Neural stem cells transplantation combined with ethyl stearate improve PD rats motor behavior by promoting NSCs migration and differentiation

Abstract Background In recent years, the ability of neural stem cells (NSCs) transplantation to treat Parkinson's disease (PD) has attracted attention. However, it is still a challenge to promote the migration of NSCs to the lesion site and their directional differentiation into dopaminergic neurons in PD. C‐C motif chemokine ligand 5 (CCL5) and C‐C motif chemokine receptor 5 (CCR5) are expressed in the brain and are important regulators of cell migration. It has been reported that ethyl stearate (PubChem CID: 8122) has a protective effect in 6‐OHDA‐induced PD rats. Methods Parkinson's disease rats were injected with 6‐hydroxydopamine (6‐OHDA) into the right substantia nigra, and striatum followed by 8 μL of an NSC cell suspension containing 100 μM ethyl stearate and 8 × 105 cells in the right striatum. The effect of transplantation NSCs combined with ethyl stearate was assessed by evaluating apomorphine (APO)‐induced turning behavior and performance in the pole test. Quantitative real‐time reverse transcription–polymerase chain reaction (qRT‐PCR), Western blotting (WB), and immunofluorescence staining were also performed. Results NSCs transplantation combined with ethyl stearate ameliorated the behavioral deficits of PD rats. PD rats that received transplantation NSCs combined with ethyl stearate exhibited increased expression of tyrosine hydroxylase (TH) and an increased number of green fluorescent protein (GFP)‐positive cells. Furthermore, GFP‐positive cells migrated into the substantia nigra and differentiated into dopaminergic neurons. The expression of CCL5 and CCR5 was significantly increased after transplantation NSCs combined with ethyl stearate. Conclusions These findings suggest that NSCs transplantation combined with ethyl stearate can improve the motor behavioral performance of PD rats by promoting NSCs migration from the striatum to the substantia nigra via CCL5/CCR5 and promoting the differentiation of NSCs into dopaminergic neurons.


| INTRODUC TI ON
Parkinson's disease (PD), as a common progressive neurodegenerative disease, 1 is characterized by the progressive loss of nigrostriatal dopaminergic neurons. 2 The central nervous system lacks repair mechanisms to replace lost dopaminergic neurons. Available treatments, which include pharmacological approaches, physiotherapy, deep brain stimulation, and stem cell-based therapeutic approaches, can improve motor and nonmotor signs and symptoms. 3 In recent years, there have been studies on the use of stem cell-based therapeutic strategies to replace lost dopaminergic neurons in PD. [4][5][6][7] Neural stem cells (NSCs) exhibit the abilities of self-renewal and differentiation potential, low immunogenicity, and good migratory ability in the context of transplantation, [8][9][10][11][12][13][14][15] thus, NSCs transplantation is one of the most promising methods for the treatment of PD.
However, most transplanted NSCs differentiate into glial cells rather than neurons. Additionally, in many studies, transplanted NSCs exhibit considerably limited migration ability, which restricts their therapeutic impact. [16][17][18][19] Chemokines are small secreted proteins with multiple physiological functions, and they have been shown to play a role in guiding the migration of neural progenitors in the developing brain 20,21 . C-C motif chemokine receptor 5 (CCR5), a seven-transmembrane-domain G-protein-coupled receptor expressed in the central nervous system, is stimulated by chemokine ligands CCL3, CCL4, and CCL5 . 22 Recently, studies have shown that NSCs can express CCR5. [23][24][25][26] Thus, NSCs can exhibit chemotactic responses to the relative chemokine ligands; however, few studies have reported the effect of CCL5/CCR5 on the migration of transplanted NSCs in PD.
In our previous studies, we found that plastrum testudinis extract (PTE) could improve the behavior of PD model rats and that its active ingredient was ethyl stearate. In this study, we treated NSCs with ethyl stearate and transplanted them into the striatum of PD model rats. The results showed that NSCs transplantation combined with ethyl stearate rescued the behavioral deficits in PD rats by promoting NSCs migration and differentiation.

