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Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated SH-SY5Y cells

Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated... Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. The oxidative stress is an important component of the pathogenesis of PD. Artemisinin (ART ) has antioxidant and neuroprotective effects. The purpose of this study is to explore the neuroprotective effect of ART on 1-methyl- 4-phenyliodine iodide (MPP )-treated SH-SY5Y cells and underlying mechanism. Methods: We used MPP -treated SH-SY5Y cells to study the neuroprotective effect of ART. Cell viability was meas- ured by MTT assay after incubating the cells with MPP and/or ART for 24 h. DCFH-DA was used to detect the level of intracellular reactive oxygen species (ROS), and WST-8 was used to detect the level of superoxide dismutase (SOD). The level of intracellular reduced glutathione (GSH) was detected with 5,5΄-dithiobis-(2-nitrobenzoic acid), and the level of malondialdehyde (MDA) was assessed based on the reaction of MDA and thiobarbituric acid. A mitochon- drial membrane potential detection kit (JC-1) was used to detect changes in the mitochondrial membrane potential (MMP), and an Annexin V-FITC cell apoptosis kit was used to detect cell apoptosis. The expression levels of caspase-3, cleaved caspase-3 and the autophagy-related proteins LC3, beclin-1, and p62 were detected by Western blotting. In addition, to verify the change in autophagy, we used immunofluorescence to detect the expression of LC3 and p62. Results: No significant cytotoxicity was observed at ART concentrations up to 40 μM. ART could significantly increase the viability of SH-SY5Y cells treated with MPP and reduce oxidative stress damage and apoptosis. In addition, the Western blotting and immunofluorescence results showed that MPP treatment could increase the protein expres- sion of beclin1 and LC3II/LC3I and decrease the protein expression of p62, indicating that MPP treatment could induce autophagy. Simultaneous treatment with ART and MPP could decrease the protein expression of beclin1 and LC3II/LC3I and increase the protein expression of p62, indicating that ART could decrease the level of autophagy induced by MPP . Conclusion: Our results indicate that ART has a protective effect on MPP -treated SH-SY5Y cells by the antioxidant, antiapoptotic activities and inhibition of autophagy. Our findings may provide new hope for the prevention and treat - ment of PD. Keywords: Artemisinin, Parkinson’s disease, Oxidative stress, Apoptosis, Autophagy Background Parkinson’s disease (PD) is a neurodegenerative disease *Correspondence: yanjq@haust.edu.cn Molecular Biology Laboratory, The First Affiliated Hospital, College characterized by the degeneration and death of dopa- of Clinical Medicine of Henan University of Science and Technology, mine (DA) neurons in the substantia nigra of the mid- Jinghua Road 24, Luoyang, Henan 471003, People’s Republic of China brain [1]. The prevalence rate of PD in China is as high Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 2 of 10 as 1.7% among people above the age of 65 years [2]. The recent years, it has been discovered that ART can pro- main manifestations of PD include resting tremor, brad- tect a variety of neurons from oxidative stress damage ykinesia, muscle rigidity and gait disturbance [3]. At [12]. Xia Zhao et  al. [13] reported that ART decreased present, Parkinson’s disease is mainly treated with drugs the hydrogen peroxide (H O )-induced oxidative damage 2 2 that principally relieve symptoms, such as dopamine ago- in SH-SY5Y cells and hippocampal neurons by activat- nists and L-DOPA. Long-term use of these drugs is often ing the AMPK pathway. In another study, ART protected accompanied by a series of adverse reactions [4]. There - rat adrenal pheochromocytoma (PC12) cells and brain fore, exploring the pathogenesis of PD and identifying primary cortical neurons from sodium nitroprusside- new drugs that can inhibit damage to DA neurons are of induced oxidative damage through ERK [14]. However, great significance for the treatment of PD. the protective effect of ART on PD has not been studied It has been reported that oxidative stress is an impor- thus far. Therefore, in this study, we studied the neuro - tant component of the pathogenesis of PD. Autopsy protective effects of ART in an in vitro model of PD and results of PD patients showed that oxidative stress attempted to reveal the potential underlying molecular was present in the substantia nigra of PD patients [5]. mechanism. Research on the antioxidant system of PD patients showed that the level of reduced glutathione (GSH) in Results the substantia nigra is significantly reduced, which ren - ART attenuated MPP ‑induced cytotoxicity in SH‑SY5Y cells ders dopamine neurons more sensitive to oxidative We treated cells with different concentrations of stress [6]. In recent years, an increasing number of stud- MPP (0.2, 0.4, 0.8, or 1.0  mM) for 24  h, and the results ies have shown that oxidative stress is the main cause of showed that MPP could significantly reduce cell viability neuronal apoptosis in PD [7, 8]. Oxidative stress leads in a dose-dependent manner (Fig.  1a). Next, we treated to an increase in intracellular reactive oxygen species cells with different concentrations of ART (2.5, 5, 10, 20, (ROS). Excessive ROS can oxidize lipids in the neuronal or 40 μM) for 24 h, and the results showed that no obvi- cell membrane, disrupt DA neuron membrane function, ous cytotoxicity was observed at ART concentrations up or directly damage DNA, ultimately leading to neuronal to 40 μM (Fig. 1b). Then, we treated cells simultaneously degeneration. Maruyama et al. [9] found that the level of with 1  mM MPP and different concentrations of ART an endogenous MPTP-like toxin was significantly higher (5, 10, 20, or 40 μM) for 24 h, and the results showed that in the cerebrospinal fluid of untreated PD patients than in compared with the MPP group, the group treated with the cerebrospinal fluid of controls. This toxin can induce MPP and 20 μM ART exhibited the highest cell viability dopaminergic SH-SY5Y cell apoptosis, and this effect can (Fig. 1c). Therefore, in the following steps, we chose ART be inhibited by antioxidants; this is an indication that at a concentration of 20 μM to study its protective effect ROS may initiate the apoptosis of dopamine neurons. In on MPP -damaged SH-SY5Y cells. general, these results directly or indirectly indicate that oxidative stress is involved in the pathogenesis of PD and ART decreased MPP ‑induced oxidative stress in SH‑SY5Y mediates apoptosis. cells Artemisinin (ART), which is extracted from the stems We investigated the effect of ART on MPP -induced oxi- and leaves of Artemisia annua, is a well-known antima- dative stress injury in SH-SY5Y cells by assessing ROS larial drug that has saved millions of lives [10, 11]. In production, SOD activity, and GSH and MDA levels. + + Fig. 1 ART reduced the MPP -induced cytotoxicity in SH-SY5Y cells. a Eec ff ts of different concentrations of MPP on the viability of SH-SY5Y cells. b Eec ff ts of different concentrations of ART on the viability of SH-SY5Y cells. c Eec ff ts of different concentrations of ART on