Neuroprotective effects of a novel peptide from Lignosus rhinocerotis against 6‐hydroxydopamine‐induced apoptosis in PC12 cells by inhibiting NF‐κB activation

Abstract According to previous studies, oxidative stress is a leading cause of dopaminergic neuron death and may contribute to the pathogenesis of Parkinson's disease (PD). In the current study, we used chromatography of gel filtration to identify a novel peptide (Lignosus rhinocerotis peptide [LRP]) from the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden. Its neuroprotective effect was evaluated using an in vitro PD model constructed by 6‐hydroxydopamine (6‐OHDA)‐stimulated to apoptosis in PC12 cells. The molecular weight of LRP is determined as 1532 Da and the secondary structure is irregular. The simple amino acid sequence of LRP is Thr‐Leu‐Ala‐Pro‐Thr‐Phe‐Leu‐Ser‐Ser‐Leu‐Gly‐Pro‐Cys‐Leu‐Leu. Notably, LRP has the ability to significantly boost the viability of PC12 cells after exposure to 6‐OHDA, as well as enhance the cellular activity of antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH‐Px). LRP also lowers the level of malondialdehyde (MDA), decreases the activation performance of Caspase‐3, and reduces 6‐OHDA‐induced apoptosis via inhibition of nuclear factor‐kappa B (NF‐κB) activation. These data indicate that LRP may have the potential to act as a neuroprotective agent.


| INTRODUC TI ON
Parkinson's disease (PD) affects 2%-3% of individuals over the age of 65, and it is regarded as the second most common neurodegenerative disease in humans (Darvas et al., 2014). Pathologically, PD is often characterized by a significant decrease of dopaminergic neurons in the substantia nigra, which leads to a reduction in dopamine biosynthesis in both the substantia nigra and striatum (Tysnes & Storstein, 2017). The mechanism of this neuronal degeneration is unclear, although many factors may contribute to this pathology, including genetic factors, environmental influences, aging, and oxidative stress (Burbulla et al., 2017). Among these, oxidative stress is considered an important cause of PD (Fahn & Cohen, 1992).
Although normal physiological activities of the human brain require lots of oxygen, antioxidant enzymes are relatively scarce, making the brain particularly vulnerable to oxidative stress-related damage (Lau et al., 2007). Indeed, prior research has demonstrated that the increase of mitochondrial oxidative stress in substantia nigra compacta dopaminergic neurons triggers dopamine-dependent toxic cascade reactions that result in lysosomal dysfunction and α-synuclein accumulation-the two main pathological features of PD (Kalia & Lang, 2015;Vandiver et al., 2013). Conventional dopamine replacement therapy (such as L-3,4-Dihydroxyphenylalanine [L-DOPA]) is the most widely prescribed medicine for the treatment of PD symptoms, but has significant side effects (Kwon et al., 2010) including reduced motor function in the patients who have undergone long-term L-DOPA treatment (Maharaj et al., 2005). This limitation prompted us to identify new neuroprotective agents to prevent and treat PD.
Since previous research suggests that oxidative stress contributes to the pathogenesis and progression of PD (Flower et al., 2005;Guo et al., 2007), we investigated whether treatment aimed at reducing oxidative stress may help slow the progression of PD.
Natural resource extraction of chemicals favorable to human nervous system health has made significant progress in recent decades (Martorell et al., 2016;Sato et al., 2022). Indeed, Salidroside, isolated from the root of Rhodiola rosea, protects apoptotic PC12 cells by inhibiting oxidative stress and inflammation (Zhou et al., 2019), and Neoechinulin A, an alkaloid from Eurotium rubrum, protects neuronal PC12 cells from peroxynitrite cytotoxicity (Kimoto et al., 2007). Furthermore, paeoniflorin (a compound found in Paeonia lactiflora) inhibits NF-κB activation and prevents 6-hydroxydopamine (6-OHDA)-induced cell death in PC12 cells (Cao et al., 2010). However, compared to plants, there are few studies on fungal neuroprotective agents. While some fungal polysaccharides have free radical scavenging activity that may play a neuroprotective effect (Giavasis, 2014), fungal polysaccharides have large molecular weights and complex structures, which make it difficult to penetrate the blood-brain barrier. Thus, their application as neuroprotective agents is limited. Nevertheless, ethanol or water extract of some fungi contains neuroprotective factors (Xiong et al., 2017). However, the components of the extracts are not clear, and the structure of functional substances needs to be identified.
The "Tiger Milk Fungus," or Lignosus rhinocerotis (Cooke) Ryvarden, is a Polyporaceae mushroom that is frequently used in Malaysian traditional medicine to increase stamina, reduce cough, and help with asthma treatment (Sabaratnam et al., 2013). Recent scientific studies have confirmed the pharmacological benefits of L. rhinocerotis and revealed its antioxidant, antitumor, antiviral, and immunomodulatory activities (Nallathamby et al., 2018). Notably, the neuritogenic properties of L. rhinocerotis promote neurite outgrowth in PC12 cells: when applied to PC12 cells, the aqueous extract of L.
rhinocerotis increased the population of neurite-bearing cells from 9.8% to 23.6% (Eik et al., 2012). However, the components in the aqueous extract are complex, and it is difficult to determine which substance or substances may be responsible for the observed neuritogenic effects.
The PC12 cell line is derived from the adult rat adrenal medullary pheochromocytoma. The cell has a reversible neuronal phenotype response to nerve growth factor (NGF); it has been widely employed as a model to investigate dopamine biosynthesis and oxidative stressinduced cytotoxicity (Rostamian Delavar et al., 2018). 6-OHDA has also been widely used to create models of PD by reactive-oxygen species (ROS)-generated damage to dopaminergic neuronal cells (Deumens et al., 2002). Recently, a novel peptide (L. rhinocerotis peptide [LRP]) was extracted from the sclerotium of L. rhinocerotis and purified using reverse-phase high-performance liquid chromatography (RP-HPLC) after being separated via molecular exclusion chromatography; its sequence was determined using liquid chromatographytandem mass spectrometry (LC-MS-MS). The function of LRP for 6-OHDA-induced PC12 cell apoptosis and the potential pathway of LRP using NF-κB as a key target was also investigated.

