Oxymatrine-mediated prevention of amyloid β-peptide-induced apoptosis on Alzheimer’s model PC12 cells: in vitro cell culture studies and in vivo cognitive assessment in rats

Alzheimer’s disease (AD) is a major neurological disease affecting elderly individuals worldwide. Existing drugs only reduce the symptoms of the disease without addressing the underlying causes. Commonly, Aβ25–35 peptide aggregation is the main reason for AD development. Recently, the discovery of multiple protein-targeting molecules has provided a new strategy for treating AD. This study demonstrates the neuroprotective potential of oxymatrine against multiple mechanisms, such as acetylcholinesterase, mitochondrial damage, and β-amyloid-induced cell toxicity. The in vitro cell culture studies showed that oxymatrine possesses significant potential to inhibit acetylcholine esterase and promotes antioxidant, antiapoptotic effects while preventing Aβ25–35 peptide aggregation in PC12 cells. Furthermore, oxymatrine protects PC12 cells against Aβ25–35-induced cytotoxicity and down-regulates the reactive oxygen species generation. The in vivo acute toxicological studies confirm the safety of oxymatrine without causing organ damage or death in animals. Overall, this study provided evidence that oxymatrine is an efficient neuroprotective agent, with a potential to be a multifunctional drug for Alzheimer’s disease treatment. These findings present a reliable and synergistic approach for treating AD.


Introduction
Alzheimer's disease (AD) is the most common neurological disorder worldwide, causing cognitive impairment. According to the World Health Organization (WHO), more than one billion people worldwide are affected by neurodegenerative disorders; 35 million people suffer from Alzheimer's disease and 50 million people suffer from epilepsy and other neurodegenerative diseases. The expected number of AD cases and incidence is 115 million by 2050 (Long and Holtzman 2019;Yiannopoulou and Papageorgiou 2020;Alexander and Karlawish 2021;AI-Atroshi et al. 2022;Folch et al. 2016;Jeyakumar et al. 2019). The amyloid peptides targeted drug discovery is a promising method for preventing β-amyloid aggregation in AD treatment. Amyloid-peptide (Aβ25-35) is a bioactive component of the Aβ peptide that leads to oxidative stress, memory loss, and apoptotic effects in brain cells. Neurotoxic short Aβ25-35 peptide fragments affect the tissues of the neurones (Jeyakumar et al. 2022;Wang et al. 2014;Zhang et al. 2018;Caruso et al. 2022;Michaels et al. 2020;Hu et al. 2021;Sun et al. 2021;Matuszyk et al. 2022). Currently, most of the scientific studies demonstrate that the aggregation of Aβ peptide causes oxidative stressmediated necrosis in neuronal cells and develops AD. To date, several natural and synthetic curative agents have been discovered for AD. Among commercial drugs, only a few provide satisfactory cholinesterase inhibition against AD. Most medicines are used only for short-term relaxation of cognitive deficit relaxation (Nirale et al. 2020; Thoe et al. 2021;Wong et al. 2019;Abeysinghe et al. 2020). Intense biomedical research is mainly interested in the prevention of Aβ peptide deposition in the brain. In contrast, medicinal plants exhibit progressive effects and symptoms in various 1 3 diseases, including AD. Many studies have evaluated neuroprotective effects and isolated active compounds from plant extracts (Peng et al. 2021;Thakur et al. 2019;Ovais et al. 2018;Gul et al. 2021). Many plant compounds, such as resveratrol, melatonin, galantamine, and catechin are known to have excellent neuroprotective effects against AD (Yan et al. 2020;Arbo et al. 2020;Roy et al. 2021;El-Ganainy et al. 2021;Okello and Mather 2020). Plant-derived alkaloids act as acetylcholinesterase (AChE) inhibitors and are considered model AD drugs (e.g., donepezil, galantamine, and rivastigmine) (Nguyen et al. 2021;Piemontese et al. 2018). Health technology assessments have undermined the adverse side effects of neuroprotective drugs in patients with AD (Terao et al. 2022). Therefore, it is imperative to discover AChEinhibiting compounds with a biocompatible central nervous system (Tan and Ismail 2020;Franchini et al. 2020;Sinha et al. 2020).
In this study, we demonstrated the multifactorial neuroprotective efficacy of the plant compound oxymatrine in a PC12 cell model of AD. The antioxidant, anticholinesterase, and Aβ peptide aggregation inhibition were evaluated by in vitro cell culture techniques. Cognitive memory improvement in oxymatrine-administered rats was examined using an in vivo step-down passive avoidance test. We believe that this investigation will be important for the future development of AD drugs. In the future, oxymatrine could be an efficient drug for the long-term prevention of AD.

