barrier integrity is ensured by the stability of the adherens junction (AJ) complexes comprised of vascular endothelial (VE)-cadherin as well as accessory proteins such as ß-catenin and p120-catenin. Disruption of the endothelial barrier due to disassembly of AJs results in tissue edema and the influx of inflammatory cells. Using three-dimensional structured illumination microscopy, we observe that the mitochondrial protein Mitofusin-2 (Mfn2) co-localizes at the plasma membrane with VE-cadherin and ß-catenin in endothelial cells during homeostasis. Upon inflammatory stimulation, Mfn2 is sulfenylated, the Mfn2/ß-catenin complex disassociates from the AJs and Mfn2 accumulates in the nucleus where Mfn2 negatively regulates the transcriptional activity of ß-catenin. Endothelial-specific deletion of Mfn2 results in inflammatory activation, indicating an anti-inflammatory role of Mfn2 in vivo. Our results suggest that Mfn2 acts in a non-canonical manner to suppress the inflammatory response by stabilizing cell-cell adherens junctions and by binding to the transcriptional activator ß-catenin.


Assuntos
Junções Aderentes/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/metabolismo , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Western Blotting , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia
8.
Zhonghua Shao Shang Za Zhi ; 37(5): 420-428, 2021 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-34044524

RESUMO

Objective: To observe the effect of interleukin-6 (IL-6) on the phenotype and function of human umbilical vein endothelial cells (HUVECs) and explore the role of IL-6 in the process of endothelial-to-mesenchymal transition (EndMT). Methods: The experimental research method was used. Fresh umbilical cord discarded after normal maternal delivery was collected. On the second day of the primary cell isolation and cultivation, the cell morphology was observed under inverted phase contrast microscope. HUVECs of the 4th passage were identified by immunofluorescence method, and 2 batches of HUVECs ofthe 3rd to 5th passages were used for the subsequent experiments. The first batch of cells were divided into 6 groups according to the random number table (the same below): blank control group, 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group. The second batch of cells were divided into 4 groups: blank control group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group,and 50 ng/mL IL-6 group; the cells in blank control group was cultured with complete culture medium only, while the cells in the other groups were added with IL-6 of the corresponding final mass concentrations.Cells from the 1st batch were cultured for 72 hours after grouping, the morphology of HUVECS in the 6 groups was observed under inverted phase contrast microscope. At 72 h after grouping culture, the positive expressions of coagulation factor Ⅷ and α vascular smooth muscle actin (α-SMA) in HUVECs in the 6 groups were detected by immunofluorescence method, and the ratio of the number of double positive cells to the number of coagulation factor Ⅷ positive cells (the ratio of double positive cells for short) was calculated, with 6 samples per group; mRNA expression levels of vascular endothelial cadherin and α-SMA of HUVECs in 6 groups were detected by reverse transcription-polymerase chain reaction, with 3 samples per group.Cells from the 2nd batch were cultured 72 hours after grouping, the protein expression levels of vascular endothelial cadherin, α-SMA, and type Ⅰ collagen in the 4 groups were detected by Western blotting, with 3 samples per group. Data were statistically analyzed with one-way analysis of variance and Bonferroni correction. Results: On the 2nd day after isolation and cultivation, the primary cells were in short spindle shape or polygon, cells of the 4th passage were identified as HUVECs by immunofluorescence method. At 72 hours of culture after grouping, the cells from the 1st batch in the 6 groups changed to long spindle shape morphologically along with the increase of IL-6 concentration, the intercellular connections decreased or disappeared with the gap between cells becoming larger. At 72 h after grouping culture, compared with that inblank control group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (P<0.01); compared with that in 5 ng/mL IL-6 group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (P<0.01); compared with that in 10 ng/mL IL-6 group, the ratio of double positive cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly increased (P<0.01); the ratio of double positive cells in 100 ng/mL IL-6 group was significantly increased compared with those in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly decreased (P<0.01 or P<0.05); compared with that in 5 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (P<0.01); compared with that in 10 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (P<0.01); compared with that in 25 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of α-SMA of cells in 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6, group, and 100 ng/mL IL-6 group were significantly increased (P<0.05 or P<0.01). Cells from the 2nd batch were cultured for 72 hours after grouping. Compared with 1.391±0.026 in blank control group, the protein expressions of vascular endothelial cadherin of cells in 10 ng/mL IL-6 group (1.185±0.063), in 25 ng/mL IL-6 group (0.717±0.078), and in 50 ng/mL IL-6 group (0.239±0.064) were significantly decreased (P<0.05); compared with that in 10 ng/mL IL-6 group, the protein expressions of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly decreased (P<0.01); compared with that in 25 ng/mL IL-6 group, the protein expression of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group was significantly decreased (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expression levels of α-SMA of cells in 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, and 50 ng/mL IL-6 group were significantly increased (P<0.01); compared with that in 10 ng/mL IL-6 group, the protein expression levels of α-SMA of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expressions of type Ⅰ collagen of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (P<0.05). Conclusions: After IL-6 treatment, the phenotype and function of HUVECS showed the characteristics of mesenchymal cells in a concentration-dependent manner. The inflammatory factor can promote the process of EndMT, and become one of the important factors regulating the mechanism of tissue fibrosis.