| Materials and chemicals
The sclerotial powder of L. rhinocerotis was contributed by Prof. Dr.

| Segregation and purification of peptides
The L. rhinocerotis sclerotial powder was collected by the passage of the material through a 200-mesh screen. Next, 100 g of powder was soaked in 2 L of phosphate-buffered saline (PBS, pH = 7.2) and stirred with a magnetic stirrer for 6 h to form a slurry. The supernatant was then recovered by centrifuging the slurry at 5000 g for 10 min to remove any remaining particles. To obtain the precipitate, ammonium sulfate was added to the supernatant after the reaction was completed. The concentration of ammonium sulfate in the supernatant increased from 40% to 80%, and all precipitates were collected. The precipitates were then dissolved in 100 ml of distilled water. The precipitate aqueous solution was dialyzed (Union Carbide Corporation, Houston, TX, USA) to separate the solids from the liquid with a cutoff molecular weight of 3500 Da. To extract the mushroom crude protein, we then condensed (Eyela, Tokyo, Japan) and lyophilized (Christ, Osterode, Germany) the dialysate. Then, the mushroom crude protein extract was introduced to a Sephadex 30 increasing column (2.6 cm × 30 cm), which was eluted with five bed volumes of distilled water (DW) at a flow rate of 0.8 ml/min. A ultraviolet (UV) detector measured the eluate, which was collected in 2.5 ml sample tubes at 215 nm. The most concentrated peak of the protein content was extracted, after which it was purified by dialysis and lyophilization to create the mushroom protein extract.
Reverse-phase high-performance liquid chromatographywas utilized to further purify the mushroom protein extract. First, 10 ml of distilled water (DW) was added to 100 mg of protein extract to yield a solution with a concentration of 10 mg/ml. Then, a PerkinElmer (Shelton, USA) N2600580 system with a C18 column set at 25°C for purification was used for RP-HPLC analysis (injection volume of 20 μl; flow rate of 1 ml/min). The distilled water (A) and acetonitrile containing 1% trifluoroacetic acid (B) were set as mobile phase with a detection wavelength of 215 nm. The gradient elution was completed as follows: A: 0-8 min, 99%-97%; 8-12 min, 97%-96%; 12-16 min, 96%-80%; 16-20 min, 80%-99%. After tying up the radical rummaging task, the absorption peak was obtained, and the peptide extract was deemed appropriate for the current experiments because of its encouraging scavenging ability.
A Triple TOF 5600 LC/MS system was used to conduct the mass spectrometry analysis in this study (SCIEX, USA). Aspiration of the peptide sample was accomplished using a preprogrammed injector, which was subsequently combined with the C18 capture pillar (5 μm, 5 × 0.3 mm); the samples were then eluted and an- and a charge number between +2 and +5 for the duration of collection. ProteinPilot was used to obtain mass spectrum data (V4.5).

