Combination Treatment of Icariin and L-DOPA Against 6-OHDA-Lesioned Dopamine Neurotoxicity

Until now, the dopamine (DA) precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), remains the gold standard effective drug therapy for Parkinson’s disease (PD) patients. Nevertheless, long-term chronic L-DOPA administration leads to the drug efficacy loss and severe adverse effects, such as L-DOPA-induced dyskinesia (LID). Icariin (ICA), a flavonoid that is extracted from Epimedium, has been proved to evoke neuroprotection against DA neuronal loss in PD animal models. Here, the present study detected the effects of ICA combined with L-DOPA on 6-hydroxydopamine (6-OHDA)-elicited DA neurotoxicity and L-DOPA-induced motor dysfunction as well. PC12 cells were applied to investigate the combination treatment of ICA and L-DOPA against 6-OHDA-lesioned neurotoxicity. In addition, rat substantia nigral stereotaxic injection of 6-OHDA-induced DA neuronal injury was performed to explore the neuroprotective effects mediated by ICA combined with L-DOPA. The pathological movement triggered by L-DOPA was determined by the abnormal involuntary movements (AIM) scores analysis. In PC12 cells, ICA combined with L-DOPA produced better neuroprotection from 6-OHDA-induced neurotoxicity than ICA or L-DOPA alone treatment. In parkinsonian 6-OHDA lesioned rats, ICA conferred DA neuroprotection as monotherapy and an enhancement benefit of L-DOPA treatment after daily administration of L-DOPA and ICA for 21 days. Moreover, ICA ameliorated the development of LID as evidenced by the lowered AIM scores without affecting L-DOPA-mediated efficacy. Furtherly, ICA attenuated neuroinflammation in 6-OHDA-induced DA neuronal loss and the development of LID in vivo. In conclusion, these findings suggest ICA might be a potential promising adjuvant to enhance L-DOPA efficacy and attenuate L-DOPA-produced adverse effects in PD.


INTRODUCTION
Parkinson's disease (PD) is among the most common neurodegenerative movement disorders characterized by the slow and progressive dopamine (DA) neuronal loss in the substantia nigra (SN) and the subsequent depletion of DA content in the striatum. This is accompanied by the behavioral and motor dysfunctions, such as resting tremor, postural instability and muscular rigidity (Fahn, 2003).
Currently, the DA precursor, L-3,4-dihydroxyphenylalanine (L-DOPA) is the most effective drug therapy for PD (Poewe et al., 2010). Nevertheless, long-term L-DOPA treatment leads to a variety of severe adverse effects, and consequently causes the development of motor fluctuation, such as L-DOPAinduced dyskinesia (LID), which limits the treatment efficacy of PD and weakens the life quality of PD patients (Jankovic, 2005). Furthermore, L-DOPA had little effects on several non-motor PD symptoms, including cognitive impairments, sleep disturbances, dysautonomia and apathy (Encarnacion and Hauser, 2008). A battery range of non-dopaminergic therapy has been implied for PD due to its symptoms diversity but has conferred the limited clinical benefits. Thus, strategies of potential synergistic actions in combination with L-DOPA might provide a novel adjuvant therapy for PD.
Icariin (ICA), a flavonoid that is extracted from Epimedium, is well studied to possess a large number of pharmacological properties, including anti-aging, free radical-scavenging and anti-inflammation (Li et al., 2016). Recent studies demonstrate ICA evoked neuroprotection against brain ischemic injury and neurodegenerative diseases (Xiao et al., 2016). In our previous work, ICA attenuated DA neuronal damage and microglia-induced neuroinflammation both in vivo and in vitro (Wang et al., 2018). Besides, ICA showed synergistic effects combined with methylprednisolone to attenuate experimental autoimmune encephalomyelitis (EAE) through enhancing anti-apoptotic actions (Wei et al., 2015). Therefore, ICA-mediated neuroprotection attracts an increasing attention.
To widen the potential neuroprotective window of ICA, this study detected the effects of ICA combined with L-DOPA on 6-hydroxydopamine (6-OHDA)-elicited DA neurotoxicity and L-DOPA-induced motor dysfunction as well. PC12 cells were applied to investigate the combination treatment of ICA and L-DOPA against 6-OHDA-lesioned neurotoxicity. In addition, rat SN stereotaxic injection of 6-OHDA-induced DA neuronal injury was performed to explore the neuroprotective effects mediated by ICA in combination with L-DOPA. The pathological movements triggered by L-DOPA were determined by the abnormal involuntary movements (AIM) scores detection. Particularly, these findings might provide a more pronounced therapeutic strategy on extrapyramidal disorders compared to monotherapy.

