Actaea racemosa L. Rhizome Protect against MPTP-Induced Neurotoxicity in Mice by Modulating Oxidative Stress and Neuroinflammation

Parkinson’s disease (PD) is a dopaminergic neuron-related neurodegenerative illness. Treatments exist that alleviate symptoms but have a variety of negative effects. Recent research has revealed that oxidative stress, along with neuroinflammation, is a major factor in the course of this disease. Therefore, the aim of our study was to observe for the first time the effects of a natural compound such as Actaea racemosa L. rhizome in an in vivo model of PD induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). For the study, mice received four injections of MPTP (20 mg/kg) for the induction of PD. Starting 24 h after the first administration of MPTP we treated mice with Actaea racemosa L. rhizome (100 mg/kg) daily for seven days. Our findings clearly demonstrated that Actaea racemosa L. rhizome treatment decreases oxidative stress by activating redox balance enzymes such as Nrf2/HO-1. We also demonstrated that Actaea racemosa L. rhizome is capable of modulating inflammatory indicators involved in PD, such as IκB-α, NF-κB, GFAP and Iba1, thus reducing the degeneration of dopaminergic neurons and motor and non-motor alterations. To summarize, Actaea racemosa L. rhizome, which is subject to fewer regulations than traditional medications, could be used as a dietary supplement to improve patients’ brain health and could be a promising nutraceutical choice to slow the course and symptoms of PD.


Introduction
Parkinson's disease (PD), which primarily affects the substantia nigra (SN) of the brain, is one of the most common neurodegenerative diseases [1]. This area is important for producing dopamine which controls motor skills. PD occurs in patients over the age of 50 with symptoms such as tremor at rest, slowed mobility and loss of balance [2]. The etiology of PD remains largely unknown. Brain injury and exposure to various environmental factors such as pesticides and neurotoxins are potential causes [3,4]. Increased oxidative stress and inflammatory processes have been linked to PD pathology in general [5]. Current treatments for PD, including the best-known, levodopa, carbinoxamine, cicrimin and carbidopa, are capable of slowing the progression of the disease and managing symptoms but have numerous side effects such as stress, apprehension and lethargy, which have a negative impact on the patient health [6,7]. Studies are therefore needed to evaluate new Furthermore, in both in vitro and in vivo study it was demonstrated that a compound containing 10 herbs including Cimicifuga heracleifolia, called Yeoldahanso-tang, can be used when modified for both prevention and treatment of neurodegenerative diseases with PD as it is in capable of enhancing autophagy and having a neuroprotective effect [49].
Therefore, based on the characteristics of Actaea racemosa L. reported in the literature and of the studies that have highlighted the neuroprotective effect of natural compounds, and in particular of Cimicifuga, we hypothesized that the rhizome of this extract compound could effectively suppress the oxidative stress and neuroinflammation typical of PD.

Animals
C57/BL6 mice (male 25-30 g; Envigo, Milan, Italy) were acclimated in a controlled setting using conventional rodent food and water. Mice were housed in steel cages in a room set at 22 ± 1 • C with 12 h day and night cycles. The study was approved by the University of Messina's Animal Care Review Board (OPBA). All in vivo tests were conducted in accordance with the new instructions of the United States, Europe, Italy, and the ARRIVE guidelines (approval code: n • 89/2021-PR).

MPTP-Induced PD and Treatment
MPTP or saline were given to mice as previously described [50]. For treatment we used Actaea racemosa L. rhizome extract (ARL rhizome) at the dose of 100 mg/kg (origin of raw material USA; the rhizome is harvested and dried; company Fontana, Canosa di Puglia, Italy, Batch Number: 82114885). Mice were given this compound orally, beginning 24 h after the 1st MPTP injection and continuing for 7 days after the last MPTP administration. For MPTP intoxication, mice received four intraperitoneal (i.p.) injections of MPTP (20 mg/kg; Sigma, St. Louis, MO, USA) in saline at 2 h intervals over the course of one day: total dose per mouse was 80 mg/kg. Only the vehicle was given to the control animals. Mice were euthanized 8 days following MPTP injection, and brains were taken and processed for biochemical and molecular examination. The MPTP (20 mg/kg) and ARL rhizome (100 mg/kg) doses used were based on research in the literature and on preliminary studies conducted in our laboratories [51,52].

