Resveratrol attenuates cigarette smoke induced endothelial apoptosis by activating Notch1 signaling mediated autophagy.

BACKGROUND: Increasing evidence shows that endothelial apoptosis contributes to cigarette smoke (CS)-induced disease progression, such as chronic obstructive pulmonary disease (COPD). Our previous studies have validated Notch1 as an anti-apoptotic signaling in CS-induced endothelial apoptosis. Resveratrol (RESV) is a naturally occurring polyphenol that exhibits an anti-apoptotic activity in endothelial cells that exposed to many kinds of destructive stimulus. However, the effects of resveratrol on Notch1 signaling in CS-induced endothelial apoptosis have not yet been fully elucidated. Therefore, the aim of this study was to examine whether RESV can protect endothelial cells from CS-induced apoptosis via regulating Notch1 signaling. METHODS: Human umbilical vein endothelial cells (HUVECs) were pretreated with RESV for 2 h, followed by cotreatment with 2.5%CSE for 24 h to explore the role of RESV in CSE induced endothelial apoptosis. 3-methyladenine (3-MA) or rapamycin was used to alter autophagic levels. Lentivirus Notch1 intracellular domain (LV-N1ICD), γ-secretase inhibitor (DAPT) and Notch1 siRNA were used to change Notch1 expression. The expression of Notch1, autophagic and apoptotic markers were examined by Western blot and the apoptosis rate was detected by Flow cytometry analysis. RESULTS: Our results showed that activating autophagy reduced CSE-induced endothelial apoptosis, while blocking autophagy promoted cell apoptosis in HUVECs. RESV pretreatment attenuated the CSE-induced endothelial apoptosis and activated Notch1 signaling. RESV pretreatment also increased LC3b-II and Beclin1 production, decreased p62 and mTOR expression. 3-MA treatment inhibited autophagy and aggravated CSE induced apoptosis, while rapamycin promoted autophagy, led to a decrease in cell apoptosis. LV-N1ICD transfection upregulated autophagy and reduced apoptosis. However, this protective effect was abolished by 3-MA treatment. In cells treated with DAPT or Notch1 siRNA, autophagy was decreased, while apoptosis was increased. RESV partly rescued the DAPT or Notch1 siRNA induced apoptosis by activating Notch1 signaling. CONCLUSION: In HUVECs, RESV attenuates CSE induced endothelial apoptosis by inducing autophagy in a Notch1-dependent manner.

miR-34a-5p up-regulates the IL-1ß/COX2/PGE2 inflammation pathway and induces the release of CGRP via inhibition of SIRT1 in rat trigeminal ganglion neurons.

Migraine is a debilitating neurological condition, with a global prevalence rate of 10.68% in men and 18.79% in women. Elucidation of the molecular mechanisms underlying migraines is of great importance for improving the quality of life of patients. The release of the neuropeptide calcitonin gene-related peptide (CGRP) from trigeminal nerve terminals is involved in the pathogenesis of migraine. Recent studies have shown that up-regulation of miR-34a-5p expression is associated with acute migraine attacks. Here, we investigated whether alteration of the expression of miR-34a-5p induces the release of the vasoactive peptide CGRP. We isolated primary rat trigeminal ganglion neurons and performed gain- and loss-of-function assays to alter the expression level of miR-34a-5p. Down-regulation of miR-34a-5p inhibited the expression of interleukin-1ß (IL-1ß)/cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2), decreased IL-1ß, PGE2 and CGRP release, and up-regulated the expression of silencing information regulator 1 (SIRT1) in trigeminal ganglion, whereas overexpression of miR-34a-5p enhanced the expression of IL-1ß/COX2/PGE2, increased the release of IL-1ß, PGE2 and CGRP, and decreased the expression of SIRT1 in trigeminal ganglion. In addition, overexpression of miR-34a-5p induced apoptosis in primary rat trigeminal neurons. In summary, these findings suggest that miR-34a-5p up-regulates the IL-1ß/COX2/PGE2 inflammation pathway, induces apoptosis and enhances release of CGRP via inhibition of SIRT1 expression in trigeminal ganglion neurons; thus, miR-34a-5p may have potential as a therapeutic target for the treatment of migraine.

