Melatonin restores neutrophil functions and prevents apoptosis amid dysfunctional glutathione redox system

Melatonin is a chronobiotic hormone, which can regulate human diseases like cancer, atherosclerosis, respiratory disorders, and microbial infections by regulating redox system. Melatonin exhibits innate immunomodulation by communicating with immune system and influencing neutrophils to fight infections and inflammation. However, sustaining redox homeostasis and reactive oxygen species (ROS) generation in neutrophils are critical during chemotaxis, oxidative burst, phagocytosis, and neutrophil extracellular trap (NET) formation. Therefore, endogenous antioxidant glutathione (GSH) redox cycle is highly vital in regulating neutrophil functions. Reduced intracellular GSH levels and glutathione reductase (GR) activity in the neutrophils during clinical conditions like autoimmune disorders, neurological disorders, diabetes, and microbial infections lead to dysfunctional neutrophils. Therefore, we hypothesized that redox modulators like melatonin can protect neutrophil health and functions under GSH and GR activity–deficient conditions. We demonstrate the dual role of melatonin, wherein it protects neutrophils from oxidative stress-induced apoptosis by reducing ROS generation; in contrast, it restores neutrophil functions like phagocytosis, degranulation, and NETosis in GSH and GR activity–deficient neutrophils by regulating ROS levels both in vitro and in vivo. Melatonin mitigates LPS-induced neutrophil dysfunctions by rejuvenating GSH redox system, specifically GR activity by acting as a parallel redox system. Our results indicate that melatonin could be a potential auxiliary therapy to treat immune dysfunction and microbial infections, including virus, under chronic disease conditions by restoring neutrophil functions. Further, melatonin could be a promising immune system booster to fight unprecedented pandemics like the current COVID-19. However, further studies are indispensable to address the clinical usage of melatonin.     

重组人白血病抑制因子受体α亚基细胞外区的分子克隆

纯化可溶性白血病抑制因子受体，获得特异性免疫原并利用衰变加速因子的附膜特性探讨ＬＩＦ受体的生物特性和功能。采用基因重组技术，将ＬＩＦ受体α亚基细胞外区的Ｃ－端与ＤＡＦＣ－端３７个氨基酸连接，再将该重组基因主ｐＧＥＸ－５Ｔ表达系统。结果：在Ｅ．ｃｏｌｉ ＸＬ１中可高度表达一种Ｎ－端为谷胱甘肽－Ｓ－转移酶和Ｃ－端为１９０ｓｏｌＤＡＦ的融合重组蛋白。     

Glutathione metabolism in skeletal muscle derived cells of the L6 line

Skeletal muscle derived L6 myoblasts possess a considerably high resting total glutathione (TGSH) pool. Exposure to l -buthionine-[S,R]-sulphoximine resulted in a 90% depletion of the intracellular TGSH pool. All the key enzymes of glutathione metabolism, especially glutathione S-transferase, were observed to be considerably active in the undifferentiated cells. Se-dependent glutathione peroxidase activity appeared to account for most of the total GSH peroxidase activity of the cells. A significant contribution of γ-glutamyl transpeptidase-independent (5 mm acivicin insensitive) mechanism to the extracellular GSH uptake capacity of the muscle cells was evident. Efflux of oxidized glutathione from the cells exposed to t-butyl hydroperoxide was rapid and appeared to be energy linked.     

冠心病病人SOD,GSH·PX活性及LPO含量的变化

测定了正常人及冠心病病人红细胞内超氧化物歧化酶(SOD)、血中谷胱甘肽过氧化物酶(GSH·PX)活性及脂质过氧化物(LPO)含量。结果表明,冠心病病人SOD,GSH·PX活性低于正常人,而LPO含量高于正常人,且心肌梗塞病人较心绞痛病人变化更为显著。SOD,GSH·PX均与LPO呈负相关。提示自由基和LPO的增多在冠心病的发病过程中起重要作用。     

AMINO ACID TRANSPORT AND CELLULAR GLUTATHIONE

Levels of cellular glutathione protective against electrophilic agents depend on the inward transport of either cysteine or cystine.     

