Introduction: Parkinson’s disease (PD) is the most common neurodegenerative disease worldwide. Studies have shown that insulin-like growth factor-binding protein 5 (IGFBP5) may contribute to methamphetamine-induced neurotoxicity and neuronal apoptosis in PC-12 cells and rat striatum. Here, we studied the expression and role of IGFBP5 in the 6-OHDA-toxicant model of PD. Methods: PC-12 and SH-SY5Y cells were exposed to 50 μm 6-OHDA for 24 h. qRT-PCR, western blotting, CCK-8 assay, EdU staining, annexin V staining, and immunofluorescence were performed to study the effects of IGFBP5-specific siRNAs. The effects of IGFBP5 on a rat 6-OHDA model of PD were confirmed by performing behavioral tests, tyrosine hydroxylase (TH) immunofluorescence staining, and western blotting. Results: In the GSE7621 dataset, IGFBP5 was highly expressed in the substantia nigra tissues of PD patients compared to healthy controls. In PC-12 and SH-SY5Y cells, IGFBP5 was upregulated following 6-OHDA exposure in a dose-dependent manner. Silencing of IGFBP5 promoted PC-12 and SH-SY5Y proliferation and inhibited apoptosis under 6-OHDA stimulation. Silencing of IGFBP5 relieved 6-OHDA-induced TH-positive neuron loss. Hedgehog signaling pathway was predicted as a downstream signaling pathway of IGFBP5. Negative regulation between IGFBP5 and sonic hedgehog (SHH) signaling pathway was confirmed in vitro. The effects of IGFBP5 silencing on SH-SY5Y cells were partially reversed using cyclopamine, a direct inhibitor of the SHH signaling pathway. In addition, silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. Conclusion: Elevated IGFBP5 expression may be involved in 6-OHDA-induced neurotoxicity through regulation of the SHH signaling pathway.

Highlights of the Study

  • IGFBP5 was highly expressed in a 6-OHDA-toxicant model of PD.

  • Silencing of IGFBP5 promotes proliferation of 6-OHDA-treated neurons, alleviates apoptosis of 6-OHDA-treated neurons, and inhibits the loss of TH-positive neurons.

  • Silencing of IGFBP5 is neuroprotective due to regulation of sonic hedgehog signaling pathway.

The etiology and pathogenesis of Parkinson's disease (PD) are complex and have not been fully defined [1]. Currently, it is believed that the occurrence and development of PD are closely related to oxidative stress, mitochondrial dysfunction, neuroinflammation, and abnormal protein folding caused by endoplasmic reticulum stress and other factors and is the result of the coordinated action of multiple mechanisms [2]. Since PD is caused by an imbalance between the cholinergic system and the dopamine system, the drugs currently used to treat PD mainly include dopaminergic and anticholinergic drugs. However, long-term use can reduce the effect of therapy and lead to a range of side effects, such as nausea, hallucinations, delusions, drowsiness, dystonia, and significant movement problems [3, 4]. Therefore, identifying the etiology and pathological mechanism of PD will provide a theoretical basis for a comprehensive understanding of PD, follow-up targeted drug screening, and individualized treatment in different stages of clinical disease.

Insulin-like growth factor-binding protein 5 (IGFBP5) is a secreted protein that functions as a key regulator of the bioavailability of IGF1 [5]. IGFBP5 is highly conserved and is frequently dysregulated in cancers, osteogenesis, and autoimmune diseases [6, 7]. In addition, its expression is involved in the development of various diseases depending on the cellular context [8]. For example, high expression of IGFBP5 enhances the inflammation of glomerular endothelial cells and promotes diabetic kidney disease progression [9]. IGFBP5 contributes to high glucose-induced cardiac fibrosis and fibroblast proliferation [10]. IGFBP5 has also been reported to be critical in mediating methamphetamine-induced neuronal apoptosis [11]. However, the expression and functional roles of IGFBP5 in the development of PD remain unclear.

