Sanggenol L alleviates Rotenone-induced Parkinson’s disease inhibits mitochondrial complex I by apoptotic via P13K/AKT/mTOR signalling

Sanggenol L alleviates Rotenone-induced Parkinson’s disease inhibits mitochondrial complex I by apoptotic via P13K/AKT/mTOR signalling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sanggenol L alleviates Rotenone-induced Parkinson’s disease inhibits mitochondrial complex I by apoptotic via P13K/AKT/mTOR signalling Nan Zhao, Menghai Wu, Jianbin Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5016013/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Parkinson’s disease (PD) is the age-associated, second most advanced neurodegenerative illness. Rotenone is an extensively used pesticide to study PD pathology and inhibits mitochondrial complex I. Reports indicate that rotenone exerts neurotoxicity by its capability to produce reactive oxygen species (ROS), which eventually leads to neuronal apoptosis. Sanggenol L (SL) is an eminent flavonoid present in the Morus alba root bark, which exhibits neuroprotective, anticancer, and antioxidant properties. Hence, we assessed the neuroprotective activity of SL (5 and 10 µM/ml) on rotenone-stimulated SK-N-SH neuroblastoma cells and elucidated the effect of the P13K/AKT/mTOR signaling. The anti-PD action of SL on proliferation, oxidative stress (OS), intracellular ROS, apoptosis, Bax, cleaved Caspase-12, 9, 3, and Cyt-c,Bcl-2and P13k/AKT/mTOR signaling was determined by MTT assay, biochemical analysis, DCFDA, AO/EB staining and western blot. It was found that SL (5 and 10 µM/ml) reduced rotenone-triggered OS, ROS levels, and apoptosis in a concentration-related way. SL alleviates Bax, cleaved caspase-12, 9, 3, and Cyt-c, while reducing Bcl-2. Furthermore, SL safer mitochondria by increase MMP and suppresses phosphorylation of P13k/AKT/mTOR pathway, thereby regulating apoptotic signalling. Our findings indicate that SL showed protective effects against rotenone-induced OS, mitochondrial complex I in neuronal cell damage, which suggests that SL might potentially serve as an anti-PD remedial candidate for PD treatment. Sanggenol L Parkinson’s disease rotenone Oxidative stress Apoptosis P13K/AKT/mTOR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Parkinson’s disease (PD) remains the supreme predominant type of neurodegenerative syndrome. There are more than five million PD patients globally (Agafonova et al.,2024). The dopaminergic neurondegeneration occurs in the region of substantia nigra pars compacta. The deprivation of the striatum containing dopamine levels is the leading cause of PD (Werner and Olanow,2022), which results in the clinical symptoms comprising tremors, postural imbalance, bradykinesia, muscular stiffness, and secondary symptoms containing impaired gait, trouble in gait, and suffering in speech (Raza et al.,2019, Dionisio et al.,2021). The key risk elements related to PD are environmental toxins including pesticides, carbon tetrachloride, heavy metals, and n -hexane, as well as genetics, gender (males are more susceptible than females), and age (Chakrabarti and Bisaglia,2023, El-Latif et al.,2023). Former reports presented an elevation of oxidative stress (OS) occurs in the PD patient’s cerebrospinal fluid and proposed that extreme generation of free radicals was associated with PD progression (Dong, et al.,2021). The dopaminergic damage led to an increase in oxidative injury causing the production of reactive oxygen species (ROS). ROS constrains the function of mitochondria, leading to protein misfolding, resulting in cell destruction and apoptotic cell death (Hassanzadeh and Rahimmi, 2018 , Hu et al., 2018 ). Numerous cellular mechanisms have been established in PD's progression, including mitochondrial dysfunction, increased iron content, OS, and anomalous folding of proteins (Desouky et al.,2023). Furthermore, studies demonstrated that the hyperproduction of ROS and extreme stimulation of interrelated caspase networks and microglial cells could direct neuronal cell death (Campolo et al.,2017, Zhu et al.,2019). Therefore, several research studies were carried out to explore the roles of natural antioxidative agents in treating various PD models (Zhao et al.,2020, Babaei et al.,2020). Sanggenol L (SL) is a famous flavonoid constituent isolated from the Morus Alba (Mulberry) root bark, which has been referred to exposed medicinal use, comprising anti-cancer (Nam et al.,2016), anti-inflammatory (Qin et al.,2015), and neuroprotective (Jung et al.,2015), properties. SL stimulates antiproliferative and apoptotic actions in ovarian carcinoma cells by activating caspases and inhibits NF-κB pathway (Nam et al.,2016). Previously, it has been explored that the protective effect of SL stimulates apoptosis that inhibits inflammatory and cell proliferative signaling pathways in DMBA-treated buccal pouch carcinogenesis in hamsters (Fu et al.,2023). A moment ago, SL alleviated inflammation and ankle joint destruction in collagen type II-stimulated arthritis in experimental rats by the subdual of the P13K/AKT pathway (Sun et al.,2023). Recently, SL caused cell cycle block and apoptosis by the initiation of p53 and repression of PI3K/Akt/mTOR pathways in prostate cancer (Won and Seoet,2020). These studies indicated SL is a potential flavonoid for PD treatment. Rotenone is a heteropentacyclic organic compound attained mainly from the Lonchocarpus plant species which is used as a pesticide and insecticide (Heinz et al.,2017). In the PD experimental in vitro and in vivo animal models, a neurotoxic compound rotenone is generally used to induce neurodegenerative alterations. Administration of rotenone inhibits the electron transport chain (ETC) system through the mitochondrial complex I binding (Erro et al.,2021). Complex-I blockers were identified as toxic to dopaminergic neurons in vitro and in vivo models (Feng and Xi,2022). Due to the lipophilic nature of rotenone, it can penetrate the blood-brain, which causes aggregation of alpha-synuclein in the brain (Flores et al.,2023). In neuronal cells, rotenone inhibits mitochondrial complex I and facilitates α-synuclein aggregation, it affects mitochondrial dysfunction and overproduction of ROS which eventually trigger neuronal cell death (Moors et al.,2017). Rotenone is related to several mechanisms including mitochondrial dysfunction, oxidative injury, α -synuclein amassing, reformed calcium signaling, and cell death. As a PD-inducing pesticide, rotenone generates neuronal indications and motor deficits reproducing human PD (Goyal et al.,2023). The vital evidence emphasizes the stimulation of PI3K/AKT/ mTOR pathway and its valuable neuroprotective role in PD (Yaoet et al.,2022, Li et al.,2023). PI3K/AKT/mTOR cascading axis, by its effect on excess proteins, has been recognized among the most fundamental pathways accomplishing neuronal survival, ameliorating neurogenesis, and suppressing apoptosis induced by neurotoxins in various PD models (Wang et al.,2022, Zhang et al.,2021). P13K/AKT/mTOR signaling has numerous biological functions such as apoptosis, autophagy, cell growth, differentiation, and protein synthesis. Earlier research has described that PI3K/Akt/mTOR signaling dysregulation contributes to the growth and development of PD (Peng et al.,2019). Taken together, our present report aimed to unravel the possible neuroprotective action of SL on rotenone-induced human neuroblastoma cells SK-N-SH; in a cellular model of PD by focusing on targeting the signaling cascade of PI3K/AKT/mTOR pathway. Materials and methods Chemicals and reagents Sanggenol L (Purity: 98.00%), Rotenone, antibiotics, DMEM, culturing reagents, FBS, glutamine, DCFH-DA, AO/EB staining, SDS, and DMSO were purchased from Ruicong Ltd (Shanghai, China). Antibodies for western blot analysis were procured from Ese‐Bio (Shanghai, China). Biochemical kits were acquired from Biomed (Badr