Correction: Licochalcone D reduces H2O2-induced SH-SY5Y cell neurotoxicity by regulating reactive oxygen species

Oxidative stress due to excessive reactive oxygen species (ROS) levels, a primary cause of nerve Oxidative stress due to excessive reactive oxygen species (ROS) levels, a primary cause of nerve damage, contributes to the development of neurodegenerative diseases such as Alzheimer's Disease, damage, contributes to the development of neurodegenerative diseases such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease, Parkinson's Disease, and stroke (Ashok et al., Amyotrophic Lateral Sclerosis, Huntington's Disease, Parkinson's Disease, and stroke (Ashok et al., 2022). Natural compounds are attracting attention as potential alternatives for treating various 2022). Natural compounds are attracting attention as potential alternatives for treating various diseases because they have fewer side effects (Zhang et al., 2020). The antioxidant defense system diseases because they have fewer side effects (Zhang et al., 2020). The antioxidant defense system plays an important role in controlling oxidative stress, and natural products can protect nerve cells plays an important role in controlling oxidative stress, and natural products can protect nerve cells from oxidative stress via various mechanisms (Jeong et al., 2021). from oxidative stress via various mechanisms (Jeong et al., 2021).A small but significant portion of oxygen, approximately 4-5%, undergoes conversion to ROS via A small but significant portion of oxygen, approximately 4-5%, undergoes conversion to ROS via biological reducing agents in the human body (Jomova et al., 2023). ROS, which include superoxide biological reducing agents in the human body (Jomova et al., 2023). ROS, which include superoxide anions, H 2 O 2 , and OH -radicals, are highly reactive (Smith et al., 2022) and play a central role in anions, H 2 O 2 , and OH -radicals, are highly reactive (Smith et al., 2022) and play a central role in neuronal degeneration by disrupting the functions of key biomolecules, causing mitochondrial neuronal degeneration by disrupting the functions of key biomolecules, causing mitochondrial dysfunction, cellular damage, and ultimately cell death (Jomova et al., 2023). Reducing ROS dysfunction, cellular damage, and ultimately cell death (Jomova et al., 2023). Reducing ROS production may therefore provide a promising strategy for preventing and treating these conditions. production may therefore provide a promising strategy for preventing and treating these conditions. Their levels are regulated by major antioxidants, including superoxide dismutases, catalase, and Their levels are regulated by major antioxidants, including superoxide dismutases, catalase, and glutathione peroxidases, thus maintaining homeostasis (Jiang et al., 2022;Jomova et al., 2023). glutathione peroxidases, thus maintaining homeostasis (Jiang et al., 2022;Jomova et al., 2023). Under oxidative stress, however, excessive amounts of ROS are maintained in the body owing to Under oxidative stress, however, excessive amounts of ROS are maintained in the body owing to unstable antioxidant mechanisms, thus disrupting homeostasis (Ashok et al., 2022). H 2 O 2 , a low-unstable antioxidant mechanisms, thus disrupting homeostasis (Ashok et al., 2022). H 2 O 2 , a lowreactivity radical and an ROS, participates in inflammatory processes and is cytotoxic toward various reactivity radical and an ROS, participates in inflammatory processes and is cytotoxic toward various types of cells (Park, 2013;Tochigi et al., 2013). It damages cells by generating the highly reactive types of cells (Park, 2013;Tochigi et al., 2013). It damages cells by generating the highly reactive OH -radical via the Fenton reaction, whereby it is reduced by metal ions such as iron or copper to OH -radical via the Fenton reaction, whereby it is reduced by metal ions such as iron or copper to form highly reactive and harmful hydroxyl radicals (Kumar et al., 2023). Although this renders H 2 O 2 form highly reactive and harmful hydroxyl radicals (Kumar et al., 2023). Although