With the development of gene microarray and sequencing technology, comparing the transcriptome differences between pathological and physiological tissues helps in the search for differentially expressed genes related to PD. In this study, differentially expressed genes between patients with PD and healthy controls were screened using the GEO database. IGFBP5 was identified as an upregulated gene in substantia nigra tissues of patients with PD. Moreover, the expression and effects of IGFBP5 in 6-OHDA-toxicant model of PD, and the underlying mechanisms were revealed. This study aimed to clarify the role of IGFBP5 in PD and to confirm whether IGFBP5 can be used as a therapeutic target.

Bioinformatics Analysis

We used the GSE7621 dataset from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) to analyze the genes differentially expressed between PD patients and healthy controls. The GSE7621 dataset comprises substantia nigra tissues from postmortem brain of normal and PD patients. The upregulated genes in PD patients were screened using the “limma” package in the R language. Gene Ontology (GO) term function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to analyze the enrichment pathways related to IGFBP5 using the ClusterProfiler package in R.

Cell Culture

The rat adrenal pheochromocytoma cell line PC-12 and the human neuroblastoma cell line SH-SY5Y were purchased from Procell (Wuhan, China). PC-12 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% horse serum (Sigma, St. Louis, MO, USA) and 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). SH-SY5Y cells were cultured in RPMI-1640 medium supplemented with 10% FBS. Cells were maintained at 37°C in an atmosphere of 95% air and 5% CO2. Highly differentiated PC-12 cells were induced using 50 ng/mL nerve growth factor (Gibco) for 4 days.

For neurotoxin exposure, PC-12 and SH-SY5Y cells were exposed to 1, 5, 10, 50, or 100 μm 6-OHDA (Sigma) for 24 h followed by two washes with phosphate-buffered saline and were used in the following experiments. To inhibit the SHH signaling pathway, cells were treated with 5 μm cyclopamine (MedChemExpress, Monmouth Junction, NJ, USA) for 24 h.

Cell Transfection

Before neurotoxin exposure, PC-12 and SH-SY5Y cells were transfected with siRNAs specific for IGFBP5 or scrambled sequences (RiboBio, Guangzhou, China). The siRNAs designed by RiboBio are as follows. Homo sapiens IGFBP5 siRNA: 5'-GCA​AGT​CAA​GAT​CGA​GAG​AGA-3'; siRNA scrambled: 5'-GAG​GAA​GTA​GAA​CCG​AAC​AGT-3'; Rattus norvegicus IGFBP5 siRNA: 5'-GGG CTC​TTT​CGT​GCA​TTG​TGA-3'; siRNA scrambled: 5'-GGT​GCG​CGT​CGG​TTT​ATT TCA-3'. For cell transfection, cultures were washed twice with phosphate-buffered saline and transfected with 30 nm siRNAs using Lipofectamine® 3000 (Invitrogen, Carlsbad, CA, USA) at 37°C for 48 h.

Cell Counting Kit-8 Assay

PC-12 and SH-SY5Y cells were seeded in 96-well plates at a density of 5,000 cells/well followed by siRNA transfection and 6-OHDA treatment. Cell viability was measured using the Cell Counting Kit-8 kit (Sangon Biotech, Shanghai, China). The absorbance of each well was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

5-Ethynyl-2'-Deoxyuridine Staining

Following siRNA transfection and 6-OHDA treatment, PC-12 and SH-SY5Y cells in 96-well plates (5,000 cells/well) were stained with 50 μm 5-ethynyl-2'-deoxyuridine (EdU) solution (Abcam, Cambridge, MA, USA) for 2 h at 37°C. EdU-stained cells were analyzed using a Nikon Eclipse Ni fluorescence microscope (Nikon, Tokyo, Japan).

Apoptosis Analysis

Following siRNA transfection and 6-OHDA treatment, PC-12 and SH-SY5Y cells in 24-well plates (2 × 105 cells/well) were analyzed using an Annexin V-binding assay kit (Solarbio, Shanghai, China), according to the manufacturer’s instructions. Annexin V-FITC-positive cells were considered apoptotic cells and were counted by a flow cytometry (BD Biosciences, San Jose, CA, USA).

