РОЛЬ НЕТОЗА В ПАТОГЕНЕЗЕ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТИ1 Юринская М.М., 2 Сусликов А.В., 1 Винокуров М.Г. 1 Институт биофизики клетки Российской академии наук -обособленное подразделение ФГБУН «Федеральный исследовательский центр «Пущинский научный центр биологических исследований» Российской академии наук», Пущино, e-mail:

—Hsp70 and hydrogen sulfide donors reduce inflammatory processes in human and animal cells. The biological action mediated by Hsp70 and H 2 S donors (GYY4137 and sodium thiosulfate) depends on their protection kinetics from cell activation by lipopolysaccharides. However, the molecular mechanisms of action of Hsp70 and H 2 S are not well understood. We studied the effect of human recombinant Hsp70 and H 2 S donors on the formation of reactive oxygen species and tumor necrosis factor-alpha induced in human cells (THP-1) by lipopolysaccharides. Transcriptomic changes occurring in these cells after LPS administration in combination with GYY4137 pretreatment were investigated. The results we obtained showed that Hsp70 and hydrogen sulfide donors reduce inflammatory processes in cells activated by the action of LPS. Hsp70 and H 2 S donors differed in the kinetics of the protective action, while hydrogen sulfide donors turned out to be more effective. The role of endocytosis in the mechanisms of protection of cells by H 2 S and Hsp70 donors from the action of LPS was studied. It has been found that GYY4137 pretreatment of LPS-exposed cells reduces the LPS-induced induction of various pro-inflammatory genes and affects the expression of genes of various intracellular signaling pathways.

АДРЕНОБЛОКАТОРЫ ПРОПРАНОЛОЛ И ФЕНТОЛАМИН МОДУЛИРУЮТ LPS-ИНДУЦИРОВАННУЮ ВНУТРИКЛЕТОЧНУЮ СИГНАЛИЗАЦИЮ НЕЙТРОФИЛОВ ЧЕЛОВЕКА1,2 Юринская М.М., 1,2 Винокуров М.Г., 2 Сусликов А

КЛЕТОЧНАЯ ГИБЕЛЬ И ФУНКЦИОНАЛЬНАЯ АКТИВНОСТЬ НЕЙТРОФИЛОВ У БОЛЬНЫХ ХРОНИЧЕСКОЙ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТЬЮ ПРИ ЭНДОТОКСЕМИИ1,2 Юринская М.М., 1,2 Винокуров М

ШАПЕРОН HSP70 МОДУЛИРУЕТ ПУТИ ЭНДОЦИТОЗА КЛЕТОК RAW264.7 ПРИ LPS-ИНДУЦИРОВАННОЙ АКТИВАЦИИ1 Юринская М.М., 2 Евгеньев М.Б., 1 Афанасьев В.Н

The effects of exogenous recombinant human heat shock protein Hsp70 and hydrogen sulfide donor GYY4137 on the mechanisms of endocytosis of lipopolysaccharide (LPS) by human neuroblastoma cells SH-SY5Ywas studied. Hsp70 and GYY4137 have been shown to significantly reduce LPS-induced production of inflammatory mediators by SH-SY5Y cells, including reactive oxygen species, nitric oxide, TNFα, IL-1β, and IL-6. Both the recombinant protein Hsp70 and the hydrogen sulfide donor GYY4137 exhibited significant protective effects; however, the combined use of these agents did not lead to a cumulative effect. It has been shown that pinocytosis, as well as clathrin-, caveolin-, tubulin- and receptor-dependent endocytosis were involved in protecting the cells by both the hydrogen sulfide donor and Hsp70 from LPS-induced production of reactive oxygen species and NO.