Assessment of cell viability using MTT assay
The cytotoxic effect of oxymatrine on neuronal cells was examined using the MTT assay (Prabhu et al. 2019). PC12 cells were seeded in a 96-well plate (1 × 105 cells/ well) and incubated at 37 °C for 24 h. The cytotoxicity of only oxymatrine was examined at different concentrations (25-200 μM/L) against PC12 cells. The IC 50 of oxymatrine was quantified from triplicate MTT cell viability assays. The oxymatrine-mediated prevention of cytotoxicity of Aβ25-35 peptide-induced PC12 cells was examined at various concentrations (1, 10, and 100 μM/L) of oxymatrine pretreatment under optimum conditions for 48 h. Subsequently, 50 μM/L of Aβ25-35 peptide was added to the culture plates, and cells were incubated for 48 h. After that, the cells were stained with 50 μl of MTT solution, and incubation was continued for another 4 h at 37 °C. Excess MTT was removed with dimethyl sulfoxide. The absorbance of the cell suspension was measured at 570 nm using an ELISA plate reader. In a comparative study, cells were treated with oxymatrine (1,10, and 100 μM/L) loaded into a well plate before Aβ25-35 treatment: where Absc-absorbance of control cells, Abst-absorbance of treated cells.

Downregulation of reactive oxygen species (ROS) levels induced by Aβ25-35 protein
The antioxidant potential of oxymatrine was examined using a DCFHDA assay (Jeyakumar et al. 2022). The fluorescent probe DCFH-DA was added to PC12 cells and incubated at 37 °C for 1 h. The treated cells were washed with phosphate buffer solution (PBS). The fluorescence intensity of DCF-HDA was recorded at 485 nm excitation and 530 nm emission ranges. Inhibition of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) was measured according to manufacturer's instructions of a commercial kit (Nanjing Jiancheng Bioengineering Institute, China). SOD, CAT, and GPx inhibition levels were quantified at 405, 412, and 550 nm, respectively.

Assessment of apoptosis from Caspase-3 activation assay
To assess the anti-apoptotic properties of oxymatrine in PC12 cells, a Caspase-3 activation assay was performed (Pei et al. 2022). Caspases-3 protease is a primary indicator of apoptosis. Caspase 3 activity in oxymatrine-treated PC12 cells was measured using the acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVDpNA) enzyme to hydrolyse the substrate p-nitroaniline (PNA). The reaction mixture contained 200 μM Ac-DEVD-pNA, and 50 μg of Aβ25-35 proteins. The reaction mixtures were loaded into 24 well plates and incubated at room temperature for 1 h. Donepezil was used as a positive control. The percentage inhibition of caspase-3 was measured at 405 nm and p-nitroaniline was used as the reference. The quantification results are expressed as μ mol of pNA/min/mg of protein. The percentage of Caspases 3 activity was calculated using the following equation:

Free radical scavenging assay
The antioxidant potential of oxymatrine was examined using the DPPH free-radical scavenging assay. The hydrogen peroxide (H 2 O 2 ) scavenging ability of oxymatrine was evaluated according to the method described by Jeyakumar et al. (Jeyakumar et al. 2019). A H 2 O 2 solution of 50 mM at pH 7.4 was added with different doses (10-50 µg/ml) of oxymatrine. The reaction mixture was incubated at room temperature for 15 min. The inhibition was quantified from the maximum absorbance of H 2 O 2 at 550 nm. PBS solution was used as a reference. L-ascorbic acid was used as a positive control. The total percentage of H 2 O 2 scavenging was calculated using the following equation: where Absc-absorbance of control, and Abst-absorbance of treated group.

Determination of mitochondrial membrane potential
Mitochondrial membrane potential was assessed by spectrofluorimetric and fluorescence microscopic analyses using JC-1 staining (Raju et al. 2022). PC12 cells were treated for 24 h in the IC 50 concentration 82.43 ± 0.02 μM/L of oxymatrine. After 24 h of exposure, 5 μL of JC-1 stain was added and the cells were incubated for 20 min. Stained PC12 cells were washed with PBS to remove the free-floating dye. The fluorescence intensity of the cell culture medium was recorded at excitation and emission wavelengths of 480 and 530 nm, respectively. Cellular changes were monitored, and images were captured using a fluorescence microscope at 20 × magnification.