the viability of + # ## + MPP -treated SH-SY5Y cells. The results are presented as the mean ± SD (n = 5), *p < 0.05, ***p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 3 of 10 DCFH-DA is an indicator of general oxidative stress that ART alleviated MPP ‑induced Mitochondrial Membrane was used to measure the level of intracellular ROS pro- Potential (MMP) damage in SH‑SY5Y cells duction. As shown in Fig.  2a–d, ART significantly JC-1 is an ideal fluorescent probe that is widely used to reduced the production of intracellular ROS induced by detect the MMP, and the transition of JC-1 from red fluo - MPP . The fluorescence intensity shown in Fig.  2a–d was rescence to green fluorescence can be used as an indica - analyzed, and the results are shown in Fig. 2e. As shown tor of early apoptosis. As shown in Fig. 3a, in the control in Fig.  2e, ROS production increased to 2.48 ± 0.20 after group and ART-treated group, JC-1 staining showed treatment with MPP , which was clearly different from weak green fluorescence and bright red fluorescence. the ROS production in the control cells of 1.00 ± 0.13 In the MPP treatment group, JC-1 staining showed (p < 0.001, n = 5) (Fig.  2e). However, compared with increased green fluorescence and decreased red fluo - MPP treatment alone, simultaneous treatment with rescence, which indicated a significant decrease in the ART and MPP obviously reduced ROS production to MMP. However, simultaneous treatment with ART and 1.67 ± 0.16 (p < 0.01, n = 5) (Fig. 2e). MPP reduced the dissipation of the MMP, resulting in As shown in Fig. 2f and Fig. 2g, compared with the cor- increased red fluorescence and decreased green fluores - responding controls, MPP clearly decreased the level of cence. The ratio of red fluorescence to green fluorescence −1 −1 SOD from 29.9 ± 2.15 U mg to 17.7 ± 1.53 U mg and represents the degree of MMP depolarization (Fig. 3b). −1 decreased the level of GSH from 6.5 ± 0.5 μmol  mg to −1 + 3.23 ± 0.31 μmol  mg (p < 0.001, n = 5). However, com- ART decreased MPP ‑induced apoptosis in SH‑SY5Y cells pared with MPP alone, simultaneous treatment with To study whether ART can reduce the apoptosis of SH- + + ART and MPP clearly increased the level of SOD from SY5Y cells injured by MPP , we detected the protein −1 −1 17.7 ± 1.53 U mg to 23.3 ± 2.08 U mg and increased expression of caspase-3 and cleaved caspase-3 and per- −1 the level of GSH from 3.23 ± 0.31 μmol  mg to 4.9 ± 0.36 formed flow cytometry analysis. The results showed that, −1 + U mg . compared with the control, MPP significantly increased As shown in Fig.  2h, the content of MDA clearly the relative expression of cleaved caspase-3. However, −1 + + increased to 1.24 ± 0.13 nmol  mg in the MPP group, compared with MPP , simultaneous treatment with ART −1 + while this content was only 0.61 ± 0.13 nmol  mg and MPP significantly reduced the protein expression of in the control group (p < 0.01, n = 5). Compared with cleaved caspase-3 (Fig.  4a–b). In addition, we used flow MPP alone, simultaneous treatment with ART and cytometry to detect cell apoptosis. In the control and MPP significantly reduced the MDA content to ART treatment groups, the levels of apoptosis were very −1 + 0.91 ± 0.09 nmol  mg (p < 0.05, n = 5). low. After MPP insult, the apoptosis rate increased from 1.22% to 27.35%. However, after simultaneous treatment Fig. 2 ART reduced the oxidative stress injury induced by MPP . The level of intracellular ROS production was measured using a DCFH-DA fluorescent probe (a–d), and the fluorescence intensity was analyzed by ImageJ (e). The fluorescence intensity of the control group was set to 1, and the fluorescence intensity of the other groups was compared with that of the control group. ART increased the SOD activity (f) and GSH production (g) and decreased the MDA production (h) compared with MPP . Scale bar = 100 μm. The results are presented as the mean ± SD * ** *** # ## + (n = 5), p < 0.05, p < 0.01, p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 4 of 10 Fig. 3 ART alleviated the MPP -induced mitochondrial damage. In the JC-1 staining data, red fluorescence represents JC-1 aggregates and indicates that the MMP is normal, and green fluorescence represents JC-1 monomers and indicates that the MMP is decreased (a). The ratio of red fluorescence intensity to green fluorescence intensity was analyzed by ImageJ (b). The results are presented as the mean ± SD (n = 5), Scale *** ## + bar = 50 μm. p < 0.001 vs. control; p < 0.01 vs. MPP Fig. 4 ART reduced the MPP -induced apoptosis in SH-SY5Y cells. The protein expression of caspase-3 and cleaved caspase-3 was detected by Western blotting, and β-actin was used as an equal loading control (a, b). Flow cytometry was used to detect cell apoptosis after double staining ** *** # ## + with annexin V-FITC and PI (c, d). The data are presented as the mean ± SD (n = 3). p < 0.01, p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 5 of 10 + + with ART and MPP , the percentage of apoptotic cells compared with the control, MPP significantly increased decreased to 15.82% (Fig. 4c–d). the expression of LC3 and decreased the expression of p62. However, compared with MPP alone, simultaneous + + ART decreased the level of autophagy induced by  MPP treatment with ART and MPP decreased the expression To study the effect of ART on autophagy in MPP -treated of LC3 and increased the expression of p62. These results SH-SY5Y cells, we detected the expression of beclin-1, were consistent with the Western blotting results. p62 and LC3, which are marker proteins of autophagy. As shown in Fig. 5a–d, compared with the control, MPP Discussion treatment significantly increased the protein expression MPP , an active metabolic byproduct of MPTP, is a of beclin-1 and the ratio of LC3II/LC3I but decreased neurotoxin that can be taken up by dopamine trans- the expression of p62, indicating that MPP treatment porters into the mitochondria of dopaminergic neurons induced autophagy. However, after simultaneous treat- and inhibit the activity of mitochondrial complex I. The ment with ART and MPP , the protein expression of reduction in mitochondrial complex I activity can lead to beclin-1 and the ratio of LC3II/LC3I decreased, but the oxidative stress damage to neuronal cells and ultimately expression of p62 significantly increased. Interestingly, lead to dopaminergic neuron death [15]. That is, MPP ART showed the same effects as 3-MA in regulating can selectively destroy dopaminergic neurons. In fact, autophagy-related protein expression. These results sug - treatment of SH-SY5Y cells with MPP to induce dam- gested that ART could inhibit the autophagy induced by age is a common method used to establish PD models MPP . in vitro [16]. To verify the results described above, we performed It is well known that ART is one of the best drugs immunofluorescence staining. As shown in Fig.  6a–d, for treating malaria, and its use has been promoted Fig. 5 ART inhibited the autophagy induced by MPP in SH-SY5Y cells. The protein expression of beclin-1, p62 and LC3 was detected by Western *** # blotting, and β-actin was used as an equal loading control (a–d). The data are presented as the mean ± SD (n = 3). p < 0.001 vs. control; p < 0.05, ## + p < 0.01 