Immunofluorescence

Tyrosine hydroxylase (TH)-positive cells were detected following a standard immunohistochemical protocol as reported before [12]. Briefly, cells were fixed with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 at room temperature. After blocking with 5% FBS for 1 h at room temperature, cells were incubated with primary anti-TH antibody (1:100; Abcam) at 4°C overnight. The cells were then incubated with a fluorescent Alexa Fluor 488 conjugated secondary anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.

Rat brain tissues were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm thick sections. Substantia nigra were incubated with primary anti-TH antibody (1: 50; Cell Signaling Technology) at 4°C overnight, followed by incubation with a fluorescent Alexa Fluor 555 conjugated secondary antibody (1:200; Invitrogen) for 1 h at room temperature. Nuclei were labeled with DAPI. A Nikon Eclipse Ni fluorescence microscope (Nikon, Tokyo, Japan) was used to capture the stained tissue sections.

Real-Time Quantitative RT-PCR

Total RNA was extracted using TRIzol reagent (Sangon Biotech). Total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Real-time PCR was performed with the SYBR Green PCR kit (SparkJade, Shandong, China) on a Real-Time PCR System (Bio-Rad) with β-actin gene being used as an internal control. The 2-ΔΔCt method was used to analyze the data. The primer sequences used for qPCR are listed below. H. sapiens IGFBP5: 5'-CGA​GAA​AGC​CCT​CTC​CAT​GT-3' (forward), 5'-ACG​GGA​GTC​TCT​CTC​GAT​CT-3' (reverse); H. sapiens β-actin: 5'-ACA​GAG​CCT​CGC​CTT​TGC​C-3' (forward), 5'-GAT​ATC​ATC​ATC​CAT​GGT​GAG​CTG​G-3' (reverse); Rattus norvegicus IGFBP5: 5'-GCG​AGC​AAA​CCA​AGA​TAG​AGA​G-3' (forward), 5'-GGA​GTA​GGT​CTC​CTC​AGC​CA-3' (reverse); R. norvegicus β-actin: 5'-CCC​GCG​AGT​ACA​ACC​TTC​TTG-3' (forward), 5'-GTC​ATC​CAT​GGC​GAA​CTG​GTG-3' (reverse).

Western Blotting

A protein extraction kit (Solarbio) was used to extract the total proteins from cells and tissues. The protein concentration was measured using a BCA protein assay kit (Sangon Biotech). Proteins from each sample were separated by electrophoresis on 10%–12% SDS-PAGE gels, transferred onto polyvinylidene fluoride membranes, blocked with Tris-buffered saline with Tween-20 containing 5% FBS at 25°C for 1 h, and then incubated at 4°C overnight with primary antibodies against IGFBP5, Bcl-2, Bax, caspase-3, TH, SHH, SMO, Gli1, and β-actin (dilution 1:1,000, Abcam). Later, samples were incubated with horseradish peroxidase-conjugated secondary antibodies (dilution 1:1,000, Abcam) at 25°C for 1 h. The protein expression was detected with ECL reagents (Beyotime, Shanghai, China) using a chemiluminescent imaging system (Bio-Rad).

Animals

Eight-week-old Wistar male rats (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in standard controlled cages with 12 h light/dark cycle, 22 ± 2°C, ∼60% humidity, and free access to food and water. All procedures were approved by the Animal Care and Use Ethics Committee of Shaanxi Provincial People’s Hospital and performed in accordance with the standards of Animal Care and Use and the Animal Welfare Act.