| Morphology observation
To analyze the shape of the peptide, scanning electron microscopy (SEM) and atomic force microscopes (AFMs) were utilized. The peptide powder (2 mg) was secured to the SEM reinforcement table and sputter-coated with gold using a vacuum-based sputter coater (Q150R Plus; Quorum, UK). The peptide was then examined under a scanning electron microscope (Quanta FEG 250; Oregon, USA) to determine the quality. All samples were subjected to an accelerating voltage of 20.0 kV during the examination. Micrographs of the peptide were obtained at 20000× and 5000× magnification.
The peptide solution was diluted to a concentration of 1 mg/ ml, then mixed with Milli-Q water. Then, 10 μl of the diluted solution was placed on a freshly split mica platform and washed with Milli-Q water to remove any remaining untarnished peptide residue.
After air-drying, the sample was scanned using an AFM straightaway (Hitachi SPM400, Japan).

| Circular dichroism spectroscopy
To prepare the solution at our desired concentration of 0.25 mg/ ml, we dissolved the peptide in water. The circular dichroism (CD) spectra measurement was performed on a Chirascan V100 (Applied Photophysics Limited, Surrey, UK) at 25°C using a 2 mm quartz cuvette (Qiu et al., 2009). The CD spectra were collected from 190 to 260 nm three times for the average at 1 nm intervals and 1 nm bandwidths.

| Cell culture and treatment
PC12 cells were planted in the environment of RPMI medium 1640 supplemented with 5% FBS, 10% horse serum, 100 μg/ml streptomycin, and 100 U/ml penicillin in an incubator at 37°C with 5% carbon dioxide for 2 days. PC12 cells were then induced to differentiate with nerve growth factor beta (NGFβ) at a final concentration of 50 ng/ml for 7 days. Afterward, cells were pretreated with LRP at different concentrations (10, 20, 40, 80, and 160 μM) for 6 h, followed by 6-OHDA.

| Cell viability assay
The MTT assay was used to evaluate the cytoprotective efficacy of LRP against cell damage produced by 6-OHDA. Each well was filled

| Reactive oxygen species measurement
A DCFDA/H2DCFDA-Cellular ROS Assay Kit was used to measure cellular ROS. Briefly, cells were washed twice with prewarmed PBS in a 96-well plate after drug treatment. Forty-five minutes of exposure to DCFDA was used to stain the cells. After washing, absorbance was detected at 535 nm in a microplate reader (SpectraMax plus 384; Molecular Devices, San Jose, CA, USA). In the control group, the amount of ROS was normalized to the amount of generated fluorescence.

| Determination of malondialdehyde (MDA) content and antioxidant enzymes' activity
Lignosus rhinocerotis peptide (of varying concentrations) was applied to PC12 cells for 6 h, after which the cells were treated with 6-OHDA (100 μM) for 24 h. Following a PBS wash, cells were collected, exposed to 50 μl lysate, and centrifuged for 10 min at 12,000 g to obtain the supernatant for subsequent determination. MDA measurement was performed according to manufacturer's instructions using the 2-thiobarbituric acid (TBA) technique (Beyotime, Shanghai, China). A commercial kit was then used to measure the endogenous antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity (Beyotime, Shanghai, China).