Apoptotic and live cell staining
The Acridine orange and ethidium bromide (AO/EtBr) dualstaining method was used to evaluate apoptotic and live/dead cells (Raju et al. 2022). PC12 cells were pretreated with different concentrations of oxymatrine (1, 10, and 20 μM/L) and Aβ25-35 peptide (50 μM/L) based on the MTT assay.
Specific activity (%) Treated cells were incubated at 37 °C under a carbon dioxide incubator. Treated cells were washed with PBS and transferred onto microscopic glass slides. Next, AO/EtBr dual stains (10 µl) were added to treated cells, and incubation was continued for another 10 min. Excess stain was washed with PBS. Images were captured using a fluorescence microscope (Nikon ECLIPSE Ti-E, Japan).

Assessment of anti-cholinesterase activity
The AChE inhibitory potential of oxymatrine was examined as previously reported (Jeyakumar et al. 2019). Briefly, two different concentrations (5 and 10 µg/ml) of oxymatrine were incubated with Aβ25-35 peptide (20 βL) at room temperature. After completion of the incubation period, 1 mM of DTNB (100 µl) was added with Tris-HCl buffer solution at pH 8.0 and made up the total volume of 1 mL. Furthermore, Acetylthiocholine iodide (50 µl) was added to the reaction mixture. Finally, 1 mM 5-thio-2-nitrobenzoate anion was added for yellow colour formation. The absorbance of the reaction solution was recorded at 405 nm using UV-Vis spectroscopy. The experiment was performed in triplicate. Donepezil was used as a positive control. Percentage of acetylcholine inhibition was calculated using the following formula: where Sc is the specific activity of the control and St is the specific activity of the treated group.

Aβ25-35 peptide anti-aggregation assay
The Aβ25-35 peptide anti-aggregative potential of oxymatrine was quantified using oligomer and fibril antiaggregation methods (Jeyakumar et al. 2022). Next, 0.5 mg of Aβ25-35 powder was dissolved in 1 ml of hexafluoro-2-propanol and left at room temperature for 24 h to form monomers.

Phase I
Inhibition of oligomer aggregation: in this experiment, Aβ25-35 monomers (100 μM) were dissolved in 100 mM Tris-HCL buffer and incubated for 24 h. After the incubation oxymatrine (5 and 10 μg/ml) was treated with oligomer and incubated for another 24 and 48 h. Galantamine (100 µg/ ml) was considered a positive control. Finally, the treated samples were subjected to Thioflavin-T assay.

Phase II
Inhibition of fibrils aggregation: in this experiment, Aβ25-35 powder (100 μM) was dissolved in 50 mM of Tris-HCL buffer solution and incubated at 37 °C for 96 h to allow fibril maturation. The aggregated fibrils were treated with different concentrations of oxymatrine (5 and 10 μg/ml) and Galantamine (100 µg/ml) and incubated for another 96 h and 9 days, respectively. The total mature fibrils formation was quantified using a T assay.

Assessment of apoptosis in Aβ25-35-induced PC12 cell by flow cytometry
The anti-apoptotic effect of oxymatrine in PC12 cells was examined using Annexin V-FITC/PI staining (Pei et al. 2022). The IC 50 concentration of oxymatrine treated cells was 82.43 ± 0.02 μM/L and 2.5 µl of annexin V-FITC/PI was used to stain the cells. The total percentage of apoptotic cells was quantified using the CytoFLEX flow cytometer. Early/late apoptotic cells were quantified using the FlowJo software.

Animal maintenance and groupings
The healthy 12-week-old Sprague-Dawley (SD) rats (180-250 g weight) were obtained from the Medicine Centre of Jinzhou Medical University (Jinzhou, China). Rats were maintained at 25 °C in 12 h dark/light cycle conditions with 60 − 70% humidity. The animals experiments were approved by the Experimental Animal Ethics Committee of Jinzhou Medical University, China. Experimental animals were divided into four groups. Toxicity tests were performed according to the Organization for Economic Cooperation and Development test guidelines (OECD Guideline 423) for acute oral toxicity tests. Before starting the experiment, the body weights of the animals were individually recorded to calculate the appropriate treatment dosage. The volume was adjusted depending on the body weight of the rat using 10 ml/kg to be used in the rat as mentioned. SD rats were administered standard rat pellets and reverse osmosis (RO) water ad libitum. They were acclimatised to laboratory conditions for 7 days before the experiments and were housed in groups of three for acute oral toxicity. The rats were maintained at a room temperature of 25 °C, with a 12 h light/dark cycle.