vs. MPP Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 6 of 10 Fig. 6 The protein expression of LC3 based on immunofluorescence staining (a), and the fluorescence intensity was analyzed by ImageJ (b). The protein expression of p62 based on immunofluorescence staining (c) and the fluorescence intensity was analyzed by ImageJ (d). The fluorescence intensity of the control group was set to 1, and the fluorescence intensity of the other groups was compared with that of the control group. The * ** *** # ## + data are presented as the mean ± SD (n = 3), Scale bar = 50 μm. p < 0.05, p < 0.01, p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP worldwide [17]. In recent years, studies have found that Autophagy is a process by which cells degrade their in addition to its antimalarial effects, ART also has neu - own organelles and misfolded proteins [20]. A large num- roprotective effects [18]. In fact, the antioxidant and ber of studies have shown that autophagy plays impor- neuroprotective effects of ART have been studied in the tant roles in the occurrence and development of PD (e.g. context of another neurodegenerative disease, Alzhei- [21, 22]). However, the specific mechanism by which mer’s disease. Sarina et al. [19] reported that ART pro- autophagy functions in PD models is unclear. One study tects PC12 cells from β-amyloid-induced apoptosis by showed that there was a substantial loss of dopaminergic neurons in the substantia nigra and a substantial degree activating the ERK1/2 signaling pathway. In our study, of autophagy in a MPTP-induced model of PD in rhesus we found that ART could significantly increase the via - monkeys [23]. These results were further proved in C6 bility of MPP -damaged SH-SY5Y cells, significantly cells. Another study showed that MPP reduce oxidative stress damage and reduce MMP depo- disrupted the larization. In the apoptosis study, we found that ART autophagic flux in a PC12 cell model, leading to a signifi - reduced the protein expression of cleaved caspase-3 cant increase in the number of LC3-positive autophagic and reduced the rate of cell apoptosis caused by MPP , vesicles [24]. These results showed that the activation of which were consistent with previous studies. These autophagy may be a part of the programmed cell death results indicated that ART is a potential neuroprotec- triggered by neurotoxins [25], which is called "autophagic cell death—ACD". ACD can also be observed in ani tive drug that can inhibit the apoptosis of dopamine - neurons caused by oxidative stress. mals and patients with PD [25]. Our study showed that Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 7 of 10 the protein expression of the autophagy markers LC3 Logan, UT, USA) with 10% fetal bovine serum (Gibco, and beclin-1 increased in MPP -treated SH-SY5Y cells, Grand Island, NY, USA) and 1% glutamine in a humidi- while the expression of p62 decreased. This result indi - fied incubator with 5% CO2 at 37°C. We changed the cated that MPP treatment induced the occurrence of culture medium every two days and subcultured the autophagy, and this conclusion is the same as that of pre- cells when the density reached 80%. (1) To study the vious studies [26, 27]. In addition, we found that ART effect of MPP and/or ART on cell viability, we treated treatment could reverse the increase in the autophagy the cells with the indicated concentrations of MPP and/ levels caused by MPP , indicating that the antioxidant or ART for 24  h and performed a CCK-8 assay. (2) To + + effect of ART in MPP -treated SH-SY5Y cells may be study the protective effect of ART on MPP -treated SH- related to autophagy. Therefore, we hypothesize that SY5Y cells, we treated the cells with PBS, ART, MPP , the antioxidant and antiapoptotic effects of ART in or ART + MPP for 24  h. The concentration of ART + + MPP -treated SH-SY5Y cells may be related to ACD. was 20  μM, and the concentration of MPP was 1  mM. However, the specific mechanism of autophagy in PD still (3) To study changes in autophagy, we treated the cells + + + requires further research. with PBS, ART, MPP , ART + MPP or 3-MA + MPP At present, the drug treatments for PD are limited to for 24 h. The concentration of ART was 20 μM, the con - dopaminergic agonists and cholinergic antagonists, but centration of MPP was 1  mM, and the concentration long-term use of these drugs is associated with many of 3-MA was 5  mM. MPP was purchased from Sigma adverse reactions. Antioxidants can prevent the neuronal (Sigma, D048, St Louis, USA). ART was purchased from damage caused by oxidative stress. Therefore, identifying Nanjing  DASF biotechnology  co., Ltd (Nanjing, China). high-efficiency, low-cost, low-toxicity and even nontoxic The autophagy inhibitor 3-methyladenine (3-MA) was natural antioxidants to prevent or treat neurodegen- purchased from Sigma (Sigma, 189490, St Louis, USA). erative diseases has attracted substantial attention. ART exhibits low toxicity. Clinical studies and meta-analyses Cell viability showed that no serious side effects were observed dur - Cell viability was measured by CCK-8 assay (Solarbio, ing the use of artemisinin, and serious adverse reactions CA1210, Beijing, China), according to the manufacturer’s have not been reported after long-term use [28]. To the instructions. Briefly, 5 × 10 SH-SY5Y cells per well were best of our knowledge, our study is the first to reveal that plated in 96-well plates and incubated for 24 h. Then, the ART exerts a neuroprotective effect in cell models of PD cells were treated with MPP and/or ART for another through its antioxidant activity, but our research still has 24  h. Then, 100  μl culture medium containing 10  mM some limitations. More specifically, (1) our research was CCK-8 was added to each well and incubated at 37°C conducted under in vitro conditions. Therefore, it is nec - for 2 h. The absorbance was measured at 450 nm with a essary to conduct in  vivo experiments in future studies; multimode microplate reader (EnSpire, PerkinElmer, Sin- (2) it showed that autophagy was involved in the mecha- gapore). The cell viability of the control group was set to nism underlying the neuroprotective effect of ART. Due 100%, and the cell viability of the other groups was com- to time and other reasons, we were unable to conduct pared with that of the control group. further studies on this specific mechanism. However, our future research will explore this mechanism. Intracellular reactive oxygen species (ROS) detection The levels of intracellular ROS were measured by a ROS Conclusion assay kit (Beyotime Biotechnology, S0033, Shanghai, In conclusion, our results indicate that ART protects China), according to the manufacturer’s instructions. dopaminergic neurons from MPP -induced damage by After drug treatment, the SH-SY5Y cells were incubated reducing oxidative stress and apoptosis, and these effects with serum-free medium containing 10  μM DCFH-DA may be related to the inhibition of autophagy. Therefore, at 37  °C for 20  min and then washed with serum-free our research reveals a new drug with potential protec- culture medium three times. After adding 1  ml fresh tive effects on dopamine neurons in PD, which may pro - medium to each well, the fluorescence was observed vide hope for the prevention and treatment of