this renders H 2 O 2 cytotoxic to nerve cells, antioxidants can reverse this (Jeong et al., 2021;Ashok et al., 2022). cytotoxic to nerve cells, antioxidants can reverse this (Jeong et al., 2021;Ashok et al., 2022).Licorice is a traditional herbal medicine commonly used in East Asia for the treatment of various Licorice is a traditional herbal medicine commonly used in East Asia for the treatment of various diseases (Jiang et al., 2020). Licorice extract (1 g of extract per kg body weight) has been reported to diseases (Jiang et al., 2020). Licorice extract (1 g of extract per kg body weight) has been reported to have neuroprotective effects (Zhou et al., 2017). Licochalcone D (LCD), extracted from licorice have neuroprotective effects (Zhou et al., 2017). Licochalcone D (LCD), extracted from licorice Glycyrrhiza inflata, is a flavonoid-based biologically active ingredient (Maharajan et al., 2021; Deng Glycyrrhiza inflata, is a flavonoid-based biologically active ingredient (Maharajan et al., 2021;Deng et al., 2023). LCD exerts anti-cancer effects via various pathways in cancers such as skin, oral, and et al., 2023). LCD exerts anti-cancer effects via various pathways in cancers such as skin, oral, and lung cancer (Deng et al., 2023). Via its AMPK activity, it exerts antioxidant, anti-aging, and lung cancer (Deng et al., 2023). Via its AMPK activity, it exerts antioxidant, anti-aging, and cardioprotective effects (Maharajan et al., 2021). Here, we examined its neuroprotective effects cardioprotective effects (Maharajan et al., 2021). Here, we examined its neuroprotective effects against H 2 O 2 -induced neurotoxicity in vitro. against H 2 O 2 -induced neurotoxicity in vitro.The reagents and materials were sourced as follows The SH-SY5Y human neuroblastoma cell line was obtained from the Korean Cell Bank (Seoul, The SH-SY5Y human neuroblastoma cell line was obtained from the Korean Cell Bank (Seoul, Republic of Korea). The cells were cultured for 24 h in DMEM supplemented with growth medium Republic of Korea). The cells were cultured for 24 h in DMEM supplemented with growth medium (10% FBS and 1% penicillin/streptomycin) in a 37 °C humidified atmosphere containing 5% CO 2 .(10% FBS and 1% penicillin/streptomycin) in a 37 °C humidified atmosphere containing 5% CO 2 .The medium was then replaced with differentiation medium (DMEM containing 1% FBS and 10 μM The medium was then replaced with differentiation medium (DMEM containing 1% FBS and 10 μM RA), and the cells were differentiated for five days. The differentiation medium was replaced after RA), and the cells were differentiated for five days. The differentiation medium was replaced after three days. The differentiated cells were pretreated with LCD for 3 h, and then exposed to H 2 O 2. three days. The differentiated cells were pretreated with LCD for 3 h, and then exposed to H 2 O 2.Cell viability was assessed using a CCK-8 assay (Dojindo). SH-SY5Y cells (3.5×10 4 cells/well) were Cell viability was assessed using a CCK-8 assay (Dojindo). SH-SY5Y cells (3.5×10 4 cells/well) were cultured and differentiated in a 96-well plate, and pre-treated with 0.5, 1, or 2 μM LCD for 3 h, cultured and differentiated in a 96-well plate, and pre-treated with 0.5, 1, or 2 μM LCD for 3 h, followed by incubation with 25 μM H 2 O 2 for 24 h. Then, 10 μL of CCK-8 solution was added to each followed by incubation with 25 μM H 2 O 2 for 24 h. Then, 10 μL of CCK-8 solution was added to each well, followed by incubation at 37 °C with 5% CO 2 for 3 h. Absorbance was measured using a well, followed by incubation at 37 °C with 5% CO 2 for 3 h. Absorbance was measured using a microplate reader (Cytation 5, BioTek, Winooski, VT, USA) at 450 nm. microplate reader (Cytation 5, BioTek, Winooski, VT, USA) at 450 nm.Cytotoxicity was evaluated by measuring LDH release from the cytoplasm into the culture medium Cytotoxicity was evaluated by measuring LDH release from the cytoplasm into the culture medium upon plasma-membrane damage. The cells were plated at 3.5×10 4 cells per well in a 96-well plate upon plasma-membrane damage. The cells were plated