Assuntos
Interleucina-6 , Células-Tronco Mesenquimais , Western Blotting , Colágeno Tipo I , Células Endoteliais da Veia Umbilical Humana , Humanos
9.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33909550

RESUMO

Introduction. Macrophages polarization is essential in infection control. Llipopolysaccharide (LPS) plays an essential role in host innate immune system-pathogen interaction. The LPS structure of Pseudomonas aeruginosa modifies in the adaptation of this pathogen to biofilm-related chronic infection.Gap statement. There have been several studies on LPS induced polarization of human and mouse macrophages with different results. And it was reported that the lipid A structure of the LPS derived from biofilm-forming Pseudomonas aeruginosa strain PAO1 was modified.Aim. This study aimed to investigate the effect and the involved pathway of LPS from biofilm-forming PAO1 on human and murine macrophage polarization.Methodology. LPS was isolated from biofilm-forming and planktonic PAO1 and quantified. Then the LPS was added to PMA-differentiated human macrophage THP-1 cells and Raw264.7 murine macrophage cells. The expression of iNOS, Arg-1, IL4, TNF-α, CCL3, and CCL22 was analysed in the different cell lines. The expression of TICAM-1 and MyD88 in human THP-1 macrophages was quantified by Western blot. PAO1 infected macrophages at different polarization states, and the intracellular bacterial growth in macrophages was evaluated.Results. LPS from biofilm-forming PAO1 induced more marked hyperinflammatory responses in THP-1 and Raw264.7 macrophages than LPS derived from planktonic PAO1, and these responses were related to the up-regulation of MyD88. Intracellular growth of PAO1 was significantly increased in THP-1 macrophages polarized by LPS from biofilm-forming PAO1, but decreased both in THP-1 and Raw264.7 macrophages polarized by LPS from planktonic PAO1.Conclusion. The presented in vitro study indicates that LPS derived from biofilm-forming PAO1 induces enhanced M1 polarization in human and murine macrophage cell lines than LPS from planktonic PAO1.


Assuntos
Biofilmes/crescimento & desenvolvimento , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Pseudomonas aeruginosa/química , Animais , Western Blotting , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Macrófagos/patologia , Camundongos , Microscopia de Fluorescência , Pseudomonas aeruginosa/fisiologia , Células RAW 264.7 , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Células THP-1
10.
Int J Mol Sci ; 22(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921647

