Neuroprotective effects of triterpenoid saponins from Medicago sativa L. against H2O2-induced oxidative stress in SH-SY5Y cells

Medicago sativa L. is a forage legume plant widely distributed in all continents. Six new triterpenoid saponins, Medicagosides A-F (1–6) and five known ones (7–11) were isolated from M. sativa. Their structures were determined via HRESIMS, 1D and 2D NMR analysis. Biologically, all the isolates displayed neuroprotective activities against H₂O₂-induced damage in SH-SY5Y cells. Among them, compounds 1, 3–5 and 10 exhibited striking neuroprotective activities at 100 μM, restoring cell viability range from 79.66% to 89.03%, relative to 79.46% (100 μM) of Trolox used as the positive control.     

Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.     

Artemisianins A-D,new stereoisomers of seco-guaianolide involved heterodimeric [4+2] adducts from Artemisia argyi induce apoptosis via enhancement of endoplasmic reticulum stress

Artemisianins A-D (1–4), four stereoisomers of sesquiterpenoid dimers, forming via [4+2] cycloaddition from a 1, 10-seco-guaianolide dienophile and a guaianolide diene, along with two biosynthetically related precurors 5 and 6, were isolated from the famous traditional Chinese medicine Artemisia argyi. The structures of 1–4, including their absolute configurations, were elucidated by extensive spectroscopic data and ECD/TDDFT calculation analysis. Compounds 1–4 exhibited cytotoxicity with IC₅₀ values ranging from 7.2 to 23.3 μM. The accumulation of Ca²⁺ in cytoplasm and enlarged endoplasmic reticulum (ER) indicated that 1 mediated HT-29 cancer cell apoptosis through improvement of ER-stress, which was further proved by unfolded protein response (UPR) pathway on basis of the upregulation of IRE1α, p-PERK, ATF6, and CHOP.     

Characteristics of lysophosphatidylcholine-induced Ca2+ response in human neuroblastoma SH-SY5Y cells

Lysophosphatidylcholine (LPC) is an important bioactive lipid. In the nervous system, elevated levels of LPC have been shown to produce demyelination. In the present study, we examined the effect of exogenous LPC on intracellular Ca2+ mobilization in human neuroblastoma SH-SY5Y cells. In Ca2+-containing medium, introduction of LPC induced a steady rise in cytosolic Ca2+ levels ([Ca2+]i) in a dose-dependent manner, and this rise was provoked by LPC itself, not by its hydrolysis product produced by lysophospholipase. The increase in [Ca2+]i was reduced by 36% by removal of extracellular Ca2+, while preincubation of the cells with verapamil, an L-type Ca2+ channel blocker, inhibited the response by 23%, part of the Ca2+ influx. Conversely, Ni2+, which inhibits the Na+-Ca2+ exchanger, or Na+-deprivation did not affect LPC-induced Ca2+ influx. In Ca2+-free medium, depletion of Ca2+ stores in the endoplasmic reticulum (ER) by thapsigargin, an ER Ca2+-ATPase inhibitor, abolished the Ca2+ increase. Moreover, LPC-induced [Ca2+]i increase was fully blocked by ruthenium red and procaine, inhibitors of ryanodine receptor (RyR), but was not affected by 2-aminoethoxydiphenyl borate, an inhibitor of inositol triphosphate receptor, or by pertussis toxin, a G(i/o) protein inhibitor. Combined treatment with verapamil plus thapsigargin markedly inhibited but did not abolish the LPC-induced Ca2+ response. These findings indicate that LPC-induced [Ca2+]i increase depends on both external Ca2+ influx and Ca2+ release from ER Ca2+ stores, in which L-type Ca2+ channels and RyRs may be involved. However, in digitonin-permeabilized SH-SY5Y cells, LPC could not induce any [Ca2+]i increase in Ca2+-free medium, suggesting that LPC may act indirectly on RyRs of ER.     

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

Mobilization of Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Stores Supports Bradykinin- and Muscarinic-Evoked Release of [3H]Noradrenaline from SH-SY5Y Cells

Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [³H]-noradrenaline ([³H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca²⁺] ([Ca²⁺]_e) was buffered to ～50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ～50% with EC₅₀ values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log₁₀), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular free [Ca²⁺] ([Ca²⁺]_i). These elevations were reduced at low [Ca²⁺]_e and under these conditions the EC₅₀ values for peak elevation of [Ca²⁺]_i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC₅₀ determinations). At low [Ca²⁺]_e, depletion of nonmitochondrial intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca²⁺]_i and a minor release of [³H]NA. At low [Ca²⁺]_e, thapsigargin abolished elevation of [Ca²⁺]_i in response to methacholine and bradykinin and completely inhibited their stimulation of [³H]NA release. It is proposed, therefore, that Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [³H]NA release in SH-SY5Y cells.     

Cytoprotective effect of polypeptide from Chlamys farreri on neuroblastoma (SH-SY5Y) cells following H2O2 exposure involves scavenging ROS and inhibition JNK phosphorylation

Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Treatment with antioxidants is a promising approach for slowing disease progression. In this study, we used the neuroblastoma SH-SY5Y cells as an in vitro model to first assess the effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, on H₂O₂-induced neuronal cell death. Pre-treatment of SH-SY5Y cells with PCF inhibited H₂O₂-induced cell death in a concentration-dependent manner. In parallel, intracellular reactive oxygen species generation and lipid peroxidation were inhibited by PCF. Under severe H₂O₂ insult, PCF promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. PCF also protected DNA from oxidative damage and enhanced the removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA. Further, we found that PCF potentially prevented H₂O₂–induced cell apoptosis. When investigated mitogen-activated protein kinase signaling pathway, we found that pre-treatment of cells with PCF significantly blocked H₂O₂–induced phosphorylation of c- Jun N-terminal kinase of the mitogen-activated protein kinase family. However, PCF had little inhibitory effect on the H₂O₂–induced activation of extracellular signal-regulated kinase. Taken together, these data demonstrate that PCF prevents oxidative stress-induced reactive oxygen species production and c- Jun N-terminal kinase activation and may be useful in the treatment of neurodegenerative diseases.