Reduced levels of glutathione in liver and tumour tissue of the mouse after administration of eupatoriopicrin

In this study the effect of the cytostatic acting sesquiterpene lactone eupatoriopicrin, isolated from Eupatorium cannabinum L., on gluthathione (GSH) levels in liver and tumour tissue of the mouse is described. C57Bl mice, bearing a solidly growing Lewis Lung carcinoma or a FIO 26 fibrosarcoma, with a volume between 500–1000 μL, were injected with eupatoriopicrin 20 or 40 mg/kg, intraperitoneally (i.p.) or intravenously (i.v.). At different time points, between 0–48 h after administration, the GSH content in liver and tumour tissue was determined. A dose dependent reduction of the GSH content was found. No difference in reduction was seen between i.p. and i.v. administration, but the route of administration appeared to be of great importance regarding acute toxicity: 40 mg/kg i.p. was lethal within 48 h, whereas mice receiving the same dose i.v. survived for over three months.     

前列腺素E1联合黄芪对大鼠心肌缺血再灌注损伤的作用

目的：观察前列腺素E1（PGE1）与黄芪（AM）两药合用对大鼠心肌缺血再灌注损伤的保护作用。方法：建立大鼠心肌缺血再灌注模型，结扎冠状动脉左室支30min后在松扎的同时分别给予等容量生理盐水（模型对照组）、PGE1（PGE1组）、AM（AM组）及PGE1＋AM（PGE1＋AM组）60min。用黄嘌呤氧化酶法及硫代巴比妥酸法测定两药单用及半量合用对再灌注心肌组织中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH－Px)活力，丙二醛（MDA）、Ca^2 含量及血清肌酸磷酸激酶同功酶（CK－MB）含量和心律失常情况的影响。结果：与模型对照组及AM组相比，两药合用可显著提高再灌注心肌组织中SOD、GSH－Px活力（P＜0．01－0．05），降低MDA、Ca^2 含量及血清CK－MB含量（P＜0．01－0．05）；显著减轻再灌注室性心律失常（P＜0．01）。结论：PGE1与AM合用在保护心肌缺血再灌注损伤中有显著的协同作用，其保护作用主要与清除自由基有关。     

SPONTANEOUS OCCURRENCE OF PLACENTAL GLUTATHIONE S-TRANSFERASE-POSITIVE FOCI IN THE LIVERS OF LEC RATS

Spontaneous occurrence of placental glutathione S-transferase (GST-P)-positive foci was observed in the livers of 5-month-old LEC rats. Quantitative studies revealed that GST-P foci appeared after the onset of hepatitis. The number and size of GST-P foci increased with age and more foci were induced in males than in females. No sex difference, however, was found in the incidence of hepatitis. Although hepatitis is necessary for the induction of GST-P foci, it is insufficient for their further growth. Since hereditary hepatitis first appears at around 4 months of age, leading to a high incidence of hepatocellular carcinomas in later life, the spontaneous occurrence of the foci may be related to the development of hepatocellular carcinoma.     

The use of mass spectrometry in the study of chemically-reactive drug metabolises. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

Glutathione metabolism in activated human neutrophils: stimulation of glutathione synthesis and consumption of glutathione by reactive oxygen species

Since glutathione (GSH) is involved in the modulation of the function of polymorphonuclear leucocytes (PMN) such as phagocytosis and production of reactive oxygen species, the metabolism of GSH was studied in human PMN. The concentration of GSH in resting PMN amounted to 13.3 nmol 10(-7) PMN and remained stable over 100 min of incubation. Upon activation of PMN with phorbol myristate acetate intracellular GSH decreased to 50% of the resting concentration within 80 min. In the presence of buthionine sulfoximine, which inhibits the synthesis of GSH, the depletion of intracellular GSH was dramatically accelerated, indicating that activation of PMN is associated with a marked stimulation of GSH synthesis. Since a similar depletion of GSH was seen in the presence of propargylglycine, an inhibitor of the cystathionine pathway, most of the cysteine required for the resynthesis of GSH must originate from methionine and not from cysteine generated by the catabolism of GSH. Further studies showed that GSH is sequentially oxidized by O2-. and HOCl, first to GSSG and then to an unidentified compound, most likely a chloramine. In the presence of an adequate supply of GSH and NADPH which is required for the reduction of GSSG by glutathione reductase this further oxidation of GSSG was prevented. Thus, the highly toxic HOCl generated by PMN can be detoxified by the glutathione reducatase system. The capacity of PMN to re-synthesize GSH may be an important determinant of PMN function.