City, Egypt). Biochemicals and solvents analytical grades were used. Cell Culture The neuroblastoma cells SK-N-SH were procured from GenScript (USA). These were cultivated in DMEM together with glutamine (2mM), streptomycin (100 U/mL), gentamicin (100 𝜇g/mL) penicillin (100U/mL), and 10% FBS, and changed the medium every two days. The incubator environments were 5% CO 2 , saturated humidity, at 37°C. Fresh DMSO (0.05%) was used to dissolve SL and rotenone before the experiment. SL pre-treated for 4 h before rotenone administration. For the first experiment, neuroblastoma cells were preserved with various quantities (5, 25, 50, 100, and 200 nM/ml) of rotenone for one day, and an MTT test was accomplished to identify the IC 50 data. For the second test, neuroblastoma cells were pre-administered with various dosages (2.5, 5 and 10µM) of SL preserved for four hours followed by incubation with an IC 50 value of rotenone for one day. The selected doses of SL were used to detect the possible protection against rotenone-induced neurotoxicity. MTT test The viability of neuroblastoma cells administered with various quantities of rotenone (5, 25, 50, 100 and 200 nM/ml) and SL (2.5, 5 and 10 µM/ml) was estimated by the test of MTT according to the exposure of live cells containing mitochondrial dehydrogenase reaction (Tamilselvam et al.,2013). MTT was treated in all wells, and the dishes were sustained for 4 h at 37 o C. Then, the treated cells were centrifuged for 10min, and the supernatant with DMSO (200 µL) was administered into the separate well, determining the absorbance at 595 nm in a microplate reader (BD Biosciences, NJ, USA). Biochemical assays Previously described methods (Jiang and Peng,2021) used to follow the assay of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activity were estimated by using assay kits provided by Biomed (Badr City, Egypt) affording to the company’s guidelines. Estimation of ROS intracellular level The production of ROS was estimated by adding 2, 7-diacetyl dichlorofluorescein (DCFH-DA) as a non-fluorescent probe that can move in the intracellular matrix. Determination of ROS percentage was in the control, SL (5 and 10 µM), and rotenone (100nM)-administered human neuroblastoma cells SK-N-SH. Concisely, 8×10 6 cells/ml isolated cells were converted into 2 ml as an ultimate volume with PBS. Afterward, 1ml cellaliquot was taken, treated with 100 𝜇L of 10 𝜇M DCFH-DA, and maintained at 37 o C for 30 min. Estimate the fluorescence by a multimode reader as described previously (Velu et al., 2020 ). Evaluation of Apoptosis by AO/EB dual staining The apoptotic effect of SL (5 and 10 µM) against SK-N-SH cells were examined by AO/EB dual staining as described previously (Velu et al., 2020 a ). SK-N-SHcells were added with SL (5 and 10 µM) incubated for 24h. Control and treated groups were administered with a mixture of dyes containing AO/EB (100 µg/ml per dye). They were kept under the dark; ensuring unbinding dye was eliminated by PBS and observed by a fluorescence microscope (Olympus, Tokyo, Japan). Analysis of Western blot Human neuroblastoma cells SK-N-SH were PBS washed twofold, and isolated proteins from the cells were separated on SDS-PAGE. Subsequently, the gel was relocated to PVDF film. Blotting was carried out with primary antibodies (anti-Bcl-2, anti-Bax, anti-Cyt-c, anti-caspase-12, anti-caspase-3, anti-caspase-9, anti-P13K, anti-p-Akt, anti-p-mTOR, and anti- β-actin) kept throughout the night at 4°C. Then, PBS was washed and preserved with HRP-conjugated secondary antibodies for 1h. Then, the protein bands were stained and imagined for protein identification. Quantified the protein bands through densitometry with software Image J and homogeneous to β-actin expression. Statistical examination Data were exhibited as mean ± SD for four tests and the statistical comparisons were conducted by Graph Pad Prism software version 8.0.1 was employed to perform an analysis of variance (ANOVA) and subsequently Duncan’s analysis. Student T-test has been conducted and P < 0.05 was exhibited as significant. Results Impact of SL on neuroblastoma cellproliferation Control cells showed 100% cell proliferation. Different concentrations of (5, 25, 50,100 and 200 nM) rotenone-treated SK-N-SH cell proliferation were decreased in an amount-related mode (Fig. 1 A). Administered with 100 nM of rotenone showed 48.17% of SK-N-SH cell proliferation. 10 𝜇M SL treated group revealed almost 122.44% protection after 24h (Fig. 1 B). Hence, 5 and 10 𝜇M SL were selected for further studies. Administration of SL (5 and 10𝜇M) increased the variations made by 100nM rotenone-induced cell viability (Fig. 1 C). SL reduced rotenone-induced oxidative stress and ROS formation PD growth and progression contributed by OS hence estimated the concentration of OS enzyme markers such as LDH, MDA, GPx, and SOD in neuroblastoma cells. Rotenone-induced neuroblastoma cells revealed remarkably increased (p < 0.05) MDA and LDH contents, while considerably diminished (p < 0.05) the SOD and GPx activities against control. Pre-treatment with SL (5 and 10 µM) could attenuate the MDA and LDH contents, whereas significantly enhanced (p < 0.05) the SOD and GPx activities in a concentration-related manner. Our data established that SL could attenuate MDA and LDH however, the SOD and GPx activities were increased in rotenone-induced neuroblastoma cells, thereby reducing OS and ROS formation (Fig. 2 ). SL reduces ROS production at the intracellular level The intracellular ROS development of rotenone-stimulated neuroblastoma cells was considerably elevated when compared to the control. Pre-treated SL (5 and 10 µM) could decrease rotenone (100 nM) stimulated ROS generation in a concentration-dependent way (Fig. 3 ). SL attenuates rotenone-induced apoptosis and elevates cell viability Rotenone and SL (5 and 10 µM/ml) treated SK-N-SH cells, with AO/EB dual staining were used to determine the apoptosis. The control SH-N-SH cells showed green bright fluorescence with green nuclei and normal cell morphology. In contrast, with rotenone (100 nM) exposure, SK-N-SH cells exhibited orange luminescent apoptotic body formation than control. Administration of SL (5 and 10 µM/ml) dose- dependently increased cell viability and reduced apoptotic cell death when compared to cells exposed only rotenone (Fig. 4 ). Impact of SL on apoptosis-associated proteins on rotenone-induced neuroblastoma cells Rotenone-induced neuroblastoma cells unveiled an elevated protein expression of Cyt-c and Bax while reducing the Bcl-2 protein expression against control. Pre-treated SL (5 and 10 µM) to rotenone (100 nM)-stimulated neuroblastoma cells attenuated the protein expression of Bax and Cyt-c, but Bcl-2 protein expression was sharply increased in a concentration-related manner (Fig. 5 ). This data indicates that SL shows inhibitory effect on apoptosis against rotenone-stimulated SK-N-SH cells through regulating apoptotic-associated proteins. Effect of SL on caspase apoptotic signaling pathway on rotenone-induced neuroblastoma cells Rotenone-induced neuroblastoma cells exhibited an elevated protein expression of Cleaved Caspase-12, 9, and 3 as compared to the control. Pre-treated SL (5 and 10 µM) to rotenone-treated SK-N-SH cells attenuated the protein expression of cleaved caspase-12, 9, and 3 in a concentration-related way (Fig. 6 ). SL could inhibit apoptosis of neuroblastoma cells via regulating caspase-12 apoptotic pathway. SL protects rotenone-stimulated neuroblastoma cells by the stimulation of the PI3K/Akt/mTOR pathway Rotenone-induced neuroblastoma cells down-regulated p-PI3K, p-Akt, and p-mTOR protein expression more than control. Pre-treated SL (5 and 10 µM) with rotenone-induced SK-N-SH cells up-regulation of p-PI3K, p-Akt, and p-mTOR protein expression levels in an amount-dependent way. Furthermore, PI3K, Akt, and mTOR protein expression had no variation between