PD in the using the GFP channel of an inverted fluorescence micro - future. scope (Nikon,  ECLIPSE Ti, Tokyo,  Japan). The fluores - cence intensity was analyzed by ImageJ. Methods Cell culture and drug treatment Superoxide dismutase (SOD) activity assay The human neuroblastoma cell line SH-SY5Y was The intracellular SOD activity was measured with a total obtained from Sun Yat-Sen University (Guangzhou, SOD activity detection kit (Beyotime Biotechnology, China) and cultured in DMEM/H medium (HyClone, S0101, Shanghai, China), according to the manufacturer’s Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 8 of 10 instructions. After drug treatment, the cells were washed cells were incubated at room temperature for 20 min, and once with PBS and collected by centrifugation. The cells then, the fluorescence was detected by flow cytometry were fully lysed in SOD sample preparation solution, (BD, Accuri C6, New York, USA). and the supernatants were collected at 12,000 × g at 4°C for 5  min. The absorbance was detected at 450  nm and Western blot assay 600 nm (reference wavelength) with a multimode micro- Cell lysates were prepared, and the protein concen- plate reader, and the SOD activity was calculated. trations were quantified with a BCA protein assay kit (CWBIO, CW2011S, Beijing,  China). Samples with equal amounts of proteins were separated by 12% Glutathione (GSH) assay SDS-PAGE and transferred to PVDF membranes. The content of GSH was measured with a glutathione After blocking with 5% skim milk at room tempera- assay kit (Beyotime Biotechnology, S0052, Shang- ture for 1  h, the membranes were incubated at 4°C hai, China), according to the manufacturer’s instructions. overnight with primary antibodies (1:1000) against After drug treatment, the cells were washed once with cleaved caspase-3 (Cell Signaling Technology, Aps175, PBS and collected by centrifugation. After the samples Boston,  USA), caspase-3 (Abcam, ab90437, Cam- and standards had been prepared, the absorbance was bridge,  UK), LC3 (Abcam, ab128025, Cambridge, UK), detected at 412  nm. We generated a standard curve and Beclin1 (BD, 612113, New York,  USA), p62 (Abcam, calculated the contents of GSH in the samples based on ab56416, Cambridge, UK) or β-actin (1:5000; CWBIO, this standard curve. CW0096M, Beijing,  China). The next day, the mem - branes were washed with TBST three times and incu- Malondialdehyde (MDA) assay bated with HRP-conjugated anti-rabbit or anti-mouse The intracellular MDA levels were measured with an secondary antibodies (1:5000,  CWBIO,  Beijing, China) MDA detection kit (Beyotime Biotechnology, S0131, at room temperature for 1  h. The blots were visualized Shanghai,  China), according to the manufacturer’s with a cECL Western blot kit (CWBIO, CW0049M, instructions. After drug treatment, the cells were col- Beijing,  China), and the images were analyzed by lected. The cells were lysed, and the supernatants were ImageJ. collected at 10,000 × g for 10  min. After the samples and standards had been prepared, the absorbance was detected at 532 and 450  nm (reference wavelength). The Immunofluorescence staining MDA contents in the samples were quantified based on SH-SY5Y cells were seeded on slides in 12-well culture the standard curve. plates. After drug treatment, the cells were fixed with ice-cold methanol for 5  min, blocked with 1% bovine Mitochondrial membrane potential (MMP) assay serum albumin (BSA) for 30  min, and then incubated To monitor the mitochondrial integrity, a mitochondrial with primary antibodies against LC3 (1:200) and P62 membrane potential assay kit with JC-1 (Beyotime Bio- (1:100) at 4°C overnight. The next day, the slides were technology, C2006, Shanghai,  China) was used, accord- incubated with Alexa Fluor 488 (Bioss Antibodies, bs- ing to the manufacturer’s instructions. Briefly, after drug 0295G-AF488, Beijing, China) or Alexa Fluor 555 (Bioss treatment, the SH-SY5Y cells were incubated with JC-1 Antibodies, bs-0296G-AF555, Beijing,  China) second- working solution at 37°C for 20  min and washed twice ary antibodies (1:500) at 37°C for 1  h and incubated with JC-1 buffer. Red fluorescence and green fluores - with DAPI (Boster, AR1177, Wuhan, China) for 5  min. cence were observed with the GFP channel and TRITC Fluorescence was observed with a fluorescence  micro - channel of an inverted fluorescence microscope (Nikon, scope (Olympus, BX53, Tokyo, Japan). ECLIPSE Ti, Japan). The fluorescence intensity was ana - lyzed by ImageJ. Statistical analysis Flow cytometry assay The results are expressed as the mean ± standard devia- We used an Annexin V-FITC cell apoptosis detection kit tion (mean ± SD). Comparisons among multiple groups (Beyotime Biotechnology, C1062M, Shanghai,  China) were performed with one-way ANOVA followed by to detect cell apoptosis, according to the manufactur- Tukey’s post hoc test, and p < 0.05 was considered sta- er’s instructions. After drug treatment, the cells were tistically significant. harvested and resuspended in PBS. A total of 50,000 to 100,000 cells were collected and resuspended in 195  μl Annexin V-FITC binding buffer, and then, 5  μl Annexin V-FITC and 10 μl PI were added with gentle mixing. The Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 9 of 10 Abbreviations 7. Liu H, Wu H, Zhu N, Xu Z, Wang Y, Qu Y, et al. Lactoferrin protects against PD: Parkinson’s disease; ART : Artemisinin; MPP : 1-Methyl-4-phenyliodine iron dysregulation, oxidative stress, and apoptosis in 1-methyl-4-phenyl- iodide; DCFH-DA: 2′,7′-Dichloro-dihydro-fluorescein diacetate; ROS: Reac- 1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease in mice. J tive oxygen species; SOD: Superoxide dismutase; GSH: Glutathione; MDA: Neurochem. 2020;152(3):397–415. https ://doi.org/10.1111/jnc.14857 . Malondialdehyde; MMP: Mitochondrial membrane potential; DA: Dopamine; 8. Radi E, Formichi P, Battisti C, Federico A. Apoptosis and oxidative stress in H2O2: Hydrogen peroxide; PC12: Rat adrenal pheochromocytoma; MA: neurodegenerative diseases. J Alzheimers Dis. 2014;42(Suppl 3):S125–52. 3-Methyladenine; MDA: Malondialdehyde; BSA: Bovine serum albumin; MPTP: https ://doi.org/10.3233/jad-13273 8. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ACD: Autophagic cell death; 9. Maruyama W, Abe T, Tohgi H, Naoi M. An endogenous MPTP-like L-DOPA: Levodopa; MPTP: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; WST- dopaminergic neurotoxin, N-methyl(R)salsolinol, in the cerebrospinal 8: 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- fluid decreases with progression of Parkinson’s disease. Neurosci Let. tetrazolium sodium salt. 1999;262(1):13–6. https ://doi.org/10.1016/s0304 -3940(99)00003 -8. 10. Cui L, Su XZ. Discovery, mechanisms of action and combination therapy Acknowledgements of artemisinin. Expert Rev Anti Infect Ther. 2009;7(8):999–1013. https :// Not applicable. doi.org/10.1586/eri.09.68. 11. Meshnick SR. Artemisinin: mechanisms of action, resistance and toxic- Authors’ contributions ity. Int J Parasitol. 2002;32(13):1655–60. https ://doi.org/10.1016/S0020 HM and XL were involved in the manuscript preparation. JH, MS and AL were -7519(02)00194 -7. involved in accessing the data. JH and WS were involved in proofreading the 12. Yan F, Wang H, Gao Y, Xu J, Zheng W. Artemisinin protects retinal neuronal manuscript. JY and YZ were involved in the design, writing and organization. cells against oxidative stress and restores rat retinal physiological function All authors read and approved the final manuscript. from light exposed damage. ACS Chem Neurosci. 2017;8(8):1713–23. https ://doi.org/10.1021/acsch emneu ro.7b000 21. Funding 13. Zhao X, Fang J, Li S, Gaur U, Xing X, Wang H, et al. Artemisinin attenuated Medical science and technology research in Henan Province (LHGJ20190560). hydrogen peroxide (H(2)O(2))-induced oxidative injury in SH-SY5Y and hippocampal neurons via the activation of AMPK pathway. Int J Mol Sci. Availability of data and materials 2019. https ://doi.org/10.3390/ijms2 01126 80. All the data generated or analyzed during this study are included in this 14. Zheng W, Chong CM, Wang H, Zhou X, Zhang L, Wang R, et al. 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Artemisinin attenuated oxidative stress and apoptosis by inhibiting autophagy in MPP+-treated SH-SY5Y cells