| Animals
Specific-pathogen-free (SPF) male Sprague-Dawley (SD) rats weighing 180-220 g were used for in vivo experiments, and pregnant rats with 14-to 17-day embryos were used for NSCs isolation. All experimental animals were provided by the Laboratory Animal Research Center of Guangzhou University of Chinese Medicine (ARCG-UCM).
The rats were housed at ARCG-UCM at a temperature of 24-26°C and humidity of 40-60% on a 12/12-hour light/dark cycle and provided access to food and water. The rats were allowed to acclimate to the laboratory environment for 1 week before the experiment was started. After the completion of the in vivo experiment, rats from each group were sacrificed for tissue collection via injection of sodium pentobarbital. Brains were removed rapidly from decapitated rats and cooled immediately in ice-cold 0.9% NaCl solution.
We removed the cortex and dissected the striatum and substantia nigra according to the rat brain atlas.

| NSCs culture and preparation of green fluorescent protein (GFP)-NSCs
Embryos were collected at embryonic days 14-17 from pregnant SD rats, and NSCs were isolated from the brains of the embryos by mechanical dissociation and trypsin digestion under aseptic conditions. The digestion of the dissected tissues was terminated by using DMEM/F12 (Gibco) containing 10% fetal bovine serum. After being washed twice with phosphate-buffered saline (PBS, Gibco), the cells were plated in culture bottles in serum-free DMEM/F12 supplemented with 20 ng/mL epidermal growth factor (EGF, GenScrip), 20 ng/mL basic fibroblast growth factor (bFGF, GenScrip) and 2% B27 (v/v, Gibco) as well as 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco); they were then cultured at 37°C in an incubator containing 5% CO 2 . NSCs were cultured and passaged with serum-free medium, and they were used at passage two in the subsequent experiment. Immunofluorescence staining, Western blotting (WB), quantitative real-time reverse-PCR (qRT-PCR), and analysis of NSC migration were performed.

| In vitro experiment
NSCs in passage three neurospheres cultured in proliferation medium were identified by immunofluorescence staining for the marker Nestin. The neurospheres were divided into different groups and uniformly seeded in 6-well culture plates. Then they were stimulated with different doses of ethyl stearate. The groups were the blank control group (0 μM group), 10 μM group, 100 μM group, and 1000 μM group. The cell migration distance and expression of CCR5 in these groups were evaluated.
For in vitro differentiation, NSCs were dissociated into singlecell suspensions in DMEM/F12 supplemented with 10% fetal bovine serum as well as 100 units/mL penicillin and 100 μg/mL streptomycin K E Y W O R D S C-C motif chemokine ligand 5, C-C motif chemokine receptor 5, ethyl stearate, neural stem cells, Parkinson's disease and then plated in 6-well culture plates. Different doses of ethyl stearate were added to induce cell differentiation. After 5 days of induction, Immunofluorescence staining, WB analysis, and qRT-PCR were performed.

| Quantification of the cell migration distance
Neurospheres were uniformly plated in poly-lysine-coated 6-well plates at a low density (10-15 neurospheres per dish) to ensure a large distance between the individual spheres and cultured in the proliferation medium. The neurospheres were then treated with ethyl stearate (0, 10, 100, or 1000 μM) for 1-3 days, and the migratory behavior of NSCs was evaluated by measuring the end-to-end distance of the extended processes. The lengths of the 10 longest processes were estimated from the edge of the neurosphere or cell body to the tip of the processes. Distance measurements were performed by using ImageJ. 27

| PD model preparation and analysis of apomorphine (APO)-induced rotation
Specific-pathogen-free male SD rats were randomly divided into two groups: the sham-operated group and the model group. Rats in the model group received unilateral stereotaxic injections of 8 μL 6-hydroxydopamine (6-OHDA, 4 μg/μl, H873296, Macklin, Shanghai, China) in the substantia nigra and striatum as previously described. 28 The following coordinates were used to target the Beginning a week after surgery, the rats were intraperitoneally injected with APO (0.5 mg/kg, 017-18,321, Wako, Japan) once a week, and rotation behavior was assessed for 30 min. The model establishment was considered successful if the rats that were injected with 6-OHDA exhibited more than 6 rotations per minute within 6 weeks of observation.