RESUMO

Platyphyllenone is a type of diarylheptanoid that exhibits anti-inflammatory and chemoprotective effects. However, its effect on oral cancer remains unclear. In this study, we investigated whether platyphyllenone can promote apoptosis and autophagy in SCC-9 and SCC-47 cells. We found that it dose-dependently promoted the cleavage of PARP; caspase-3, -8, and -9 protein expression; and also led to cell cycle arrest at the G2/M phase. Platyphyllenone up-regulated LC3-II and p62 protein expression in both SCC-9 and SCC-47 cell lines, implying that it can induce autophagy. Furthermore, the results demonstrated that platyphyllenone significantly decreased p-AKT and increased p-JNK1/2 mitogen-activated protein kinase (MAPK) signaling pathway in a dose-dependent manner. The specific inhibitors of p-JNK1/2 also reduced platyphyllenone-induced cleavage of PARP, caspase-3, and caspase -8, LC3-II and p62 protein expression. These findings are the first to demonstrate that platyphyllenone can induce both autophagy and apoptosis in oral cancers, and it is expected to provide a therapeutic option as a chemopreventive agent against oral cancer proliferation.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cetonas/farmacologia , Neoplasias Bucais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Western Blotting , Caspase 3 , Caspase 8/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
11.
Int J Mol Sci ; 22(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921734

RESUMO

Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in ß-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Genisteína/uso terapêutico , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/metabolismo , Androstenos/uso terapêutico , Animais , Western Blotting , Linhagem Celular Tumoral , Colesterol/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Doenças por Armazenamento dos Lisossomos , Lisossomos/metabolismo , Proteína C1 de Niemann-Pick/metabolismo
12.
Molecules ; 26(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923441

RESUMO

Salmonella typhimurium infection is associated with gastrointestinal disorder and cellular injury in the liver of both humans and animals. Cinnamaldehyde, the main component of essential oil from cinnamon, has been reported to have anti-inflammatory, anti-oxidative, and anti-apoptotic effects. However, it remains unknown whether cinnamaldehyde can alleviate Salmonella typhimurium infection-induced liver injury in mice. In the present study, we found that cinnamaldehyde attenuated Salmonella typhimurium-induced body weight loss, the increase of organ (liver and spleen) indexes, hepatocyte apoptosis, and the mortality rate in mice. Further study showed that cinnamaldehyde significantly alleviated Salmonella typhimurium-induced liver injury as shown by activities of alanine transaminase, aspartate transaminase, and myeloperoxidase, as well as malondialdehyde. The increased mRNA level of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and chemokines (CCL2 and CCL3) induced by Salmonella typhimurium were significantly abolished by cinnamaldehyde supplementation. These alterations were associated with a regulatory effect of cinnamaldehyde on TLR2, TLR4, and MyD88. 16S rDNA sequence analysis showed that Salmonella typhimurium infection led to upregulation of the abundances of genera Akkermansia, Bacteroides, Alistipes, Muribaculum, and Prevotellaceae UCG-001, and downregulation of the abundances of genera Lactobacillus, Enterorhabdus, and Eggerthellaceae (unclassified). These alterations were reversed by cinnamaldehyde supplementation. In conclusion, cinnamaldehyde attenuated the inflammatory response, oxidative stress, and apoptosis in the liver of Salmonella typhimurium-infected mice. Supplementation of cinnamaldehyde might be a preventive strategy to alleviate liver injury caused by Salmonella typhimurium infection in humans and animals.


Assuntos
Acroleína/análogos & derivados , Acroleína/química , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , DNA Ribossômico/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Marcação In Situ das Extremidades Cortadas , Inflamação/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Salmonella typhimurium/patogenicidade , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
13.
Molecules ; 26(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923651