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Abstract

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. The oxidative stress is an important component of the pathogenesis of PD. Artemisinin (ART ) has antioxidant and neuroprotective effects. The purpose of this study is to explore the neuroprotective effect of ART on 1-methyl- 4-phenyliodine iodide (MPP )-treated SH-SY5Y cells and underlying mechanism. Methods: We used MPP -treated SH-SY5Y cells to study the neuroprotective effect of ART. Cell viability was meas- ured by MTT assay after incubating the cells with MPP and/or ART for 24 h. DCFH-DA was used to detect the level of intracellular reactive oxygen species (ROS), and WST-8 was used to detect the level of superoxide dismutase (SOD). The level of intracellular reduced glutathione (GSH) was detected with 5,5΄-dithiobis-(2-nitrobenzoic acid), and the level of malondialdehyde (MDA) was assessed based on the reaction of MDA and thiobarbituric acid. A mitochon- drial membrane potential detection kit (JC-1) was used to detect changes in the mitochondrial membrane potential (MMP), and an Annexin V-FITC cell apoptosis kit was used to detect cell apoptosis. The expression levels of caspase-3, cleaved caspase-3 and the autophagy-related proteins LC3, beclin-1, and p62 were detected by Western blotting. In addition, to verify the change in autophagy, we used immunofluorescence to detect the expression of LC3 and p62. Results: No significant cytotoxicity was observed at ART concentrations up to 40 μM. ART could significantly increase the viability of SH-SY5Y cells treated with MPP and reduce oxidative stress damage and apoptosis. In addition, the Western blotting and immunofluorescence results showed that MPP treatment could increase the protein expres- sion of beclin1 and LC3II/LC3I and decrease the protein expression of p62, indicating that MPP treatment could induce autophagy. Simultaneous treatment with ART and MPP could decrease the protein expression of beclin1 and LC3II/LC3I and increase the protein expression of p62, indicating that ART could decrease the level of autophagy induced by MPP . Conclusion: Our results indicate that ART has a protective effect on MPP -treated SH-SY5Y cells by the antioxidant, antiapoptotic activities and inhibition of autophagy. Our findings may provide new hope for the prevention and treat - ment of PD. Keywords: Artemisinin, Parkinson’s disease, Oxidative stress, Apoptosis, Autophagy Background Parkinson’s disease (PD) is a neurodegenerative disease *Correspondence: yanjq@haust.edu.cn Molecular Biology Laboratory, The First Affiliated Hospital, College characterized by the degeneration and death of dopa- of Clinical Medicine of Henan University of Science and Technology, mine (DA) neurons in the substantia nigra of the mid- Jinghua Road 24, Luoyang, Henan 471003, People’s Republic of China brain [1]. The prevalence rate of PD in China is as high Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 2 of 10 as 1.7% among people above the age of 65 years [2]. The recent years, it has been discovered that ART can pro- main manifestations of PD include resting tremor, brad- tect a variety of neurons from oxidative stress damage ykinesia, muscle rigidity and gait disturbance [3]. At [12]. Xia Zhao et  al. [13] reported that ART decreased present, Parkinson’s disease is mainly treated with drugs the hydrogen peroxide (H O )-induced oxidative damage 2 2 that principally relieve symptoms, such as dopamine ago- in SH-SY5Y cells and hippocampal neurons by activat- nists and L-DOPA. Long-term use of these drugs is often ing the AMPK pathway. In another study, ART protected accompanied by a series of adverse reactions [4]. There - rat adrenal pheochromocytoma (PC12) cells and brain fore, exploring the pathogenesis of PD and identifying primary cortical neurons from sodium nitroprusside- new drugs that can inhibit damage to DA neurons are of induced oxidative damage through ERK [14]. However, great significance for the treatment of PD. the protective effect of ART on PD has not been studied It has been reported that oxidative stress is an impor- thus far. Therefore, in this study, we studied the neuro - tant component of the pathogenesis of PD. Autopsy protective effects of ART in an in vitro model of PD and results of PD patients showed that oxidative stress attempted to reveal the potential underlying molecular was present in the substantia nigra of PD patients [5]. mechanism. Research on the antioxidant system of PD patients showed that the level of reduced glutathione (GSH) in Results the substantia nigra is significantly reduced, which ren - ART attenuated MPP ‑induced cytotoxicity in SH‑SY5Y cells ders dopamine neurons more sensitive to oxidative We treated cells with different concentrations of stress [6]. In recent years, an increasing number of stud- MPP (0.2, 0.4, 0.8, or 1.0  mM) for 24  h, and the results ies have shown that oxidative stress is the main cause of showed that MPP could significantly reduce cell viability neuronal apoptosis in PD [7, 8]. Oxidative stress leads in a dose-dependent manner (Fig.  1a). Next, we treated to an increase in intracellular reactive oxygen species cells with different concentrations of ART (2.5, 5, 10, 20, (ROS). Excessive ROS can oxidize lipids in the neuronal or 40 μM) for 24 h, and the results showed that no obvi- cell membrane, disrupt DA neuron membrane function, ous cytotoxicity was observed at ART concentrations up or directly damage DNA, ultimately leading to neuronal to 40 μM (Fig. 1b). Then, we treated cells simultaneously degeneration. Maruyama et al. [9] found that the level of with 1  mM MPP and different concentrations of ART an endogenous MPTP-like toxin was significantly higher (5, 10, 20, or 40 μM) for 24 h, and the results showed that in the cerebrospinal fluid of untreated PD patients than in compared with the MPP group, the group treated with the cerebrospinal fluid of controls. This toxin can induce MPP and 20 μM ART exhibited the highest cell viability dopaminergic SH-SY5Y cell apoptosis, and this effect can (Fig. 1c). Therefore, in the following steps, we chose ART be inhibited by antioxidants; this is an indication that at a concentration of 20 μM to