Therapeutic effect of TO901317 on 6-OHDA-induced Parkinson rats in vitro and in vivo

doi:10.21203/rs.3.rs-15406/v1

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Therapeutic effect of TO901317 on 6-OHDA-induced Parkinson rats in vitro and in vivo

https://doi.org/10.21203/rs.3.rs-15406/v1

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Background: Stem cells from different sources could differentiate into dopamine-producing cells and ameliorate behavioral deficits in Parkinsonian models. Especially, human bone marrow mesenchymal stem cells (hBMSCs) have many advantages without ethical dispute. Liver X receptor s (LXRs) are involved in the maintenance of the normal function of the central nervous system myelin. We have reported the induction of cocktail-induced da phenotypes from adult rat BMSCs by using sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF) and TO901317 (agonist of LXRs) with 87.42% of efficiency in 6 days of period of induction. But the previous work did not verify whether the induced cells had the corresponding neural function.

Methods: In this study, we demonstrated that TO901317 could promote the differentiation of hBMSCs into dopaminergic neurons. Neuronal markers (Tuj1, Neun and Nestin), dopamine neuron markers (tyrosine hydroxylase, TH), LXRa and LXRb were detected by immunofluorescence. RT-qPCR was used to measure the mRNA expressions of adenosine triphosphate-binding cassette transporter A1 (ABCA1). Western Blotting detected the changes of LXRa, LXRb and TH expression.

Results: TO901317 significantly enhanced the differentiation from hBMSCs to DA neurons. Only the LXR+GF group released dopamine by the result of enzyme linked immunosorbent assay (ELISA). Compared with the control group and GF group, the optimal time for differentiation of hBMSCs treated by 0.5mM TO901317 combined with GF was six days. And the maximum induction efficiency was 91.67%. After transplanting induced-cells into Parkinson's disease rats, the symptoms of Parkinson's rats decreased, and the number of dopamine neurons increased in the substantia nigra and striatum.

Conclusions: TO901317 promoted differentiation of hBMSCs into dopamine neurons may be related to activation of LXR-ABCA1 signaling pathway. These data suggest that TO901317 may serve as a potential therapeutic methods for Parkinson's disease.

Stem Cell & Developmental Cell Biology

Human bone marrow mesenchymal stem cells

Parkinson’s disease

Liver X receptor agonist

TO901317

Differentiation

Cell transplantation

Parkinson’s disease (PD) is a complex, age-related neurodegenerative disease with early prominent death and loss of dopaminergic (DA) neurons in the substantia nigra parscompacta (SNpc) ¹. The main feature of PD is progressive, extensive dopaminergic neuron loss in the substantia nigra-striatum ². The main clinical manifestations are static tremor, slow movement and reduction, increased muscle tone, unstable posture. PD is also closely related with various non-motor symptoms, such as cognitive dysfunction, mood and psychotic disorder ³. A considerable number of patients have cognitive impairment, and late stage of PD may have dementia and depression. In the degenerative diseases of the nervous system, the incidence of PD in the 65-year-old population is more than 1%, second only to Alzheimer's disease, and it is the second most common neurodegenerative disorder, bringing an escalating burden on economic terms and quality of life to these patients, their families and society.

