Nrf2 signaling contributes to the neuroprotective effect of Cicadidae Periostracum against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Periostracum has various pharmacological effects, including neuroprotective potential via nuclear receptor related-1 (NURR1) protein signaling. However, there are no studies on its antioxidative effect. The neuroprotective effect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage to dopaminergic neurons was evaluated and explored the mechanisms of its antioxidant action, focusing on nuclear factor E2-related factor 2 (Nrf2) in a mouse model of Parkinson's disease. We measured NURR1-related neurotrophic signaling and the levels of antioxidant factors in vitro and in vivo . The effects of Cicadidae Periostracum against MPTP-induced neurotoxicity were associated with inhibition of the neurotrophic signaling pathways and up-regulation of Nrf2 pathways. Thus, Cicadidae Periostracum mitigates neurotoxicity via neurotrophic signaling not only by increasing Nrf2 activity but also by increasing antioxidant activity. tive effect of Periostracum against


Introduction
Parkinson's disease is a neurodegenerative disease caused by diminished dopamine due to damage to dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain. The first-line treatment strategy of Parkinson's disease is dopamine replacement therapy (Dauer and Przedborski, 2003) using L-DOPA, a dopamine precursor. However, this agent cannot be used for long periods due to adverse effects including loss of appetite and vomiting (Lannielli et al., 2018). Thus, it is necessary to expand treatment strategies.
Drugs derived from natural products can have fewer adverse effects from long-term use than synthetic drugs. Many researchers are focusing their research and development efforts on such products based on traditional evidence of their usage that has been described in medical books.
Nuclear factor E2-related factor 2 (Nrf2) plays an important role in neurodegenerative diseases, including Parkinson's disease (Tonelli et al., 2018;Wang et al., 2019). It binds to antioxidant response elements (AREs) to promote the transcription of phase 2 enzyme-related genes (Wang et al., 2019). These Nrf2 signaling changes are associated with abnormal redox homeostasis (Kerins and Ooi, 2018). The loss of Nrf2-mediated transcription exacerbates the vulnerability of dopaminergic neurons to oxidative damage (Kim et al., 2020). Nrf2 knockout mice show a greater loss of dopaminergic neurons than wild-type mice when exposed to 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) . Thus, Nrf2 could serve as an important signaling molecule in neural protection strategies for Parkinson's disease pathogenesis. al., 2019b). Cicadidae Periostracum has been used to treat various disorders including convulsions, itchy rashes, and eczema . Although the neuro-protective effects have been demonstrated, its effect on Nrf2 regulation has not been examined, and no study has investigated whether it can influence oxidative damage or stress via Nrf2 regulation in MPTP models of Parkinson's disease.
Here, we investigated the inhibitory effects of Cicadidae Periostracum on 1-methyl-4-phenylpyridinium (MPP+)-induced reactive oxygen species (ROS), and the expression and regulation of Nrf2, in vitro. To confirm whether Cicadidae Periostracum directly affected Nrf2, ML385, an Nrf2 transcriptional inhibitor, was used. We verified that Nrf2 pre-inhibition with ML385 neutralized the effects of Cicadidae Periostracum on the neurotrophic factors induced by MPTP in vivo.

Preparation of the extract and treatments of differentiated PC12 cells
Standardized Cicadidae Periostracum extracts were prepared according to previously published methods and were stored at 4°C (3-18-0038 code) in the Korea Institute of Oriental Medicine (Lim et al., 2019;Park et al., 2021). The PC12 cell line was obtained from the Korean Cell Line Bank and maintained in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. To differentiate PC12 cells, the culturing medium was changed every 2-3 days, and cultured cells were treated with nerve growth factor for 7 days. Differentiated PC12 cells were then pretreated with Cicadidae Periostracum (0.1-50 μg/ mL) for 1 hour, followed by MPP+ (100 μg/mL) for 23 hours.

Measurement of ROS and cytotoxicity
The release of lactic acid dehydrogenase (LDH) was used to assess cytotoxicity. LDH was determined using a CytoScan according to the manufacturer's instructions. The experiment was conducted using a culture medium obtained after Cicadidae Periostracum treatment. Briefly, the culture medium (100 μL) was removed, centrifuged, transferred to an analysis tube, then reacted with the dye solution (100 μL) in the dark for 30 min. The absorbance of the solution was measured at 490 nm. For the ROS assay, differentiated PC12 cells were pre-treated with Cicadidae Periostracum (0.1 -50 μg/mL) for 1 hour, stimulated with MPP+ (100 μg/ mL), then incubated with 20 μM 2,7-dichlorofluorescein diacetate at 30 min at 37°C. The fluorescence was measured at 485/530 nm.

