褪黑素对大鼠腹膜透析相关性腹膜纤维化的影响

目的观察褪黑素(melatonin,MT)对大鼠腹膜透析相关性腹膜纤维化的影响。方法将38只雄性SD大鼠分为对照组(6只)、腹膜纤维化模型组(8只)、模型+低剂量MT(5mg·kg~(-1)·d~(-1))腹腔给药组(8只)、模型+中剂量MT(10mg·kg~(-1)·d~(-1))腹腔给药组(8只)、模型+高剂量MT(20 mg·kg~(-1)·d~(-1))腹腔给药组(8只)。腹腔注射4.25%腹膜透析液100 ml·kg~(-1)·d~(-1)建立腹膜纤维化大鼠模型。实验第28天行4h腹膜平衡实验(peritoneal equilibration test,PET),量取超滤量(ultrafiltration,UF),检测腹透液尿素氮浓度(dialysate urea nitrogen concentration,D)、血浆尿素氮浓度(plasma urea nitrogen concentration,Purea)、初始腹膜透析液葡萄糖浓度(D0)、4 h后透出液葡萄糖浓度(D4),并计算D/Purea、D4/D0。取大鼠壁层腹膜组织行HE和Masson染色,采用免疫组化方法检测转化生长因子β1(transforming growth factorβ1,TGF-β1),胶原Ⅰ(collagenⅠ,Col-Ⅰ)和α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达。结果 4 h PET显示模型组与对照组相比,UF和D4/D0明显减少(P0.05),D/Purea明显增加(P0.05);MT各给药组与模型组比较,UF和D4/D0明显增加(P0.05),D/Purea明显减少(P0.05);HE和Masson染色显示对照组腹膜菲薄,间皮细胞连接紧密,模型组腹膜明显增厚,部分可见间皮细胞缺失,间皮下可见胶原沉积,MT各给药组上述腹膜病理改变较模型组明显减轻;免疫组化显示模型组TGF-β1、Col-Ⅰ、α-SMA表达明显高于对照组(P0.05),MT各给药组表达明显低于模型组(P0.05)。结论 MT能通过改善腹膜的结构和功能来发挥其对抗大鼠腹膜纤维化的作用并能抑制TGF-β1、Col-Ⅰ和α-SMA表达。     

褪黑激素对增生性瘢痕成纤维细胞增殖的影响及机制研究

目的：探讨褪黑激素对人增生性瘢痕成纤维细胞增殖的影响及作用机制。方法收集人皮肤增生性瘢痕标本,原代培养人皮肤增生性瘢痕成纤维细胞(HSFBs)。将HSFBs细胞根据不同的处理方法分为空白对照组、低浓度褪黑激素组、中浓度褪黑激素组、高浓度褪黑激素组、NVP-BEZ235处理组、NVP-BEZ235+褪黑激素组、褪黑激素+siRNA-PTEN组、褪黑激素+siRNA-scramble组。MTT法检测各组细胞的增殖情况,qRT-PCR和Western blot检测各组细胞中磷酸酶与张力蛋白同源物(PTEN)、CyclinD1和Caspase-3的表达以及PI3k/AKt/mTOR通路相关蛋白p-Akt和p-mTOR的蛋白水平。结果与空白对照组比较,从低到高浓度褪黑激素均能够减少HSFBs细胞同一时间点OD值,上调PTEN和Caspase-3表达量,同时抑制CyclinD1、p-Akt和p-mTOR表达水平,组间差异具有统计学意义(P<0.05)。同时与高浓度褪黑激素组比较,NVP-BEZ235+褪黑激素组中细胞增殖显著被抑制,差异具有统计学意义(P<0.05);沉默PTEN后拮抗褪黑激素对HSFBs细胞增殖和PI3K/Akt/mTOR信号通路有抑制作用,差异具有统计学意义(P<0.05)。结论褪黑激素通过上调PTEN表达,抑制PI3K/Akt/mTOR信号通路活化从而抑制HSFBs细胞增殖。     

Long-term study of dendritic spines from hippocampal CA1 pyramidal cells, after neuroprotective melatonin treatment following global cerebral ischemia in rats