| Apoptosis analysis
The apoptosis of PC12 cells was detected using flow cytometry. In

| Measurement of Caspase activity
The activities of Caspase-9 and Caspase-3 were determined using commercial colorimetric assay kits following the manufacturers' instructions. After coculture with 6-OHDA, PC12 cells were harvested and suspended for 5 min in lysis buffer on ice. For Caspase-3, the supernatant was collected via centrifugation and mixed gently after the addition of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA, 2 mM). For Caspase-9, acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA, 2 mM) was added. The mixture was incubated at 37°C for 60 min and the absorbance was read at a wavelength of 405 nm using a Microplate Reader (Molecular Devices, San Jose, CA, USA).

| Statistical analysis
All results are expressed as mean ± standard deviation (SD) of triplicate values. One-way analysis of variance (ANOVA) was applied to determine significant differences between the groups using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Significance was assigned at p < .05 by Newman-Keuls tests (Duncan's Multiple Range Test [DMRT]).

| Isolation and purification of LRP
The protein extract was subjected to stepwise ammonium sulfate precipitation followed by centrifugation to remove impurities. After freeze-drying, the protein extract (9.82 g) was obtained from 100 g of dry powder of L. rhinocerotis. The extract was separated and fractionated using a Sephadex 30 column (2.6 cm × 30 cm), and then washed with distilled water to obtain a crude protein extract (Figure 1a). After three exposures to bed column volume, four elution peaks (A 1-4 ) were obtained. There was a mass of 1.92 g for the A 1 component, and 1.58, 0.98, and 2.34 g for the A 3-4 components, respectively. Based on the appearance time of the characteristic peak, A 4 was selected as the protein extract. The protein extract was further separated by RP-HPLC (Figure 1b), and three characteristic absorption peaks were identified (B 1-3 ). One of them, B 1 , weighed 0.45 g; the others, B 2-3 , weighed 0.62 and 0.58 g, respectively. According to the HPLC analysis, the purity of B 1-3 was more than 95%, with B 1 having a purity of 97.42%, B2 having a purity of 97.04%, and B3 having a purity of 95.48% (Detailed test methods can be found in the supporting information). The B 2 component had the best free radical scavenging activity (data not shown) and was therefore selected for LC-MS-MS analysis and subsequent harvest of the L. rhinocerotis peptide (LRP). LRP had a relative molecular weight of 1532 Da and contained 15 amino acids. The amino acid sequence was Thr-Leu-Ala-Pro-Thr-Phe-Leu-Ser-Ser-Leu-Gly-Pro-Cys-Leu-Leu.

| Morphology observation
Lignosus rhinocerotis peptide had an uneven flaky structure ( Figure 2a), with curls appearing at the edges (Figure 2b), as demonstrated by scanning electron microscopy (SEM). In an aqueous solution (10 μM), LRP presents as a tight, granular structure. Notably, in the present study, more than 90% of the particles measured between 3 and 4 nm in height, with the tip of the particle pointing upward ( Figure 2D). The average height of the particle tip was 3.51 nm.

| Secondary structure of LRP
The secondary structure of the LRP is shown in Figure 3. A strong negative band near 198 nm and a weak positive band near 220 nm were observed by far-ultraviolet CD spectra; these bands are considered by some researchers to be characteristics of an irregular secondary structure (Liu et al., 2011). Further statistical analysis confirmed that 53.9% of LRP showed random coil and 34.6% showed β-turn in aqueous solution.

| Effect of LRP on viability of 6-OHDA-induced PC12 cells
Lignosus rhinocerotis peptide (0-160 μM) had no significant effect on PC12 cell viability (data not shown). However, 6-OHDA had a significant inhibitory effect on the viability of PC12 cells in a concentration-and time-dependent treatment manner (Figure 4a Hence, 40 μM LRP was used in subsequent experiments.

| Effects of LRP and 6-OHDA on apoptosis of PC12 cells
After 6-OHDA treatment, the ROS content in PC12 cells increased significantly (Figure 5a), and the resulting oxidative stress induced cell apoptosis (Figure 6a). However, the apoptotic rate of cells that were incubated with LRP before 6-OHDA treatment was significantly reduced (Figure 6b-d).