The mainstay of Parkinson’s disease managements are drug therapy, surgical treatment and stem cell replacement therapy. Drugs based management strategies are carefully to considerations of the side-effects and tolerance levels of the patient ⁴. Surgical treatments include thalamicectomy and deep brain stimulation (DBS). Thalamotomy has strong synaptic connections with the thalamus and striatum, but in PD patient's physiological regulations it seems to alter and results in motor function impairments ⁵. As for DBS, patients have to undergo surgeries again and again, which increase the risk of infection and socioeconomic burdens ⁶. Although currently available pharmacological or surgical treatments may significantly improve the quality of life of many patients with PD, these are only symptomatic treatments that do not slow or stop the progressive course of the disease. At present, replacement of the progressively degenerated DA neurons through cell transplantation is considered to be the most potential therapy ⁷. Some data suggested a more immediate and reachable goal of cell transplantation may be neuronal protection ⁸. Currently, embryonic stem cells (ESCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and bone marrow mesenchymal stem cells (BMSCs) are available for stem cell replacement therapy ⁹. The use of ESCs and NSCs existed ethical problems inherent. So much hope is placed on iPSCs which induced human fibroblasts into a source of patient-specific and disease-specific neurons, especially as, in theory, this approach would avoid many of the ethical issues associated with using ESCs ^{10, 11}. Cell replacement therapy is derived from the patient's own cells, avoiding immune rejection. In autologous transplantation, it is necessary to establish iPSCs from each patient, and current technical operations take much time and high cost, so it is difficult to spread to general treatment. In addition, the use of iPSCs with the patient's own genetic factors, the sensitivity of the disease may be high. It also has the risk of introducing cancer cells ¹². Specifically, iPSCs often obtained chromosomal abnormalities, with gains or losses of whole chromosomes ¹³. Different from these stem cells, BMSCs come from patients themselves without ethics dispute. Especially, BMSCs have multi-directional differentiation potential ¹⁴, own low risk of tumorigenesis ¹⁵ and are rich in source, easy to extract, separate and purify. Many studies in vitro and preclinical strongly proved the therapeutic potential of BMSCs when be applied as a treatment for different pathological conditions ^{16, 17}.

There have been different methods involved in the differentiation of BMSCs, including cell growth factors ^{18, 19}, chemical inducer ²⁰ and lentiviral transduction ²¹. Currently, in vitro induction of stem cells using growth factors with sonic hedgehog (SHH) and fibroblast growth factors (FGFs), succeeded in inducing adult human BMSCs into DA neurons with 67% of efficiency in 12 days ^{22, 23}. This is the current maximum induction efficiency and the shortest induction time.

Liver X receptors (LXRs) including LXRα and LXRβ are members of the nuclear receptor supergene family of ligand-activated transcription factors ²⁴, and play a key role in the regulation of cholesterol, fatty acid homeostasis ²⁵ and central nervous system (CNS) ^{26, 27}. Deletion of the LXRβ in mice caused hypoplasia in the dentate gyrus, including abnormalities in the formation of progenitor cells and granule cell differentiation ^{26, 27}. Both LXRα and LXRβ are expressed in the epithelial cells of the choroid plexus. It has been found that LXRs played crucial roles in regulation of genes involved in CSF production and structural integrity of choroid plexus ²⁹. LXRs are involved in the processes of myelination and remyelination ³⁰. Activation of LXRs can promote the regeneration and survival of motor neurons ³¹.

Thus, LXR agonists induce transcriptional activity of LXR target genes, plays therapeutic role in different neurodegeneration animal models^{32 − 34}. Based on our previous work and others have found the induction of cocktail-induced da phenotypes in adult rat BMSCs by using SHH, fibroblast growth factor 8 (FGF8), basic fibroblast growth factor (bFGF) and TO901317 with 87.42% of efficiency in 6 days of period of induction ³⁵. LXR agonist significantly shortened induction time and improved induction efficiency compared to methods which had been reported. But these previous work did not investigate whether induced-cells released dopamine and had the corresponding dopaminergic neurons. We did not sure if the induced cells have a therapeutic effect on Parkinson's disease.

In this study we investigated the effect of TO901317 on the differentiation of hBMSCs into dopamine neurons. We also explored whether induced-cells had dopamine neuronal function and the possible mechanism. Finally, we transplanted the induced-cells into PD rats to observe the therapeutic effect.

Materials

6-hydroxydopamine (6-OHDA) was purchased from Selleck. Human bone marrow mesenchymal stem cells (hBMSCs) was purchased from Cyagen Biosciences. LXR agonist-TO901317 (N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-tri-fluoro1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide) was purchased from Sigma-Aldrich. Apomorphine hydrochloride was purchased from Pharmaceutical Factory of Qinghai, China.