at 3.5×10 4 cells per well in a 96-well plate and cultured for 24 h, and then treated with RA diluted in medium containing 1% FBS and and cultured for 24 h, and then treated with RA diluted in medium containing 1% FBS and differentiated for five days. Prior to treatment with H 2 O 2 for 24 h, the cells were pre-treated with differentiated for five days. Prior to treatment with H 2 O 2 for 24 h, the cells were pre-treated with LCD at various concentrations for 3 h. The positive controls were lysed with lysis buffer for 40 min LCD at various concentrations for 3 h. The positive controls were lysed with lysis buffer for 40 min at 37 °C in an incubator. Each sample aliquot was incubated with 50 μl of CytoTox 96 Reagent for 30 at 37 °C in an incubator. Each sample aliquot was incubated with 50 μl of CytoTox 96 Reagent for 30 min at 25 °C in the dark. To terminate the reaction, 50 μl of Stop Solution was added to each well. min at 25 °C in the dark. To terminate the reaction, 50 μl of Stop Solution was added to each well.Absorbance was measured at 490 nm or 492 nm within 1 h after the reaction was stopped.Absorbance was measured at 490 nm or 492 nm within 1 h after the reaction was stopped.Intracellular ROS activity was assessed using the cell-permeating fluorescent redox probe 2′,7′-Intracellular ROS activity was assessed using the cell-permeating fluorescent redox probe 2′,7′dichlorofluorescein diacetate (DCFH-DA). The cells were seeded in 96-well plates at a density of dichlorofluorescein diacetate (DCFH-DA). The cells were seeded in 96-well plates at a density of 3.5×10 4 cells/well and differentiated with 10 μM retinoic acid (RA) for five days. The cells were 3.5×10 4 cells/well and differentiated with 10 μM retinoic acid (RA) for five days. The cells were pretreated with vehicle or LCD for 3 h, and 20 μM DCFH-DA was added to each well for 30 min. pretreated with vehicle or LCD for 3 h, and 20 μM DCFH-DA was added to each well for 30 min. treatment was using the as the cells were treatment was using the as the cells were with and treated with 25 μM H 2 O 2 and 50 μM was with and treated with 25 μM H 2 O 2 and 50 μM was as a positive to the incubation for 1 h at 37 as a positive to the incubation for 1 h at 37 ROS levels were measured at and nm using a microplate reader (Cytation 5, ROS levels were measured at and nm using a microplate reader (Cytation 5, was assessed by mitochondrial potential using a was assessed by mitochondrial potential using a fluorescent fluorescent The cells were pretreated LCD or H 2 O 2 or with LCD at The cells were pretreated LCD or H 2 O 2 or with LCD at various concentrations 1, and 2 for 3 h, and then with and incubated with various concentrations 1, and 2 for 3 h, and then with and incubated with for min at 37 the cells were with and treated for min at 37 the cells were with and treated with 20 μM of or 25 μM of H 2 O 2 for 1 with 20 μM of or 25 μM of H 2 O 2 for 1 h at 37 treatment was using the as was as a h at 37 treatment was using the as was as a positive was using a microplate reader (Cytation positive was using a microplate reader (Cytation was at nm and was at nm and at nm. at and and were seeded in cell culture plates at 24 h, the culture medium was were seeded in cell culture plates at 24 h, the culture medium was and replaced with differentiation medium containing were pretreated with various and replaced with differentiation medium containing were pretreated with various concentrations of LCD to 2 then exposed to H 2 O 2 was extracted using concentrations of LCD to 2 then exposed to H 2 O 2 was extracted using the to the the to the of was into using of was into using the The were then the The were then using a system USA) using a system USA) with the was measured at The with the was measured at The are in To neuronal and are in To neuronal and and we neuronal and we neuronal and was in using the and was in using the and and was to of the and and was to of the were in in for and then three with were in in for and then three with was using containing for min at was using containing for min at To the cells were for 1 h