groups (Fig. 7 ). Discussion PD is an advanced neurodegenerative syndrome frequently perceived among the elderly, nevertheless, there are no effective cures. The Pathological representative of PD is the loss of dopaminergic neurons or dopamine deficiency (Agafonov et al.,2024 Werner and Olanow,2022). Furthermore, the dopaminergic neuron loss generates motor symptoms of PD due to either putamen dopamine loss or dopaminergic neuron loss (Raza et al.,2019, Dionisio etal.,2021, Chakrabarti and Bisaglia,2023). The generation of ROS causes OS and mitochondrial dysfunction the common pathogenic mechanisms associated with progressive neurodegenerative diseases (Hassanzadeh and Rahimmi,2018, Hu et al.,2018, Desouky et al.,2023). Dopaminergic neurodegeneration consequences from OS transformed the morphology of mitochondria, which directs mitochondrial dysfunction (Hu et al.,2018, Desouky et al.,2023). Thus, the discovery of innovative beneficial ingredients against PD by aiming ROS formation and OS is clinically important. In the current report, we presented a neuroprotective effect of SL (5 and 10 µM) against PD using rotenone-stimulated SK-N-SH cells, an in vitro model of PD. SL decreased rotenone-induced ROS generation, OS, and apoptosis in neuroblastoma cells in a concentration-related way. As well, SL enhances rotenone-reduced SK-N-SH cell proliferation, MMP, and induced apoptotic protein expression. We then further explored the protective molecular mechanism of SL on rotenone-stimulated apoptotic cell death of neuroblastoma cells. Rotenone is a strong lipophilic pesticide targeting the mitochondrial complex consequently harming neuronal development and triggering neurochemical pathological and behavioral changes (Erro et al.,2021, Ibarra et al,2023). Recently, rotenone weakened the behavioral parameters with a steady elevation in the akinesia and catalepsy and a drop in locomotor activities (Ibarra et al.,2023, El-Shamarka et al.,2023). Dopaminergic injury may be linked with behavioral parameters (Ibarra et al.,2023, El-Shamarka et al.,2023 a ). Rotenone is allied to mitochondrial dysfunction, α -synuclein accretion, oxidative injury, and cell apoptosis. In this study, SL (5 and 10 µM) could attenuate rotenone-stimulated mitochondrial impairment, OS, and apoptosis in neuroblastoma cells and showed a neuroprotective effect. Previously, it has been demonstrated that SL exerts neuroprotective activity (Jung et al.,2015). This research established that rotenone is toxic to neuroblastoma cells, as documented in earlier investigations (Tamilselvam et al.,2013). Here in, SL (5 and 10 µM) could reduce dose-related way of the toxic sequence of rotenone-stimulated cells. The MTT experiment is extensively utilized to determine cell viability by reducing the MTT tetrazolium salt to formazan. This reaction is primarily catalyzed by proliferating cells containing mitochondrial dehydrogenases (Tamilselvam et al.,2013 a ). Cell viability is determined as per the mitochondrial optimal function, therefore it plays a crucial part in controlling cell death signaling by the contribution of ROS production, regulating cellular energy metabolism, and the discharge of apoptotic mediators into the cytosol (Wang et al.,2022, Zhang et al.,2021). Findings acquired from the MTT test in this current work propose a conventional neuroprotective effect of SL on rotenone-facilitated mitochondrial dysfunction. Mitochondria show a vital part in the progression and apoptosis of cells, which are the major sources of intracellular ROS and also the key targets of OS (Moors et al.,2017). ROS formed due to oxidative injury caused by the PD pathogenesis (Grootveld,2022). Mitochondrial dysfunction originates from ROS formation which destructively affects cellular structures such as DNA, lipids, and proteins (Grootveld,2022, Grootveld et al.,2019). High-level production of ROS and cell death has been established in the former PD models (Moors et al.,2017, Goyal et al.,2023). Our findings also exhibit that rotenone induction can trigger the overproduction of ROS in neuroblastoma cells, as substantiated by prior investigations (Tamilselvam et al.,2013, Grootveld, 2022 , Juan et al.,2021). Herein, rotenone treatment exposed high contents of LDH and MDA, however, decreasing the activities of GPx and SOD in the neuroblastoma cells. Hence, it has been established that rotenone is directed to anabatic apoptosis, hypergeneration of ROS, and higher OS in the PD model. Moreover, it is demonstrated that the ROS creation and apoptotic capability of rotenone-administered neuroblastoma cells were attenuated by SL (5 and 10 µM) in a concentration-related mode, meanwhile, we also observed that SL decreased the LDH and MDA and ameliorated the GPx and SOD actions in this in vitro model of PD. To the greatest of our understanding, we were the first to prove that SL has the role of averting apoptotic cell death, diminishing ROS creation, inhibiting OS, and elevating MMP in rotenone-stimulated neuroblastoma cells. Furthermore, we explored the apoptotic mechanisms in an in vitro PD model treatment with SL. Bcl-2, as an anti-apoptotic protein reduces cell death activated via numerous latent mechanisms including OS, elevated caspases and Bax will trigger neuronal cell death in PD (Li et al.,2020, Li et al.,2023). In this study, rotenone declined the Bcl-2 protein expression, while elevating the protein level of Bax and caspases. As a result of rotenone induction, increasing the discharge of Cyt-c from the mitochondria, which activates caspases-12, 9, and 3 terminating with apoptosis (41–43). SL treatment elevates the mitochondrial permeability, and averts the cyt c discharge from the mitochondria, thereby preventing caspases12, 3, and 9, thus restoring the imbalance in the expression profiles of Bax and Bcl-2, and preventing cell death. Furthermore, over expression of Bcl-2 interrupts the pro-apoptotic proteins of Bax and averts the mitochondrial release of cyt c, thus suppressing the stimulation of caspases, and apoptosis (Li et al.,2020, Li et al.,2023, Wu et al.,2018). It has been documented that complex I anticipation by rotenone may be the consequence of the mitochondrial permeability transition pores (PTP) opening, which makes a precise conformational transformation of complex I and a huge generation of ROS (Li et al.,2023, Wu et al.,2018). Rise in ROS within the mitochondria are recognized to root auxiliary mitochondrial membrane depolarization and discharge of ROS. In the existing work, we demonstrated that SL expressively suppressed Cyt-c, Bax, and Caspase-12pathways, while enhancing MMP and Bcl-2 protein expression in this PD model. Therefore, we established that SL repressed the apoptosis by regulating the apoptosis-related protein expressions. Earlier reports have exposed that the dysregulation of the PI3K/Akt/mTOR network has been linked to the injury of dopaminergic neurons in PD (Yao et al 2022 , Li et al.,2023). Our data established that rotenone alleviated PI3K, Akt, and mTOR phosphorylation in neuroblastoma cells, which is related to the preceding documents (Yao et al 2022 , Li et al.,2023, Wang et al.,2022). Administration of neuroblastoma cells with SL (5 and 10 µM), the p-PI3K, p-Akt, and p-mTOR protein expression were markedly upregulated dose-dependably than the in vitro model of PD. These findings specified that SL activated the PI3K, Akt, and mTOR phosphorylation, and augmented the PI3K/Akt/mTOR pathway in this PD cellular model. Activated PI3K/AKT/mTOR signaling subdues cell apoptosis and autophagy, thus defending the cells from OS damage (Wang et al.,2022, Zhang et al.,2021, Peng et al.,2019). Agreeing with the above results, we rationally accomplish that SL protects the SK-N-SH cells by triggering the PI3K/ Akt/mTOR signaling. In conclusion, SL could increase cell proliferation of rotenone-induced SK-N-SH neuroblastoma cells, MMP, while reducing neuronal injury, mitochondrial dysfunction, ROS generation, and apoptosis through regulating apoptotic proteins. PI3K/AKT/mTOR signaling pathway suppresses cell apoptosis and promotes the stimulation of autophagy, thus protecting the cells from OS damage. Here, we found that rotenone down-regulated the phosphorylation level of PI3K, Akt, and mTOR in SK-N-SH cells. Furthermore, SL (5 and 10 µM/ml) ameliorates the PI3K/Akt/ mTOR pathway in the in vitro PD model in a dose-related way. These results emphasize that SL might be a possible remedial component for PD, which could promote further research on animal models and clinical trials as a new therapeutic agent. Declarations Acknowledgments None. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nan Zhao, Menghai Wu, Jianbin Zhang. The first draft of the manuscript was written by Jianbin Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethical approval Not Applicable Conflict of interest statement There are no conflicts of interest. References Agafonova I, Chingizova E, Chaikina E, Menchinskaya E, Kozlovskiy S, Likhatskaya G, Sabutski Y, Polonik S, Aminin D, Pislyagin E (2024) Protection Activity of 1,4- Naphthoquinones in Rotenone-Induced Models of Neurotoxicity. 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Drug Des Devel Ther 15:87–97 Peng T, Liu X, Wang J, Liu Y, Fu Z, Ma X, Li J, Sun G, Ji Y, Lu J, Wan W, Lu H (2019) Long noncoding RNA HAGLROS regulates apoptosis and autophagy in Parkinson's disease via regulating miR-100/ATG10 axis and PI3K/Akt/mTOR pathway activation. Artif Cells Nanomed Biotechnol 47(1):2764–2774 Tamilselvam K, Braidy N, Manivasagam T, Essa MM, Prasad NR, Karthikeyan S, Thenmozhi AJ, Selvaraju S, Guillemin GJ (2013) Neuroprotective effects of hesperidin, a plant flavanone, on rotenone-induced oxidative stress and apoptosis in a cellular model for Parkinson's disease. Oxid Med Cell Longev. 2013:102741 Jiang D, Peng Y (2021) The protective effect of decoction of Rehmanniae via PI3K/Akt/mTOR pathway in MPP+-induced Parkinson's disease model cells. J Recept Signal Transduct Res 41(1):74–84 Velu P, Vijayalakshmi A, Vinothkumar V (2020) Inhibiting the PI3K/Akt, NF-κB signalling pathways with syringic acid for attenuating the development of oral squamous cell carcinoma cells SCC131. J Pharm Pharmacol 72(11):1595–1606 Ibarra-Gutiérrez MT, Serrano-García N, Orozco-Ibarra M (2023) Rotenone-Induced Model of Parkinson's Disease: Beyond Mitochondrial Complex I Inhibition. Mol Neurobiol 60(4):1929–1948 El-Shamarka ME, Abdel-Salam OM, Shafee N, Zeidan HM (2023) Curcumin modulation of L-dopa and rasagiline-induced neuroprotection in rotenone model of Parkinson's disease. Iran J basic Med Sci 26(2):139–147 Grootveld M (2022) Evidence-Based Challenges to the Continued Recommendation and Use of Peroxidatively-Susceptible Polyunsaturated Fatty Acid-Rich Culinary Oils for High-Temperature Frying Practises: Experimental Revelations Focused on Toxic Aldehydic Lipid Oxidation Products. Front Nutr 8:711640 Trist BG, Hare DJ, Double KL (2019) Oxidative stress in the aging substantia nigra and the etiology of Parkinson's disease. Aging Cell 18(6):e13031 Juan CA, de la Pérez JM, Plou FJ, Pérez-Lebeña E (2021) The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies. Int J Mol Sci 22(9):4642 Li H, Xue X, Li L, Li Y, Wang Y, Huang T, Wang Y, Meng H, Pan B, Niu Q (2020) Aluminum-Induced Synaptic Plasticity Impairment via PI3K-Akt-mTOR Signaling Pathway. Neurotox Res 37(4):996–1008 Li W, Yin X, Yan Y, Liu C, Li G (2020) Kurarinone attenuates hydrogen peroxide-induced oxidative stress and apoptosis through activating the PI3K/Akt signaling by upregulating IGF1 expression in human ovarian granulosa cells. Environ Toxicol 38(1):28–38 Wu X, Liang Y, Jing X, Lin D, Chen Y, Zhou T, Peng S, Zheng D, Zeng Z, Lei M, Huang K, Tao E (2020) Rifampicin Prevents SH-SY5Y Cells from Rotenone-Induced Apoptosis via the PI3K/Akt/GSK-3β/CREB Signaling Pathway. Neurochem Res 43(4):886–893 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5016013","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":350036219,"identity":"5393b798-25c2-4f2b-8eae-ad8bf59d4b79","order_by":0,"name":"Nan Zhao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Zhao","suffix":""},{"id":350036220,"identity":"9df1129b-4f9d-44da-88bd-4e91c6461b06","order_by":1,"name":"Menghai Wu","email":"","orcid":"","institution":"Jinan People’s Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Menghai","middleName":"","lastName":"Wu","suffix":""},{"id":350036223,"identity":"1cc0cfe4-ec67-4e24-a6b4-d2c048d55bc8","order_by":2,"name":"Jianbin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYDACCRDBAyQlGBsYEipsmPkZgAzitXw4k8Yu2UCUFiiDcWbbYX6DAwTcJT+7+dnDLzIW8ubSzW2PeduYpY3PH2578IPBTk4Xh2WMc46ZG8vwSBjunHOw3ZjnHJux2Y3EdsMehmRjMxzWMUskmElL8EgwbriR2CbNU8aTbHaDsU2Ch+FA4jYcWtgk0r+BtNhDtLBJ1G/uP9gm+QePFh6JHDPJDzwSiSAtkjPaDJgNGEB68WiRkMgpkwZqTN5w52CbxIczCcwSIOtkDHD7RX5G+jbJnz11thtutz+TSKj4z8zff/yZ5JsKOzlcWsBBwNuDIWaAWzkIMP74gV/BKBgFo2AUjHAAALfKWlJQWDRlAAAAAElFTkSuQmCC","orcid":"","institution":"Shandong Provincial Third Hospital, Shandong University，Jinan","correspondingAuthor":true,"prefix":"","firstName":"Jianbin","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-09-02 07:02:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5016013/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5016013/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65948587,"identity":"d6853a85-e0ac-45e2-8f22-4e5d1e70a153","added_by":"auto","created_at":"2024-10-04 18:37:26","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211094,"visible":true,"origin":"","legend":"\u003cp\u003eSL alleviates rotenone-treated SK-N-SH cell viability reduction in neuroblastoma cells. (a) MTT assay was used to evaluate the concentration-related outcome of rotenone (5, 25, 50, 100 and 200 nM/ml) on cell viability. (b) MTT test was used to determine the concentration-related effect of SL (2.5, 5, and 10 μM/ml) on rotenone-stimulated variations of SK-N-SH cell viability. C) MTT test was used to determine the concentration-related effect of SL (5, and 10 μM/ml) on rotenone-stimulated variations of SK-N-SH cell viability. Results are exhibited as mean ± SD of four trials. \u003csup\u003e#,∗ \u003c/sup\u003edenotes significance (p\u0026lt;0.05) related to untreated SK-N-SH cells. Data exhibited as mean ± SD of three experiments.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/8b3acd911dcbed7922494d4e.jpeg"},{"id":65948588,"identity":"71a12a88-4c2b-422b-8354-98316b5fdedf","added_by":"auto","created_at":"2024-10-04 18:37:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18097,"visible":true,"origin":"","legend":"\u003cp\u003eSL attenuates rotenone-stimulated oxidative stress in neuroblastoma cells. (A) MDA (B) LDH (C) SOD (D) GPx.\u003cstrong\u003e \u003c/strong\u003eUntreated human neuroblastoma control cells, rotenone-induced SK-N-SH cells were pre-treated with 5 and 10 µM/ml of SL for one day. Data are exhibited as mean ± SD of four experiments. Results are exhibited as mean ± SD of four trials. \u003csup\u003e#,∗ \u003c/sup\u003edenotes significance (p\u0026lt;0.05) related to untreated SK-N-SH cells. Data exhibited as mean ± SD of three experiments.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/0e3da8269da300f0cedb089e.png"},{"id":65948592,"identity":"d75715c7-1822-4670-99c8-c751288f56da","added_by":"auto","created_at":"2024-10-04 18:37:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123241,"visible":true,"origin":"","legend":"\u003cp\u003eSL attenuates the intracellular ROS in the rotenone-induced neuroblastoma cells. Untreated human neuroblastoma control cells, rotenone-induced SK-N-SH cells were pre-administered with 5 and 10 µM/ml of SL for one day. The generation of intracellular ROS was determined by DCFDA staining.