Evaluation of acute toxicity
The healthy 12-week-old SD rats (180-250 g weight) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). Rats were maintained at 25 °C in 12 h dark/light cycle conditions with 60 − 70% humidity. The animals were handled according to the guidelines of the Animal Ethics Committee of China Pharmaceutical University. The animals were then divided into corresponding groups (Al-Afifi et al. 2018). Oxymatrine was orally administered to the test animals 5 days before euthanization. Testing compound oxymatrine was dissolved in 0.5% of carboxymethylcellulose sodium salt solution (500, 1000, and 1500 mg/kg) and fed via oral administration. After oxymatrine administration, behavioural changes and mortality were continuously monitored for the first 24 h. One set of animals was occasionally observed for another 14 days to assess delayed acute toxicity. The PBS-treated rat group was used as the control group. All oxymatrine-treated rats were sacrificed on the 15th day. Organ damage in the heart, liver, and kidneys was evaluated macroscopically. In vivo acute toxic effects of oxymatrine in rats were examined using eosin-haematoxylin staining. The slides were observed and images were captured using an Olympus fluorescence microscope (SZX7 Zoom Stereo Microscope).

In vivo step-down passive avoidance test
A modified step-down passive avoidance test was performed to examine the learning ability and cognitive improvement in rats (Karimi et al. 2022). A total of 30 rats were used in the experiment (six rats/group. The experimental apparatus consisted of a plastic box divided into five equal compartment, separated by spaced steel bars measuring 1 cm. Rats were subjected to separate training and recall trials. Before the training trials, the animals were allowed to habituate for 5 min to the test environment. An electric current (24 V, 0.5 mA) was delivered to the apparatus platform, and the testing animals were gently placed on the platform. When the rats stepped down onto the platform, electric shock intermittently allowed them to return. Different doses of oxymatrine (20, 40, and 80 mg/kg) or a standard drug donepezil (5.0 mg/kg) were orally administrated prior to training trial. Donepezil was used as a positive control. After 30 min, scopolamine (3 mg/kg)-induced cognitive impairment. The learning and memory performance of the animals was evaluated from the latency step-down of first-time errors within 300 s of the shock.

Statistical analysis
Statistical analysis of all experimental data was performed using GraphPad Prism 8 software. The results were expressed as mean ± standard deviation (SD) and the comparisons were performed using one-way analysis of variance (ANOVA). P values of < 0.05 were considered to be statistically significant.

Cytotoxicity of oxymatrine and Aβ25-35 peptide against PC12 cells
Prevention of cell death is essential for AD control. Enhancement of neuronal cell viability can protect against neurological disorders (Calvo-Rodriguez and Bacskai 2021). In this study, the cytotoxicity of oxymatrine in PC12 cells was examined using the MTT assay. The IC 50 of oxymatrine alone was calculated to be 82.43 ± 0.02 μM/L. Aβ25-35 peptide treatment significantly (29.05 ± 1.2%) reduced the viability and density of PC12 cells (Fig. 1A) at the concentration of 50 μM/L. In this context, we investigated oxymatrine-treated PC12 cells, which showed non-toxic effect in PC12 cells. Furthermore, cells were pre-treated with oxymatrine at 1, 10, and 100 μM/L concentrations for 3 h. Aβ25-35 peptidetreated cells were cultured for another 24 h. The result revealed that treatment of oxymatrine prevented the cytotoxic effects of Aβ25-35 peptide, as shown in Fig. 1B, and mediation of oxymatrine rehabilitated the Aβ25-35 peptide-induced cell death. When the cells were treated at 100 μM/L, oxymatrine showed 86.50 ± 0.5% of viable cells, which was much higher than in the Aβ25-35 peptide. Morphological changes of Aβ25-35 peptide and oxymatrine were observed, and images were captured under an inverted phase contrast microscope. The control and oxymatrine-treated cells showed a clear cell structure and high cell density. In Aβ25-35, peptide-treated cells showed irregular morphology and a reduction in cell density.
The incorporation of Aβ25-35 peptide damaged the cell wall region and lead to cell shrinkage post-exposure. Oxymatrine-loaded cells were similar to the control cells. When the concentration of oxymatrine was increased, cell density and viability were also enhanced (Fig. 2). The cell viability assay confirmed that the incorporation of oxymatrine prevented the cytotoxic effect of Aβ25-35 in a dose-dependent manner. Therefore, oxymatrine is suitable for neuroprotective applications in cellular studies.