Experimental Groups
The animals were randomly distributed into the following groups: Group 1. Sham + Veh: vehicle solution (saline) was injected i.p. during the 1st day, as in MPTP protocol. Group 2. Sham + ARL rhizome: identical to Group 1, but ARL rhizome extract (100 mg/kg body weight, orally) was given starting 24 h after the first saline solution injection and continuing for 7 days after the last administration of vehicle solution (data are not shown in all results because of no significant differences with Group 1). Group 3. MPTP + Veh: MPTP was administered as described for Group 1. Group 4. MPTP+ ARL rhizome: same as Group 3, but Actaea racemosa L. rhizome extract (at a dose of 100 mg/kg, orally) was administered, starting 24 h after the 1st MPTP administration and continuing through 7 additional days after the last dose of MPTP.

Immunofluorescence Co-Localization for TH/α-Syn and DNA Fragmentation (TUNEL) Assay for Dopaminergic Neurons
The immunofluorescence analysis was carried out according to the methodology published previously [61]. Anti-TH antibody (1:100; Invitrogen, 185, Waltham, MA, USA) and anti-α-syn (1:100; Invitrogen, PA5-85791) were incubated in a humidified chamber at 37 • C O/N on the brain sections. After washing with PBS, sections were incubated for 1 h at 37 • C with secondary antibodies, TEXAS RED-conjugated anti-rabbit Alexa Fluor-594 (1:1000 in PBS, v/v Molecular Probes, Eugene, OR, USA) and FITC-conjugated anti-mouse Alexa Fluor-488 (1:2000 v/v Molecular Probes, Eugene, OR, USA). Nuclei were stained by adding 2 µg/mL 40,60-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS. To identify dopaminergic neurons subjected to DNA fragmentation, some sections were sequentially doubled by immunostaining with anti-TH followed by TUNEL. Sections were equilibrated in labeling buffer for 10 min at room temperature immediately after the last immunostaining rinse, and then incubated in the TUNEL staining solution according to a Roche technique [62,63]. Sections were washed with PBS and were incubated with secondary antibody FITC-conjugated anti-mouse Alexa Fluor-488 antibody (1:2000 v/v Molecular Probes, Eugene, OR, USA) for 1 h at 37 • C. The sections were analyzed using a fluorescence microscope (Leica DM2000, Wetzlar, Germany). Each photograph was scanned with an 8-bit resolution in a 2560 × 1920 pixel array. The collected photos were then cut out and processed for figure assembly using Adobe Photoshop 7.0. (Adobe Systems, Palo Alto, CA, USA) [64].

Histological Analysis
Eight days after MPTP injection, brain tissue was collected. As previously described, the sections were stained with Hematoxylin/Eosin (H&E) and studied using light microscopy interfaced to an imaging system [65,66]. Blinded observation was used for histological assessment, and slides were graded for the severity of pathological outlines following H&E staining using a semiquantitative 5-point scale: 0 = no pathology; 1 = mild pathology; 2 = moderate pathology; 3 = severe pathology; 4 = extremely severe pathology.

Behavioral Tests
Behavioral studies on mice were conducted one day before MPTP injection and eight days following PD induction. All behavioral studies were conducted in a blinded manner.

Analysis for Motor Symptoms -
Open Field (OF) Using a previously published technique [67,68], locomotor activity was observed in an OF for 5 min. The animals' actions were recorded. The number of line crossings and the amount of time spent in the center were calculated and scored. - Rotarod Test (RT) Motor incoordination was assessed with a rotary rod apparatus using a protocol previously described [50,69]. Each mouse's total time on the RT and total number of falls were recorded.

-Catalepsy Test (CT)
Catalepsy, defined as a decreased ability to initiate movement and a failure to maintain proper posture, was measured as previously described [70,71]. The time the mice remained in this position was timed using a timer up to 180 s. The duration the mice stayed in this position was timed up to 180 s. Cataleptic mice were those who remained in this position for 30 s or longer. - Pole test (PT) The PT was performed as previously described [53,72]. Two criteria were evaluated: time until the mouse turned by 180 • , and time until the mouse descended to the floor.