study its protective effect ROS may initiate the apoptosis of dopamine neurons. In on MPP -damaged SH-SY5Y cells. general, these results directly or indirectly indicate that oxidative stress is involved in the pathogenesis of PD and ART decreased MPP ‑induced oxidative stress in SH‑SY5Y mediates apoptosis. cells Artemisinin (ART), which is extracted from the stems We investigated the effect of ART on MPP -induced oxi- and leaves of Artemisia annua, is a well-known antima- dative stress injury in SH-SY5Y cells by assessing ROS larial drug that has saved millions of lives [10, 11]. In production, SOD activity, and GSH and MDA levels. + + Fig. 1 ART reduced the MPP -induced cytotoxicity in SH-SY5Y cells. a Eec ff ts of different concentrations of MPP on the viability of SH-SY5Y cells. b Eec ff ts of different concentrations of ART on the viability of SH-SY5Y cells. c Eec ff ts of different concentrations of ART on the viability of + # ## + MPP -treated SH-SY5Y cells. The results are presented as the mean ± SD (n = 5), *p < 0.05, ***p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 3 of 10 DCFH-DA is an indicator of general oxidative stress that ART alleviated MPP ‑induced Mitochondrial Membrane was used to measure the level of intracellular ROS pro- Potential (MMP) damage in SH‑SY5Y cells duction. As shown in Fig.  2a–d, ART significantly JC-1 is an ideal fluorescent probe that is widely used to reduced the production of intracellular ROS induced by detect the MMP, and the transition of JC-1 from red fluo - MPP . The fluorescence intensity shown in Fig.  2a–d was rescence to green fluorescence can be used as an indica - analyzed, and the results are shown in Fig. 2e. As shown tor of early apoptosis. As shown in Fig. 3a, in the control in Fig.  2e, ROS production increased to 2.48 ± 0.20 after group and ART-treated group, JC-1 staining showed treatment with MPP , which was clearly different from weak green fluorescence and bright red fluorescence. the ROS production in the control cells of 1.00 ± 0.13 In the MPP treatment group, JC-1 staining showed (p < 0.001, n = 5) (Fig.  2e). However, compared with increased green fluorescence and decreased red fluo - MPP treatment alone, simultaneous treatment with rescence, which indicated a significant decrease in the ART and MPP obviously reduced ROS production to MMP. However, simultaneous treatment with ART and 1.67 ± 0.16 (p < 0.01, n = 5) (Fig. 2e). MPP reduced the dissipation of the MMP, resulting in As shown in Fig. 2f and Fig. 2g, compared with the cor- increased red fluorescence and decreased green fluores - responding controls, MPP clearly decreased the level of cence. The ratio of red fluorescence to green fluorescence −1 −1 SOD from 29.9 ± 2.15 U mg to 17.7 ± 1.53 U mg and represents the degree of MMP depolarization (Fig. 3b). −1 decreased the level of GSH from 6.5 ± 0.5 μmol  mg to −1 + 3.23 ± 0.31 μmol  mg (p < 0.001, n = 5). However, com- ART decreased MPP ‑induced apoptosis in SH‑SY5Y cells pared with MPP alone, simultaneous treatment with To study whether ART can reduce the apoptosis of SH- + + ART and MPP clearly increased the level of SOD from SY5Y cells injured by MPP , we detected the protein −1 −1 17.7 ± 1.53 U mg to 23.3 ± 2.08 U mg and increased expression of caspase-3 and cleaved caspase-3 and per- −1 the level of GSH from 3.23 ± 0.31 μmol  mg to 4.9 ± 0.36 formed flow cytometry analysis. The results showed that, −1 + U mg . compared with the control, MPP significantly increased As shown in Fig.  2h, the content of MDA clearly the relative expression of cleaved caspase-3. However, −1 + + increased to 1.24 ± 0.13 nmol  mg in the MPP group, compared with MPP , simultaneous treatment with ART −1 + while this content was only 0.61 ± 0.13 nmol  mg and MPP significantly reduced the protein expression of in the control group (p < 0.01, n = 5). Compared with cleaved caspase-3 (Fig.  4a–b). In addition, we used flow MPP alone, simultaneous treatment with ART and cytometry to detect cell apoptosis. In the control and MPP significantly reduced the MDA content to ART treatment groups, the levels of apoptosis were very −1 + 0.91 ± 0.09 nmol  mg (p < 0.05, n = 5). low. After MPP insult, the apoptosis rate increased from 1.22% to 27.35%. However, after simultaneous treatment Fig. 2 ART reduced the oxidative stress injury induced by MPP . The level of intracellular ROS production was measured using a DCFH-DA fluorescent probe (a–d), and the fluorescence intensity was analyzed by ImageJ (e). The fluorescence intensity of the control group was set to 1, and the fluorescence intensity of the other groups was compared with that of the control group. ART increased the SOD activity (f) and GSH production (g) and decreased the MDA production (h) compared with MPP . Scale bar = 100 μm. The results are presented as the mean ± SD * ** *** # ## + (n = 5), p < 0.05, p < 0.01, p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 4 of 10 Fig. 3 ART alleviated the MPP -induced mitochondrial damage. In the JC-1 staining data, red fluorescence represents JC-1 aggregates and indicates that the MMP is normal, and green fluorescence represents JC-1 monomers and indicates that the MMP is decreased (a). The ratio of red fluorescence intensity to green fluorescence intensity was analyzed by ImageJ (b). The results are presented as the mean ± SD (n = 5), Scale *** ## + bar = 50 μm. p < 0.001 vs. control; p < 0.01 vs. MPP Fig. 4 ART reduced the MPP -induced apoptosis in SH-SY5Y cells. The protein expression of caspase-3 and cleaved caspase-3 was detected by Western blotting, and β-actin was used as an equal loading control (a, b). Flow cytometry was used to detect cell apoptosis after double staining ** *** # ## + with annexin V-FITC and PI (c, d). The data are presented as the mean ± SD (n = 3). p < 0.01, p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 5 of 10 + + with ART and MPP , the percentage of apoptotic cells compared with the control, MPP significantly increased decreased to 15.82% (Fig. 4c–d). the expression of LC3 and decreased the expression of p62. However, compared with MPP alone, simultaneous + + ART decreased the level of autophagy induced by  MPP treatment with ART and MPP decreased the expression To study the effect of ART on autophagy in MPP -treated of LC3 and increased the expression of p62. These results SH-SY5Y cells, we detected the expression of beclin-1, were consistent with the Western blotting results. p62 and LC3, which are marker proteins of autophagy. As shown in Fig. 5a–d, compared with the control, MPP Discussion treatment significantly increased the protein expression MPP , an active metabolic byproduct of MPTP, is a of beclin-1 and the ratio of LC3II/LC3I but decreased neurotoxin that can be taken up by dopamine trans- the expression of p62, indicating that MPP treatment porters into the mitochondria of dopaminergic neurons induced autophagy. However, after simultaneous treat- and inhibit the activity of mitochondrial complex I. The ment with ART and MPP , the protein expression of reduction in mitochondrial complex I activity can lead to beclin-1 and the ratio of LC3II/LC3I decreased, but the oxidative stress damage to neuronal cells and ultimately expression of p62 significantly increased. Interestingly, lead