6-OHDA Lesion and AAV Infection

Rats were randomly divided into four groups (eight rats per group): Sham, 6-OHDA model, 6-OHDA+AAV-shScr, and 6-OHDA+AAV-shRNA groups. Rats were anesthetized with 80/20 mg/kg ketamine and toluene thiazide intraperitoneally. The head of rats was fixed horizontally on the brain stereoscope, the hair was shaved, the skin was disinfected with iodine and alcohol, the scalp was cut along the midline, and the fontanel of the skull was fully exposed. Rats in the 6-OHDA model group were delivered with 20 μg 6-OHDA (in 4 μL normal saline with 0.2 mg/mL ascorbic acid), which was injected into the right medial forebrain bundle. The sham group of rats was injected with the same volume of vehicle. Three weeks following 6-OHDA injection, 6-OHDA+AAV-shScr and 6-OHDA+AAV-shRNA group of rats were, respectively, injected with 1 μL AAV-shScr (1 × 1012 vg/mL; AAV2/5 expressing scramble shRNA) or AAV-shRNA (1 × 1012 vg/mL; AAV2/5 expressing shIGFBP5) into the substantia nigra. Three months later, behavioral tests were performed and the rats were sacrificed for immunofluorescent staining.

Behavioral Tests

Before injection with 6-OHDA, all rats were trained on the behavioral tests for 2 days. For apomorphine-induced rotation, rats were subcutaneously injected with 0.25 mg/kg apomorphine, which was dissolved in a 0.2 mg/mL ascorbic acid in normal saline. A fixed camera was used to record the ipsilateral and contralateral rotations for 20 min. For rotarod and narrow beam tests, rats were trained 5 times per day for 2 days and were performed as previously described [13].

Statistical Analysis

The results are presented as the mean ± SD from three independent experiments. GraphPad Prism software (version 8.0) was used to perform statistical analysis and mapping. Comparisons between the two groups were conducted using a t-test. Comparisons between three and more measurements were conducted using one-way or two-way analysis of variance, followed by a Bonferroni post hoc test. Data were considered significantly different at p < 0.05.

IGFBP5 Was Highly Expressed in 6-OHDA-Treated PC12 and SH-SY5Y Cell Lines

GSE7621 was screened from the GEO database as a PD-related dataset. In the GSE7621 dataset, IGFBP5 was found to be upregulated in the substantia nigra tissues of PD patients compared with healthy controls (p < 0.05, Fig. 1a). To confirm the upregulation of IGFBP5 during PD development, PC-12 and SH-SY5Y cells were exposed to various doses of 6-OHDA. qRT-PCR data showed that IGFBP5 gene levels were significantly increased in PC-12 and SH-SY5Y cells exposed to 6-OHDA, and the increase induced by 6-OHDA was dose-dependent (p < 0.05, Fig. 1b, c). In addition, western blotting analysis showed that the protein levels of IGFBP5 were increased by 6-OHDA exposure in a dose-dependent manner (p < 0.05, Fig. 1d, e).

Fig. 1.

Expression of IGFBP5 in 6-OHDA-treated PC12 and SH-SY5Y cell lines. a GEO data analysis showed the expression of IGFBP5 in GSE7621 dataset that comprises substantia nigra tissue from 16 PD patients and 9 healthy controls. b-e PC-12 and SH-SY5Y cells were treated with 1, 5, 10, 50, or 100 μm 6-OHDA for 24 h. qRT-PCR data showing the expression of IGFBP5 gene in PC-12 (b) and SH-SY5Y cells (c). Western blotting of IGFBP5 proteins in PC-12 (d) and SH-SY5Y cells (e). n = 3. n.s. indicates no significance, *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

Fig. 1.

Expression of IGFBP5 in 6-OHDA-treated PC12 and SH-SY5Y cell lines. a GEO data analysis showed the expression of IGFBP5 in GSE7621 dataset that comprises substantia nigra tissue from 16 PD patients and 9 healthy controls. b-e PC-12 and SH-SY5Y cells were treated with 1, 5, 10, 50, or 100 μm 6-OHDA for 24 h. qRT-PCR data showing the expression of IGFBP5 gene in PC-12 (b) and SH-SY5Y cells (c). Western blotting of IGFBP5 proteins in PC-12 (d) and SH-SY5Y cells (e). n = 3. n.s. indicates no significance, *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

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Silencing of IGFBP5 Promotes Proliferation of PC12 and SH-SY5Y Cell Lines

