Abstract Aims Inadequate autophagy contributed to endothelial dysfunction in diabetic patients. We aimed to investigate the relationship between inadequate autophagy and endothelial cells (ECs) apoptosis in diabetes and its underlying mechanism. Methods and results Aortic intima and ECs were isolated from diabetic patients. Cultured human aortic endothelial cells (HAECs) were stimulated with advanced glycation end products (AGEs). The expression of autophagy and apoptosis-related proteins were determined by western blotting. Autophagosomes were observed by electron microscopy. The fusion of autophagosome and lysosomes was detected by immunofluorescence. Compared with non-diabetic subjects, the levels of LC3-II, p62, FoxO1, and Ac-FoxO1 were increased in ECs from diabetic patients, accompanied by the decreased expressions of Atg14, STX17, and co-localization of LC3-II/LAMP2 and Atg14/STX17. Long-term stimulation with AGEs up-regulated LC3-II and p62 expression and the number of autophagosomes with decreased level of Atg14, STX17, Ras-related protein 7 (Rab7), and co-localization of LC3-II/LAMP2 and Atg14/STX17 in HAECs. The apoptosis rates were increased with elevated cleaved-caspase-3 and declined Bcl-2 expression. Inhibition of autophagy with 3-methyladenine could reduce long-term AGEs-induced apoptosis. Higher levels of FoxO1, Ac-FoxO1, and Ac-FoxO1 binding to Atg7 were detected in AGEs-treated HAECs. AGEs-induced FoxO1 enhanced Akt activity, decreased SIRT1-deacetylase activity by phosphorylation and elevated Ac-FoxO1. Knockout of FoxO1 reduced AGEs-induced autophagy and promoted the expression of Atg14 and the co-localization of LC3-II/LAMP 2 and Atg14/STX17. Conclusion Inadequate autophagy with impaired autophagosome-lysosomal fusion exists in aortic intima and ECs from diabetic patients. FoxO1 mediates AGEs-induced ECs autophagic apoptosis through impairing autophagosome-lysosomes fusion by inhibiting Atg14 expression.
Objective
To investigate the diagnostic value of pyruvate kinase M2(PKM2)gene expression in papillary thyroid carcinoma(PTC).
Methods
Quantitative real-time PCR(RT-qPCR)was used to detect the expression of PKM2 mRNA in benign thyroid nodules, PTC, and normal thyroid tissues around nodules of fine-needle aspiration(FNA)specimens. Immunohistochemistry(IHC)was used to detect the expression of PKM2 protein in thyroid tissue after thyroidectomy. The receiver operating characteristic curve was constructed to evaluate the diagnostic value of PKM2 in PTC.
Results
The expression of PKM2 mRNA was detectable in FNA specimens of thyroid nodules, higher in PTC than those in normal thyroid tissue and benign thyroid nodules(P 0.05). The expression level of PKM2 mRNA in FNA specimens of thyroid nodules was paralleled with the expression level of PKM2 protein in postoperation pathological tissues. The accuracy, sensitivity, specificity of PKM2 gene in the diagnosis of PTC were 62.8%, 46.9%, and 95.7%, respectively. The accuracy and sensitivity of PKM2 combined with BRAFV600E were increased to 87.6% and 83.7%, significantly higher than those of single detection (P<0.05) .
Conclusion
Detection of PKM2 gene in FNA specimens is highly specific in the diagnosis of PTC, making it a valuable molecular marker for preoperative diagnosis. The combination of PKM2 and BRAFV600E detection shows a higher diagnosis efficiency.
Key words:
Papillary thyroid carcinoma; Pyruvate kinase M2; Fine-needle aspiration
Pacritinib, an oral JAK kinase inhibitor, has recently been granted approval for the treatment of myelofibrosis and thrombocytopenia, making it a newly authorized drug within the pharmaceutical industry. In this study, a quantitative detection method for pacritinib in rat plasma was established by LC-MS/MS with Ibrutinib as an internal standard. Subsequently, this method was applied to pharmacokinetic and drug-drug interaction studies in rats. The established method showed good linearity in the range of 1-1500 ng/mL, with a lower limit of quantification (LLOQ) of 1 ng/mL. Furthermore, both intra-day and inter-day precision RSD% of Pacritinib were less than 10.69%, while the accuracy range was -2.31% to 2.08%. The recovery, matrix effect, and stability were consistent with the FDA guidelines. This method proved effective in quantitatively detecting pacritinib content in rat plasma. In the pharmacokinetic studies, we observed that isavuconazole significantly inhibited the metabolism of pacritinib compared to voriconazole. This led to a 2.5-fold increase in AUC(0-t), a 2.3-fold increase in AUC(0-∞), a 4.4-fold increase in CLz/F, and a 3.4-fold increase in Cmax, respectively. This study successfully established a reliable LC-MS/MS method for detecting pacritinib plasma drug concentration in rats. Pharmacokinetic studies showed that isavuconazole is more likely to increase the blood exposure of pacritinib than voriconazole.
During pregnancy, maternal pancreatic β-cells undergo a compensatory expansion in response to the state of insulin resistance, where prolactin (PRL) plays a major role. Retinoblastoma protein (Rb) has been shown to critically regulate islet proliferation and function. The aim of the study was to explore the role of Rb in β-cell mass expansion during pregnancy. Expression of pocket protein family and E2Fs were examined in mouse islets during pregnancy and in insulinoma cells (INS-1) stimulated by PRL. PRL-stimulated INS-1 cells were used to explore the signaling pathway that regulates Rb downstream of the PRL receptor. Pancreas-specific Rb-knockout (Rb-KO) mice were assessed to evaluate the in vivo function of Rb in β-cell proliferation during pregnancy. During pregnancy, expression of Rb, phospho-Rb (p-Rb), p107, and E2F1 increased, while p130 decreased in maternal islets. With PRL stimulation, induction of Rb expression occurred mainly in the nucleus, while p-Rb was predominantly in the cytoplasm. Inhibition of STAT5 significantly restrained the expression of CDK4, Rb, p-Rb, and E2F1 in PRL-stimulated INS-1 cells with attenuation in cell cycle progression. Reduction of Rb phosphorylation by CDK4 inhibition blocked PRL-mediated proliferation of INS-1 cells. On the other hand, knockdown of Rb using siRNA led to an induction in E2F1 leading to cell cycle progression from G1 to S and G2/M phase, similar to the effects of PRL-mediated induction of p-Rb that led to cell proliferation. With Rb knockdown, PRL did not lead to further increase in cell cycle progression. Similarly, while Rb-KO pregnant mice displayed better glucose tolerance and higher insulin secretion, they had similar β-cell mass and proliferation to wild-type pregnant controls, supporting the essential role of Rb suppression in augmenting β-cell proliferation during pregnancy. Rb-E2F1 regulation plays a pivotal role in PRL-stimulated β-cell proliferation. PRL promotes Rb phosphorylation and E2F1 upregulation via STAT5-cyclin D/CDK4 pathway during pregnancy.
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