F I G U R E 1 Isolation and purification of Lignosus rhinocerotis peptide (LRP). (a) Gel chromatography; (b)
Reverse-phase highperformance liquid chromatography (RP-HPLC); (c) Amino acid sequence of LRP. The crude protein extract of L. rhinocerotis was purified by using ammonium sulfate precipitation. Purification of the crude protein extract was accomplished through the use of Sephadex 30 chromatography, which yielded four characteristic absorption peaks (A 1-4 ). The A 4 component was further separated using RP-HPLC, resulting in the separation of three fine components (B 1-3 ). To evaluate the amino acid composition of LRP, the B 2 component was subjected to a further round of analysis using liquid chromatography-tandem mass spectrometry (LC-MS-MS).
Furthermore, 6-OHDA treatment altered the ratio of B-cell lymphoma 2:Bcl-2 associated protein (Bcl-2:Bax) in the cell, which may act as a molecular switch that dictates whether apoptosis is initiated.

| Nuclear NFκ B in 6-OHDA-treated cells
Since ROS overproduction was induced by 6-OHDA exposure, the content of NF-κB was significantly increased-a change that occurred within 1 h after 6-OHDA treatment. Furthermore, the amount of NF-κB remained high for 12 h, although LRP pretreatment slowed NF-κB activation and reduced overall content. Interestingly in turn promoted the activation of NF-κB. However, LRP treatment slowed the phosphorylation of I-κBα, resulting in lower levels of cytoplasmic p-I-κBα. In the nuclear protein of normal PC12 cells, the content of I-κBα was low, although it increased significantly beginning 4 h after LRP treatment and continuing until 12 h after treatment ( Figure 8).

| DISCUSS ION
Mushrooms are high in protein and provide numerous medicinal benefits, making them a good source of natural active peptides for research and development. A polypeptide derived from Pleurotus eryngii mycelium (PEMP) has anticancer, antioxidant, and immunostimulatory activities (Sun et al., 2017). Cordymin, a peptide derived from the medicinal fungus, Cordyceps sinensis, exhibits neuroprotective effects on the rat ischemic brain, inhibits inflammation, and increases antioxidant activity related to pathological changes (Wang et al., 2012). Because of their simple structure, reduced immunogenicity, and ease of artificial synthesis, mushroom-based peptides are a significant component of contemporary medication development. Ion-exchange chromatography and gel filtration chromatography are commonly used to separate physiologically active peptides from protein hydrolysates. Ion-exchange chromatography F I G U R E 3 Circular dichroism (CD) spectra of Lignosus rhinocerotis peptide (LRP). In the vicinity of 198 and 210 nm, there were a strong negative band and a weak positive band, respectively, which indicates the presence of an irregular secondary structure for LRP.