Cells Culture and Treatment
PC12 cells were bought from the Cell Culture Center in the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in RPMI-1640 medium containing 5% horse serum, 5% fetal bovine serum and streptomycin/penicillin on an atmosphere with 5% CO 2 at 37 • C (Klymov et al., 2015). PC12 cells were treated with different concentrations of ICA and L-DOPA for 24 h before 6-OHDA administration for another 24 h.

MTT Assay
PC12 cells were cultured in 1 × 10 5 /well in 96-well plates and incubated in an environment with 5% CO 2 at 37 • C for 24 h. After 6-OHDA, ICA and L-DOPA treatment, the culture supernatant was removed and cells were incubated with MTT (5 g/L) for 4 h at 37 • C. Formazan crystals in the cells were solubilized using dimethyl sulfoxide (DMSO) and the absorbance was detected by an automatic plate reader (Clinibio 128C, Austria) within a 490-nm wavelength.

LDH Assay
PC12 cells were added LDH test working fluid mix. LDH activity was measured by detecting the absorbance at 490 nm after incubation for 30 min in the dark at 25 • C.

Flow Cytometric Assessment of Cell Apoptosis
PC12 cells were washed with cold PBS and resuspended in the binding buffer. FITC-labeled Annexin V (5 µl) and PI (5 µl) were added to PC12 cells followed by the incubation at room temperature in the dark for 15 min. Cell apoptosis was assessed by flow cytometry.

Animals and Treatment
Male Sprague-Dawley rats (200-250 g) were obtained from the Experimental Animal Center of the Third Military Medical University (Chongqing, China; Specific pathogen-free Grade II; Certificate No. SCXK 2012-0005). Rats were housed in the standard conditions of the maintained temperature (25 ± 2 • C), humidity (60 ± 4%) and under 12-h light/12-h dark cycle. The experimental uses of animals were performed in accordance with the National Institute of Health Guideline for the Animal Care and Use of Laboratory Animal and approved by the Animal Care and Use Committee of Zunyi Medical University (Zunyi, China). Animals were anesthetized by 7% chloral hydrate (0.5 ml/100 g, v/w) and mounted on a stereotaxic apparatus (Narishige, USA) coupled with a rat adaptor. Rats received a single 6-OHDA (8 µg in 4 µl saline) unilateral injection into the SN pars compacts on the left side of the brain, followed by the coordinates: 5.2 mm posterior to brahma, 2.2 mm lateral to the midline and 8.0 mm ventral to the surface of the skull. Three weeks later, rats were daily treated with L-DOPA (25 mg/kg, i.p.) and ICA (20 mg/kg, p.o.) for 3 weeks (Mo et al., 2010). ICA was daily given 30 min before L-DOPA treatment. Normal control animals received equivolume injections of saline.

HPLC Coupled With Electrochemical Detection
Animal striatal levels of DA, DOPAC and HVA were measured by HPLC coupled with electrochemical detection. Rat striatal tissue was sonicated in perchloric acid with the internal standards. Then, the homogenate was centrifuged and an aliquot of the supernatant was directly injected onto HPLC equipped with a C 18 column (Dionex, Germering, Germany). The mobile phase consisted of tetrahydrofuran, monochloroacetic acid and acetonitrile with sodium octyl sulfate and EDTA. The DA, DOPAC and HVA levels were measured by the comparison of peak height ratio of tissue sample with standards and indicated in the quality of wet weight of tissue (Zhang et al., 2006).