in containing To the cells were for 1 h in containing FBS and were then incubated at 4 °C with the primary FBS and were then incubated at 4 °C with the primary diluted in with with containing diluted in with with containing the cells were incubated for 1 h with an the cells were incubated for 1 h with an and in containing and in containing was measured using the was measured using the USA) with a were using USA) with a were using which various for which various for cells were lysed using buffer supplemented with a The cells were lysed using buffer supplemented with a and and concentrations were using a of per was concentrations were using a of per was to or followed by to or followed by The were The were with in containing 1% 20 for h at and then with in containing 1% 20 for h at and then incubated at 4 °C with primary incubated at 4 °C with primary or diluted or diluted with containing 1% the were treated with with containing 1% the were treated with or for 2 h at or for 2 h at was using the was using the were using the system were using the system are as the of the from three or The are as the of the from three or were using were using USA) for was assessed using followed by USA) for was assessed using followed by were significant at were significant at cell growth and and LCD were measured and evaluated SH-SY5Y cell growth and and LCD were measured and evaluated using CCK-8 and LDH LCD cell viability The cells were using CCK-8 and LDH LCD cell viability The cells were differentiated in differentiation medium containing RA for five and treated with H 2 O 2 and LCD differentiated in differentiation medium containing RA for five and treated with H 2 O 2 and LCD for 24 h to cell viability and H 2 O 2 reduced cell in a for 24 h to cell viability and H 2 O 2 reduced cell in a to the H 2 O 2 cell with to the H 2 O 2 cell with 50 of μM at 24 h. in 25 μM H 2 O 2 was used to 50 of μM at 24 h. in 25 μM H 2 O 2 was used to The LCD 50 was to μM The LCD 50 was to μM LCD was the H 2 O 2 treated LCD LCD was the H 2 O 2 treated LCD at 0.5, 1, or 2 μM cell viability in a at 0.5, 1, or 2 μM cell viability in a treated with H 2 O 2 LDH activity, as the treated with H 2 O 2 LDH activity, as the positive the H 2 O 2 treated LCD LDH activity, in a positive the H 2 O 2 treated LCD LDH activity, in a H 2 O 2 treatment reduced cell density H 2 O 2 treatment reduced cell density to the and LCD treatment LCD the H 2 O 2 -induced to the and LCD treatment LCD the H 2 O 2 -induced in cell density in cell density the neuroprotective effects of was measured using a To the neuroprotective effects of was measured using a a H 2 O 2 was significant a H 2 O 2 was significant in cell was reduced by The LCD in cell was reduced by The LCD and H 2 O 2 to the in of cell and and H 2 O 2 to the in of cell and H 2 O 2 at 25 μM reduced the cell and growth H 2 O 2 at 25 μM reduced the cell and growth to its effects cell viability Under LCD and H 2 O 2 to its effects cell viability Under LCD and H 2 O 2 cell and growth in to the LCD cell and growth in to the LCD with and with and and H 2 O 2 treatment reduced and H 2 O 2 treatment reduced with and by in the LCD with and by in the LCD the of these was to in the Under LCD and H 2 O 2 of these was to in the Under LCD and H 2 O 2 the of the with the LCD the of the with the LCD with H 2 O 2 and 2 μM and was to in the treatment with H 2 O 2 and 2 μM and was to in the of and was by and of and was by and to in the to in the the H 2 O 2 -induced and neurotoxicity in SH-SY5Y cells is due to ROS To the H 2 O 2 -induced and neurotoxicity in SH-SY5Y cells is due to ROS we evaluated cell viability and with and the antioxidant we evaluated cell viability and with and the antioxidant treated with the were with in the treated with the were with in the H 2 O 2 treatment reduced cell viability to and H 2 O 2 to H 2 O 2 treatment reduced cell viability to and H 2 O 2 to cell viability treatment with H 2 O 2 Cytotoxicity was in the cell viability treatment with H 2 O 2 Cytotoxicity was in the the and the LCD H 2 O 2 the and the LCD H 2 O 2 H 2 O 2 to in the positive H 2 O 2 to