Melatonin reduces pyramidal neuronal death in the hippocampus and prevents the impairment of place learning and memory in the Morris water maze, otherwise occurring following global cerebral ischemia. The cytoarchitectonic characteristics of the hippocampal CA1 remaining pyramidal neurons in brains of rats submitted 120 days earlier to acute global cerebral ischemia (15-min four vessel occlusion, and melatonin 10mg/(kg h 6h), i.v. or vehicle administration) were compared to those of intact control rats in order to gain information concerning the neural substrate underlying preservation of hippocampal functioning. Hippocampi were processed according to a modification of the Golgi method. Dendritic bifurcations from pyramidal neurons in both the oriens-alveus and the striatum radiatum; as well as spine density and proportions of thin, stubby, mushroom-shaped, wide, ramified, and double spines in a 50 microm length segment of an oblique dendrite branching from the apical dendrite of the hippocampal CA1 remaining pyramidal neurons were evaluated. No impregnated CA1 pyramidal neurons were found in the ischemic-vehicle-treated rats. CA1 pyramidal neurons from ischemic-melatonin-treated rats showed stick-like and less ramified dendrites than those seen in intact control neurons. In addition, lesser density of spines, lower proportional density of thin spines, and higher proportional density of mushroom spines were counted in ischemic-melatonin-treated animals than those in the sinuously branched dendrites of the intact control group. These cytoarchitectural arrangements seem to be compatible with place learning and memory functions long after ischemia and melatonin neuroprotection.     

Melatonin protects against epirubicin-induced cardiotoxicity

We investigated the cytoprotective effect of melatonin in epirubicin-induced cardiotoxicity using four experimental groups of male Wistar rats: untreated control rats, epirubicin-treated rats, epirubicin+melatonin-treated rats, and melatonin-treated rats. We examined the histopathological and biochemical effects of melatonin on the epirubicin-induced changes and measured the levels of the lipid peroxidation end-product (malondialdehyde, MDA), an indicator of nitric oxide (NO) synthesis (nitrite/nitrate production), and reduced glutathione (GSH) in the heart. We also studied the extracellular matrix components (fibronectin, laminin) in the heart. Vacuole formation, mitochondrial deformation and degeneration, and disordered myofibrillary structures were detected ultrastructurally in the epirubicin-treated group. The degeneration was reduced in the heart tissues of the epirubicin+melatonin group. Epirubicin increased the nitrite/nitrate production, but did not change the MDA and GSH levels significantly. Melatonin treatment lowered the nitrite/nitrate concentrations, while increasing the GSH levels, which exceeded the levels in epirubicin+melatonin-treated rats. We conclude that the epirubicin increased the nitrozative stress, not the oxidative stress, in heart tissue, and the cardioprotective effect of melatonin was partially attributed to the suppression of epirubicin-induced nitrozative stress. These results suggest that melatonin partially protects against epirubicin-induced cardiotoxicity.     

不同光照方式对慢性阻塞性肺疾病急性加重 患者血清褪黑素水平及近期预后的影响

目的 探讨不同光照方式对慢性阻塞性肺疾病急性加重（acute exacerbation of chronic obstructive pulmonary disease,AECOPD）患者血清褪黑素（melatonin,Mel）及近期预后的影响。方法 选取2019年7月至2020年12月浙江省人民医院收治的96例AECOPD患者为研究对象,按照随机数字表法分为对照组（n=46）和观察组（n=50）。对照组患者给予24h持续光照,观察组患者采用间断光照方式,8:00至20:00给予持续光照,20:00至次日8:00关闭光照并为患者佩戴黑色眼罩,连续观察10d。比较两组患者血清Mel及尿6-羟基硫酸褪黑素（6-sulphatoxymelatonin,6-SMT）水平变化及撤机成功率、重症监护室（intensive care unit,ICU）停留时间、多器官功能障碍综合征（multiple organ dysfunction syndrome,MODS）发生率、住院28 d生存率差异。结果 观察组患者7:00和22:00的血清Mel及尿6-SMT水平均呈现升高趋势,且入院后第5天和第10天上述指标均显著高于同期对照组（P<0.05）。观察组患者撤机成功率显著高于对照组,ICU停留时间显著短于对照组,MODS发生率显著低于对照组（P<0.05）。观察组患者住院28d的生存率略高于对照组,但两组比较差异无统计学意义（84.0% vs 73.9%,χ2=0.390,P=0.532）。结论 对AECOPD患者实施间断光照能够明显提高患者体内Mel及其代谢产物水平,改善近期预后,值得在临床中推广应用。     

Determination of melatonin content in traditional Thai herbal remedies used as sleeping aids

Background

Melatonin content was screened in leaves of seven edible herbs used as sleeping aids in Thai traditional medicine. These plants are Piper nigrum L, Sesbania glandiflora (L.) Desv., Sesbania sesban (L.) Merr., Senna tora (L.) Roxb., Moringa oleifera Lam., Momordica charantia L. and Baccaurea ramiflora Lour. Dried leaves were extracted by sonication in methanol for six hours at room temperature, and then melatonin was purified by C18 solid phase extraction (SPE). Melatonin was then quantified by a validated RP-C18 HPLC method with fluorescent detection.

Findings

Melatonin contents in extracts of B. ramiflora, S. glandiflora, M. charantia, S. tora and S. sesban were 43.2, 26.3, 21.4, 10.5 and 8.7 ng/g of dry sample weight, respectively. The highest melatonin content was from P. nigrum extract (1092.7 ng/g of dry sample weight). Melatonin was not detected in the extract of M. oleifera. Melatonin identification was confirmed by ELISA.