Analysis for Non-Motor Symptoms -
Elevated plus maze (EPM) As previously mentioned, the EPM test was used to assess anxiety-like behavior [73,74]. The marble burying test is a defensive behavioral test for mice used to determine anxiety levels. The analysis was carried out exactly as detailed in a prior paper [75]. For 30 min, the mice were placed in a cage with a 5 cm thick coating of pine and 20 little glass marbles (15 mm in diameter) set in 4 evenly spaced rows of 5 marbles each. A significant number of marbles buried suggests increased concern. As a result, an animal is considered worried if at least 2/3rd of the marble is covered in litter.

-Tail suspension test (TST)
We used the TST for additional neuropsychological tests, such as depression. This behavioral test was carried out exactly as previously reported [76]. Except for breathing movements, all body motions were recorded for 6 min.

Statistical Analysis
All values in the figures and text are expressed as mean ± standard deviation (SD) of N observations. For the in vivo studies, N represents the number of animals studied. In experiments involving histology, immunohistochemistry, and immunofluorescence the figures shown are representative of at least three experiments performed on different days on tissue sections collected from all animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant [77].

Results
3.1. Effects of Actaea racemosa L. Rhizome on Nrf2/HO-1/NQO1 Pathway In general, oxidative stress in the brain is associated with increased expression of genes involved in free radical detoxification as well as genes responding to cell survival stress. In this regard, we wanted to see how ARL rhizome treatment affected Nrf2 activation and, as a result, the expression of HO-1 and NQO1. When MPTP-treated mice were compared to Sham mice, Western blot analysis demonstrated a slight increase in Nrf2. This demonstrates that the animal's body attempts to activate an antioxidant response following treatment with MPTP even if this is not significant. Treatment with ARL rhizome was able to enhance the antioxidant response by significantly increasing Nrf2 levels ( Figure 1A and densitometric analysis Figure 1A'). Furthermore, the same results were observed for HO-1 and NQO1. Thus, a nonsignificant increase was found in the MPTP group, while in mice treated with ARL rhizome the levels of these proteins were significantly increased ( Figure 1B, and densitometric analysis Figure 1B').

Effect of Actaea racemosa L. Rhizome Treatment on Neuroinflammation
We used Western blot analysis on midbrain tissues to assess the anti-inflammatory effect of ARL rhizome treatment on the development of PD. When MPTP-injured animals were compared to Sham mice, Ikb-a degradation was triggered, whereas ARL rhizome treatment elevated Ikb-a cytosolic expression (Figure 2A, and densitometric analysis Figure 2A'). As a result of increased Ikb-a degradation, we saw a considerable increase in nuclear translocation of NF-kB in the MPTP-treated group, but ARL rhizome treatment reduced its expression in the nucleus ( Figure 2B, and densitometric analysis Figure 2B'). In addition, immunohistochemistry labeling for GFAP and Iba-1 expression was used to examine astrocyte and microglial cell activity in connection to PD. The levels of GFAP and Iba-1 were very low in the Sham group but significantly higher in the MPTP-treated mice. ARL rhizome treatment effectively reduced the elevated expression of GFAP and Iba-1 under these conditions ( Figure 2C-F for GFAP and Figure 2G-J for Iba-1).

Effect of Actaea racemosa L. Rhizome Treatment on Apoptosis
To assess the effect of ARL rhizome treatment on MPTP-induced apoptosis, we used Western blot analysis to look at the expression of Bax and Bcl-2. Tissues from MPTP-treated animals had higher levels of Bax expression than the Sham group. This expression was diminished by ARL rhizome treatment ( Figure 3A and densitometric analysis Figure 3A'). Tissues from Sham mice, on the other hand, displayed basal levels of Bcl-2; MPTP injection reduced this expression, while ARL rhizome treatment restored Bcl-2 expression to baseline ( Figure 3B, and densitometric analysis Figure 3B').
We used double TH/TUNEL labeling to see if dopaminergic neurons were responsible for cell death. We detected considerable colocalization in the MPTP group compared to the Sham group by labeling dopaminergic neurons with the TH antibody, and then performed the TUNEL assay, whereas the ARL rhizome treatment considerably decreased the death of these neurons ( Figure 4A,B).