Animals

Sprague-Dawley (SD) rats were accommodated in the barrier housing facility, in keeping with the national standard of “Laboratory Animal-Requirements of Environment and Housing Facilities.” The care of the laboratory animal and the animal experimental operation conform to the “Chongqing Administration Rule of Laboratory Animal.” The experimental procedures were approved by the animal laboratory administrative center and the institutional ethics committee of Chongqing Medical University. All male SD rats were weighted 220 g-250 g. To establish the rat model of PD, 30 male rats received a 6-OHDA lesion of the medial forebrain bundle (MFB) on the right side and 10 rats were as control group only injected with solvent which was used to dissolve 6-OHDA ³⁶.

Differentiation of hBMSCs

Human BMSCs were divided into 3 groups and plated onto 24 well plates where each plate contained 2.0 × 10⁴ cells and cells were cultured at 37 °C with 5% CO2.

Control group: Cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, BI).

Growth factors treated group (GF): hBMSCs were cultured in DMEM/F-12 containing 10% FBS with 24 hours. After that, the medium was replaced with neurobasal medium (Invitrogen/Gibco, USA) and 0.5% B27 supplement (Invitrogen/Gibco, USA). The cells were induced only once with a cocktail of 250 ng/ml Recombinant Human SHH (PeproTech, Rocky Hill, NJ, USA), 100 ng/ml Recombinant Human FGF8 (PeproTech, Rocky Hill, NJ, USA), and 50 ng/ml Recombinant Human basic-FGF (bFGF; PeproTech, Rocky Hill, NJ, USA). The medium was not replaced in 12 days.

TO901317 and growth factors treated group (LXR + GF): On the basis induction of GF, effects of TO901317 on the differentiation of hBMSCs into DA neurons in the time-dependent and concentration-dependent manner were investigated. According to the results of (a) (b) and (c), the cells were induced only once with the cocktail and the medium was not replaced during the induction period.

Explore the best concentration of TO901317 added: Different concentrations of TO901317 (0.125, 0.25, 0.5, 1 and 2 μM) were added with GF (250ng/ml SHH, 100ng/ml FGF8, 50ng/ml bFGF).
Explore the best time to add TO901317 during growth factor induction period: On the basis of GF, TO901317 were added on the first day, the third day, the sixth day, and the ninth day, respectively, to induce differentiation for 12 days.
Explore the induction time of the TO901317 in combination with GF: According to the result of (a) (b), 0.5 μM TO901317 was added to the culture medium, and the cell morphology was observed every 3 days.

Cell Counting kit-8 assay

A Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay was used to test the growth rate by following the manufacturer’s instructions. 3000 cells/well of hBMSCs were cultured into a 96-well plate and allowed to adhere overnight. At 3-day intervals, the growth rates of cells in each group were measured by application of CCK-8 kit and optical density (OD) was determined at 450 nm using a microplate reader (Thermo Scientific, USA).

Parkinson's Disease rats

All male SD rats were fed to 220–250 g. Rats were intraperitoneally injected with 0.5 mg/kg apomorphine. Rats without rotation behavior were selected to establish PD model. To generate unilateral PD models, 6-OHDA was injected into MFB in 30 rats ³⁷.

Immunofluorescence

In brief, cells of each group were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton-X100 and blocked in phosphate buffer solution (PBS) containing 5% normal goat serum. Cells were incubated with monoclonal antibodies overnight with 4℃. Antibodies’ dilutions were as follows: β III tubulin, 1:200 (Tuj1; Abcam, Cambridge, UK); Tyrosine hydroxylase, 1:200 (TH, Abcam, Cambridge, UK); Nestin, 1:200 (Abcam, Cambridge, UK); Neun, 1:200 (Abcam, Cambridge, UK); LXR α receptor, 1:200 (Abcam, Cambridge, UK); LXR β receptor, 1:200 (Gene Tex, USA). After extensive washing for 3 times in PBS, suitable secondary antibodies anti-mouse IgG-Alexa Fluor 488, anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Cy3, anti-rabbit IgG-Alexa Fluor 594 were diluted at 1:200 in PBS, and then suitable secondary antibodies were added and incubated in darkness for 1 h at room temperature. Then nuclear stain 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) was used for nuclear staining.