Rotarod Test
Rotarod test is performed to study the muscular coordination. It contained cylindrical arrangement of thin steel rods. The rods were divided into two parts through compartmentalization to detect two rats at the same time. Upon the training session, the speed was at 10 cycles/min and the cut-off time was 3 min during the 5-min test duration. Before the test started, animals were trained on rods till they stayed on rod at least for the cut-off time. Then, rats were permitted to retain stationary for a while at 0 rpm. The rotation speed was steadily increased to 10 rpm in 20-s interval until animals fell off from rungs. Rat behavior changes were determined and the mean duration time stayed on rod was recorded (Khuwaja et al., 2011).

AIM Scores Assessment
AIM scores were employed to assess the properties of L-DOPA and ICA on rat behavior changes 1, 7, 14 and 21 days after L-DOPA and ICA treatment, respectively. AIM scores are considered to be comparable to LID assessments in patients with PD (Ostock et al., 2015). Rat behavior was detected and scored every 20 min during the 2-h period. Then, the scores of the three AIM subtypes (axial, limb and orolingual) were summed. For each subtype, the dyskinesia severity was scored by a four-point scale (0 = absence, 1 = presence during less than half of the observation time, 2 = presence for more than half of the observation time, 3 = presence all the times but suppressible by external stimulus, 4 = presence all the times and not suppressible by external stimulus). The total AIM scores were calculated by adding each of the axial, limb and orolingual AIM scores (Xie et al., 2014).

Forepaw Adjusting Steps Test
Rodent akinesia presents as deficient stepping ability in the side of the body contralateral to brain lesion via the forepaw adjusting steps (FAS) test (Lindenbach et al., 2011). Since L-DOPA reversed the stepping deficits, FAS could be applied to investigate whether an adjunctive treatment was associated with L-DOPA-mediated antiparkinsonian properties (Eskow et al., 2007). To perform the test, rat rear torso and one forelimb were held, when the free forelimb was forced to bear the body weight. Rats were then laterally moved through a table in a steady rate of 90 cm/10 s and the number of adjusting steps taken by each forelimb to compensate for lateral movement was quantified. Every session was composed of six trials per forelimb alternating among directions, in which forehand steps were defined as weight-bearing steps taken towards the body and backhand steps were defined as steps moved away from the body. The stepping results were shown as % intact stepping (lesioned steps/ intact steps). The lower % intact stepping score indicated the greater forelimb akinesia.

Immunohistochemistry Staining and DA Neuronal Counting in SN
Rat brains were cut into 35-µm transverse free-floating sections via a horizontal sliding microtome. A total of 36 consecutive brain slices through the entire SN was collected and every sixth section was performed for the immunocytochemistry staining . DA neurons were identified by an anti-TH antibody. Digital images of SN TH-positive neurons were obtained through an Olympus microscope (Olympus , Tokyo, Japan). A region of interest was established by outlining SN via Optimas version 6.51 (Media Cybernetics Inc., Rockville, MD, USA). Quantification of DA neurons was assessed by blindly visual counting the number of TH-positive neuronal cell bodies by two investigators and the result was analyzed from the average. Then, the mean values of TH-positive neuronal numbers were deduced by averaging the counts of six sections for each brain.

Statistical Analysis
Results were indicated as mean ± standard error of the mean (SEM). Statistical significance was analyzed by oneor two-way ANOVA through the GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Upon ANOVA demonstrating the significant differences, pairwise comparison between means was evaluated by Bonferroni's post hoc test with correction. A value of p < 0.05 was considered statistically significant.