F I G U R E 4
Lignosus rhinocerotis peptide (LRP) increased the cell viability of PC12 cells exposed to 6-hydroxydopamine (6-OHDA). (a) Cell viability determination was performed of PC12 cells cocultured with different concentrations of 6-OHDA for 24 h. (b) Cell viability of PC12 cells cocultured with 6-OHDA at a concentration of 100 μM for a different time. (c) In the presence or absence of 100 μM 6-OHDA pretreatment for 24 h, the PC12 cells were treated with the specified dose of LRP to determine cell viability. In a and c, different lowercase letters represent significant differences at the 5% level. In panel B, *p < .05, **p < .01 compared with PC12 cells not exposed to 6-OHDA.
distinguishes substances based on the charge of ions and polar molecules, whereas gel filtration chromatography distinguishes substances based on the sizes of individual molecules. However, no standard method to extract peptides from mushrooms currently exists. Indeed, peptides extracted from mushrooms are typically separated using different techniques, which makes it challenging to promote and use mushroom peptides in a consistent way.
In contrast to the mushroom polysaccharide (which can be extracted using a standard approach), the extraction of mushroom peptides requires further investigation. Polysaccharides are one of the most representative fungal active substances from L. rhinocerotis, which have been proven by many researchers to have antioxidant, antitumor, anti-inflammatory, and immune-enhancing activities (Keong et al., 2016;Lee et al., 2014;Seow et al., 2017). However, in this study, we have isolated a novel peptide, LRP, and it was shown to contain no polysaccharides as determined by the phenol-sulfuric acid method. Hence, we confirmed that the peptide contributed to the anti-apoptotic effect in PC12 cells in this study.
The degeneration and cell death of dopaminergic neurons in the substantia nigra and striatum lead to dopamine deficiency, collectively manifested PD. Thus, dopamine replacement therapy (such as L-3,4-Dihydroxyphenylalanine, L-DOPA) is currently one of the best treatment methods for idiopathic PD and effectively masks or reduces disease symptoms. However, long-term L-DOPA treatment may be associated with adverse motor effects, particularly after the initial response phase. In addition, some researchers have detected 6-OHDA in the urine of PD patients who have been taking L-DOPA for extended periods (Kwon et al., 2010). Here, we found that the presence of 6-OHDA induced cells to produce ROS and cause oxidative damage, although long-term L-DOPA treatment produces more 6-OHDA, which may negatively regulate PD treatment. Therefore, functional ingredients from natural products may aid in treating PD by removing active oxygen and relieving oxidative stress. According to this, peptides from Malaysian traditional medicinal fungi (L. rhinocerotis) were identified and purified for the preventive and adjuvant therapy of Parkinson's disease.
Paradoxically, eukaryotic aerobic creatures cannot survive without oxygen, but oxygen is fundamentally harmful to their survival (Davies, 1995). At the same time, the oxidation reaction provides energy, and superoxide anion radicals and hydroxyl radicals generated during the reaction cause oxidative damage. Endogenous antioxidant enzymes (among others) balance the production and consumption of ROS (Xiong et al., 2016). However, in elderly individuals, increased ROS production outpaces the antioxidative system's capacity to safeguard. Endogenous antioxidant enzymes (including SOD, CAT, and GSH-Px) act as the body's natural resistance to the F I G U R E 5 The effect of Lignosus rhinocerotis peptide (LRP) on 6-hydroxydopamine (6-OHDA)induced oxidative stress and endogenous antioxidant enzymes was investigated in PC12 cells. (a). The oxidant-sensitive fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to monitor the generation of reactive oxygen species (ROS). (b) The effect of LRP on lipid peroxidation. (c-e) The effects of LRP on the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), respectively. The percentage was used to express the change compared with the control group and the bar represents the mean ± SD (n = 3), ## p < .01 compared with the control group. *p < .05, **p < .01 compared with the 6-OHDA group. oxidative stress defense system (Liu et al., 2020). The effect of LRP on the activities of Caspase-3 and Caspase-9 in PC12 cells that had been exposed to 6-OHDA. Various doses of LRP were used to pretreat cells for 6 h before they were exposed to 100 M 6-OHDA for 24 h. The bar represents the mean ± SD (n = 3) and ## p < .01 compared with the control group, *p < .05, **p < .01 compared with the 6-OHDA group.