Reduction of ROS production against Aβ25-35-induced PC12 cells
Oxidative damage is closely associated with neurodegenerative diseases, such as AD, dementia, and Parkinson's disease, contributing to cellular inflammation and nitratedprotein dysfunctions (Good et al. 1996). The total production of ROS and cell morphology changes of Aβ25-35 and oxymatrine-treated PC12 cells was evaluated using DCFH-DA assay. The exposure of Aβ25-35 peptide at the dose of 50 μg/ml significantly induces ROS production in PC12 cells. The results of the DCFH-DA assay revealed maximum levels of SOD, GPx, and CAT enzyme activities, with some decreases compared to the control group (Fig. 3). The oxymatrine-pretreated cells showed low level of ROS production and enhanced SOD, GPx, and CAT activities. Presence of oxymatrine prevented oxidative stress in Aβ25-35 treated groups. The group treated with 10 g/mL oxymatrine exhibited similar levels of protective activity to that of commercial drug donepezil. The oxymatrine-treated group exhibited increased enzymatic activity in all three phases. CAT activity did not differ from that of the control. Whereas,

The anti-apoptotic potential of oxymatrine against Aβ25-35 peptide
Caspase-3 activity mediates cellular apoptosis The cleavage of cellular proteins in the Aβ25-35 treated PC12 cells and oxymatrine-treated cells were examined by caspase-3 activation assay. The results of PC12 cells with Aβ25-35 (50 μg/ ml) treatment exposed 0.824 ± 0.007 μM of pNA/min/mg of protein increase in caspase-3. Control cells exhibited only 0.384 ± 0.040 μM of pNA/min/mg of protein increase in caspase-3 activity. Compared with a control group, Aβ25-35 treated cells showed a significant amount of caspase-3 activity-mediated apoptosis. In this context, cells were pretreated with oxymatrine and the commercial drug donepezil ( Fig. 4D) showed a significant decrease in caspase-3 values such as 0.559 ± 0.016, 0.5789 ± 0.006, and 0.533 ± 0.015 μM of pNA/min/mg of protein. The results of the caspase-3 activation assay suggested that the oxymatrine prevented the Aβ25-35-induced apoptotic cell death in PC12 cells.

Reduction of oxidative stress by oxymatrine pre-treatment
Free radical scavengers prevent the ROS forming and eliminate them from cell damage. Plant-derived compounds are well-known antioxidants. Antioxidants neutralize the free radical scavenging of cells by donating their electrons (Raju and Natarajan 2021;Raju et al. 2020). They act as natural switches that control free radicals. The antioxidant potential of oxymatrine was evaluated using the DPPH assay. The results showed excellent H 2 O 2 scavenging activity under ambient conditions. Oxymatrine exhibited significant free radical reducing potential at the concentration of 50 µg/ ml. Positive control L-ascorbic acid showed 67.70 ± 1.32% and oxymatrine was 68.01 ± 1.21%, respectively (Fig. 5A). Compared to L-ascorbic acid, oxymatrine exhibited an equal hydrogen peroxide inhibition potential.

Enhancement of mitochondrial membrane potential by oxymatrine
The shape of the mitochondria is controlled by a balance of fusion-fission events. Damage to these mechanisms is closely associated with cell state. Loss of extracellular glucose supply leads to mitochondrial membrane potential (MMP) damage. Damage to the mitochondrial membrane potential harnesses cell protein production and energy flow (Wang et al. 2019;Misrani et al. 2021). In this experiment, fluorometric and microscopic changes of Aβ25-35 and oxymatrine-treated PC12 cells were assessed by Rh 123 staining technique. Aβ25-35 (50 μg/ ml) treated PC12 cells showed a significant decline in MMP, as evidenced by a 59.92 ± 2.64% of cellular fluorescence decrease of Rhodamine 123. On the contrary, the cells pre-treated with oxymatrine exhibit an increased fluorescence intensity, such as 95.92 ± 2.53% and 96.91 ± 3.65%, respectively. Oxymatrine treatment maintains mitochondrial integrity and protects cells from MMP damage. However, the effect of oxymatrine pretreatment on cellular mitochondrial membrane integrity was similar to that in the control group (Fig. 5B). The experimental outputs confirmed that oxymatrine protected the mitochondria from Aβ25-35-induced MMP damage-mediated neurotoxicity.