in the positive LCD treatment ROS production LCD treatment ROS production ROS was treatment with H 2 O 2 in ROS was treatment with H 2 O 2 in the positive of LCD and neuroprotective the positive of LCD and neuroprotective and effects at concentrations at which was and and effects at concentrations at which was and in effects from effects in effects from effects mitochondrial is an important in neurodegenerative mitochondrial is an important in neurodegenerative (Ashok et al., et al., 2022). to the effects (Ashok et al., et al., 2022). to the effects of H 2 O 2 and LCD LCD mitochondrial of H 2 O 2 and LCD LCD mitochondrial the of to treatment the of to treatment with H 2 O 2 H 2 O 2 and LCD this in an H 2 O 2 H 2 O 2 and LCD this in an a was H 2 O 2 and a was H 2 O 2 and to the effects of mitochondrial levels, to the effects of mitochondrial levels, LCD and the levels reduced by H 2 O 2 LCD and the levels reduced by H 2 O 2 reduced ROS production production in a reduced ROS production the cell the mechanisms of of we the of H 2 O 2 the mechanisms of of we the of H 2 O via H 2 O 2 via H 2 O 2 LCD reduced it H 2 O 2 the of the LCD reduced it H 2 O 2 the of the and reduced of the these and reduced of the these effects were LCD and H 2 O 2 was reduced effects were LCD and H 2 O 2 was reduced of was H 2 O 2 reduced 3 of was H 2 O 2 reduced 3 with LCD it LCD with LCD it LCD H 2 O 2 -induced H 2 O 2 -induced SH-SY5Y cells, we examined the neuroprotective effects of LCD and its potential mechanisms SH-SY5Y cells, we examined the neuroprotective effects of LCD and its potential mechanisms of in H 2 O 2 -induced oxidative LCD for 24 h of in H 2 O 2 -induced oxidative LCD for 24 h significant in SH-SY5Y cells is with LCD significant in SH-SY5Y cells is with LCD at concentrations of μM the viability of human at concentrations of μM the viability of human cells (Maharajan et al., 2021). H 2 O 2 in cells (Maharajan et al., 2021). H 2 O 2 in reducing cell viability to at 25 μM H 2 O 2 reducing cell viability to at 25 μM H 2 O 2 LCD the H 2 O 2 -induced in cell viability LCD the H 2 O 2 -induced in cell viability system is highly to the of various et The system is highly to the of various et al., cell have been used in to as al., cell have been used in to as they are to and and they are to and and cell can differentiated into cells with by growth cell can differentiated into cells with by growth or by et al., therefore treated the SH-SY5Y cell line or by et al., therefore treated the SH-SY5Y cell line with RA to differentiation into a neuronal cell a of with RA to differentiation into a neuronal cell a of is a key used to neurotoxicity et al., is a key used to neurotoxicity et al., this differentiated SH-SY5Y cell we the neuroprotective effects of H 2 O 2 this differentiated SH-SY5Y cell we the neuroprotective effects of H 2 O 2 neurotoxicity at and with neurotoxicity at and with LCD this LCD H 2 O 2 LCD this LCD H 2 O 2 LCD may in oxidative LCD may in oxidative in of and of the et al., 2022). which is in of the et al., 2022). which is in plays important in neuronal and (Jiang and plays important in neuronal and (Jiang and participates in the neuronal and is used as a cell participates in the neuronal and is used as a cell (Deng and a used plays an important role in neuronal (Deng and a used plays an important role in neuronal processes such as neuronal by with and processes such as neuronal by with and and which plays an important role in and which plays an important role in and neuronal is in the of with Parkinson's and neuronal is in the of with Parkinson's et al., 2020). H 2 O 2 et al., 2020). H 2 O 2 neurotoxicity pathways to and neurotoxicity pathways to and as by the reduced of as by the reduced of to the of it may important to the to the of it may important to the levels of and and thus levels of and and thus and and et and and et al., The of LCD used is within the in in al., The of LCD used is within the in in neuronal cell from the LCD neuronal cell from the LCD concentrations in the may is to concentrations in the may is to the and of to its the and of to its the of