RESUMO

Curcumin is a natural compound that has been widely used as a food additive and medicine in Asian countries. Over several decades, diverse biological effects of curcumin have been elucidated, such as anti-inflammatory and anti-oxidative activities. Monocyte chemoattractant protein-1 (MCP-1) is a key inflammatory marker during the development of atherosclerosis, and curcumin blocks MCP-1 expression stimulated by various ligands. Hence, we studied the action of curcumin on lysophosphatidic acid (LPA) mediated MCP-1 expression and explored the specific underlying mechanisms. In human vascular smooth muscle cells, LPA induces Rho-associated protein kinase (ROCK) dependent transforming growth factor receptor (TGFBR1) transactivation, leading to glycosaminoglycan chain elongation. We found that LPA also signals via the TGFBR1 transactivation pathway to regulate MCP-1 expression. Curcumin blocks LPA mediated TGFBR1 transactivation and subsequent MCP-1 expression by blocking the ROCK signalling. In the vasculature, ROCK signalling regulates smooth muscle cell contraction, inflammatory cell recruitment, endothelial dysfunction and vascular remodelling. Therefore, curcumin as a ROCK signalling inhibitor has the potential to prevent atherogenesis via multiple ways.


Assuntos
Quimiocina CCL2/metabolismo , Curcumina/farmacologia , Lisofosfolipídeos/farmacologia , Quinases Associadas a rho/metabolismo , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923922

RESUMO

Doxorubicin increases endothelial permeability, hence increasing cardiomyocytes' exposure to doxorubicin (DOX) and exposing myocytes to more immediate damage. Reactive oxygen species are major effector molecules of doxorubicin's activity. Mangiferin (MGN) is a xanthone derivative that consists of C-glucosylxanthone with additional antioxidant properties. This particular study assessed the effects of MGN on DOX-induced cytotoxicity in human umbilical vein endothelial cells' (HUVECs') signaling networks. Mechanistically, MGN dramatically elevated Nrf2 expression at both the messenger RNA and protein levels through the upregulation of the PI3K/AKT pathway, leading to an increase in Nrf2-downstream genes. Cell apoptosis was assessed with a caspase-3 activity assay, transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining was performed to assess DNA fragmentation, and protein expression was determined by Western blot analysis. DOX markedly increased the generation of reactive oxygen species, PARP, caspase-3, and TUNEL-positive cell numbers, but reduced the expression of Bcl-2 and antioxidants' intracellular concentrations. These were effectively antagonized with MGN (20 µM), which led to HUVECs being protected against DOX-induced apoptosis, partly through the PI3K/AKT-mediated NRF2/HO-1 signaling pathway, which could theoretically protect the vessels from severe DOX toxicity.


Assuntos
Doxorrubicina/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Xantonas/farmacologia , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fragmentação do DNA/efeitos dos fármacos , Imunofluorescência , Humanos , Marcação In Situ das Extremidades Cortadas , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
15.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924053

RESUMO

Previous studies have investigated the inhibitory effect of BMI-1026 on cyclin-dependent kinase 1 in vitro. However, the molecular mechanisms by which BMI-1026 treatment leads to cancer cell death remain unclear. This study was conducted to investigate the anticancer mechanisms of BMI-1026 on human renal carcinoma Caki cells. BMI-1026 induced apoptosis in association with the cleavage of poly(ADP-ribose) polymerase and pro-caspase-3 and the release of apoptosis-inducing factor and cytochrome c from mitochondria in Caki cells. BMI-1026-induced apoptosis was inhibited by the pan-caspase inhibitor z-VAD-fmk. Furthermore, BMI-1026 downregulated Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) at the transcriptional level and Mcl-1 (L) and cellular FADD-like IL-1ß-converting enzyme inhibitory protein (c-FLIP (L)) at the post-transcriptional level. Interestingly, Mcl-1 (L) and c-FLIP (L), but not Bcl-2 or XIAP, played important roles in BMI-1026-induced Caki cell apoptosis. Although the constitutively active form of Akt did not attenuate BMI-1026-induced apoptosis, blockade of the PI3K/Akt pathway using a subcytotoxic concentration of the PI3K/Akt inhibitor LY294002 enhanced Caki cell apoptosis induced by BMI-1026. Electrophysiological safety was confirmed by determining the cardiotoxicity of BMI-1026 via left ventricular pressure analysis. These results suggest that BMI-1026 is a potent multitarget anticancer agent with electrophysiological safety and should be further investigated.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma de Células Renais/metabolismo , Fenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Cromonas/farmacologia , Regulação para Baixo , Citometria de Fluxo , Células HCT116 , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo
16.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924098