Conclusions

Melatonin was found in six of the seven herbs in the traditional Thai sleeping recipe. One of these, P. nigrum, exhibited an encouragingly high amount of melatonin.     

Potential use of melatonin supplementation to protect vitrified testicular grafts from hypoxic–ischaemic damage

This study aimed to assess the morphological changes in neonate vitrified testicular grafts after host treatment with melatonin. Neonate vitrified testes, candidates for transplantation to treated and nontreated groups receiving melatonin, were thawed in media containing or not containing 100 μm melatonin. Following transplantation, melatonin (20 mg kg^?1 per day) and saline were given to the treated and nontreated groups for up to 9 weeks. The testicular status was assayed by terminal deoxynucleotidyl transferase (TdT)‐mediated deoxyuridine triphosphate (dUTP)‐biotin nick‐end labelling TUNEL, semi‐thin section and ultra‐structure studies. Chi‐squared test was used to compare categorical variables between the groups. Overall, the degenerative and apoptosis changes in the vitrified testis parenchyma were not significant. However, atrophic seminiferous cords and jumbled appearance of the interstitial space were more often observed in the nontreated group than in the treated ones. Semi‐thin sections showed that the germinal epithelium was taken in a normal arrangement on the testicular grafts of both groups. Nevertheless, ultrastructural analysis revealed that the characteristics of interstitial space cells, basement membrane BM and epithelial cells of seminiferous tubules in the treated group were better than those in the nontreated group. The study revealed a beneficial effect of melatonin on vitrified neonatal testes and after that, on restoring testicular grafts.     

Melatonin alterations and brain acetylcholine lesions in sleep disorders in Cockayne syndrome

Background: Cockayne syndrome (CS) is a genetic disorder caused by deficient nucleotide excision repair. Patients with CS exhibit progeroid features, developmental delay, and various neurological disorders; they are also known to suffer from sleep problems, which have never been investigated in detail. Objective: The aim of this study is to investigate the pathogenesis of sleep disorders in patients with CS. Methods: We performed a questionnaire survey of the families of patients with CS, enzyme-linked immunosorbent analyses of the melatonin metabolite, 6-sulphatoxymelatonin (6-SM), in the patients’ urine, and immunohistochemistry in the hypothalamus, the basal nucleus of Meynert (NbM), and the pedunculopontine tegmental nucleus (PPN) in four autopsy cases. Results: Sleep–wakefulness rhythms were disturbed in patients with CS, and these disturbances seemed to be related to a reduced urinary excretion of 6-SM. In addition, although the hypothalamic nuclei were comparatively preserved, acetylcholine neurons (AchNs) were severely decreased in the NbM and PPN. Conclusions: AchNs modulate both arousal and rapid eye movement sleep, and selective lesions of AchNs in the PPN and/or NbM in combination with disturbed melatonin metabolism might be involved in the sleep disorders in CS.     

The melatonin analog 5-MCA-NAT increases endogenous dopamine levels by binding NRH:quinone reductase enzyme in the developing chick retina

NRH:quinone reductase (QR2) is present in the retinas of embryonic and post-hatched (PH) chicks. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a QR2 ligand that increases cAMP levels in developing retinas, but it does not affect cAMP levels in CHO-QR2 cells. The dopamine quinone reductase activity of QR2 retrieves dopamine, which increases cAMP levels in developing retinas. The objective of the present study was to investigate whether 5-MCA-NAT increases endogenous dopamine levels in retinas from chick embryos and post-hatched chicks. Endogenous dopamine was measured by enzyme-linked immunosorbent assay (ELISA). 5-MCA-NAT increased retinal endogenous dopamine levels at all developmental stages studied and in PH chicks (−log EC50 = 11.62 ± 0.34 M). This effect was inhibited by non-selective antagonists of receptors and melatonin binding sites N-acetyl-2-benzyltryptamine (luzindole, 5 μM), but it was not inhibited by the Mel1b melatonin receptor antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT, 10 nM). The QR2 cosubstrate, N-methyl-dihydronicotinamide (NMH) (−log EC50 = 6.74 ± 0.26 M), increased endogenous dopamine levels in controls and in retinas stimulated with 5-MCA-NAT (3 nM). The QR2 inhibitor benzo[e]pyrene inhibited endogenous dopamine levels in both control (−log IC50 = 7.4 ± 0.28 M) and NMH-stimulated (at 100 nM and 1 μM benzo[e]pyrene concentrations) retinas. Theoretical studies using Molegro Virtual Docking software corroborated these experimental results. We conclude that 5-MCA-NAT increases the level of endogenous dopamine via QR2. We suggest that this enzyme triggers double reduction of the dopamine quinone, recovering dopamine in retinal development.