to dopaminergic neuron death [15]. That is, MPP ART showed the same effects as 3-MA in regulating can selectively destroy dopaminergic neurons. In fact, autophagy-related protein expression. These results sug - treatment of SH-SY5Y cells with MPP to induce dam- gested that ART could inhibit the autophagy induced by age is a common method used to establish PD models MPP . in vitro [16]. To verify the results described above, we performed It is well known that ART is one of the best drugs immunofluorescence staining. As shown in Fig.  6a–d, for treating malaria, and its use has been promoted Fig. 5 ART inhibited the autophagy induced by MPP in SH-SY5Y cells. The protein expression of beclin-1, p62 and LC3 was detected by Western *** # blotting, and β-actin was used as an equal loading control (a–d). The data are presented as the mean ± SD (n = 3). p < 0.001 vs. control; p < 0.05, ## + p < 0.01 vs. MPP Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 6 of 10 Fig. 6 The protein expression of LC3 based on immunofluorescence staining (a), and the fluorescence intensity was analyzed by ImageJ (b). The protein expression of p62 based on immunofluorescence staining (c) and the fluorescence intensity was analyzed by ImageJ (d). The fluorescence intensity of the control group was set to 1, and the fluorescence intensity of the other groups was compared with that of the control group. The * ** *** # ## + data are presented as the mean ± SD (n = 3), Scale bar = 50 μm. p < 0.05, p < 0.01, p < 0.001 vs. control; p < 0.05, p < 0.01 vs. MPP worldwide [17]. In recent years, studies have found that Autophagy is a process by which cells degrade their in addition to its antimalarial effects, ART also has neu - own organelles and misfolded proteins [20]. A large num- roprotective effects [18]. In fact, the antioxidant and ber of studies have shown that autophagy plays impor- neuroprotective effects of ART have been studied in the tant roles in the occurrence and development of PD (e.g. context of another neurodegenerative disease, Alzhei- [21, 22]). However, the specific mechanism by which mer’s disease. Sarina et al. [19] reported that ART pro- autophagy functions in PD models is unclear. One study tects PC12 cells from β-amyloid-induced apoptosis by showed that there was a substantial loss of dopaminergic neurons in the substantia nigra and a substantial degree activating the ERK1/2 signaling pathway. In our study, of autophagy in a MPTP-induced model of PD in rhesus we found that ART could significantly increase the via - monkeys [23]. These results were further proved in C6 bility of MPP -damaged SH-SY5Y cells, significantly cells. Another study showed that MPP reduce oxidative stress damage and reduce MMP depo- disrupted the larization. In the apoptosis study, we found that ART autophagic flux in a PC12 cell model, leading to a signifi - reduced the protein expression of cleaved caspase-3 cant increase in the number of LC3-positive autophagic and reduced the rate of cell apoptosis caused by MPP , vesicles [24]. These results showed that the activation of which were consistent with previous studies. These autophagy may be a part of the programmed cell death results indicated that ART is a potential neuroprotec- triggered by neurotoxins [25], which is called "autophagic cell death—ACD". ACD can also be observed in ani tive drug that can inhibit the apoptosis of dopamine - neurons caused by oxidative stress. mals and patients with PD [25]. Our study showed that Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 7 of 10 the protein expression of the autophagy markers LC3 Logan, UT, USA) with 10% fetal bovine serum (Gibco, and beclin-1 increased in MPP -treated SH-SY5Y cells, Grand Island, NY, USA) and 1% glutamine in a humidi- while the expression of p62 decreased. This result indi - fied incubator with 5% CO2 at 37°C. We changed the cated that MPP treatment induced the occurrence of culture medium every two days and subcultured the autophagy, and this conclusion is the same as that of pre- cells when the density reached 80%. (1) To study the vious studies [26, 27]. In addition, we found that ART effect of MPP and/or ART on cell viability, we treated treatment could reverse the increase in the autophagy the cells with the indicated concentrations of MPP and/ levels caused by MPP , indicating that the antioxidant or ART for 24  h and performed a CCK-8 assay. (2) To + + effect of ART in MPP -treated SH-SY5Y cells may be study the protective effect of ART on MPP -treated SH- related to autophagy. Therefore, we hypothesize that SY5Y cells, we treated the cells with PBS, ART, MPP , the antioxidant and antiapoptotic effects of ART in or ART + MPP for 24  h. The concentration of ART + + MPP -treated SH-SY5Y cells may be related to ACD. was 20  μM, and the concentration of MPP was 1  mM. However, the specific mechanism of autophagy in PD still (3) To study changes in autophagy, we treated the cells + + + requires further research. with PBS, ART, MPP , ART + MPP or 3-MA + MPP At present, the drug treatments for PD are limited to for 24 h. The concentration of ART was 20 μM, the con - dopaminergic agonists and cholinergic antagonists, but centration of MPP was 1  mM, and the concentration long-term use of these drugs is associated with many of 3-MA was 5  mM. MPP was purchased from Sigma adverse reactions. Antioxidants can prevent the neuronal (Sigma, D048, St Louis, USA). ART was purchased from damage caused by oxidative stress. Therefore, identifying Nanjing  DASF biotechnology  co., Ltd (Nanjing, China). high-efficiency, low-cost, low-toxicity and even nontoxic The autophagy inhibitor 3-methyladenine (3-MA) was natural antioxidants to prevent or treat neurodegen- purchased from Sigma (Sigma, 189490, St Louis, USA). erative diseases has attracted substantial attention. ART exhibits low toxicity. Clinical studies and meta-analyses Cell viability showed that no serious side effects were observed dur - Cell viability was measured by CCK-8 assay (Solarbio, ing the use of artemisinin, and serious adverse reactions CA1210, Beijing, China), according to the manufacturer’s have not been reported after long-term use [28]. To the instructions. Briefly, 5 × 10 SH-SY5Y cells per well were best of our knowledge, our study is the first to reveal that plated in 96-well plates and incubated for 24 h. Then, the ART exerts a neuroprotective effect in cell models of PD cells were treated with MPP and/or ART for another through its antioxidant activity, but our research still has 24  h. Then, 100  μl culture medium containing 10  mM some limitations. More specifically, (1) our research was CCK-8 was added to each well and incubated at 37°C conducted under in vitro conditions. Therefore, it is nec - for 2 h. The absorbance was measured at 450 nm with a essary to conduct in  vivo experiments in future studies; multimode microplate reader (EnSpire, PerkinElmer, Sin- (2) it showed that autophagy was involved in the mecha- gapore). The cell viability of the control group was set to nism underlying the neuroprotective effect of ART. Due 100%, and the cell viability of the other groups was com- to time and other reasons, we were unable to conduct pared with that of the control group. further studies on this specific mechanism. However, our