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/e0388828ff0477fdef9e9cb7.png"},{"id":65948590,"identity":"9cd225f5-9708-4916-8ae4-f5321defc731","added_by":"auto","created_at":"2024-10-04 18:37:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173932,"visible":true,"origin":"","legend":"\u003cp\u003eSL alleviates apoptosis in the rotenone-induced neuroblastoma cells.\u003cstrong\u003e \u003c/strong\u003eHuman neuroblastoma control cells, rotenone-induced SK-N-SH cells were pre-administered with control, 5, and 10 µM/ml of SL for one day. The apoptosis was estimated by the AO/EB dual staining and imaged by a fluorescence microscope.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/9fc1cab1f5092797e095e76c.png"},{"id":65948594,"identity":"e66ab756-f149-4c65-aa7d-1c1956aae661","added_by":"auto","created_at":"2024-10-04 18:37:26","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":283183,"visible":true,"origin":"","legend":"\u003cp\u003eSL reduced rotenone-stimulated SK-N-SH cell apoptosis.\u003cstrong\u003e \u003c/strong\u003eUntreated human neuroblastoma control cells, rotenone-induced SK-N-SH cells were pre-administered with 5 and 10µM of SL for one day. The protein expression of Bax, Cyt-c, and Bcl-2 were estimated by Western blot analysis.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/0a1a42ac075dc91822e6dec9.jpeg"},{"id":65949274,"identity":"1cf71592-b1e9-4515-8e33-bdcabb2b083a","added_by":"auto","created_at":"2024-10-04 18:53:26","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":370626,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of SL on rotenone-treated SK-N-SH cell Caspase pathway.\u003cstrong\u003e \u003c/strong\u003eUntreated human neuroblastoma control cells, rotenone-induced SK-N-SH cells were pre-treated with 5 and 10µM of SL for one day. The protein expression of cleaved caspase-12, 9, and 3 were determined by Western blot. Data are exhibited as mean ± SD of four experiments in each group. ∗denotes significance (p\u0026lt;0.05) compared to untreated SK-N-SH cells. ∗∗denotes significance (p\u0026lt;0.05) compared to rotenone-administered SK-N-SH cells.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/8d4d9d4c3fd6ebc5f91b6534.jpeg"},{"id":65948982,"identity":"a8d66e42-7f9a-46b2-9287-04d3567388b1","added_by":"auto","created_at":"2024-10-04 18:45:26","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":370344,"visible":true,"origin":"","legend":"\u003cp\u003eSL activates the PI3K/Akt/mTOR pathway on rotenone-treated neuroblastoma cells.\u003cstrong\u003e \u003c/strong\u003eUntreated human neuroblastoma control cells, rotenone-induced SK-N-SH cells were pre-administered with 5 and 10 µM/ml of SL for one day. The expression of p-P13K, P13K, p-Akt, Akt, p-mTOR, and mTOR protein were estimated by Western blot.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/e0ad7069870b1a20bb91349a.jpeg"},{"id":69324124,"identity":"eab07d7d-baae-4dca-adab-406fc634b4be","added_by":"auto","created_at":"2024-11-19 07:38:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2079819,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5016013/v1/2f27b97b-1691-4494-955f-4ebb3946ff3f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sanggenol L alleviates Rotenone-induced Parkinson’s disease inhibits mitochondrial complex I by apoptotic via P13K/AKT/mTOR signalling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) remains the supreme predominant type of neurodegenerative syndrome. There are more than five million PD patients globally (Agafonova et al.,2024). The dopaminergic neurondegeneration occurs in the region of \u003cem\u003esubstantia nigra\u003c/em\u003e pars compacta. The deprivation of the striatum containing dopamine levels is the leading cause of PD (Werner and Olanow,2022), which results in the clinical symptoms comprising tremors, postural imbalance, bradykinesia, muscular stiffness, and secondary symptoms containing impaired gait, trouble in gait, and suffering in speech (Raza et al.,2019, Dionisio et al.,2021). The key risk elements related to PD are environmental toxins including pesticides, carbon tetrachloride, heavy metals, and \u003cem\u003en\u003c/em\u003e-hexane, as well as genetics, gender (males are more susceptible than females), and age (Chakrabarti and Bisaglia,2023, El-Latif et al.,2023). Former reports presented an elevation of oxidative stress (OS) occurs in the PD patient\u0026rsquo;s cerebrospinal fluid and proposed that extreme generation of free radicals was associated with PD progression (Dong, et al.,2021). The dopaminergic damage led to an increase in oxidative injury causing the production of reactive oxygen species (ROS). ROS constrains the function of mitochondria, leading to protein misfolding, resulting in cell destruction and apoptotic cell death (Hassanzadeh and Rahimmi, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Hu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Numerous cellular mechanisms have been established in PD's progression, including mitochondrial dysfunction, increased iron content, OS, and anomalous folding of proteins (Desouky et al.,2023). Furthermore, studies demonstrated that the hyperproduction of ROS and extreme stimulation of interrelated caspase networks and microglial cells could direct neuronal cell death (Campolo et al.,2017, Zhu et al.,2019). Therefore, several research studies were carried out to explore the roles of natural antioxidative agents in treating various PD models (Zhao et al.,2020, Babaei et al.,2020).\u003c/p\u003e \u003cp\u003eSanggenol L (SL) is a famous flavonoid constituent isolated from the \u003cem\u003eMorus Alba\u003c/em\u003e (Mulberry) root bark, which has been referred to exposed medicinal use, comprising anti-cancer (Nam et al.,2016), anti-inflammatory (Qin et al.,2015), and neuroprotective (Jung et al.,2015), properties. SL stimulates antiproliferative and apoptotic actions in ovarian carcinoma cells by activating caspases and inhibits NF-κB pathway (Nam et al.,2016). Previously, it has been explored that the protective effect of SL stimulates apoptosis that inhibits inflammatory and cell proliferative signaling pathways in DMBA-treated buccal pouch carcinogenesis in hamsters (Fu et al.,2023). A moment ago, SL alleviated inflammation and ankle joint destruction in collagen type II-stimulated arthritis in experimental rats by the subdual of the P13K/AKT pathway (Sun et al.,2023). Recently, SL caused cell cycle block and apoptosis by the initiation of p53 and repression of PI3K/Akt/mTOR pathways in prostate cancer (Won and Seoet,2020). These studies indicated SL is a potential flavonoid for PD treatment.\u003c/p\u003e \u003cp\u003eRotenone is a heteropentacyclic organic compound attained mainly from the \u003cem\u003eLonchocarpus\u003c/em\u003e plant species which is used as a pesticide and insecticide (Heinz et al.,2017). In the PD experimental \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e animal models, a neurotoxic compound rotenone is generally used to induce neurodegenerative alterations. Administration of rotenone inhibits the electron transport chain (ETC) system through the mitochondrial complex I binding (Erro et al.,2021). Complex-I blockers were identified as toxic to dopaminergic neurons \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models (Feng and Xi,2022). Due to the lipophilic nature of rotenone, it can penetrate the blood-brain, which causes aggregation of alpha-synuclein in the brain (Flores et al.,2023). In neuronal cells, rotenone inhibits mitochondrial complex I and facilitates α-synuclein aggregation, it affects mitochondrial dysfunction and overproduction of ROS which eventually trigger neuronal cell death (Moors et al.,2017). Rotenone is related to several mechanisms including mitochondrial dysfunction, oxidative injury, \u003cem\u003eα\u003c/em\u003e-synuclein amassing, reformed calcium signaling, and cell death. As a PD-inducing pesticide, rotenone generates neuronal indications and motor deficits reproducing human PD (Goyal et al.,2023).