RESUMO

Neurogranin (Ng) is a brain-specific postsynaptic protein, whose role in modulating Ca2+/calmodulin signaling in glutamatergic neurons has been linked to enhancement in synaptic plasticity and cognitive functions. Accordingly, Ng knock-out (Ng-ko) mice display hippocampal-dependent learning and memory impairments associated with a deficit in long-term potentiation induction. In the adult olfactory bulb (OB), Ng is expressed by a large population of GABAergic granule cells (GCs) that are continuously generated during adult life, undergo high synaptic remodeling in response to the sensory context, and play a key role in odor processing. However, the possible implication of Ng in OB plasticity and function is yet to be investigated. Here, we show that Ng expression in the OB is associated with the mature state of adult-born GCs, where its active-phosphorylated form is concentrated at post-synaptic sites. Constitutive loss of Ng in Ng-ko mice resulted in defective spine density in adult-born GCs, while their survival remained unaltered. Moreover, Ng-ko mice show an impaired odor-reward associative memory coupled with reduced expression of the activity-dependent transcription factor Zif268 in olfactory GCs. Overall, our data support a role for Ng in the molecular mechanisms underlying GC plasticity and the formation of olfactory associative memory.


Assuntos
Neurogranina/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Interneurônios/metabolismo , Camundongos , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Percepção Olfatória/fisiologia , Fosforilação
17.
Molecules ; 26(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924385

RESUMO

The role of Kupffer cells (KCs) in liver regeneration is complicated and controversial. To investigate the distinct role of F4/80+ KCs at the different stages of the regeneration process, two-thirds partial hepatectomy (PHx) was performed in mice to induce physiological liver regeneration. In pre- or post-PHx, the clearance of KCs by intraperitoneal injection of the anti-F4/80 antibody (α-F4/80) was performed to study the distinct role of F4/80+ KCs during the regenerative process. In RNA sequencing of isolated F4/80+ KCs, the initiation phase was compared with the progression phase. Immunohistochemistry and immunofluorescence staining of Ki67, HNF-4α, CD-31, and F4/80 and Western blot of the TGF-ß2 pathway were performed. Depletion of F4/80+ KCs in pre-PHx delayed the peak of hepatocyte proliferation from 48 h to 120 h, whereas depletion in post-PHx unexpectedly led to persistent inhibition of hepatocyte proliferation, indicating the distinct role of F4/80+ KCs in the initiation and progression phases of liver regeneration. F4/80+ KC depletion in post-PHx could significantly increase TGF-ß2 serum levels, while TGF-ßRI partially rescued the impaired proliferation of hepatocytes. Additionally, F4/80+ KC depletion in post-PHx significantly lowered the expression of oncostatin M (OSM), a key downstream mediator of interleukin-6, which is required for hepatocyte proliferation during liver regeneration. In vivo, recombinant OSM (r-OSM) treatment alleviated the inhibitory effect of α-F4/80 on the regenerative progression. Collectively, F4/80+ KCs release OSM to inhibit TGF-ß2 activation, sustaining hepatocyte proliferation by releasing a proliferative brake.


Assuntos
Antígenos de Diferenciação/metabolismo , Macrófagos do Fígado/metabolismo , Regeneração Hepática/fisiologia , Fígado/metabolismo , Fígado/cirurgia , Oncostatina M/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Antígenos de Diferenciação/imunologia , Western Blotting , Células Cultivadas , Hepatectomia , Hepatócitos/metabolismo , Imuno-Histoquímica , Fígado/citologia , Regeneração Hepática/genética , Masculino , Camundongos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Fator de Crescimento Transformador beta2/genética
18.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925027

RESUMO

Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations.


