Evaluation of Effects of Melatonin in Reducing Postoperative Pain and Oxidative Stress in Children Undergoing Surgery,. Trought Circulating miR-34 and miR-124a, 4-Hydroxynonenal and Sirtuin 1 (SIRT1) Plasma Concentrations.
Evaluation of Effects of Melatonin in Reducing Postoperative Pain and Oxidative Stress in Children Undergoing Surgery,. Trought Circulating miR-34 and miR-124a, 4-Hydroxynonenal and Sirtuin 1 (SIRT1) Plasma Concentrations.
Background. Long-term consequences of postoperative pain are detrimental in children. We tested the hypothesis that melatonin reduces postoperative pain and oxidative stress involving sirtuin pathway in children undergoing surgery.
Methods. Thirty-one children were randomly assigned to oral supplementation with melatonin or placebo, before surgery. Plasma levels of 4-hydroxynonenal (4-HNE), melatonin, sirtuin 1 (SIRT1), and circulating miR-34 and miR-124a were analyzed at T0 (pre-hospitalization), T1 (before surgery), and T2 (1 h after the end of the surgery).
Melatonin shows a potential role in protecting neonates undergoing surgery from the deleterious effects of oxidative stress (OS). After administration of exogenous melatonin, a significant reduction in lipid and protein peroxidation in the postoperative period has been reported in neonates. Recently, Song et al. reported that sirtuin 1 (SIRT1) also plays a critical role in the pathogenesis of pain, showing analgesic effects in chronic pain conditions such as neuropathic and inflammatory pain. In this prospective, randomized, double-blind pilot study, we test the hypothesis that melatonin reduces OS and postoperative pain involving the sirtuin pathway in children undergoing surgery.
Melatonin (Dicoson, Dicofarm, Italy, 5 drops = 1 mg) was administered orally. The product is listed in the Register of Dietary Supplements on the website of the Ministry of Health (http://www.ministerosalute.it/alimenti/dietetica) and is classified with the following code: 943314283. This product is subject to the European Directive on Foodstuffs according to DL n. 169 of May 21, 2004, and not to the European Directive on Medicines 2001/20/EC transposed at the Italian level with D.L. n. 211 of June 24, 2003. Melatonin administration has a good safety profile, with no known adverse effects.
Melatonin plasma levels were assessed at each experimental time point (T0, T1, T2) using a competitive enzyme-linked immunosorbent assay (cELISA) kit from Antibodies.com (A87093) according to the manufacturer's instructions. T1 and T2 plasma samples from Mel treated children suspected of containing concentrations higher than the highest standard (500 pg/mL) were diluted 1:100 (v/v) with sample diluent prior to analysis. Color development was monitored at 450 nm in a Thermo Scientific (MultiSkan FC) microplate reader, and a standard curve (range 7.813-500 pg/mL) was generated using a four-parameter logistic (4-PL) curve fit. The sensitivity of the assay was 4.688 pg/mL; the intra- and inter-assay coefficients of variation were <8% and <10%, respectively.
4-Hydroxynonenal (4-HNE) as a marker of lipid peroxidation was measured to evaluate OS by using a cELISA kit from Antibodies.com (A86962). Plasma concentrations were calculated by reading the absorbance at 450 nm and referring to the standard curve (range 31.25-2000 pg/mL). The sensitivity of the assay was 18.75 pg/mL; the intra- and inter-assay coefficients of variation were <8% and <10%, respectively.
SIRT1 was quantified using an ELISA kit from Invitrogen (EH427RB). Plasma samples were diluted 1:2 as indicated by the Manufacturer before analysis. SIRT1 concentrations were calculated by absorbance reading at 450 nm and referring to the standard curve (range 1.23-300 ng/mL). The sensitivity of the assay was 1.23 ng/mL; the intra- and inter-assay coefficients of variation were <10% and <12%, respectively.
We performed microRNA analyses based on the quali-quantitative plasma sample available (N=3 at each time point, T0, T1, and T2). MicroRNAs (miR-34 and miR-124a) were isolated from plasma using the Norgen total RNA isolation kit13.The Plasma microRNAs and spike-in cel-miR-39 expressions were evaluated using the TaqMan miRNA assay. The TaqMan miRNA reverse transcription kit was used to reverse transcribe miRNAs. Subsequently, RT-qPCR was performed in 20 μL of PCR mix containing 1 μL of 20× TaqMan miRNA assay, which contained PCR primers and probes (5'-FAM), 10 μL of 2×TaqMan Universal PCR Master Mix No Amp Erase UNG and 5 μL of reverse-transcribed product. The reaction was first incubated at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The quantitative real-time PCR (RT-qPCR) was performed on a ABIPRISM 7500 Real Time PCR System. Data was analyzed by a 7500-system software (1 1.4.0) with the automatic comparative threshold (Ct) setting for adapting baseline. Detection thresholds were set at 35 Ct. The relative amounts of miR-34 and miR-124a were calculated using the Ct method: ΔCt = Ct (miR-34/miR-124a) - Ct (refence miRNA); 2-ΔCt.
Inclusion Criteria:
Children between 3 and 5 years of age scheduled for elective surgery, parental consent
Exclusion Criteria:
Children with cerebral malformations and/or injuries, or surgery in the afternoon or at night to eliminate conditions that could affect melatonin production, or denial of parental consent