\u003c/p\u003e \u003cp\u003eThe vital evidence emphasizes the stimulation of PI3K/AKT/ mTOR pathway and its valuable neuroprotective role in PD (Yaoet et al.,2022, Li et al.,2023). PI3K/AKT/mTOR cascading axis, by its effect on excess proteins, has been recognized among the most fundamental pathways accomplishing neuronal survival, ameliorating neurogenesis, and suppressing apoptosis induced by neurotoxins in various PD models (Wang et al.,2022, Zhang et al.,2021). P13K/AKT/mTOR signaling has numerous biological functions such as apoptosis, autophagy, cell growth, differentiation, and protein synthesis. Earlier research has described that PI3K/Akt/mTOR signaling dysregulation contributes to the growth and development of PD (Peng et al.,2019). Taken together, our present report aimed to unravel the possible neuroprotective action of SL on rotenone-induced human neuroblastoma cells SK-N-SH; in a cellular model of PD by focusing on targeting the signaling cascade of PI3K/AKT/mTOR pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eSanggenol L (Purity: 98.00%), Rotenone, antibiotics, DMEM, culturing reagents, FBS, glutamine, DCFH-DA, AO/EB staining, SDS, and DMSO were purchased from Ruicong Ltd (Shanghai, China). Antibodies for western blot analysis were procured from Ese‐Bio (Shanghai, China). Biochemical kits were acquired from Biomed (Badr City, Egypt). Biochemicals and solvents analytical grades were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eThe neuroblastoma cells SK-N-SH were procured from GenScript (USA). These were cultivated in DMEM together with glutamine (2mM), streptomycin (100 U/mL), gentamicin (100 \u0026#120583;g/mL) penicillin (100U/mL), and 10% FBS, and changed the medium every two days. The incubator environments were 5% CO\u003csub\u003e2\u003c/sub\u003e, saturated humidity, at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eFresh DMSO (0.05%) was used to dissolve SL and rotenone before the experiment. SL pre-treated for 4 h before rotenone administration. For the first experiment, neuroblastoma cells were preserved with various quantities (5, 25, 50, 100, and 200 nM/ml) of rotenone for one day, and an MTT test was accomplished to identify the IC\u003csub\u003e50\u003c/sub\u003e data. For the second test, neuroblastoma cells were pre-administered with various dosages (2.5, 5 and 10\u0026micro;M) of SL preserved for four hours followed by incubation with an IC\u003csub\u003e50\u003c/sub\u003e value of rotenone for one day. The selected doses of SL were used to detect the possible protection against rotenone-induced neurotoxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMTT test\u003c/h2\u003e \u003cp\u003eThe viability of neuroblastoma cells administered with various quantities of rotenone (5, 25, 50, 100 and 200 nM/ml) and SL (2.5, 5 and 10 \u0026micro;M/ml) was estimated by the test of MTT according to the exposure of live cells containing mitochondrial dehydrogenase reaction (Tamilselvam et al.,2013). MTT was treated in all wells, and the dishes were sustained for 4 h at 37\u003csup\u003eo\u003c/sup\u003eC. Then, the treated cells were centrifuged for 10min, and the supernatant with DMSO (200 \u0026micro;L) was administered into the separate well, determining the absorbance at 595 nm in a microplate reader (BD Biosciences, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical assays\u003c/h2\u003e \u003cp\u003ePreviously described methods (Jiang and Peng,2021) used to follow the assay of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activity were estimated by using assay kits provided by Biomed (Badr City, Egypt) affording to the company\u0026rsquo;s guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of ROS intracellular level\u003c/h2\u003e \u003cp\u003eThe production of ROS was estimated by adding 2, 7-diacetyl dichlorofluorescein (DCFH-DA) as a non-fluorescent probe that can move in the intracellular matrix. Determination of ROS percentage was in the control, SL (5 and 10 \u0026micro;M), and rotenone (100nM)-administered human neuroblastoma cells SK-N-SH. Concisely, 8\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml isolated cells were converted into 2 ml as an ultimate volume with PBS. Afterward, 1ml cellaliquot was taken, treated with 100 \u0026#120583;L of 10 \u0026#120583;M DCFH-DA, and maintained at 37\u003csup\u003eo\u003c/sup\u003eC for 30 min. Estimate the fluorescence by a multimode reader as described previously (Velu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Apoptosis by AO/EB dual staining\u003c/h2\u003e \u003cp\u003eThe apoptotic effect of SL (5 and 10 \u0026micro;M) against SK-N-SH cells were examined by AO/EB dual staining as described previously (Velu et al., 2020\u003csup\u003ea\u003c/sup\u003e). SK-N-SHcells were added with SL (5 and 10 \u0026micro;M) incubated for 24h. Control and treated groups were administered with a mixture of dyes containing AO/EB (100 \u0026micro;g/ml per dye). They were kept under the dark; ensuring unbinding dye was eliminated by PBS and observed by a fluorescence microscope (Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Western blot\u003c/h2\u003e \u003cp\u003eHuman neuroblastoma cells SK-N-SH were PBS washed twofold, and isolated proteins from the cells were separated on SDS-PAGE. Subsequently, the gel was relocated to PVDF film. Blotting was carried out with primary antibodies (anti-Bcl-2, anti-Bax, anti-Cyt-c, anti-caspase-12, anti-caspase-3, anti-caspase-9, anti-P13K, anti-p-Akt, anti-p-mTOR, and anti- β-actin) kept throughout the night at 4\u0026deg;C. Then, PBS was washed and preserved with HRP-conjugated secondary antibodies for 1h. Then, the protein bands were stained and imagined for protein identification. Quantified the protein bands through densitometry with software Image J and homogeneous to β-actin expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical examination\u003c/h2\u003e \u003cp\u003eData were exhibited as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for four tests and the statistical comparisons were conducted by Graph Pad Prism software version 8.0.1 was employed to perform an analysis of variance (ANOVA) and subsequently Duncan\u0026rsquo;s analysis. Student T-test has been conducted and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was exhibited as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImpact of SL on neuroblastoma cellproliferation\u003c/h2\u003e \u003cp\u003eControl cells showed 100% cell proliferation. Different concentrations of (5, 25, 50,100 and 200 nM) rotenone-treated SK-N-SH cell proliferation were decreased in an amount-related mode (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Administered with 100 nM of rotenone showed 48.17% of SK-N-SH cell proliferation. 