Antioxidant protein level assays
Differentiated PC12 cells or SNpc from brain samples were lysed in RIPA lysis and extraction Buffer containing a protease inhibitor cocktail. Protein concentrations in the tissue extracts were determined using the Bradford method. Western blots were prepared similarly to previously published methods (Lim et al., 2019a,b;Lim et al., 2020). Glutathione (GSH) levels were measured by an enzyme-linked immunosorbent assay.

Trans-AM Nrf2 DNA-binding activity and dopamine contents
The efficiency of Nrf2 DNA-binding activity was evaluated using a commercially available Trans-AM Nrf2 kit (50296; Active Motif, USA). Briefly, the nuclear extract was incubated with competitor oligonucleotides for the ARE. Then, the primary Nrf2 antibody was added at 1:1000, washed after 1 hour, and the secondary horseradish peroxidase-conjugated antibody was added at 1:1000 for 1 hour. After reacting by adding the developing solution for x hour, the stop solution was added and the absorbance was measured at 450 nm.
Striatal dopamine contents were assessed using the 2-CAT (A-N) Research ELISA fluorometric assay kit following the manufacturer's protocol. Briefly, each tissue sample was assessed separately according to its volume. When the volume was less than 100 μL, ultrapure water was added to reach a final volume of 100 μL. We then used the TE buffer and the acylation buffer for acylation. Afterward, samples and standards were added to each well of a 96-well microtiter plate and incubated with the enzyme solution. After substrate treatment to form the conjugate and incubation for 20 min, the stop solution was added the absorbance was measured at 450 nm.

Statistical analyses
All statistical parameters were calculated using Prism 5.0 software (Graphpad Software, USA). Values are expressed as means ± standard error of the mean. Statistical comparisons between the different treatments were performed using a one-way analysis of variance with Tukey's multiple comparison post-test. A p-value <0.05 was considered to be statistically significant.

Effects on MPP+-induced neurotoxicity in differentiated PC12 cells
Treatment with Cicadidae Periostracum at a concentration of 100 μg/mL, but not at 1-50 μg/mL, decreased the viability of PC12 and differentiated PC12 cells 24 hours after treatment (data not shown). Thus, further experiments were performed with 1-50 μg/mL Cicadidae Periostracum. To investigate the effects on cytotoxicity, we determined the LDH level in differentiated PC12 cells. Cells exposed to 100 µg/mL MPP+ a showed a significant increase in the LDH level (153 ± 4.5%) compared with control cells. In contrast, cells treated with 0.1-50 μg/mL Cicadidae Periostracum before exposure to MPP+ were protected against injury (125 ± 6.6% -103 ± 2.4%) compared with control cells ( Figure 1A).

Effect on MPP+-induced ROS generation in differentiated PC12 cells
Exposure to 100 µg/mL MPP+ significantly elevated

Principle
Western blotting is an experimental technique used to detect target proteins using antibodies that bind to specific amino acid sequences called epitopes. The assay is based on the principle of immunochromatography in which proteins are separated according to their molecular weights using polyacrylamide gel electrophoresis.

Procedure
Step 1: Sample preparation The protein extract was mixed with 5× SDS-polyacrylamide gel loading buffer and dissolution buffer, then heated at 90°C for 10 min and cooled on ice for 10 min.
Step 2: Electrophoresis Equivalent amounts of protein (recommended: 10-50 μg/ lane) were loaded into each well of the 4-20% SDS-polyacrylamide gel and electrophoresed at 100 V for 1.5 hour.
Step 3: Transfer After electrophoresis, the proteins were transferred from the gel to a polyvinylidene fluoride membrane at 2.5A, 20V for 3 min using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratory).
Step 4: Blocking and primary antibody Membranes were blocked with 3% BSA or 5% skim milk in TBST for 1 hour at room temperature. Then, the primary antibody was diluted in 3% BSA or 3% skim milk to the appropriate dilution factor, and the membranes were incubated in the primary antibody solution at 4°C overnight.
Step 5: Washing The membrane was then gently shaken and washed three times with TBST for 15 min each.
Step 6: Secondary antibody The secondary horseradish peroxidase conjugated antibody was diluted in 3% BSA or 5% skim milk. The membrane was incubated with this mixture for 1 hour at room temperature.
Step 7: Washing (same as step 5) Step 8: Detection Visualization of the immunoreactive proteins was achieved using a chemiluminescence kit, and images were obtained using the ChemiDoc XRS+ system (Bio-Rad Laboratories). In addition, the relative expression level of each protein was calculated using the expression level of the housekeeping protein (β-actin) with the ChemiDoc band analysis system (Bio-Rad Laboratories). The optimal exposure time was identified by using various exposure lengths, which can generally be adjusted by the automation system of the device. the ROS levels in differentiated PC12 cells (166 ± 4.0%) compared with control cells. Cicadidae Periostracum pre-treatment at 1-50 µg/mL inhibited ROS generation (132 ± 6.4% -82.4 ± 3.4%) compared with the control cells ( Figure 1B).