Assuntos
Cromatografia em Gel/métodos , Vesículas Extracelulares/ultraestrutura , Ultracentrifugação/métodos , Biomarcadores/sangue , Western Blotting , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Exossomos/química , Exossomos/ultraestrutura , Vesículas Extracelulares/química , Citometria de Fluxo , Humanos , Lipoproteínas/sangue , Lipoproteínas/isolamento & purificação , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Soro/química
19.
Nutrients ; 13(3)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805703

RESUMO

The food industry commonly uses milk ingredients as technological aids in an uncounted number of products. On the other hand, milk contains allergenic proteins causing adverse allergic reactions in sensitized/allergic individuals. This work intends to evaluate the effect of autoclaving and in vitro digestion on the allergenicity of milk proteins incurred in meat products. Protein profiles of raw and autoclaved sausages without and with the addition of 10% of milk protein concentrates were analyzed by gel electrophoresis and liquid chromatography-mass spectrometry. Additionally, residual IgE-reactivity was evaluated by immunoblot analysis using pooled sera of cow's-milk-allergic individuals followed by bioinformatic analysis. Results showed that autoclaving led to an increase in protein fragmentation (higher number of short peptides) and consequently to a higher digestion rate, that was found to be more pronounced in ß-casein. The IgE-binding capacity of milk proteins seems to be reduced after autoclaving prior to digestion, with a residual reactivity in caseins, but was eliminated following digestion. This study highlights the importance of autoclaving as a processing strategy to produce hypoallergenic formulas.


Assuntos
Digestão/fisiologia , Temperatura Alta , Imunoglobulina E/metabolismo , Produtos da Carne , Hipersensibilidade a Leite/prevenção & controle , Proteínas do Leite/metabolismo , Animais , Western Blotting , Cromatografia Líquida , Duodeno , Eletroforese em Gel de Poliacrilamida , Imunoglobulina E/imunologia , Técnicas In Vitro , Espectrometria de Massas , Hipersensibilidade a Leite/imunologia , Proteínas do Leite/imunologia
20.
Nutrients ; 13(3)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809593

RESUMO

The addition of plant oils such as soybean oil (S) to a diet rich in saturated fatty acids is discussed as a possible route to prevent or diminish the development of metabolic disease. Here, we assessed whether a butterfat-rich diet fortified with S affects the development of early non-alcoholic steatohepatitis (NASH) and glucose intolerance. Female C57BL/6J mice were fed a standard-control diet (C); a fat-, fructose-, and cholesterol-rich diet (FFC, 25E% butterfat, 50% (wt./wt.) fructose, 0.16% (wt./wt.) cholesterol); or FFC supplemented with S (FFC + S, 21E% butterfat + 4E% S) for 13 weeks. Indicators of liver damage, inflammation, intestinal barrier function, and glucose metabolism were measured. Lipopolysaccharide (LPS)-challenged J774A.1 cells were incubated with linolenic and linoleic acids (ratio 1:7.1, equivalent to S). The development of early NASH and glucose intolerance was significantly attenuated in FFC + S-fed mice compared to FFC-fed mice associated with lower hepatic toll-like receptor-4 mRNA expression, while markers of intestinal barrier function were significantly higher than in C-fed mice. Linolenic and linoleic acid significantly attenuated LPS-induced formation of reactive nitrogen species and interleukin-1 beta mRNA expression in J774A.1 cells. Our results indicate that fortifying butterfat with S may attenuate the development of NASH and glucose intolerance in mice.


Assuntos
Manteiga/efeitos adversos , Alimentos Fortificados , Intolerância à Glucose/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Óleo de Soja/uso terapêutico , Animais , Arginase/metabolismo , Western Blotting , Gorduras na Dieta/efeitos adversos , Endotoxinas/sangue , Ácidos Graxos não Esterificados/sangue , Feminino , Intolerância à Glucose/etiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR gama/sangue , Peroxidase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Óleo de Soja/administração & dosagem , Fator de Necrose Tumoral alfa/sangue
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