10 \u0026#120583;M SL treated group revealed almost 122.44% protection after 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Hence, 5 and 10 \u0026#120583;M SL were selected for further studies. Administration of SL (5 and 10\u0026#120583;M) increased the variations made by 100nM rotenone-induced cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSL reduced rotenone-induced oxidative stress and ROS formation\u003c/h2\u003e \u003cp\u003ePD growth and progression contributed by OS hence estimated the concentration of OS enzyme markers such as LDH, MDA, GPx, and SOD in neuroblastoma cells. Rotenone-induced neuroblastoma cells revealed remarkably increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) MDA and LDH contents, while considerably diminished (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the SOD and GPx activities against control. Pre-treatment with SL (5 and 10 \u0026micro;M) could attenuate the MDA and LDH contents, whereas significantly enhanced (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the SOD and GPx activities in a concentration-related manner. Our data established that SL could attenuate MDA and LDH however, the SOD and GPx activities were increased in rotenone-induced neuroblastoma cells, thereby reducing OS and ROS formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSL reduces ROS production at the intracellular level\u003c/h2\u003e \u003cp\u003eThe intracellular ROS development of rotenone-stimulated neuroblastoma cells was considerably elevated when compared to the control. Pre-treated SL (5 and 10 \u0026micro;M) could decrease rotenone (100 nM) stimulated ROS generation in a concentration-dependent way (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSL attenuates rotenone-induced apoptosis and elevates cell viability\u003c/h2\u003e \u003cp\u003eRotenone and SL (5 and 10 \u0026micro;M/ml) treated SK-N-SH cells, with AO/EB dual staining were used to determine the apoptosis. The control SH-N-SH cells showed green bright fluorescence with green nuclei and normal cell morphology. In contrast, with rotenone (100 nM) exposure, SK-N-SH cells exhibited orange luminescent apoptotic body formation than control. Administration of SL (5 and 10 \u0026micro;M/ml) dose- dependently increased cell viability and reduced apoptotic cell death when compared to cells exposed only rotenone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImpact of SL on apoptosis-associated proteins on rotenone-induced neuroblastoma cells\u003c/h2\u003e \u003cp\u003eRotenone-induced neuroblastoma cells unveiled an elevated protein expression of Cyt-c and Bax while reducing the Bcl-2 protein expression against control. Pre-treated SL (5 and 10 \u0026micro;M) to rotenone (100 nM)-stimulated neuroblastoma cells attenuated the protein expression of Bax and Cyt-c, but Bcl-2 protein expression was sharply increased in a concentration-related manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This data indicates that SL shows inhibitory effect on apoptosis against rotenone-stimulated SK-N-SH cells through regulating apoptotic-associated proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SL on caspase apoptotic signaling pathway on rotenone-induced neuroblastoma cells\u003c/h2\u003e \u003cp\u003eRotenone-induced neuroblastoma cells exhibited an elevated protein expression of Cleaved Caspase-12, 9, and 3 as compared to the control. Pre-treated SL (5 and 10 \u0026micro;M) to rotenone-treated SK-N-SH cells attenuated the protein expression of cleaved caspase-12, 9, and 3 in a concentration-related way (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). SL could inhibit apoptosis of neuroblastoma cells via regulating caspase-12 apoptotic pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSL protects rotenone-stimulated neuroblastoma cells by the stimulation of the PI3K/Akt/mTOR pathway\u003c/h2\u003e \u003cp\u003eRotenone-induced neuroblastoma cells down-regulated p-PI3K, p-Akt, and p-mTOR protein expression more than control. Pre-treated SL (5 and 10 \u0026micro;M) with rotenone-induced SK-N-SH cells up-regulation of p-PI3K, p-Akt, and p-mTOR protein expression levels in an amount-dependent way. Furthermore, PI3K, Akt, and mTOR protein expression had no variation between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePD is an advanced neurodegenerative syndrome frequently perceived among the elderly, nevertheless, there are no effective cures. The Pathological representative of PD is the loss of dopaminergic neurons or dopamine deficiency (Agafonov et al.,2024 Werner and Olanow,2022). Furthermore, the dopaminergic neuron loss generates motor symptoms of PD due to either putamen dopamine loss or dopaminergic neuron loss (Raza et al.,2019, Dionisio etal.,2021, Chakrabarti and Bisaglia,2023). The generation of ROS causes OS and mitochondrial dysfunction the common pathogenic mechanisms associated with progressive neurodegenerative diseases (Hassanzadeh and Rahimmi,2018, Hu et al.,2018, Desouky et al.,2023). Dopaminergic neurodegeneration consequences from OS transformed the morphology of mitochondria, which directs mitochondrial dysfunction (Hu et al.,2018, Desouky et al.,2023). Thus, the discovery of innovative beneficial ingredients against PD by aiming ROS formation and OS is clinically important. In the current report, we presented a neuroprotective effect of SL (5 and 10 \u0026micro;M) against PD using rotenone-stimulated SK-N-SH cells, an \u003cem\u003ein vitro\u003c/em\u003e model of PD. SL decreased rotenone-induced ROS generation, OS, and apoptosis in neuroblastoma cells in a concentration-related way. As well, SL enhances rotenone-reduced SK-N-SH cell proliferation, MMP, and induced apoptotic protein expression. We then further explored the protective molecular mechanism of SL on rotenone-stimulated apoptotic cell death of neuroblastoma cells.\u003c/p\u003e \u003cp\u003eRotenone is a strong lipophilic pesticide targeting the mitochondrial complex consequently harming neuronal development and triggering neurochemical pathological and behavioral changes (Erro et al.,2021, Ibarra et al,2023). Recently, rotenone weakened the behavioral parameters with a steady elevation in the akinesia and catalepsy and a drop in locomotor activities (Ibarra et al.,2023, El-Shamarka et al.,2023). Dopaminergic injury may be linked with behavioral parameters (Ibarra et al.,2023, El-Shamarka et al.,2023\u003csup\u003ea\u003c/sup\u003e). Rotenone is allied to mitochondrial dysfunction, \u003cem\u003eα\u003c/em\u003e-synuclein accretion, oxidative injury, and cell apoptosis. In this study, SL (5 and 10 \u0026micro;M) could attenuate rotenone-stimulated mitochondrial impairment, OS, and apoptosis in neuroblastoma cells and showed a neuroprotective effect. Previously, it has been demonstrated that SL exerts neuroprotective activity (Jung et al.,2015).\u003c/p\u003e \u003cp\u003eThis research established that rotenone is toxic to neuroblastoma cells, as documented in earlier investigations (Tamilselvam et al.,2013). Here in, SL (5 and 10 \u0026micro;M) could reduce dose-related way of the toxic sequence of rotenone-stimulated cells. The MTT experiment is extensively utilized to determine cell viability by reducing the MTT tetrazolium salt to formazan. This reaction is primarily catalyzed by proliferating cells containing mitochondrial dehydrogenases (Tamilselvam et al.,2013\u003csup\u003ea\u003c/sup\u003e). Cell viability is determined as per the mitochondrial optimal function, therefore it plays a crucial part in controlling cell death signaling by the contribution of ROS production, regulating cellular energy metabolism, and the discharge of apoptotic mediators into the cytosol (Wang et al.,2022, Zhang et al.,2021). Findings acquired from the MTT test in this current work propose a conventional neuroprotective effect of SL on rotenone-facilitated mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003eMitochondria show a vital part in the progression and apoptosis of cells, which are the major sources of intracellular ROS and also the key targets of OS (Moors et al.,2017). ROS formed due to oxidative injury caused by the PD pathogenesis (Grootveld,2022). Mitochondrial dysfunction originates from ROS formation which destructively affects cellular structures such as DNA, lipids, and proteins (Grootveld,2022, Grootveld et al.,2019). High-level production of ROS and cell death has been established in the former PD models (Moors et al.,2017, Goyal et al.,2023). Our findings also exhibit that rotenone induction can trigger the overproduction of ROS in neuroblastoma cells, as substantiated by prior investigations (Tamilselvam et al.,2013, Grootveld, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Juan et al.,2021). Herein, rotenone treatment exposed high contents of LDH and MDA, however, decreasing the activities of GPx and SOD in the neuroblastoma cells. Hence, it has been established that rotenone is directed to anabatic apoptosis, hypergeneration of ROS, and higher OS in the PD model. Moreover, it is demonstrated that the ROS creation and apoptotic capability of rotenone-administered neuroblastoma cells were attenuated by SL (5 and 10 \u0026micro;M) in a concentration-related mode, meanwhile, we also observed that SL decreased the LDH and MDA and ameliorated the GPx and SOD actions in this \u003cem\u003ein vitro\u003c/em\u003e model of PD. To the greatest of our understanding, we were the first to prove that SL has the role of averting apoptotic cell death, diminishing ROS creation, inhibiting OS, and elevating MMP in rotenone-stimulated neuroblastoma cells.