RESUMO
The deposition of aggregated human islet amyloid polypeptide (hIAPP) in the pancreas, that has been associated with ß-cell dysfunction, is one of the common pathological features of patients with type 2 diabetes (T2D). Therefore, hIAPP aggregation inhibitors hold a promising therapeutic schedule for T2D. Chitosan oligosaccharides (COS) have been reported to exhibit a potential antidiabetic effect, but the function of COS on hIAPP amyloid formation remains elusive. Here, we show that COS inhibited the aggregation of hIAPP and disassembled preformed hIAPP fibrils in a dose-dependent manner by thioflavin T fluorescence assay, circular dichroism spectroscopy, and transmission electron microscope. Furthermore, COS protected mouse ß-cells from cytotoxicity of amyloidogenic hIAPP, as well as apoptosis and cycle arrest. There was no direct binding of COS and hIAPP, as revealed by surface plasmon resonance analysis. In addition, both chitin-oligosaccharide and the acetylated monosaccharide of COS and glucosamine had no inhibition effect on hIAPP amyloid formation. It is presumed that, mechanistically, COS regulate hIAPP amyloid formation relating to the positive charge and degree of polymerization. These findings highlight the potential role of COS as inhibitors of hIAPP amyloid formation and provide a new insight into the mechanism of COS against diabetes.
Assuntos
Amiloide/metabolismo , Quitosana/farmacologia , Citoproteção/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Oligossacarídeos/farmacologia , Animais , Benzotiazóis/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quitosana/síntese química , Quitosana/química , Quitosana/isolamento & purificação , Fluorescência , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Cinética , Camundongos , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Agregados Proteicos/efeitos dos fármacos , Estrutura Secundária de ProteínaRESUMO
In the process of sepsis, excessive occurrence of pyroptosis, a form of programmed cell death acting as a defense mechanism against pathogens, can disrupt immune responses, thus leading to tissue damage and organ dysfunction. Chitosan oligosaccharide (COS), derived from chitosan degradation, has demonstrated diverse beneficial effects. However, its impact on sepsis-induced pyroptosis remains unexplored. In the present study, ATP/LPS was utilized to induce canonical-pyroptosis in THP-1 cells, while bacterial outer membrane vesicles (OMV) were employed to trigger non-canonical pyroptosis in RAW264.7 cells. Our results revealed a dose-dependent effect of COS on both types of pyroptosis. This was evidenced by a reduction in the expression of pro-inflammatory cytokines, as well as crucial regulatory proteins involved in pyroptosis. In addition, COS inhibited the cleavage of caspase-1 and GSDMD, and reduced ASC oligomerization. The underlying mechanism revealed that COS acts an antioxidant, reducing the release of pyroptosis-induced ROS and malondialdehyde (MDA) by upregulation the expression and promoting the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), which led to an elevation of glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD). Notably, the actions of COS were completely reversed by the Nrf2 inhibitor. Consequently, COS intervention increased the survival rate of sepsis.
RESUMO
AIMS AND OBJECTIVES: The aim of this study is to discuss the influence of endotoxin on insulin amyloid formation, to provide guidance for therapeutic insulin preparation and storage. MATERIALS AND METHODS: The ThT and ANS binding assays were applied to characterize the dynamics curve of insulin amyloid formation with the presence or absence of endotoxin. The morphological structures of intermediate and mature insulin fibrils were observed with SEM and TEM. Secondary structural changes of insulin during fibriliation were examined with CD, FTIR and Raman spectral analysis. The cytotoxic effects of oligomeric and amyloidogenic insulin aggregates were detected using a cck-8 cell viability assay kit. The influence of endotoxin on insulin efficacy was analyzed by monitoring the activation of insulin signal transduction. KEY FINDINGS: ThT analysis showed that endotoxin, regardless of species, accelerated insulin fibrils formation in a dose-dependent manner, as observed with a shorter lag phase. ANS binding assay demonstrated endotoxin provoked the exposure of insulin hydrophobic patches. The results of SEM and TEM data displayed that endotoxin drove insulin to cluster into dense and viscous form, with thicker and stronger filaments. Based on CD, FTIR and Raman spectra, endotoxin promoted the transition of α-helix to random coil and ß-strand secondary structures during insulin aggregation. Insulins in both oligomeric and amyloidogenic forms were cytotoxic to HepG2 cells, with the former being more severe. Finally, the efficacy of endotoxin treated insulin obviously decreased. SIGNIFICANCE: Our studies revealed that endotoxin disrupts the structural integrity of insulin and promotes its amyloidosis. These findings offered theoretical guidance for insulin storage and safe utilization, as well as pointing up a new direction for insulin resistance research.