Anti-apoptotic activity of oxymatrine by AO/EtBr dual staining
Apoptosis is a highly sophisticated energy-dependent molecular event. Apoptosis triggers cellular injury and the disposal of damaged cells in an orderly fashion Chu et al. 2020). In this AO/EtBr dual staining method, here Aβ25-35 treated cells exposed 40.90 ± 2.56% of dead cells with apoptotic effects. Control cells showed only 5.45 ± 2.07% of dead cells. In contrast, cells pre-treated with oxymatrine showed a significant decrease in apoptotic activity (10.66 ± 1.47%) compared with the Aβ25-35 treated group, i.e., threefold lesser (Fig. 6). Morphological changes and nuclear damage were evaluated using fluorescence microscopy. The microscopic images of AO/EtBr dual staining, Aβ25-35-induced cells showed a higher level of red fluorescence which represented the apoptotic cells. Cells pretreated with oxymatrine can also prevent apoptotic cell death and exhibit a regular cell structure with bright fluorescence. These fluorometric and microscopic evaluations suggested that oxymatrine have an excellent potential to prevent apoptotic cell death in brains affected by AD.

Anticholinesterase activity of oxymatrine in PC12 cells
Acetylcholinesterase (AChE) plays a vital role in the regulation of cholinergic mechanisms and neurotransmission. Decreased cholinergic activity is a common cause of neuronal cell death. According to cholinergic kinetics, a declining level of acetylcholine in the brain is the leading cause of memory destruction in AD (Lao et al. 2019;Stanciu et al. 2019;Majdi et al. 2020). The results revealed that oxymatrine enhanced the cholinergic activity and prevented the aggregation of Aβ25-35 peptide by AChE inhibition. Oxymatrine higher dose (50 µg/ml) showed 98.56 ± 0.21% of AChE inhibition. Standard drug donepezil showed 99.65 ± 0.837% of inhibition (Fig. 7A). Recent reports Fig. 6 Evaluation of the anti-apoptotic effect of oxymatrine against Aβ25-35 treated PC12 cells. Fluorescence microscopic images of AO/EtBr double stained PC12 cells after treatment (20×). The bar graph represents the apoptotic percentage of Aβ25-35 and oxymatrine-treated cells. The data represent Mean ± SD of triplicates with significance (P < 0.05) compared to the control demonstrate phytocompounds as the new bio-functional scaffold for cholinesterase and β amyloid aggregation targeting. The Aβ25-35 treated cells exhibited a notable expansion in AChE level with enzyme activities (0.0078 ± 0.0001 U/mg of protein segregation. In this case, oxymatrine co-treated cells exhibited a significant level of AChE reduction with specific activities of (0.0035 ± 0.0002 and 0.0037 ± 0.0005 U/mg) of protein increase. The oxymatrine co-treatment effectively enhanced the cholinergic activity and prevented the Aβ25-35 peptide aggregations (Fig. 7B). The results of the anticholinesterase activity experiment confirmed that oxymatrine was a promising anti-cholinergic agent for the treatment of AD.