\u003c/p\u003e \u003cp\u003eFurthermore, we explored the apoptotic mechanisms in an \u003cem\u003ein vitro\u003c/em\u003e PD model treatment with SL. Bcl-2, as an anti-apoptotic protein reduces cell death activated via numerous latent mechanisms including OS, elevated caspases and Bax will trigger neuronal cell death in PD (Li et al.,2020, Li et al.,2023). In this study, rotenone declined the Bcl-2 protein expression, while elevating the protein level of Bax and caspases. As a result of rotenone induction, increasing the discharge of Cyt-c from the mitochondria, which activates caspases-12, 9, and 3 terminating with apoptosis (41\u0026ndash;43). SL treatment elevates the mitochondrial permeability, and averts the cyt c discharge from the mitochondria, thereby preventing caspases12, 3, and 9, thus restoring the imbalance in the expression profiles of Bax and Bcl-2, and preventing cell death. Furthermore, over expression of Bcl-2 interrupts the pro-apoptotic proteins of Bax and averts the mitochondrial release of cyt c, thus suppressing the stimulation of caspases, and apoptosis (Li et al.,2020, Li et al.,2023, Wu et al.,2018). It has been documented that complex I anticipation by rotenone may be the consequence of the mitochondrial permeability transition pores (PTP) opening, which makes a precise conformational transformation of complex I and a huge generation of ROS (Li et al.,2023, Wu et al.,2018). Rise in ROS within the mitochondria are recognized to root auxiliary mitochondrial membrane depolarization and discharge of ROS. In the existing work, we demonstrated that SL expressively suppressed Cyt-c, Bax, and Caspase-12pathways, while enhancing MMP and Bcl-2 protein expression in this PD model. Therefore, we established that SL repressed the apoptosis by regulating the apoptosis-related protein expressions.\u003c/p\u003e \u003cp\u003eEarlier reports have exposed that the dysregulation of the PI3K/Akt/mTOR network has been linked to the injury of dopaminergic neurons in PD (Yao et al \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Li et al.,2023). Our data established that rotenone alleviated PI3K, Akt, and mTOR phosphorylation in neuroblastoma cells, which is related to the preceding documents (Yao et al \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Li et al.,2023, Wang et al.,2022). Administration of neuroblastoma cells with SL (5 and 10 \u0026micro;M), the p-PI3K, p-Akt, and p-mTOR protein expression were markedly upregulated dose-dependably than the \u003cem\u003ein vitro\u003c/em\u003e model of PD. These findings specified that SL activated the PI3K, Akt, and mTOR phosphorylation, and augmented the PI3K/Akt/mTOR pathway in this PD cellular model. Activated PI3K/AKT/mTOR signaling subdues cell apoptosis and autophagy, thus defending the cells from OS damage (Wang et al.,2022, Zhang et al.,2021, Peng et al.,2019). Agreeing with the above results, we rationally accomplish that SL protects the SK-N-SH cells by triggering the PI3K/ Akt/mTOR signaling.\u003c/p\u003e \u003cp\u003eIn conclusion, SL could increase cell proliferation of rotenone-induced SK-N-SH neuroblastoma cells, MMP, while reducing neuronal injury, mitochondrial dysfunction, ROS generation, and apoptosis through regulating apoptotic proteins. PI3K/AKT/mTOR signaling pathway suppresses cell apoptosis and promotes the stimulation of autophagy, thus protecting the cells from OS damage. Here, we found that rotenone down-regulated the phosphorylation level of PI3K, Akt, and mTOR in SK-N-SH cells. Furthermore, SL (5 and 10 \u0026micro;M/ml) ameliorates the PI3K/Akt/ mTOR pathway in the \u003cem\u003ein vitro\u003c/em\u003e PD model in a dose-related way. These results emphasize that SL might be a possible remedial component for PD, which could promote further research on animal models and clinical trials as a new therapeutic agent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments \u0026nbsp;\u003c/strong\u003e None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nan Zhao, Menghai Wu, Jianbin Zhang. The first draft of the manuscript was written by Jianbin Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp; \u0026nbsp;\u003c/strong\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u0026nbsp; \u0026nbsp;The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u0026nbsp; \u0026nbsp;Not Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u0026nbsp; \u0026nbsp; There are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgafonova I, Chingizova E, Chaikina E, Menchinskaya E, Kozlovskiy S, Likhatskaya G, Sabutski Y, Polonik S, Aminin D, Pislyagin E (2024) Protection Activity of 1,4- Naphthoquinones in Rotenone-Induced Models of Neurotoxicity. 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Neurochem Res 43(4):886\u0026ndash;893\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sanggenol L, Parkinson’s disease, rotenone, Oxidative stress, Apoptosis, P13K/AKT/mTOR","lastPublishedDoi":"10.21203/rs.3.rs-5016013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5016013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is the age-associated, second most advanced neurodegenerative illness. Rotenone is an extensively used pesticide to study PD pathology and inhibits mitochondrial complex I. Reports indicate that rotenone exerts neurotoxicity by its capability to produce reactive oxygen species (ROS), which eventually leads to neuronal apoptosis. Sanggenol L (SL) is an eminent flavonoid present in the \u003cem\u003eMorus alba\u003c/em\u003e root bark, which exhibits neuroprotective, anticancer, and antioxidant properties. Hence, we assessed the neuroprotective activity of SL (5 and 10 \u0026micro;M/ml) on rotenone-stimulated SK-N-SH neuroblastoma cells and elucidated the effect of the P13K/AKT/mTOR signaling. The anti-PD action of SL on proliferation, oxidative stress (OS), intracellular ROS, apoptosis, Bax, cleaved Caspase-12, 9, 3, and Cyt-c,Bcl-2and P13k/AKT/mTOR signaling was determined by MTT assay, biochemical analysis, DCFDA, AO/EB staining and western blot. It was found that SL (5 and 10 \u0026micro;M/ml) reduced rotenone-triggered OS, ROS levels, and apoptosis in a concentration-related way. SL alleviates Bax, cleaved caspase-12, 9, 3, and Cyt-c, while reducing Bcl-2. Furthermore, SL safer mitochondria by increase MMP and suppresses phosphorylation of P13k/AKT/mTOR pathway, thereby regulating apoptotic signalling. Our findings indicate that SL showed protective effects against rotenone-induced OS, mitochondrial complex I in neuronal cell damage, which suggests that SL might potentially serve as an anti-PD remedial candidate for PD treatment.\u003c/p\u003e","manuscriptTitle":"Sanggenol L alleviates Rotenone-induced Parkinson’s disease inhibits mitochondrial complex I by apoptotic via P13K/AKT/mTOR signalling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 18:37:21","doi":"10.21203/rs.3.rs-5016013/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e4c4f398-bbe3-42f7-b17d-c86101604698","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-19T07:38:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-04 18:37:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5016013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5016013","identity":"rs-5016013","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}