Actaea racemosa L. Rhizome Treatment Reduced Loss of TH and DAT Expression and a-Syn Aggregation
We looked at the expression of TH and DAT to see how ARL rhizome administration affected the dopamine pathway. A considerable loss of TH-positive cells was visible in the midbrain 8 days after MPTP intoxication ( Figure 5B, densitometry analysis Figure 5D) compared to the Sham group ( Figure 5A, densitometry analysis Figure 5D), while ARL rhizome treatment appreciably reduced the loss of TH-positive neurons in the midbrain ( Figure 5C, densitometry analysis Figure 5D). We also noticed a similar pattern in DAT expression. In summary, a major decrease of DAT was detected in MPTP-injected mice ( Figure 5F, densitometry analysis Figure 5H) compared to the Sham group ( Figure 5E, densitometry analysis Figure 5H), but the ARL rhizome treatment significantly restored DAT levels ( Figure 5G, densitometry analysis Figure 5H). To determine the ability of the ARL rhizome to counteract the neurodegenerative process we wanted to evaluate the expression of α-syn, as the accumulation of this protein is a characteristic of PD and represents the main constituent of intraneuronal protein aggregates known as Lewy bodies.
In comparison to the Sham group, our immunohistochemical study revealed significant immunoreactivity in MPTP-damaged mice ( Figure 6B, densitometry analysis Figure 6D) ( Figure 6A, densitometry analysis Figure 6D). ARL rhizome treatment, on the other hand, drastically reduced α-syn expression ( Figure 6C, densitometry analysis Figure 6D).
Furthermore, to show that a-syn accumulation occurred in dopaminergic neurons, we used immunofluorescence analysis to doubly stain TH (green) and α-syn (red). We found no α-syn in TH-positive dopaminergic neurons in the Sham group ( Figure 6E), but there was an increase in α-syn accumulation in TH-positive neurons following MPTP intoxication ( Figure 6F). In dopaminergic neurons, ARL rhizome treatment inhibited α-syn aggregation ( Figure 6G).

Effect of Actaea racemosa L. Rhizome Treatment on Histological Changes and Behavioral Impairments Induced by MPTP Administration
H&E staining revealed that MPTP administration caused histological alterations in the midbrain. Sham mice had normal brain architecture and a normal number of neurons ( Figure 7A,D). Instead, MPTP-treated mice displayed significant changes in normal brain architecture as well as a visible loss of neuronal cells ( Figure 7B,D). In contrast, ARL rhizome-treated animals had a significant drop in the number of cells with pycnotic nuclei in the region of interest ( Figure 7C,D).
The data at time 0 are not shown since no significant changes between the groups were found. Motor coordination was assessed using the RT. MPTP-treated mice demonstrated severe motor incoordination after 8 days of PD induction, as evidenced by a decrease in time spent on the Rotarod and an increase in the number of falls. In contrast, the motor impairments in the ARL rhizome-treated mice were significantly reduced ( Figure 7E). MPTP also had a significant cataleptic effect in mice. In fact, 8 days after receiving MPTP, the mice displayed a considerable rise in cataleptic symptoms. Daily ARL rhizome administration, on the other hand, dramatically shortened the duration of MPTP-induced catalepsy ( Figure 7F). We used the PT to assess MPTP-induced bradykinesia. When compared to the Sham group, "turn time" and "total time" increased dramatically following MPTP injection. Treatment with ARL rhizome considerably reduced "total time" and "turnaround time", implying a considerable reduction in bradykinesia ( Figure 7G,G1).
Furthermore, at the 8th day after MPTP intoxication, mice had non-motor PD signs. In the EPM, mice were observed for anxiety-like behavior. The behavioral test that we conducted revealed a significant increase in the duration spent in the open arms and the number of entries in the open arms following ARL rhizome treatment as compared to the MPTP group ( Figure 7H,H1). This increase in anxiety observed in MPTP-treated mice was also confirmed by the MBT. In this test, MPTP-treated mice tended to leave significantly more glass beads unburied than mice from the Sham group. ARL rhizome treatment reduced the animals' anxiety state, demonstrated by the number of buried marbles found, which was similar to the control group ( Figure 7I). The depression that often accompanies anxiety is another important non-motor feature of PD. TST was used to measure depression-like behavior on day 8 after MPTP intoxication. When compared to Sham mice, these mice were much more immobile during the 6 min interval. Treatment with ARL rhizome reduced immobility time to values comparable to the Sham group ( Figure 7J).