Enzyme-Linked Immunosorbent Assay (ELISA)

To investigate whether cells in each group release dopamine. Culture supernatant and cells of control group, GF group and LXR + GF group were collected. Cells were added with PBS, then grinded on ice. The mixture was centrifuged at 3000 g for 20 min at 4 °C and collected the supernatants. Dopamine was detected by ELISA kits (n = 6) (Mei biao, Jiangsu, China). The samples and standards were tested according to the instructions.

Western Blotting Test

Cells were plated on six-well plate for 24 hours with 10% FBS, then grown in induction medium for six days to be used for preparation of whole cell extract. After removing the media, cells were washed three times with PBS. Then cells in each well were added 150∝ l lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and cracked on ice for 30 minutes. The mixture was centrifuged at 12,000 g for 20 min at 4 °C and collected the supernatants. The protein concentration was determined by BCA Protein Assay Kit (Beyotime, China). The protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, United States). The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated with specific primar antibodies, TH, 1:500 (Abcam, Cambridge, UK); LXR α receptor, 1:500 (Abcam, Cambridge, UK); LXR β receptor, 1:500 (GeneTex, United States) were included and overnight at 4℃. The membranes were rinsed three times in TBST and incubated with HRP conjugated secondary antibodies at room temperature for 1 h. Then after washed three times in TBST, protein signals were visualized by ECL (Bio-Rad, United States).

Quantitative real-time PCR (qPCR)

Total RNA was isolated from the cells in control group, GF group and LXR + GF group by Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Then mRNA was subjected to reverse transcription using HiScript Q Select RT SuperMix (Vazyme, Nanjing, China). SYBR Green II (Biomake, USA) incorporation method was used to detect the amount of mRNA. Negative controls were used as no template cDNA reactions and melting curves were used to confirm the results. The results were normalized using GAPDH concentration of each sample.

Histopathological Examination

Hematoxylin and eosin (HE) staining was performed to show pathological histological damage in the substantia nigra pars compacta (SNc) and striatum. The rats of control group, 6-OHDA group and 6-OHDA + Cells group were anesthetized with sodium chloral hydrate and perfused with PBS, and then perfusion with 4% paraformaldehyde. After that, the brains were dehydrated in a graded series of alcohols and embedded in paraffin. A series of 5-µm-thick sections were cut from the brain. Finally, the sections were stained with HE reagents for histopathological examination.

Statistical analysis

All results were expressed as the means ± standard deviation (SD) and the statistical significance of differences was analyzed by GraphPad Prism (GraphPad Software, La Jolla, CA, USA). For the comparison of multiple groups, statistical analysis was performed using one-way analysis of variance (ANOVA) with Bonferroni’s posthoc test. Probability values less than 0.05 (p < 0.05) were considered to be statistically significant.

Culture of hBMSCs

hBMSCs were purchased in Cyagen Biosciences. Cells exhibited a long fusiform and vortex arrangement (FIGURE 1).

Effect of different concentrations of TO901317 on cell survival

CCK8 kit was used to detect the survival rate of cells in control group, GF group and LXR + GF group. In addition, the concentration of TO901317 in the LXR + GF group was given to 0.125∝M, 0.25∝M, 0.5∝M, 1∝M and 2∝M respectively. After three days, the survival rate of cells in each group did not show significant difference (FIGURE 2A). The survival rate significantly increased in GF group and LXR + GF group compared with control group when cells were cultured for six days, nine days and twelve days. Interestingly, there was no significant difference in cell survival rate when cells were cultured with different concentrations of TO901317 (FIGURE 2).

Determination of the concentration of TO901317

Immunofluorescence was used to detect the expression of neuronal markers (Neun, Nestin and Tuj1) and dopamine neuron markers (TH).

In this study, TO901317 was used of five concentrations of 0.125∝M, 0.25∝M, 0.5∝M, 1∝M and 2∝M. We found that when GF combined with 0.5∝M TO901317, the number of TH⁺ positive cells reached the maximum (^**P < 0.01) (FIGURE 3).