To study the functional effects of IGFBP5 on the development of PD, the expression of IGFBP5 in PC-12 and SH-SY5Y cells was silenced by transfection with specific siRNAs. Compared to the scrambled control, IGFBP5 siRNAs remarkably reduced IGFBP5 expression in both PC-12 and SH-SY5Y cells (Fig. 2a). Cell Counting Kit-8 data showed that 6-OHDA exposure induced significant viability loss in PC-12 and SH-SY5Y cells, and the loss of viability was attenuated by transfection with IGFBP5 siRNAs (p < 0.05, Fig. 2b, c). In addition, 6-OHDA significantly reduced the percentage of EdU-positive cells, indicating the inhibitory effects of 6-OHDA on PC-12 and SH-SY5Y cell proliferation (p < 0.05). However, compared with the scrambled control group, the cells in the IGFBP5 siRNA groups showed higher proliferation rates (p < 0.05, Fig. 2d, e). These data suggest that IGFBP5 silencing promotes neuronal proliferation under 6-OHDA stimulation.

Fig. 2.

Effects of IGFBP5 silencing on the proliferation of PC12 and SH-SY5Y cell lines. a-e PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. a Western blotting of IGFBP5 proteins in PC-12 and SH-SY5Y cells. Survival of PC-12 (b) and SH-SY5Y cells (c) was measured by CCK-8 kits. Proliferation of PC-12 (d) and SH-SY5Y cells (e) was measured by EdU staining. n = 3. **p < 0.01, ***p < 0.001 compared to the indicated group. CCK-8, Cell Counting Kit-8.

Fig. 2.

Effects of IGFBP5 silencing on the proliferation of PC12 and SH-SY5Y cell lines. a-e PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. a Western blotting of IGFBP5 proteins in PC-12 and SH-SY5Y cells. Survival of PC-12 (b) and SH-SY5Y cells (c) was measured by CCK-8 kits. Proliferation of PC-12 (d) and SH-SY5Y cells (e) was measured by EdU staining. n = 3. **p < 0.01, ***p < 0.001 compared to the indicated group. CCK-8, Cell Counting Kit-8.

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Silencing of IGFBP5 Alleviates Apoptosis of PC12 and SH-SY5Y Cell Lines

We studied the effects of IGFBP5 silencing on apoptosis in PC12 and SH-SY5Y cells. As shown in Fig. 3a, b, 6-OHDA induced significant increases in PC-12 and SH-SY5Y cell apoptosis (p < 0.05), while IGFBP5 siRNAs significantly reduced the apoptotic cell rates (p < 0.05). Consistently, 6-OHDA treatment induced Bcl-2 downregulation, Bax upregulation, and the activation of caspase-3, whereas IGFBP5 siRNAs significantly reversed the effects of 6-OHDA on these protein expression (p < 0.05, Fig. 3c, d). These data demonstrate the antiapoptotic effects of IGFBP5 silencing in PC12 and SH-SY5Y cell lines.

Fig. 3.

Effects of IGFBP5 silencing on the apoptosis of PC12 and SH-SY5Y cell lines. a-d PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. Flow cytometry detection was performed to show the apoptosis of PC-12 (a) and SH-SY5Y cells (b). Annexin V-FITC-positive cells, the cells in UR (upper right) and LR (lower right), were counted as apoptotic cells. Western blotting of Bcl-2, Bax, and caspase-3 proteins in PC-12 (c) and SH-SY5Y cells (d). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

Fig. 3.

Effects of IGFBP5 silencing on the apoptosis of PC12 and SH-SY5Y cell lines. a-d PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. Flow cytometry detection was performed to show the apoptosis of PC-12 (a) and SH-SY5Y cells (b). Annexin V-FITC-positive cells, the cells in UR (upper right) and LR (lower right), were counted as apoptotic cells. Western blotting of Bcl-2, Bax, and caspase-3 proteins in PC-12 (c) and SH-SY5Y cells (d). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

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Silencing of IGFBP5 Inhibits TH-Positive Neuron Loss in PC12 and SH-SY5Y Cell Lines

TH is the rate-limiting enzyme of the biosynthesis of dopamine. TH-positive neurons are considered dopaminergic neurons, the loss of which is one of the main classical symptoms of PD. In this study, immunofluorescence assay results showed that 6-OHDA treatment remarkably reduced the number of TH-positive cells, while IGFBP5 siRNAs increased the number of TH-positive cells (Fig. 4a, b). This result was further confirmed by detecting the protein expression of TH via western blotting analysis (p < 0.05, Fig. 4c, d). Taken together, silencing of IGFBP5 was shown to be an effective way for inhibiting 6-OHDA-induced TH-positive neuron loss.