Discussion
Numerous investigations have found a clear role for oxidative stress and neuroinflammation in the etiology of PD [78]. In this regard, several researchers are trying to develop anti-inflammatory and antioxidant drugs capable of counteracting these processes that play a crucial role in PD. In recent years, it has been shown that natural compounds have beneficial effects on health, as many of these products can act on aging and neurodegeneration [67,[79][80][81]. Given the analgesic, antioxidant, anti-inflammatory, antipyretic and antiviral properties of Actaea racemosa L. [82], in the present study we wanted to evaluate its beneficial effects for the first time in an experimental model of MPTP-induced PD.
Our findings suggest that ARL rhizome can inhibit not just oxidative stress but also the inflammatory response and apoptosis, hence preventing a-syn buildup, dopaminergic neuron loss, and behavioral impairments.
Nrf2, a transcription factor that activates genes with cytoprotective functions, is involved in antioxidant and anti-inflammatory reactions [83,84]. Several phase II detoxification enzymes, including NQO1, HO-1, and many others, are encoded by these cytoprotective genes [85,86]. In an in vivo study, Actaea racemosa L. rhizome has already been shown to upregulate the Nrf2/HO-1 pathway [52]. Therefore, our theory is that ARL rhizome can perform its neuroprotective action through the activation of the Nrf2/HO-1/NQO1 axis. In comparison to the MPTP group, our data demonstrated that ARL rhizome dramatically increased Nrf2, HO-1, and NQO1 levels. Therefore, ARL rhizome activates the antioxidant response by restoring the redox balance.
Excessive oxidative stress induces activation of NF-kB, indicating a relationship between these two pathways [87,88]. NF-kB is a transcription factor that regulates the inflammatory response by activating several genes that code for pro-inflammatory cytokines and immunoregulatory mediators [89]. Our findings show that ARL rhizome can block NF-kB nuclear translocation and IkB-α degradation. This shows that Nrf2 regulation of redox homeostasis likely contributes to the modulation of NF-kB activity and the inflammatory response seen in PD. This pathology is also characterized by neuroinflammatory changes associated with the activation of astrocytes and microglia [50,51,90]. Several previous studies have demonstrated the phenomenon of astrogliosis and microgliosis in the MPTP-induced PD model. Our ARL rhizome treatment was able to contain the activation of GFAP and Iba-1. Once we observed the effects of ARL rhizome on oxidative stress and neuroinflammation, we moved on to the evaluation of the apoptotic process by investigating two specific markers such as Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptotic protein). Our study found that by limiting the redox imbalance and the activation of inflammatory processes induced by MPTP, the ARL rhizome was able to reduce neuronal death and in particular the death of dopaminergic neurons, as demonstrated by the double staining TUNEL/TH. Degeneration is usually accompanied by a-syn buildup and a decrease in dopamine due to the death of dopaminergic neurons [91]. Given the protective role of ARL rhizome on the latter, we evaluated the ability of the ARL rhizome to reduce the loss of dopamine and the accumulation of a-synuclein by analyzing specific markers such as DAT and α-syn. The results obtained revealed that ARL rhizome is also able to act on these specific parameters of PD and on the motor and cognitive alterations that follow.

Conclusions and Limitations
In conclusion, our results demonstrated that ARL rhizome, probably acting through the Nrf2 pathway, is able to act as a potent antioxidant, reducing MPTP-induced oxidative stress, and is able to work through an indirect mechanism of acting on neuroinflammation by preventing the death of dopaminergic neurons and the aggregation of α-syn.
Obviously, the administration of ARL rhizome does not cure PD, but we can certainly say that the ARL rhizome represents a nutritional product that could limit the progression of the disease and improve the symptoms associated with this pathology.
It is obvious that the present study has limitations related to the characterization of the ARL rhizome. In fact, the goal of future studies will be to identify which components of ARL are responsible for the antioxidant and anti-inflammatory action in PD. We hypothesize that an important role may be played by hydroxycinnamic acid derivatives such as caffeic acid, ferulic and isoferulic acid, but also by triterpenes. Naturally, this hypothesis will have to be verified with specific studies both in vitro and in vivo. Data Availability Statement: Based upon the rules of our laboratory, the datasets used in the current study are available from the corresponding author (dimpellizzeri@unime.it) on reasonable request.