Assuntos
Amiloidose , Insulina , Humanos , Amiloide/química , Amiloidose/metabolismo , Insulina/metabolismo , Estrutura Secundária de Proteína , Transdução de Sinais , EndotoxinasRESUMO
AIM: To investigate the antioxidant mechanisms of propylene glycol mannate sulfate (PGMS) in hyperlipidemic rats. METHODS: Male Wistar rats were given high lipid emulsion diet to establish hyperlipidemic model. PGMS was given every day at different doses (37.8 and 75.6 mg.kg-1, ig) to hyperlipidemic rats for three weeks. In addition, diethyldithiocarbamate (DDC) was given 200 mg.kg-1.3 d-1 (i.p.) to inhibit SOD activity. Then, the MDA content was examined using TBA method to show the oxidation level, and the activities of SOD, GSH-Px and CAT were examined following the kit protocols to indicate the capability of eliminating OFR. RT-PCR was applied to study the expression of Cu, Zn-SOD mRNA in rat liver. RESULTS: The MDA content of PGMS treatment groups decreased markedly compared with hyperlipidemic group, and the activities of SOD, GSH-Px and CAT increased distinctly. Cu, Zn-SOD mRNA expression was significantly increased by PGMS treatment. Furthermore, the application of DDC(the SOD inhibitor) reduced total SOD activity and Cu, Zn-SOD mRNA expression induced by PGMS, and the content of MDA increased correspondingly. CONCLUSION: PGMS can induce the activities of antioxidant enzymes and the mRNA expression of Cu, Zn-SOD, which contribute to the elimination of oxygen free radical. This may explain the molecular mechanism of antioxidant effects of PGMS.
Assuntos
Antioxidantes/farmacologia , Hiperlipidemias/enzimologia , Propilenoglicóis/farmacologia , Animais , Catalase/metabolismo , Sequestradores de Radicais Livres/farmacologia , Hiperlipidemias/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
AIM: To study the effects of prophylene glycol mannate sulfate (PGMS) on monocyte chemoattractant protein-1 (MCP-1) mRNA expression in hyperlipidemic rat aorta and to clarify the molecular mechanism of PGMS for the prevention of atherosclerosis. METHODS: PGMS (37.8 and 75.6 mg.kg-1.d-1, ig) or PGMS (37.8 and 75.6 mg.kg-1.d-1, ig) combined with diethyldithiocarbamate (DDC, an inhibitor of SOD, 200 mg.kg-1 every three days, i.p.) were given to hyperlipidemic rats for three weeks. The MDA content and SOD activity were determined after 12 h of starvation, and MCP-1 mRNA expression in aorta was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: There was significant decrease (29.46% or 58.40)% of MCP-1 mRNA expression in aortic after the therapy. The SOD activity increased markedly and the MDA content decreased at the same time. After treatment with DDC, the SOD activity was inhibited and the MDA content increased, but with no significant effect on MCP-1 mRNA expression. CONCLUSION: PGMS inhibited MCP-1 mRNA expression with no relation to its effect on decreasing MDA content.
Assuntos
Aorta Torácica/efeitos dos fármacos , Quimiocina CCL2/biossíntese , Expressão Gênica/efeitos dos fármacos , Hipolipemiantes/farmacologia , Propilenoglicóis/farmacologia , Animais , Aorta Torácica/metabolismo , Quimiocina CCL2/genética , Hiperlipidemias/sangue , Hiperlipidemias/patologia , Masculino , Malondialdeído/sangue , Malondialdeído/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Wistar , Superóxido Dismutase/sangue , Superóxido Dismutase/metabolismoRESUMO
AIM: To study the effect of propylene glycol mannate sulfate (PGMS) on blood lipids and lipoprotein lipase in hyperlipidemic rat, and its anti-hyperlipidemic mechanism. METHODS: PGMS was administered ig at different doses (37.8 mg.kg-1.d-1 and 75.6 mg.kg-1.d-1) to hyperlipidemic rats for three weeks and blood serum was obtained after starved 12 h. Total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were examined. The mRNA expression of lipoprotein lipase (LPL) in liver, spleen and artery was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: PGMS significantly decreased the levels of TC, TG and LDL-C and increased that of HDL-C in hyperlipidemic serum dose-dependently. PGMS was shown to increase the level of LPL mRNA expression, which is related directly to the controlling effects of PGMS on blood lipids. CONCLUSION: PGMS modulated blood lipids by promoting mRNA expression of LPL. This may be one important mechanism of PGMS to modulate blood lipids.