Fig. 4.

Effects of IGFBP5 on TH-positive neuron loss in PC12 and SH-SY5Y cell lines. a-d PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. Immunofluorescence assay results were performed to show TH-positive PC-12 (a) and SH-SY5Y cells (b). Western blotting of TH proteins in PC-12 (c) and SH-SY5Y cells (d). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

Fig. 4.

Effects of IGFBP5 on TH-positive neuron loss in PC12 and SH-SY5Y cell lines. a-d PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. Immunofluorescence assay results were performed to show TH-positive PC-12 (a) and SH-SY5Y cells (b). Western blotting of TH proteins in PC-12 (c) and SH-SY5Y cells (d). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

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IGFBP5 Inhibits Sonic Hedgehog Signaling Pathway

GO and KEGG analyses were performed to screen for IGFBP5-related signaling pathways. The Hedgehog signaling pathway was found to be a downstream signaling pathway of IGFBP5, which is negatively regulated by IGFBP5 (Fig. 5a). The enrichment genes are shown in Figure 5b. Western blot analysis showed that 6-OHDA significantly downregulated the expression of SHH, SMO, and Gli1 (p < 0.05). Compared with the scrambled control, transfection of cells with IGFBP5 siRNAs significantly upregulated SHH, SMO, and Gli1 expression (p < 0.05, Fig. 5c, d). Collectively, these data suggest that sonic hedgehog is a downstream signaling pathway of IGFBP5 in PC12 and SH-SY5Y cell lines.

Fig. 5.

Regulation of IGFBP5 on sonic hedgehog signaling pathway. a GO and KEGG analyses were performed to screen IGFBP5-related signaling pathways and hedgehog signaling pathway was identified. b The enrichment genes. c, d PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. The expression of SHH, SMO, and Gli1 in PC-12 (c) and SH-SY5Y cells (d) was measured by western blotting analysis. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

Fig. 5.

Regulation of IGFBP5 on sonic hedgehog signaling pathway. a GO and KEGG analyses were performed to screen IGFBP5-related signaling pathways and hedgehog signaling pathway was identified. b The enrichment genes. c, d PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h. The expression of SHH, SMO, and Gli1 in PC-12 (c) and SH-SY5Y cells (d) was measured by western blotting analysis. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

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Silencing of IGFBP5 Is Neuroprotective via Regulation of Sonic Hedgehog Signaling Pathway

Cyclopamine, a direct inhibitor of the sonic hedgehog signaling pathway [14], was used to treat cells to investigate the involvement of the sonic hedgehog signaling pathway in IGFBP5-mediated PD progression. As shown in Figure 6a, IGFBP5 siRNA significantly promoted SH-SY5Y cell proliferation in the absence and presence of cyclopamine (p < 0.05). Moreover, the treatment of cells with cyclopamine effectively inhibited the proliferation rate induced by IGFBP5 siRNAs (p < 0.05). In addition, IGFBP5 siRNAs inhibited SH-SY5Y cell apoptosis, whereas cyclopamine treatment reversed the antiapoptotic effects of IGFBP5 siRNAs (p < 0.05, Fig. 6b). The protein levels of TH were increased by IGFBP5 siRNAs, and cyclopamine treatment decreased TH protein levels (p < 0.05, Fig. 6c). These results suggest that IGFBP5 silencing exerts neuroprotective effects, possibly by regulating sonic hedgehog signaling.

Fig. 6.