| Transplantation and behavioral test
Model rats were randomly distributed into three groups: the model group, the NSCs group, and the ethyl stearate+NSCs group (abbreviated as "ethyl stearate group"). Before transplantation, NSCs were transfected with lentivirus expressing GFP for 7 days and then dis-  Table 1.

| Immunofluorescence staining
Six weeks after transplantation, the rats were sacrificed by injection of sodium pentobarbital, and the brain tissues were fixed with 4% paraformaldehyde. After being immersed in 15% and 30% sucrose in PBS, the brain tissues were sliced with a freezing microtome (20 μm thick). Cultured cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The fixed cells and brain sections were then permeabilized with 0.5% Triton X-100 for 15 min at 37°C, blocked in 10% goat serum for 30 min at 37°C, and incubated overnight at 4°C with primary antibodies. They were then incubated with secondary antibodies in the dark at room temperature for 1 h. Then, the stained samples were counterstained with DAPI (P0131, Beyotime

| Identification of NSCs and preparation of GFP-NSCs
After being cultured for 5 days, the cells formed large spherical colonies, which were immunopositive for Nestin ( Figure 1A,D). Later, these neurospheres were collected and dissociated into single cells.
The single cells adhered to the plate and showed a long fusiform shape with prominent protuberances ( Figure 1A). The cells derived from NSCs were stained with neuron-specific enolase (NSE, a marker of neurons) and glial fibrillary acidic protein (GFAP, a marker of astroglia) ( Figure 1B,C). Then, the NSCs were transfected with lentiviral vectors expressing GFP for subsequent experiments. After the transfected cells were cultured for 5 days, GFP was detected in the neurospheres and differentiated cells ( Figure 1E,F). The above results illustrate that we successfully isolated and transfected NSCs.

| Ethyl stearate promotes NSCs differentiation into dopaminergic neurons and NSCs migration in vitro
To confirm that ethyl stearate could promote NSCs differentiation into dopaminergic neurons, neurospheres were dissociated into single cells for the following experiment. Five days after ethyl stearateinduced NSCs differentiation, we assessed TH expression, which is considered as a biomarker related to dopaminergic neurons. 2 WB showed that TH protein expression was significantly increased in the 100 μM group (Figure 2A). Subsequently, we used qRT-PCR to measure the Th mRNA level in the different groups. The results showed that Th mRNA expression was significantly upregulated in the 100 μM group compared with the 0 μM group ( Figure 2B).
Moreover, there were more TH-positive cells in the 100 μM group ( Figure 2C).
Next, to examine whether ethyl stearate can promote NSCs migration, the distance traveled by the cells was measured. The cells were observed to migrate out from the neurospheres, and 100 μM ethyl stearate treatment significantly increased the migration distance ( Figure 3A,B). Moreover, the immunofluorescence staining results showed that there were more CCR5-positive cells in the 100 μM ethyl stearate group ( Figure 3C).
These results suggested that 100 μM ethyl stearate promoted

NSCs differentiation into dopaminergic neurons and promoted
NSCs migration to lesions by enhancing the expression of CCR5, indicating that NSCs stimulated with 100 μM ethyl stearate might have therapeutic potential as a cell replacement strategy in PD rats.