apoptosis-inhibiting genes and apoptosis-promoting genes in the Bcl-2 protein family. Notably, the Bax protein (produced by the Bax gene) forms a heterodimer with the Bcl-2 protein and inhibits Bcl-2.
Researchers have reported that apoptosis is influenced by the ratio of Bax to Bcl-2 proteins, and the degree of the inhibitory effect on apoptosis is a significant element in determining the strength of the effect itself (Zhang et al., 2019). Mechanistically, Bax induces apoptosis by entering mitochondria under the action of tBid (truncated Bid [BH3 interacting-domain death agonist]), and increasing the permeability of the mitochondrial membrane prior to releasing cytochrome C. The release of cytochrome C is a critical step in the endogenous pathway of apoptosis, which occurs by targeting and activating Caspase-9 (further leading to the activation of effector Caspases such as Caspase-3; Shen et al., 2016). To evaluate the protective effect of LRP on 6-OHDA-induced apoptosis, we measured the expression levels of Bcl-2 and Bax and the subsequent activity of Caspases-3 and -9. Surprisingly, pretreatment of 6-OHDA with LRP considerably enhanced Bcl-2 levels and decreased Bax levels.
Furthermore, Caspase-3 and Caspase-9 activities were both suppressed, suggesting that LRP may have an anti-apoptotic effect by altering the proportion of Bcl-2:Bax and Caspase-3/-9.
Continuous oxidative stress may cause NF-κB to become active, and some researchers believe that the activation of NF-κB is involved in the dopamine-induced apoptosis of PC12 cells, which contributes to substantia nigra neurodegeneration in patients with PD . Here, we found that 6-OHDA treatment quickly activated NF-κB in PC12 cells. Specifically, NF-κB in the cytoplasm significantly decreased, while NF-κB in the nucleus increased rapidly in a short time. We also verified the mechanism of 6-OHDA in activating NF-κB through the classic activation pathway. In most cells, the NF-κB/Rel transcription complex exists in the cytoplasm in an unactivated form bound to the inhibitor I-κB. When the cell is stimulated (i.e., by ROS, as was the case in the current study), a series of phosphorylation and ubiquitination reactions lead to the degradation of I-κB, and the NF-κB transcription factor enters the nucleus (Zhang et al., 2020). In PC12 cells, 6-OHDA treatment F I G U R E 8 The mechanism of neuroprotective effects of Lignosus rhinocerotis peptide (LRP) on 6-hydroxydopamine (6-OHDA)-induced PC12 cells. (a) The expression of nuclear factor-kappa B (NF-κB), I-κBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), and phosphorylated I-κBα (p-I-κBα) protein was identified via Western blotting. (b) The intensities of relative protein levels were quantified from (a). The ratio represents the expression level as compared to the respective internal control protein (%). PC12 cells were pretreated with LRP with a concentration of 40 μM for 6 h, following with 6-OHDA at 100 μM for different durations. The protein expression of NF-κB and I-κBα in the nucleus, as well as I-κBα and p-I-κBα in cytoplasm was semiquantitatively analyzed by Western blotting. *p < .05, **p < .01 compared with the 6-OHDA group.
produces ROS, which further promotes the phosphorylation of I-κB.
After I-κB dissociates, NF-κB dimers enter the nucleus and bind to specific DNA sequences to regulate the expression of specific proteins that may affect cell apoptosis. Interestingly, we also observed that I-κB phosphorylated and degraded in the cytoplasm rapidly re-synthesized and entered the nucleus. We further confirmed that I-κB binds to NF-κB to form a complex of NF-κB-I-κB, which causes NF-κB to dissociate from its binding DNA κB site and retranslocate to the cytoplasm, completing the cycle of activation and inactivation of NF-κB. In summary, pretreatment of LRP effectively inhibits phosphorylation of I-κB, slows NF-κB nuclear membrane translocation, and inhibits NF-κB activation.

| CON CLUS ION
Lignosus rhinocerotis peptide has a strong inhibitory effect on 6-OHDA-induced apoptosis in PC12 cells. Indeed, LRP partially restores endogenous antioxidant enzyme activities, including SOD, CAT, and GSH-Pxa. LRP also acts as an endogenous antioxidant enhancer by decreasing ROS levels and MDA generation. Besides, it also adjusts the ratio of Bax:Bcl-2, inhibits the expression of Caspases-9 and -3, and blocks mitochondrial pathway apoptosis.
Moreover, LRP inhibits the activation of the NF-κB pathway and attenuates oxidative stress injury induced by 6-OHDA in PC12 cells.
These findings show LRP's promise and lay the groundwork for more research into how it can be utilized to treat neurologic illnesses.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original image of the target protein was uploaded as supporting information and all data can be obtained by contacting Dr. Pu