Explore the adding time of TO901317

In this study, we used 0.5∝M TO901317 in combination with GF to induce hBMSCs into dopaminergic neurons. On the premise of GF addition during 12 days, TO901317 was added on the first day, third day, sixth day, and ninth day. Expression of TH (Cy3, red) and Tuj1 (Alexa Fluor 488, green) were determined by immunofluorescence. The results showed that the expression of TH was the highest when TO901317 was added on the first day (FIGURE 4).

Explore the time period induced by TO901317

Immunofluorescence was used to detect the expression changes of Tuj1 and TH after three days, six days, nine days and twelve days of induction period. The control group only expressed Tuj1 without TH. While GF group and LXR + GF group expressed Tuj1 and TH simultaneously (FIGURE 5A). In the LXR + GF group, TH⁺ cells reached maximum after six days of induction (FIGURE 5B). Expression changes of TH was detected by Western blotting in Control group, GF group and LXR + GF group. Compared with control and GF groups, the expression of TH was significantly increased in LXR + GF group (FIGURE 5D, E). Cells showed typical neuronal morphology with extended long cell processes and enlarged cell bodies in GF group and LXR group under the bright field (FIGURE 5C). In particular, in the LXR + GF group, the morphology of the neurons expressed by the cells was more obvious (FIGURE 5F).

Dopaminergic neuron properties of inducing cells

Nestin, a neuroectodermal marker, seems to be a prerequisite for acquisition of the aptness to progress towards the neural lineage ^{38, 39}. The cells in the control group probed with nestin and neun antibodies were negative and just revealed nuclear staining DAPI. Both GF group and LXR + GF group, cells showed staining with neun and nestin (FIGURE 6A, 6B).

To study whether differentiated hBMSCs release dopamine, a dopamine kit was used. Cells in control group and GF group did not secrete dopamine. Only the LXR + GF group secreted dopamine (FIGURE 6C).

The role of LXRs in cell differentiation and its possible mechanism

Expression of LXRα and LXRβ between control group, GF group and LXR + GF group did not show significant difference (FIGURE 7A, B, E, F). Western blot result showed that expression of LXRα and LXRβ were decreased significantly in LXR + GF group compared to control group and GF group (FIGURE 7C, D, G, H).

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) is the target gene of LXRs. The lack of ABCA1 leads to transport disorders of central nervous system cholesterol, which in turn leads to defects in neuronal structure and function. Compared with control group and GF group, the mRNA of ABCA1 of LXR + GF group was significantly elevated (FIGURE7I).

Establishment of Parkinson's disease model

All SD rats (220 g-250 g) received intraperitonal injection of apomorphine dissolved in sterile (0.5 mg/kg). Choosing rat without rotation behavior received a 6-OHDA lesion of the MFB ³⁷. The 6-OHDA were dissolved in saline with 0.02% ascorbic acid solution containing. Briefly, rats were anesthetized with chloral hydrate (4% chloral hydrate and 96% saline solution) by intraperitoneal injection. The MFB was targeted with an injection of 6-OHDA with a total amount of 14 µg. The lesion to stereotaxic coordinates were adjusted to the age and weight of the animals with the help of brain stereotaxic instrument (RWD, Shenzhen, China). After two weeks during one month, to determine whether the models were successful, we used apomorphine to induced rotation and behaviour was recorded over a period of 90 min. Judging that the model is successful is that the number of rotations to the healthy side is greater than 7 rotations per minute (FIGURE 8).

The vivo effects of inducing cells

Four weeks after 6-OHDA infusion, rats were divided randomly into three groups as follows:the control group as solvent group (n = 10), the model group as 6-OHDA group (n = 10) and the therapy group as 6-OHDA + cells group (n = 20). 1 × 10⁵ induced dopaminergic neurons in 2 µL transplantation buffers (phosphate buffer saline) were injected into the right side of the SNc (A/P -4.6 mm, M/L − 2.2 mm, D/V − 7 mm). The 6-OHDA group was only injected transplantation buffers at the same place as 6-OHDA + Cells group.