Assuntos
Hiperlipidemias/tratamento farmacológico , Lipase Lipoproteica/biossíntese , Propilenoglicóis/uso terapêutico , Animais , HDL-Colesterol/sangue , Modelos Animais de Doenças , Hiperlipidemias/sangue , Hiperlipidemias/enzimologia , Lipase Lipoproteica/genética , Masculino , RNA Mensageiro/biossíntese , Distribuição Aleatória , Ratos , Ratos Wistar , Triglicerídeos/sangueRESUMO
AIM: To study the effect of propylene glycol mannate sulfate (PGMS) on induction of CuZn-SOD. METHODS: Wistar rats were given PGMS p.o. at different doses (0, 18.9, 37.8 and 75.6 mg.kg-1.d) for ten days. Then the rats were sacrificed and the total RNA was extracted from the livers. The total RNA samples were loaded on a 1% agarose gel to detect the quality of total RNA. RT-PCR was applied to study the expression of CuZn-SOD mRNA in rat livers. The amplified products were detected by the 1.5% agarose gel electrophoresis. Simultaneously, the CuZn-SOD activities in rat liver were determined by nitrite method. RESULTS: The total RNA extracted from rat livers was integrated without being decomposed by RNase. The level of CuZn-SOD mRNA of the high-dosage group (75.6 mg.kg-1.d) was higher than that of the control group (0 mg.kg-1.d) (P < 0.01); the CuZn-SOD activities of the high-dosage group were significantly higher than those of the control group (P < 0.001) and the CuZn-SOD activities of the middle- (37.8 mg.kg-1.d) and low-dosage groups (18.9 mg.kg-1.d) were higher than those of the control group (P < 0.01). CONCLUSION: PGMS can increase the CuZn-SOD activities as well as CuZn-SOD on mRNA level. Therefore, it is possible for PGMS to counteract Atherosclerosis (AS) by inducing the expression of CuZn-SOD.
Assuntos
Fígado/efeitos dos fármacos , Propilenoglicóis/farmacologia , Superóxido Dismutase/biossíntese , Animais , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Fígado/metabolismo , Masculino , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Wistar , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
AIM: Construct prokaryotic expression vector carrying mouse TRBP (TAR RNA-binding protein) gene and test the double-stranded RNA binding ability of TRBP. METHODS: RT-PCR was used to obtain TRBP cDNA from mouse genomic DNA. Then, we built the His-tag fusion expression vector of TRBP and transformed it into E.coli BL21(DE3). Ni-NTA beads were used to isolate and purify the recombinant protein and vitro transcription was used to get Pre-miR-122. Finally, SDS-PAGE and ITC (isothermal titration calorimetry) assay were both used to validate TRBP's binding ability with Pre-miR-122. RESULTS: We purified the recombinant protein TRBP whose molecular weight is 32.4 kDa. The purified bioactive TRBP protein binding on NI-NTA beads showed that it had a strong binding capacity on Pre-miR-122. CONCLUSION: We constructed TRBP prokaryotic expression system successfully and studied the double-stranded RNA binding ability of TRBP preliminarily.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , MicroRNAs/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND & OBJECTIVE: Overexpression of heat shock protein (HSP)70 is expressed in many tumors, but the correlation of its overexpression in nasopharyngeal carcinoma (NPC) to immunoglobin A against viral capsid antigen of Epstein-Barr virus (EBV) in sera and its clinical significance are still unclear. This study was to determine the expression of HSP70 in NPC, and to analyze its correlations to EBV IgA/VCA titer and prognosis. METHODS: The expression of HSP70 in 38 specimens of stage II-III NPC was determined by SP immunohistochemistry; the content of HSP70 in the 38 specimens was detected by ELISA; the EBV IgA/VCA titer in sera of the 38 patients was detected by immunoenzymatic (IE) method. RESULTS: The positive rate of HSP70 in the 38 NPC samples was 60.5%. HSP70 expression was positively correlated to EBV IgA/VCA titer (r=0.690, P=0.001), but not to sex, age, and clinical stage (P>0.05). The 5-year survival rates of HSP70-positive group and HSP70-negative group were 65.2% and 80.0%. The 5-year tumor-free survival rates of HSP70-positive group and HSP70-negative group were 40.0% and 78.6% (P=0.04). CONCLUSION: HSP70 expression in stage II-III NPC tissues is positively correlated to EBV IgA/VCA titer. The prognosis of HSP70-positive NPC patients is poor.