including its the of including its and are for and are for its can LCD may significant potential as a its can LCD may significant potential as a for central system diseases et al., 2021). these for central system diseases et al., 2021). these provide LCD against neurotoxicity by the provide LCD against neurotoxicity by the H 2 O 2 -induced of as compounds such as H 2 O 2 -induced of as compounds such as LCD can cause effects owing to using or LCD can cause effects owing to using or may to the may to the 2 O 2 treatment ROS production and 2021). To H 2 O 2 treatment ROS production and 2021). To the of LCD in H 2 O 2 -induced oxidative stress in SH-SY5Y cells, the of LCD in H 2 O 2 -induced oxidative stress in SH-SY5Y cells, we its effects with of a commonly used we its effects with of a commonly used the H 2 O 2 -induced in cell viability and reduced the H 2 O 2 -induced release the H 2 O 2 -induced in cell viability and reduced the H 2 O 2 -induced release of LDH effects as LCD and LCD of LDH effects as LCD and LCD the excessive ROS production by H 2 O 2 the excessive ROS production by H 2 O 2 used as a to LCD its neuroprotective mechanisms by used as a to LCD its neuroprotective mechanisms by oxidative including neuroprotective oxidative including neuroprotective agents as to the of agents as to the of ROS levels can cellular such as ROS levels can cellular such as and Here, H 2 O 2 mitochondrial which was by and Here, H 2 O 2 mitochondrial which was by LCD or with LCD LCD or with LCD ROS production is to the development of such as cancer and as well as ROS production is to the development of such as cancer and as well as stroke and neurodegenerative diseases such as Parkinson's and Alzheimer's and neurodegenerative diseases such as Parkinson's and Alzheimer's and et al., 2022). the and et al., 2022). the of against the of against the in at H 2 O 2 with at in at H 2 O 2 with at concentrations is with the for LCD thus concentrations is with the for LCD thus LCD to neurotoxicity by reducing ROS LCD to neurotoxicity by reducing ROS LCD may protect from oxidative mitochondrial and LCD may protect from oxidative mitochondrial and such as H 2 O 2 can ROS, which can to cell death such as H 2 O 2 can ROS, which can to cell death processes such as and and 2021). Parkinson's processes such as and and 2021). Parkinson's oxidative stress the ultimately to oxidative stress the ultimately to et al., Here, H 2 O 2 treatment the of which was reduced by et al., Here, H 2 O 2 treatment the of which was reduced by LCD The cell death by LCD The cell death by and et al., 2017). The and et al., 2017). The including and cell by including and cell by mitochondrial by and mitochondrial by and et al., 2017). Here, H 2 O 2 and reduced of et al., 2017). Here, H 2 O 2 and reduced of and LCD these effects pathways to the of and LCD these effects pathways to the of and and and and and Here, LCD the of H 2 O 2 -induced and Here, LCD the of H 2 O 2 -induced H 2 O 2 -induced oxidative stress the H 2 O 2 -induced oxidative stress the and LCD this The oxidative and cell and LCD this The oxidative and cell effects of LCD are with reported for effects of LCD are with reported for cells et al., the neuroprotective potential and cells et al., the neuroprotective potential and of SH-SY5Y cells, LCD reduced H 2 O 2 -induced of SH-SY5Y cells, LCD reduced H 2 O 2 -induced ROS of mitochondrial and ROS of mitochondrial and antioxidant and effects are to to of the antioxidant and effects are to to of the and the and the the activity of compounds in the et al., the activity of compounds in the et al., et al., LCD may effects in various et al., LCD may effects in various including by oxidative stress or inflammatory this including by oxidative stress or inflammatory this The ROS and of the The ROS and of the a important for of the effects of LCD a important for of the effects of LCD et al., et al., et al., and of these and effects et al., et al., et al., and of these and effects in or oxidative stress provide into its in or oxidative stress provide into its was using using as an and was using using as an the of