Discussion
The nuclear receptor related-1 (Nurr1) protein regulates genes that are critical to the development, maintenance, and survival of dopaminergic neurons. In addition, Nurr1 plays a fundamental role in maintaining dopamine homeostasis by regulating the transcription of genes involved in dopamine synthesis, reabsorption, and packaging (Jankovic et al., 2005). Nurr1 is also known to regulate the survival of dopamine neurons by stimulating gene coding, anti-inflammatory reactions, and oxidative stress management (Jankovic et al., 2005). We reported previously that Cicadidae Periostracum up-regulates Nurr1 expression and regulates the expression of neurotrophic factors (Lim et al., 2019b). Further -more, effects on the activity of Nurr1 by Cicadidae Periostracum indicate that neural protection signals are associated with mitochondria-mediated apoptosis and neuroinflammation (Lim et al., 2019b). Thus, Nurr1 has a close association with dopamine neurons, and Cicadidae Periostracum could exert its anti-Parkinson's disease effect by regulating its activity (Lim et al., 2019b).
Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals that can be affected by oxidative stress to disturb the balance of Nurr1 in favor of cytoplasmic accumulation. The Nurr1 gene is also upregulated by the increased expression of ROS-removal genes such as SOD1 and Abl2. Nurr1 is an essential mediator of CREB-dependent neuroprotection under oxidative stress. Nurr1 overexpression protects neuronal stem cells from oxidative damage by inhibiting caspase-3 and -11.
Overall, Nurr1 and oxidative stress regulation are closely related. In the current study, we focused on the regulation of the oxidative stress gene, Nrf2, to demonstrate the efficacy of Cicadidae Periostracum. Nrf2, a master regulator of the antioxidant response in neurons, controls antioxidant response signaling, involving SOD, catalase, GSH, HO-1, and NQO1 (Zhao et al., 2009). Research on the dopamine transporter in Nrf2 knockout mice has shown that Nrf2 activation is a key factor in regulating cell-protective gene expression pathways (Burton et al., 2006). c d e f g the Nrf2/ARE/HO-1 and NQO1 pathways to counteract apoptosis, neuroinflammation, and neurotrophic dysfunction caused by neurotoxic agents Burton et al., 2006;Oh et al., 2013  . It inhibits Nrf2 through its ability to block Nrf2 transcriptional activity. In particular, ML385 binds to the basic leucine zipper domain (Neh1) of Nrf2 and inhibits its expression by preventing the protein complex from binding to the regulatory DNA binding sequence (Singh et al., 2016).

Conclusion
The reduction in striatal dopamine loss and neurotrophic factor expression by Cicadidae Periostracum is dependent on Nrf2 and related antioxidant activities.

Financial Support
This work was supported by a grant on the Development of Sustainable Application for Standard Herbal Resources (KSN2012320, KSN2013320, and KSN2021320) from the Korea Institute of Oriental Medicine, Republic of Korea.

Ethical Issue
These studies were reviewed and approved by the institutional animal care ethical committee of the Korea Institute of Oriental Medicine, Republic of Korea (approval numbers KIOM-20-003 and -078) and performed according to the guidelines of the Care and Use of Laboratory Animals in Ethics Committee of the Korea Institute of Oriental Medicine. Male C57BL/6 mice (8 weeks of age, 23-24 g) were purchased from Doo Yeol Biotech (Seoul, Korea) and maintained under temperatureand light-controlled conditions (20-23°C, 12-hours light/12hours dark cycle) with food and water provided ad libitum. All mice were acclimatized for 7 days before drug administration.