After four weeks, the apomorphine-induced contralateral rotation test was performed. the 6-OHDA + cells group already appeared significantly improved behavioral performance compared to the rats of the 6-OHDA group (FIGURE 9E). TH expression were detected of western blot and immunofluorescence. HE was used to test the changes of neurons in SNc and striatum of rats in each group (FIGURE 9A). Compared to the control group, cells presented significant nuclear pyknosis, vacuolization and nuclear deep staining. After cell transplantation, the nuclear deep staining of the nucleus and the vacuolization of cells were reduced (FIGURE 9A). Immunofluorescent staining results revealed that TH-positive signals were almost absent at the striatum in the 6-OHDA-lesioned rats that received no grafts (FIGURE 9C). In contrast, the rats in the 6-OHDA + cells group, TH-positive signals were greatly recovered in the striatum (FIGURE 9C). TH-positive signals also significantly increased at SNpc compared with 6-OHDA group(FIGURE 9B). Western blot detection also showed the same test results (FIGURE 9D).

PD is a complex neurodegenerative disease which is characterized by motor dysfunctions, and it also has some non-motor symptoms. These symptoms are tightly associated with the loss and death of dopaminergic neurons. In the current research, cell transplantation has become an important topic, and it has very important significance in clinical application. Neural stem cells, embryonic stem cells, induced pluripotent stem cells and bone marrow mesenchymal stem cells have been used to the treatment of Parkinson's disease. Neural stem cells and embryonic stem cells exist a serious ethical dispute. As for induced pluripotent stem cells, there is a possibility of carcinogenesis. Therefore, more and more researches have been taken on BMSCs. Compared to other stem cells, BMSCs have more significant advantages.

Some studies have shown that BMSCs expressed genes and proteins related with the neural lineage, and have been displayed to hold neurogenic differentiation potential the proper conditions in vitro ⁴⁰⁻⁴². BMSCs transplantation restrained multiple parameters of spinal neuroinflammation found in diabetic mice ⁴³. It is important that BMSCs can release neurotrophic factors, including GDNF and BDNF to protect neurons ^{44, 45}.

In the reported studies, many methods were adopted to drive BMSCs to differentiate into DA neurons. And induction time is generally 12 days or more ⁴⁶. The vital method was to induce directional differentiation of cells by using cytokines. Astrocyte-derived bFGF is required for regulation of DA differentiation of the stem cells and promotes growth and survival of midbrain DA cells ⁴⁷⁻⁵⁰. SHH participates in a broad array of neurodevelopmental processes in the vertebrate embryo, including morphogenesis, cell proliferation and specification, and axon pathfinding ^{51, 52}. SHH exists in the postnatal and adult CNS, such as modulation of neuronal activity, progenitor cells and astrocytes as well as in differentiated neurons^{53 − 55}. FGF8 is essential for the development of multiple brain regions, such as suprachiasmatic nuclei (SCN) and hypothalamo-pituitary ^{56, 57}. The development and survive of DA neurons is associated with GF-SHH and FGF8 ^{58, 59}.

ABCA1 affects cognitive function, leading to amyloid-beta (Aβ) production and apoptosis and promote neurorestoration ⁶⁰⁻⁶².