ICA in Combination With L-DOPA Decreased 6-OHDA-Induced PC12 Cell Apoptosis
The properties of ICA and L-DOPA on 6-OHDA-elicited PC12 cell apoptosis were determined by flow cytometry. As shown in Figures 2A,B (F (6,14) = 45.78, p < 0.001), compared with the control cultures with the early apoptotic rate of 1.5%, FIGURE 4 | ICA combined with L-DOPA attenuated the decrease of DA and its metabolites in rat striatum treated with 6-OHDA. The striatal tissue levels of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) were measured by HPLC coupled with electrochemical detection at 7 (A) and 21 (B) days after the first treatment of L-DOPA and ICA, respectively. Also, the DA metabolite ratio [(HVA + DOPAC)/DA * 100] was calculated. Data were shown as mean ± SEM from six rats (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with 6-OHDA group; ω p < 0.05 compared with 6-OHDA+ICA group; Ψ p < 0.05 compared with 6-OHDA+L-DOPA group.

ICA Alleviated L-DOPA-Produced Motor Dysfunctions Without Affecting L-DOPA-Mediated Efficacy
The body AIM scores using the different dyskinesia subtypes (axial, limb and orolingual AIM scores) was analyzed, respectively. In 6-OHDA-lesioned rats, the AIM was gradually aggravated after continuous administration of L-DOPA. In contrast, daily treatment ICA before L-DOPA administration in 6-OHDA-lesioned rats reduced L-DOPA-induced increase of all three subtypes scores 21 days after ICA and L-DOPA treatment shown in Figure 5A (F (4,25) = 35.91, p < 0.0001), Figure 5B (F (4,25) = 73.21, p < 0.001) and Figure 5C (F (4,25) = 27.11, p < 0.0001). Taken together, ICA significantly attenuated L-DOPA-induced development of total AIM scores at ICA and L-DOPA treatment for 21 days shown in Figure 5D (F (4,25) = 69.24, p < 0.001). These results implied that ICA could alleviate LID. In addition, to determine whether ICA treatment altered L-DOPA-mediated anti-parkinsonian efficacy, rats were treated with the anti-dyskinetic dose of ICA followed by L-DOPA and the motor performance was detected by the FAS test. As shown in Figure 5E, L-DOPA significantly reversed 6-OHDA-induced stepping deficits and ICA combined with L-DOPA maintained this anti-parkinsonian effect compared with 6-OHDA group 21 days after ICA and L-DOPA treatment (F (3,20) = 62.37, p < 0.01).