BACKGROUND:Hydrogen sulfide donors reduce inflammatory signaling in vitro and in vivo. The biological effect mediated by H2S donors depends on the kinetics of the gas release from the donor molecule. However, the molecular mechanisms of H2S-induced immunomodulation were poorly addressed. Here, we studied the effect of two different hydrogen sulfide (H2S)-producing agents on the generation of the LPS-induced inflammatory mediators. Importantly, we investigated the transcriptomic changes that take place in human cells after the LPS challenge, combined with the pretreatment with a slow-releasing H2S donor-GYY4137.METHODS:We investigated the effects of GYY4137 and sodium hydrosulfide on the release of proinflammatory molecules such as ROS, NO and TNF-α from LPS-treated human SH-SY5Y neuroblastoma and the THP-1 promonocytic cell lines. Transcriptomic and RT-qPCR studies using THP-1 cells were performed to monitor the effects of the GYY4137 on multiple signaling pathways, including various immune-related and proinflammatory genes after combined action of LPS and GYY4137.RESULTS:The GYY4137 and sodium hydrosulfide differed in the ability to reduce the production of the LPS-evoked proinflammatory mediators. The pre-treatment with GYY4137 resulted in a drastic down-regulation of many TNF-α effectors that are induced by LPS treatment in THP-1 cells. Furthermore, GYY4137 pretreatment of LPS-exposed cells ameliorates the LPS-mediated induction of multiple pro-inflammatory genes and decreases expression of immunoproteasome genes. Besides, in these experiments we detected the up-regulation of several important pathways that are inhibited by LPS.CONCLUSION:Based on the obtained results we believe that our transcriptomic analysis significantly contributes to the understanding of the molecular mechanisms of anti-inflammatory and cytoprotective activity of hydrogen sulfide donors, and highlights their potential against LPS challenges and other forms of inflammation.

Neuroinflammation plays a key role in the pathogenesis of neurodegenerative diseases. Microglial cells are the main immune cells of the central nervous system. On exposure to lipopolysaccharides (LPS, components of the cell wall of Gram-negative enterobacteria), microglia is activated to produce reactive oxygen species (ROS), cytokines, and inflammatory mediators, which may cause neuron death. Exogenous recombinant human heat shock protein 70 (HSP70) was tested for effect on the activation of human microglial and neuroblastoma cells in response to LPS from Escherichia coli. Experiments included cell cultivation separately and transferring the conditioned medium from A-172 microglial cells to SK-N-SH neuroblastoma cells to simulate the effect of microglia treated with LPS and/or HSP70. The levels of ROS, TNFα, and apoptosis in LPS-treated cells were estimated in the presence or absence of HSP70. HSP70 was found to reduce the LPS-induced ROS generation, TNFα production, apoptosis, and necrosis, in both separate cell cultures and neuroblastoma cells grown in the conditioned medium from microglial cells. Signaling pathways involving protein kinases p38MAPK, JNK, and PI3K were demonstrated to play an important role in HSP70-mediated protection of microglial and neuroblastoma cells from LPS-induced apoptosis and ROS production.

Lipopolysaccharides (LPS), components of the cell wall of Gram-negative bacteria, activate neutrophils that trigger pathological processes, including Gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellular glutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by lps and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Lipopolysaccharides (LPS), components of the cell wall of gram-negative bacteria, activate neutrophils that trigger pathological processes, including gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellularglutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by LPS and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

В различных заболеваниях млекопитающих большое значение имеют эндотоксины (ЭТ) грамотрицательных энтеробактерий, вызывающие увеличение генерации активных форм кислорода (ROS) полиморфноядерными лейкоцитами (ПМЯЛ). Индуцибельный белок теплового шока человека Бтш70 при внеклеточном применении снижает продукцию ROS полиморфноядерными лейкоцитами, индуцированную ЭТ. В работе установлено, что в механизме защиты ПМЯЛ белком Бтш70 от активации клеток ЭТ важная роль принадлежит клатринзависимому и рецепторзависимым путям эндоцитоза Бтш70.

Intact amyloid-β peptides (Aβ) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aβ present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer’s disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aβ isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aβ. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aβ, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aβ molecule from zinc-mediated interactions with the same site of another Aβ molecule can effectively inhibit the oligomerization and aggregation of Aβ. Taking into account the similarity in the structural organization of zinc-binding sites within Aβ and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aβ. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aβ and Aβ with isomerized Asp7 (isoAβ). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aβ(1–42) and isoAβ(1–42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aβ in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

Neuronal dysfunction and loss associated with the accumulation of amyloid-β (Aβ) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer's disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aβ42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aβ42 with isomerised Asp7 (isoAβ42) which is one of the most abundant Aβ isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAβ42 rather than intact Aβ42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAβ42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAβ42, being to date the only known synthetic Aβ species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAβ42 in AD progression and provide possible neurotoxicity paths for this particular isoform.