future research will explore this mechanism. Intracellular reactive oxygen species (ROS) detection The levels of intracellular ROS were measured by a ROS Conclusion assay kit (Beyotime Biotechnology, S0033, Shanghai, In conclusion, our results indicate that ART protects China), according to the manufacturer’s instructions. dopaminergic neurons from MPP -induced damage by After drug treatment, the SH-SY5Y cells were incubated reducing oxidative stress and apoptosis, and these effects with serum-free medium containing 10  μM DCFH-DA may be related to the inhibition of autophagy. Therefore, at 37  °C for 20  min and then washed with serum-free our research reveals a new drug with potential protec- culture medium three times. After adding 1  ml fresh tive effects on dopamine neurons in PD, which may pro - medium to each well, the fluorescence was observed vide hope for the prevention and treatment of PD in the using the GFP channel of an inverted fluorescence micro - future. scope (Nikon,  ECLIPSE Ti, Tokyo,  Japan). The fluores - cence intensity was analyzed by ImageJ. Methods Cell culture and drug treatment Superoxide dismutase (SOD) activity assay The human neuroblastoma cell line SH-SY5Y was The intracellular SOD activity was measured with a total obtained from Sun Yat-Sen University (Guangzhou, SOD activity detection kit (Beyotime Biotechnology, China) and cultured in DMEM/H medium (HyClone, S0101, Shanghai, China), according to the manufacturer’s Yan et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 8 of 10 instructions. After drug treatment, the cells were washed cells were incubated at room temperature for 20 min, and once with PBS and collected by centrifugation. The cells then, the fluorescence was detected by flow cytometry were fully lysed in SOD sample preparation solution, (BD, Accuri C6, New York, USA). and the supernatants were collected at 12,000 × g at 4°C for 5  min. The absorbance was detected at 450  nm and Western blot assay 600 nm (reference wavelength) with a multimode micro- Cell lysates were prepared, and the protein concen- plate reader, and the SOD activity was calculated. trations were quantified with a BCA protein assay kit (CWBIO, CW2011S, Beijing,  China). Samples with equal amounts of proteins were separated by 12% Glutathione (GSH) assay SDS-PAGE and transferred to PVDF membranes. The content of GSH was measured with a glutathione After blocking with 5% skim milk at room tempera- assay kit (Beyotime Biotechnology, S0052, Shang- ture for 1  h, the membranes were incubated at 4°C hai, China), according to the manufacturer’s instructions. overnight with primary antibodies (1:1000) against After drug treatment, the cells were washed once with cleaved caspase-3 (Cell Signaling Technology, Aps175, PBS and collected by centrifugation. After the samples Boston,  USA), caspase-3 (Abcam, ab90437, Cam- and standards had been prepared, the absorbance was bridge,  UK), LC3 (Abcam, ab128025, Cambridge, UK), detected at 412  nm. We generated a standard curve and Beclin1 (BD, 612113, New York,  USA), p62 (Abcam, calculated the contents of GSH in the samples based on ab56416, Cambridge, UK) or β-actin (1:5000; CWBIO, this standard curve. CW0096M, Beijing,  China). The next day, the mem - branes were washed with TBST three times and incu- Malondialdehyde (MDA) assay bated with HRP-conjugated anti-rabbit or anti-mouse The intracellular MDA levels were measured with an secondary antibodies (1:5000,  CWBIO,  Beijing, China) MDA detection kit (Beyotime Biotechnology, S0131, at room temperature for 1  h. The blots were visualized Shanghai,  China), according to the manufacturer’s with a cECL Western blot kit (CWBIO, CW0049M, instructions. After drug treatment, the cells were col- Beijing,  China), and the images were analyzed by lected. The cells were lysed, and the supernatants were ImageJ. collected at 10,000 × g for 10  min. After the samples and standards had been prepared, the absorbance was detected at 532 and 450  nm (reference wavelength). The Immunofluorescence staining MDA contents in the samples were quantified based on SH-SY5Y cells were seeded on slides in 12-well culture the standard curve. plates. After drug treatment, the cells were fixed with ice-cold methanol for 5  min, blocked with 1% bovine Mitochondrial membrane potential (MMP) assay serum albumin (BSA) for 30  min, and then incubated To monitor the mitochondrial integrity, a mitochondrial with primary antibodies against LC3 (1:200) and P62 membrane potential assay kit with JC-1 (Beyotime Bio- (1:100) at 4°C overnight. The next day, the slides were technology, C2006, Shanghai,  China) was used, accord- incubated with Alexa Fluor 488 (Bioss Antibodies, bs- ing to the manufacturer’s instructions. Briefly, after drug 0295G-AF488, Beijing, China) or Alexa Fluor 555 (Bioss treatment, the SH-SY5Y cells were incubated with JC-1 Antibodies, bs-0296G-AF555, Beijing,  China) second- working solution at 37°C for 20  min and washed twice ary antibodies (1:500) at 37°C for 1  h and incubated with JC-1 buffer. Red fluorescence and green fluores - with DAPI (Boster, AR1177, Wuhan, China) for 5  min. cence were observed with the GFP channel and TRITC Fluorescence was observed with a fluorescence  micro - channel of an inverted fluorescence microscope (Nikon, scope (Olympus, BX53, Tokyo, Japan). ECLIPSE Ti, Japan). The fluorescence intensity was ana - lyzed by ImageJ. Statistical analysis Flow cytometry assay The results are expressed as the mean ± standard devia- We used an Annexin V-FITC cell apoptosis detection kit tion (mean ± SD). Comparisons among multiple groups (Beyotime Biotechnology, C1062M, Shanghai,  China) were performed with one-way ANOVA followed by to detect cell apoptosis, according to the manufactur- Tukey’s post hoc test, and p < 0.05 was considered sta- er’s instructions. After drug treatment, the cells were tistically significant. harvested and resuspended in PBS. A total of 50,000 to 100,000 cells were collected and resuspended in 195  μl Annexin V-FITC binding buffer, and then, 5  μl Annexin V-FITC and 10 μl PI were added with gentle mixing. The Y an et al. J of Biol Res-Thessaloniki (2021) 28:6 Page 9 of 10 Abbreviations 7. Liu H, Wu H, Zhu N, Xu Z, Wang Y, Qu Y, et al. Lactoferrin protects against PD: Parkinson’s disease; ART : Artemisinin; MPP : 1-Methyl-4-phenyliodine iron dysregulation, oxidative stress, and apoptosis in 1-methyl-4-phenyl- iodide; DCFH-DA: 2′,7′-Dichloro-dihydro-fluorescein diacetate; ROS: Reac- 1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease in mice. J tive oxygen species; SOD: Superoxide dismutase; GSH: Glutathione; MDA: Neurochem. 2020;152(3):397–415. https ://doi.org/10.1111/jnc.14857 . Malondialdehyde; MMP: Mitochondrial membrane potential; DA: Dopamine; 8. Radi E, Formichi P, Battisti C, Federico A. Apoptosis and oxidative stress in H2O2: Hydrogen peroxide; PC12: Rat adrenal pheochromocytoma; MA: neurodegenerative diseases. J Alzheimers Dis. 2014;42(Suppl 3):S125–52. 3-Methyladenine; MDA: Malondialdehyde; BSA: Bovine serum albumin; MPTP: https ://doi.org/10.3233/jad-13273 8. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ACD: Autophagic cell death; 9. Maruyama W, Abe T, Tohgi H, Naoi M. 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