DISCUSSION
This present study indicated that ICA conferred DA neuroprotection as monotherapy and an enhancement benefit of L-DOPA treatment after daily administration of L-DOPA and ICA for 21 days in parkinsonian 6-OHDA lesioned rats. Additionally, ICA ameliorated the development of LID as evidenced by the lowered AIM scores without affecting L-DOPA-mediated efficacy via FAS test. Furthermore, ICA attenuated neuroinflammation in the development of LID in vivo. Collectively, these results suggested ICA might possess as an adjunct treatment to enhance the efficacy of L-DOPA and ameliorate LID in PD (Supplementary Figure S1).
L-DOPA holds the gold standard therapy for PD patients. However, its long-term administration resulted in severe motor complications known as LID, which severely limits the efficacy of current treatments of PD (Bordet et al., 2000). It is reported that LID influences nearly 80% of PD patients after L-DOPA treatment for 5-10 years and some of these PD patients have to stop this treatment because of serious LID (Shin et al., 2015). Although the lowered dose of L-DOPA is one strategy to attenuate the adverse effects induced by L-DOPA (Ahn et al., 2017), the efficacy could be highly decreased (Kurlan, 2005). Thus, to explore the combination treatment to compensate for this limitation might provide a new scenario for PD. ICA is a natural component confirmed to have anti-aging, anti-oxidant and anti-inflammatory activities . It has been evaluated that ICA protected against 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-induced DA neuronal damage through the regulation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MEK) signaling pathway (Chen et al., 2017). Our previous study found that ICA attenuated 6-OHDA and lipopolysaccharide (LPS)-induced DA neuronal death and thereby improved motor dysfunction (Wang et al., 2018). In this study, ICA enhanced L-DOPA-mediated DA neuroprotection and ameliorated L-DOPA-induced motor dysfunction without interfering with the therapeutic efficacy of L-DOPA, suggesting that the use of the potential neuroprotective agent in combination with available drugs might serve as promising therapeutic potential for PD treatment.
In addition, it is known that dyskinesia occurs only upon dopaminergic treatment and the presence of DA neuronal loss in the SN (Rascol and Fabre, 2001). Nevertheless, the pathogenesis of LID remains poorly understood at present. Over the last decade, concerns regarding L-DOPA-induced neurotoxicity and its potential role on PD progression and the development of L-DOPA-induced motor dysfunctions have been raised (Lipski et al., 2011). In PD pathogenesis, neuroinflammation is recognized to be one of the main contributors and might be amplified by the increased DA metabolism and L-DOPA during the continuous L-DOPA treatment. However, the contribution of such event to the development of motor side complications is incompletely elucidated. Recently, multiple lines of evidence have implied a possible contribution of L-DOPA-elicited neuroinflammatory responses to LID (Carta et al., 2017). Since preclinical studies support the role of neuroinflammation on LID, whether an exacerbated neuroinflammation might be involved in LID development of PD patients could be unilluminated until now. However, during PD patients with L-DOPA treatment in late disease stage, it is not excluded that the neuroinflammatory reactions participate in this treatment. The mechanisms how L-DOPA would promote the neuroinflammatory reactions, and how that would in turn influence the dyskinetic outcomes, warrants further investigation. In this in vivo study, ICA combined with L-DOPA decreased 6-OHDA-induced protein expressions of inflammatory factors 21 days not 7 days after ICA and L-DOPA treatment, which implied that the inhibition of neuroinflammation might be one of the contributors to ICA-attenuated LID. In parallel with our previous work, we found that ICA inhibited microglia-mediated inflammation in in vivo and in vitro studies (Wang et al., 2018). However, we couldn't rule out other factors, such as the attenuation of mitochondrial oxidative stress, participated in the mechanisms underlying ICA-mediated DA neuroprotection and attenuation of LID. Since the pathogenesis of PD and LID was still unelucidated, neuroinflammation might be involved in the process of PD and LID and the corresponding mechanisms were still not clear. This present study was the pilot investigation to indicate that ICA enhanced L-DOPAmediated DA neuroprotection and attenuated L-DOPAinduced dyskinetic actions. Thus, the mechanisms underlying ICA-mediated these beneficial effects require deep rigorous exploration.
At present, no effective treatment is available to halt PD progression. Although L-DOPA is the gold standard treatment for PD, it is frequently related to a series of side complications and unsatisfactory outcomes. Therefore, to develop the effective treatment avenues to stop the progression of PD is of paramount importance (AlDakheel et al., 2014). So far, critics have argued that neuroprotective agents couldn't fulfill the therapeutic window for PD, since patients diagnosed as PD already present more than 50-60% dopaminergic deficits in the SN (Lang et al., 2013). This study indicated that ICA conferred synergistic effects with L-DOPA against DA neuronal loss and alleviated LID. Despite this optimistic perspective for the future use of ICA potential treatment for PD, most of the findings are determined in one type of PD animal model. Therefore, to verify the translational value of ICA, this combination treatment need be rigorously corroborated in other PD animal models.

CONCLUSION
This study demonstrates that ICA has synergistic effects against DA neuronal loss combined with L-DOPA and anti-dyskinetic actions induced by L-DOPA. These findings suggest ICA might be a potential promising adjuvant to enhance L-DOPA efficacy and attenuate L-DOPA-produced adverse effects in PD.

AUTHOR CONTRIBUTIONS
FZ conceived and designed the experiments. D-SL, CC and Y-XZ participated in the experiments performance and G-QW, D-DL, JL, JS and FZ finished data analysis. FZ wrote and revised the manuscript. All the authors checked the contents of the manuscript, validated the accuracy of the data and approved the submitted manuscript.