Anti-aggregation activity of oxymatrine against Aβ25-35 in PC12 cells
Extracellular aggregation of amyloid-β peptides is a leading cause of brain damage in AD. The additional formation of Aβ oligomers and β-amyloid deposition leads to brain plaques in AD. With the urgent need for alternative therapies, natural compounds serve as vital resources for novel drug developments (Zhao et al. 2022;Gomes et al. 2020;Zhang et al. 2021). The anti-aggregation ability of oxymatrine was assessed using an in vitro Thioflavin-T assay. The experiment was performed at different time intervals (24-96 h and 9 days) and in different phases: phase I (oligomer aggregation) and phase II (fibril formation). In the phase I study, the fluorescence intensity of Thioflavin-T was . The data represent Mean ± SD of triplicates with significance (P < 0.05) compared to the control found to increase in Aβ with oxymatrine 5 μg/ml treated group from 24 h (19.49 ± 2.31 AU) to 48 h (9.26 ± 1.54) indicating the reduction of Aβ aggregation. However, the treatment of oxymatrine at a dose of10 μg/ml showed excellent Thioflavin-T fluorescence reduction at 24 h (16.70 ± 0.76 AU and 48 h (6.79 ± 0.75 AU), respectively. The standard drug galantamine showed (50 μg/ml) (15.51 ± 2.54 AU at 24 h and 9.70 ± 1.75 AU at 48 h), respectively. The results of the phase II fibril formation study showed that a reduced (ThT) fluorescence intensity in oxymatrine-treated cells and standard drug galantamine in 96 h (15.5 ± 1.03 AU, 6.83 ± 1.2 AU) and (8.82 ± 1.02 AU) and 9 days (13.79 ± 1.87AU, 9.73 ± 1.79 AU) and (15.74 ± 1.14 AU), respectively (Fig. 7C). The results showed that the pretreatment of PC12 cells with oxymatrine potentially disaggregated mature fibrils. These results highlighted the inhibitory potential of oxymatrine against Aβ25-35 aggregation and phase I and II disaggregation. These experimental results provided a novel idea for the development of natural drugs for the treatment of AD.

Assessment apoptosis by flow cytometry
The total number of Aβ25-35-and oxymatrine-induced cell death and apoptotic stages was quantified by flow cytometry. The annexin-V/FITC and PI dual stains were utilised to evaluate the Aβ25-35-induced apoptosis and oxymatrinemediated protective effects. Results showed that the control group exhibited (0.3%) apoptotic cells. However, when compared to PC12 cells treated with the dose of (50 μg/ml) Aβ25-35 showed 8.99% of early apoptosis and 16.7% of late apoptosis. Interestingly, oxymatrine (10 g/mL) pretreatment showed excellent protective effects in PC12 cells Aβ25-35 leading to apoptosis (Fig. 8). Overall, the flow cytometry study suggested that oxymatrine was an effective drug for Aβ25-35-induced neuronal cell death.

Evaluation of oxymatrine-induced acute toxicity in rats
Toxicity testing is essential for drug discovery before human use. The advantages of toxicological studies include clearly defining the genetic composition of test animals and their convenience of exposure. The toxicity of all tissues and the psychomotor activities of test animals can be evaluated using acute toxicity studies (Al-Afifi et al. 2018;Zhang et al. 2021;Tripathi et al. 2022). Acute toxicological studies also provide guidance for model drugs. In this study, oxymatrineinduced acute toxic effects in rat organelles such as the liver, heart, lungs, kidney, and spleen were investigated by histopathology ( Fig. 9). Histopathological results showed no significant differences between the oxymatrine-administered animal groups and were similar to those in the PBS-treated control group. No significant tissue changes were observed in any of the groups (500-1500 mg/kg) in either of the acute oral toxicity tests. This result revealed that oxymatrine did not cause any inflammation or adverse effects on metabolic processes in the test animals. The cell morphology of the oxymatrine-treated groups was not different from that of the control group which was administered PBS. The present acute toxicological experiment demonstrated the safety of oxymatrine, suggesting that it is a promising neuroprotective drug for the treatment of AD.

Enhancement of cognitive activities by oxymatrine in the rat model
Cognitive decline is a common cause of memory impairment in patients with Alzheimer's disease. Neuropathological damage to the cerebral cortex and limbic system leads to impaired learning, memory, and spatial skills (Karimi et al. 2022). The present study explained the improvement in cognitive ability following oxymatrine administration in rat models using a step-down passive avoidance test. The Fig. 9 Histopathological images of oxymatrine-treated rats in oral administration. Microscopic images of essential organs of experimental animal (rat) from the control and oxymatrine (500-1500 mg/kg) oral administrated groups (20×). There is no inflammation and irreg-ular cell morphology appeared in oxymatrine-treated groups. The data represent Mean ± SD of triplicates with significance (P < 0.05) compared to the control commercial anticholinergic drug scopolamine was used to induce cognitive deficits in the animal model, and the commercial Alzheimer's drug donepezil was used as a positive control. The experimental results of the scopolamine-treated groups (Fig. 10) showed a significant reduction in latency and errors. When the animals were treated with oxymatrine, latency impairment and errors were reversed. The cognitive potential of the oxymatrine-treated groups showed a dose-dependent increase in learning and visual skills. The high-dose oxymatrine group (80 mg/kg) showed highest latency (233.83 s) and very few errors (1). This was better than that of the commercial drug donepezil group (5 mg/ kg, 199.33 s, 1.2). In this context, the medium dose (40 mg/ kg) of oxymatrine treated group exhibited a similar effect (184.79 s; 1.2) to donepezil. In the low-dose group (20 mg/ kg), oxymatrine did not cause any notable differences in latency or errors (158.33 s; 1.9). The in vivo cognitive study results indicated that oxymatrine prevented cognitive deficits through anticholinergic activity-mediated brain damage.