The accumulation and aggregation of β-amyloids are major molecular events underlying the progression of Alzheimer's disease. In neural cells, recombinant HSP70 reduces the toxic effect of Aβ and its isomeric forms. Here we describe the proteome of the neuroblastoma cell line after incubation with amyloid peptides Aβ42 and isomerized Asp7 (isoAβ42) without and with human recombinant heat shock protein 70 (HSP70). Incubation of SH-SY5Y cell culture with the synthetic Aβ-peptides leads to a decrease in the levels of several cytoskeleton proteins (e.g., ACTN1, VIME, TPM3) and several chaperonines involved in the folding of actin and tubulin (TCPQ, TCPG, TCPE, TCPB). These changes are accompanied by an increase in the expression of calmodulin and the proteins involved in folding in the endoplasmic reticulum and endoplasmic cell stress response. The presence of exogenous HSP70 has led to an increase in expression of several chaperones and a few other proteins including endogenous HSP70. A combined effect of recombinant HSP70 with Aβ peptides reduced cell apoptosis and significantly decreased the level of tubulin phosphorylation caused by the addition of Aβ peptides.

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer’s disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-β (Aβ42) and its more toxic form with isomerized Asp7 (isoAβ42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAβ42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAβ42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aβ peptides, proteasomes, and HSP70.

Experimental evidences indicate that heat-shock protein 70 (HSP70) can serve as a prospective therapeutic agent to treat Alzheimer's disease (AD). It has demonstrated a neuroprotective effect in vivo on mice models of AD. Moreover, HSP70 decreases oxidative stress in neurons induced by amyloid-β (Aβ42) and its more toxic form with isomerized Asp7 (isoAβ42). The dysfunction of Ubiquitin-proteasome system (UPS) is observed in AD. UPS is responsible for the degradation of the majority of cellular proteins and plays an important role in protecting cells from oxidative stress. Here, we have shown that the incubation of human neuroblastoma cells SK-N-SH with isoAβ42 increases the activity of intracellular proteasomes, which are the principal elements of the UPS. On the contrary, the proteasomal activity was decreased in isoAβ42-treated cells in the presence of exogenous HSP70. These results highlight the existence of an interplay between Aβ peptides, proteasomes, and HSP70.

Изучено влияние гемодеривата крови телят (актовегин) на выживаемость и гибель нейробластомных клеток человека линии SK-N-SH в зависимости от длительности воздействия этого препарата. Количество живых и погибших клеток в пробах определяли методом флуоресцентной микроскопии с использованием флуоресцентных зондов Hoechst 33342 и йодида пропидия на микроскопе Keyence BZ8100, Япония. Апоптоз регистрировали по наличию фрагментации ДНК клеток. При культивировании нейробластомных клеток человека in vitro их количество через 48 и 72 ч возрастало на 8,5 % (p < 0,05) и 18,4 % (p < 0,005), соответственно. Актовегин, по сравнению с контролем, концентрационно-зависимо снижал количество живых SK-N-SH клеток и этот процесс зависел от времени его экспозиции (актовегин 1 мг/мл: через 72 ч на 23,8 %; актовегин 5 мг/мл: через 48 на 25,1 %, через 72 ч на 33,2 %, p < 0,05). Анализ клеточной гибели показал, что в контроле и при добавлении актовегина в концентрации 0,2 мг/мл на всех этапах культивирования отмечалось увеличение процента клеток, погибших как путем апоптоза, так и некроза, в результате чего вклад апоптоза и некроза в клеточную смерть не менялся через 48 и 72 ч. При добавлении актовегина в концентрациях 1 и 5 мг/мл регистрировалось увеличение вклада некроза в клеточную гибель через 48 ч, который затем уменьшался через 72 ч и смещал соотношение в пользу апоптоза. Результаты свидетельствуют о том, что актовегин тормозит деление опухолевых клеток и способен вызывать их гибель, которая происходит как по механизму некроза, так и апоптоза, а относительный вклад этих процессов, вероятно, меняется в зависимости от времени экспозиции препарата.

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.

The progress of neurodegeneration in Alzheimer's disease is closely associated with inflammatory processes in the brain tissues induced by beta-amyloid peptides (Aβ). In this paper, we showed that Aβ(1-42) and isoAβ(1-42) in human neuroblastoma cells SK-N-SH and promonocyte THP-1 activated the production of tumor necrosis factor (TNFα). Notably, isoAβ(1-42) had the strongest effect on the increase in the level of TNFα. The addition of recombinant heat-shock protein HSP70 reduces TNFα production induced by Aβ, which leads to a decrease in neuronal cell damage at the organism level.