In the present study, our aim was to explore the effects of TO901317 on the differentiation of hBMSCs into dopamine neurons. We found that the growth rate of cells in GF group and LXR + GF group was initially increased and later decreased with the extension of induction time compared to control group. But the growth rate of cells in LXR + GF group was not different with GF group. We hypothesized that TO901317 had little or no effect on cell proliferation, and TO901317 may have a special effect on cell differentiation. Some studies have explored the effect of GF on inducing BMSCs differentiation into dopaminergic neurons, but the result were designated DA neuronal progenitors without neuronal function ^{63, 64}. Our results showed that hBMSCs treated with GF alone or TO901317 combination with GF leaded to directed neuronal differentiation. TO901317 could promote differentiation of hBMSCs into dopaminergic neurons in cooperation with GF. The maximum induction efficiency was 67%, and the shortest induction time was 12 days when hBMSCs treated with GF alone. While the maximum induction efficiency was 91%, and the shortest induction time was 6 days when hBMSCs treated with 0.5 µM TO901317 in cooperation with GF. This greatly improved the efficiency of induction. Cells in GF group and LXR + GF group showed neun and nestin positive. The result of ELISA revealed cells only in LXR + GF treated secreted dopamine. This suggested that TO901317 could promote the maturation of cellular functions. All of these results together indicated that simultaneous addition of TO901317 and GF could significantly improve induction efficiency and shorten induction period of hBMSCs differentiation into DA neuron-like cells and suggested that LXR may be involved in the mechanisms of hBMSCs differentiation into DA neuron-like cells.

The lack of ABCA1 leads to transport disorders of central nervous system cholesterol, which in turn leads to defects in neuronal structure and function ⁶⁵. Our study found that ABCA1 mRNA significantly elevated and LXRs obviously decreased in LXR + GF group compared to GF group. Differentiation of hBMSCs into dopaminergic neurons may be related to LXR-ABCA1 pathway.

Induced-cells transplantation in PD rats could reduce the damage caused by 6-OHDA. Compared with the model group, the dopaminergic neurons in the treatment group had a significant increase. The results indicate that TO901317 can promote the differentiation of human bone marrow mesenchymal stem cells into dopaminergic neurons by activating the lxrs-abca1 signal pathway.

Collectively, based on GF to culture cells, 0.5 µM TO901317 could promote the differentiation of human bone marrow mesenchymal stem cells into dopaminergic neurons. Induced-cells had significant therapeutic effect of on Parkinson's disease. The mechanism of TO901317 promoting the differentiation of human bone marrow mesenchymal stem cells into dopaminergic neurons may be related to the activation of the LXR-ABCA1 signaling pathway

ABCA1

Adenosine triphosphate-binding cassette transporter A1

bFGF

Basic fibroblast growth factor

FBS

Fetal bovine serum

FGF8

Fibroblast growth factor 8

LXR

Liver X receptor

hBMSCs

Human bone marrow mesenchymal stem cells

Parkinson's Disease

SHH

Sonic hedgehog

Tyrosine hydroxylase

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

Chongqing science technology commission of China provided funding for research.

Acknowledgments

This work was supported by the Chongqing science technology commission of China (NO: cstc2017shms-zdyfX0053). We thank all of the participants of this study.

Author contributions

Junqing Yang and Hong Wang made substantial contribution to conception, design, and performance of the study. Miaomiao Li, Junqing Yang, Oumei Cheng, Zhe Peng, Yin Luo, Dongzhi Ran, Yang Yang, Pu Xiang, Haifeng Huang, Xiaodan Tan took part in all the experiments and carried out the data analysis. Miaomiao li wrote the final manuscript and all authors approved the final manuscript.

Conflicts of Interest

The authors declare that there is no competitive financial interest.

Ethics declarations

Ethics approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University and in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals.

Consent for publication

Not applicable

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Therapeutic effect of TO901317 on 6-OHDA-induced Parkinson rats in vitro and in vivo

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Abstract

Figures

Introduction

Materials And Methods

Materials

Animals

Differentiation of hBMSCs

Cell Counting kit-8 assay

Parkinson's Disease rats

Immunofluorescence

Enzyme-Linked Immunosorbent Assay (ELISA)

Western Blotting Test

Quantitative real-time PCR (qPCR)

Histopathological Examination

Statistical analysis

Result

Culture of hBMSCs

Effect of different concentrations of TO901317 on cell survival

Determination of the concentration of TO901317

Explore the adding time of TO901317

Explore the time period induced by TO901317

Dopaminergic neuron properties of inducing cells

The role of LXRs in cell differentiation and its possible mechanism

Establishment of Parkinson's disease model

The vivo effects of inducing cells

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

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