Discussion
AD, a common form of dementia in the elderly, greatly impacts human life quality. Unfortunately, there is currently no successful treatment for AD. Recent studies have implicated inflammation and immunity in the disease. Researchers have also linked neurotoxicity mediated by ROS to β-amyloid. Oxymatrine, an alkaloid compound derived from the Chinese herb Sophora flavescens, has been investigated for its effects on oxidative stress, apoptotic mechanisms, and the progression of hypoxic-ischemic brain damage. Some studies have explored its potential in the pathogenesis of AD. In this study, we aimed to investigate the neuroprotective efficacy of oxymatrine using PC12 cell lines as an in vitro model for AD. Our findings demonstrated that oxymatrine exhibited a protective role in preventing AD. We engineered an in vitro Alzheimer's model by inducing PC12 cell injury with the Aβ25-35 peptide. Pre-treatment with oxymatrine effectively protected PC12 cells from Aβ25-35 peptide-induced damage and apoptosis. While oxymatrine showed mild toxicity at a concentration of 200 µM/L, lower concentrations were found to be safe. Notably, oxymatrine pre-treatment significantly prevented cell death in Aβ25-35 peptide-induced PC12 cells. The DCFHDA assay revealed that oxymatrine inhibited ROS production in Aβ25-35 peptide-treated PC12 cells in a dose-dependent manner. We evaluated the effects of oxymatrine on caspase-3 expression in Aβ25-35-treated PC12 cells using a caspase-3 activation assay. The results showed that Aβ25-35 peptide treatment significantly increased caspase-3 expression, which was prevented by oxymatrine pre-treatment. Oxymatrine-treated cells alone exhibited minimal caspase-3 expression due to its mild toxic effect. The Amyloid-β anti-aggregation assay demonstrated that oxymatrine co-treatment reduced AChE activity in PC12 cells. Furthermore, the anti-inflammatory properties of oxymatrine protected Aβ25-35 peptideinduced PC12 cells from MMP damage and apoptosis. In in vivo assays, our acute toxicity study on rats revealed no toxic effects on major organs (liver, kidney, lungs, heart, and spleen). Rats pre-treated with oxymatrine exhibited improved cognitive development, showing significant facial memory improvement after 5 days of administration. These rats also displayed normal behaviour and reduced latency errors. Oxymatrine pre-treatment effectively prevented scopolamine-induced cognitive deficits in rats. Overall, our investigation highlighted oxymatrine's ability to protect PC12 cells from Aβ25-35 peptide-induced MMP damage, apoptosis, caspase-3 gene expression, and ROS overproduction. The cellular protection observed with oxymatrine pre-treatment supports its potential neuroprotective role in AD. Future preclinical trials may establish its efficacy as an effective neuroprotective drug for AD.

Conclusions
This study highlights the significant protective effect of oxymatrine against neuronal cell death induced by β-amyloid.
In vitro cell culture experiments demonstrate that oxymatrine pre-treated PC12 cells inhibit the mitochondrial damage and ROS generation caused by Aβ25-35 peptide.
Oxymatrine's excellent antioxidant and anti-acetylcholinesterase properties effectively prevent amyloid aggregation. The antiapoptotic potential of oxymatrine in neuronal cells is confirmed through the caspase-3 activity assay. However, it is important to note that oxymatrine does exert cytotoxic effects on PC12 cells. Furthermore, in vivo acute toxicological experiments establish the safety of oxymatrine as a neuroprotective drug. These findings collectively support the development of effective neuroprotective agents for AD treatment, with oxymatrine emerging as a promising multifunctional drug in this regard.