| NSCs Transplantation combined with ethyl stearate rescues behavioral deficits in PD rats
Parkinson's disease model rats underwent stereotaxic injection of GFP-labeled NSCs or GFP-labeled NSCs and 100 μM ethyl stearate in the ipsilateral striatum. Animals in the model group were injected with the same volume of PBS at the same site. The protocol of the in vivo experiments is shown in Figure 5A.
Six weeks after surgery, APO-induced rotation behavior was alleviated in the PD rats in the NSC group and especially the ethyl stearate group; however, this change was not observed in the animals injected with PBS. Importantly, rotation behavior was decreased to a greater extent in the ethyl stearate group than in the NSCs group ( Figure 5B).
In addition, the pole test was used to assess the coordination of

| NSCs Transplantation combined with ethyl stearate protects dopaminergic neurons in PD model rats
To transplanted NSCs into dopaminergic neurons ( Figure 6A). In addition, WB analysis showed that the protein expression of TH was increased after transplantation NSCs combined with ethyl stearate ( Figure 6B), which indicated that ethyl stearate alleviated damage to dopaminergic neurons in PD rats.
In the substantia nigra, consistent with the results described above, TH-positive cells in the ethyl stearate group were significantly increased compared with those in the model group and NSCs group. Interestingly, GFP-positive cells appeared in the substantia nigra and differentiated into TH-positive cells ( Figure 6C).
WB also showed that the protein expression of TH was increased after the transplantation of NSCs combined with ethyl stearate ( Figure 6D).
Taken together, these results implied that ethyl stearate promoted NSCs migration as well as the differentiation of NSCs into dopaminergic neurons in vivo.

| Ethyl stearate increases the expression of CCL5 and CCR5 in a PD rat model
We ( Figure 9D,E). Furthermore, the expression of CCR5 in NSCs labeled with GFP in the substantia nigra was significantly increased in the ethyl stearate group compared with the NSCs group (Figure 10), indicating that CCL5 induced CCR5-NSCs to migrate to the lesion.
Taken together, these findings suggest a potential mechanism by which NSCs transplantation combined with ethyl stearate can ameliorate PD.

| DISCUSS ION
PD is related to various factors, such as age, the environment, symptoms, but it may lead to dyskinesia after a few years. 30,31 In transplanted NSCs needs to be solved. 16 Hence, we suspect that ethyl stearate may promote NSCs migration through chemokines and their receptors. The levels of proinflammatory factors and chemokines such as IL-1β, IL-33, CCL2, and CCL5 can be increased in PD. 41 The levels of IL-15 and CCL5 were increased in the PD patients who received levodopa, compared to healthy controls and the patients with PD. 42 Our study showed that ethyl stearate apparently improved the migration of NSCs in vitro, and increased the expression of CCR5 on NSCs both in vitro and in vivo. It is worth noting that the expression of CCL5 in the substantia nigra was increased in the ethyl stearate group, which may attract the transplanted cells to migrate to and integrate with the lesion site.
We predicted that ethyl stearate might improve the behavioral deficits of PD rats by promoting NSCs differentiation into dopaminergic neurons and the migration of NSCs via CCL5 and CCR5. Indeed, this research requires further experiments. However, ethyl stearate has excellent potential for solving the inadequacy of NSCs transplantation in PD.

| CON CLUS IONS
In summary, CCL5 and CCR5 expression increased significantly after transplantation NSCs combined with ethyl stearate. Ethyl stearate may guide the migration of NSCs by regulating CCL5 and CCR5 and may promote the differentiation of NSCs into dopaminergic neurons in PD model rats.

AUTH O R CO NTR I B UTI O N S
Jiapei Huang was responsible for the conceptualization, investigation, validation, and writing of the original draft. Lan Yi reviewed and edited the manuscript. Xiaoxiao Yang investigated the study.
Qi Zheng, Jun Zhong, and Sen Ye validated the data. Xican Li F I G U R E 1 0 Immunofluorescence staining of CCR5 in the substantia nigra after transplantation. The green fluorescence represents transplanted cells, the red fluorescence represents CCR5, and the blue fluorescence indicates the nuclei. These experiments were conducted in three independent replicates. Scale bar: 20 μm.
contributed resources. Li Hui and Dongfeng Chen supervised the study. Caixia Li conceptualized the study, reviewed, and edited the manuscript, and was involved in project administration. All authors read and approved the final manuscript.

ACK N O WLE D G E M ENTS
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