[Effects of electroacupuncture on miR-34a-5p/SIRT1 signaling in the trigeminal ganglion of rats with migraine].

OBJECTIVE: To investigate the effect of electroacupuncture(EA) on miR-34a-5p, silent mating type information regulation 2 homolog-1 (SIRT1) and nuclear factor-κB subunit p65 (NF-κB p65) in the trigeminal ganglion of rats with migraine, so as to explore the mechanisms of EA underlying prevention of migraine. METHODS: Male SD rats were randomly divi-ded into normal, sham operation, model, EA, and EA plus EX527(a SIRT1 inhibitor) groups, with 10 rats in each group. The rat model of migraine was established by electrical stimulation of the trigeminal ganglion. Before modeling, EA was applied at "Waiguan"(TE5) and "Fengchi"(GB20) for 20 min each time, once a day for 5 consecutive days, and intraperitoneal injection of EX527 (5 mg/kg) every day simultaneously. Serum prostaglandin E2 (PGE2) and calcitonin gene-related peptide (CGRP) concentrations were measured by enzyme-linked immunosorbent assay. The levels of miR-34a-5p, SIRT1 and interleukin-1ß (IL-1ß) mRNA，and protein expression of SIRT1, IL-1ß, NF-κB p65, NF-κB Ac-p65 and cyclooxygenase-2 （COX2） in trigeminal ganglia were detected by real-time quantitative PCR and Western blot, separately. RESULTS: The serum concentrations of PGE2 and CGRP， the expression of miR-34a-5p, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression in the trigeminal ganglion were remarkably increased (P<0.05), while the SIRT1 mRNA and protein decreased (P<0.05) in the model group in contrast to the normal group. Following EA intervention， the serum PGE2 and CGRP concentrations, miR-34a-5p expression, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression were significantly down-regulated (P<0.05), and SIRT1 mRNA and protein significantly up-regulated (P<0.05). Compared with the EA group, the serum concentrations of PGE2 and CGRP, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expressions increased (P<0.05), and SIRT1 protein decreased (P<0.05) in the EA plus EX527 group. CONCLUSION: In migraine rats, EA can inhibit miR-34a-5p expression in the trigeminal ganglion, increase SIRT1 expression, down-regulate IL-1ß/COX2 inflammation signals, reduce PGE2 synthesis, and thus redue CGRP released from the peripheral terminals, which may be one of the mechanisms of EA in preventing migraine.

miR-34a is involved in CSE-induced apoptosis of human pulmonary microvascular endothelial cells by targeting Notch-1 receptor protein.

BACKGROUND: Abnormal apoptosis of lung endothelial cells has been observed in emphysematous lung tissue and has been suggested to be an important upstream event in the pathogenesis of chronic obstructive pulmonary disease (COPD). Studies have shown that microRNAs (miRNAs) contribute to the pathogenesis of pulmonary diseases by regulating cell apoptosis. The present study was designed to investigate the expression of microRNA-34a (miR-34a) in human pulmonary microvascular endothelial cells (HPMECs) exposed to cigarette smoke extract (CSE), and the potential regulatory role of miR-34a in endothelial cell apoptosis. RESULTS: Our results showed that the expression of miR-34a was significantly increased in CSE-treated HPMECs, and inhibiting miR-34a attenuated CSE-induced HPMEC apoptosis. Furthermore, expression of Notch-1, a receptor protein in the Notch signalling pathway, was decreased and was inversely correlated with miR-34a expression in HPMECs treated with CSE. Computational miRNA target prediction confirmed that Notch-1 is a target of miR-34a. Luciferase reporter assay further confirmed the direct interaction between miR-34a and the 3'-untranslated region (UTR) of Notch-1. Restoration of Notch-1 pathway was able to partially block the effect of miR-34a on HPMEC apoptosis. These results indicate that Notch-1 is a critical downstream target of miR-34a in regulating the CSE-induced HPMEC apoptosis. CONCLUSIONS: Our results suggest that miR-34a plays a key role in CSE-induced endothelial cell apoptosis by directly regulating its target gene Notch-1 in endothelial cells.