Involvement of sonic hedgehog signaling pathway in IGFBP5-mediated PD progression. a-c PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h in the absence or presence of 5 μm cyclopamine. a Proliferation of SH-SY5Y cells was measured by EdU staining. b Flow cytometry detection was performed to show the apoptosis of SH-SY5Y cells. c Annexin V-FITC-positive cells, the cells in UR (upper right) and LR (lower right), were counted as apoptotic cells. Western blotting of TH proteins in SH-SY5Y cells. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

Fig. 6.

Involvement of sonic hedgehog signaling pathway in IGFBP5-mediated PD progression. a-c PC-12 and SH-SY5Y cells were transfected with 30 nm scrambled siRNA or IGFBP5 siRNA for 48 h and treated with 50 μm 6-OHDA for 24 h in the absence or presence of 5 μm cyclopamine. a Proliferation of SH-SY5Y cells was measured by EdU staining. b Flow cytometry detection was performed to show the apoptosis of SH-SY5Y cells. c Annexin V-FITC-positive cells, the cells in UR (upper right) and LR (lower right), were counted as apoptotic cells. Western blotting of TH proteins in SH-SY5Y cells. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.

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Silencing of IGFBP5 Attenuated Motor Deficits and Neuronal Damage in 6-OHDA-Induced PD Rats

To evaluate the role of IGFBP5 on PD in vivo, a 6-OHDA-induced PD rat model was established. Contralateral rotations and total time on the beam were increased, while latency to fall off from rotarod was decreased in the 6-OHDA group as compared to the sham group (p < 0.05, Fig. 7a–c). As compared to AAV-shScr injection, rats injected with AAV-shRNA had lower contralateral rotations, total time on the beam, and higher latency to fall off from rotarod. As the results shown in Figures 7d, e, 6-OHDA treatment significantly reduced TH expression, while the reduced TH expression could be reversed by AAV-shRNA injection (p < 0.05). In addition, SHH, SMO, and Gli1 were downregulated by 6-OHDA treatment, while the downregulation of these proteins was reversed by AAV-shRNA injection (p < 0.05).

Fig. 7.

Silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. a-f A 6-OHDA-induced PD rat model was established, and then AAV-shScr (AAV2/5 expressing scramble shRNA) or AAV-shRNA (AAV2/5 expressing shIGFBP5) was injected into the substantia nigra. Apomorphine-induced rotation test (a), rotarod test (b), and narrow beam test (c). The expression of TH in substantia nigra was measured by immunofluorescence (d) and western blot (e). f The expression of IGFBP5 and key proteins in the sonic hedgehog signaling pathway was measured by the western blot. n = 8. ***p < 0.001 compared to the indicated group.

Fig. 7.

Silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. a-f A 6-OHDA-induced PD rat model was established, and then AAV-shScr (AAV2/5 expressing scramble shRNA) or AAV-shRNA (AAV2/5 expressing shIGFBP5) was injected into the substantia nigra. Apomorphine-induced rotation test (a), rotarod test (b), and narrow beam test (c). The expression of TH in substantia nigra was measured by immunofluorescence (d) and western blot (e). f The expression of IGFBP5 and key proteins in the sonic hedgehog signaling pathway was measured by the western blot. n = 8. ***p < 0.001 compared to the indicated group.

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This study focused on the expression and effects of IGFBP5 in the 6-OHDA-toxicant model of PD. 6-OHDA is a type of neurotoxin that forms several toxic molecules, such as ROS, hydrogen peroxide and superoxide, and dopamine quinone to induce dopaminergic neuron death, and thus has been frequently used to mimic PD models [15]. IGFBP5 was upregulated in 6-OHDA-treated PC-12 and SH-SY5Y cells and a 6-OHDA-induced PD rat model. Silencing of IGFBP5 using specific siRNAs relieves 6-OHDA-induced neurotoxicity. In addition, the effects of IGFBP5 on neurons may be mediated via regulation of the sonic hedgehog signaling pathway.

IGFBP5 is a secretory protein that is widely expressed in the body, including lungs, bones, muscles, kidneys, and mammary glands [16]. IGFBP5 has been reported to be highly expressed in a variety of human diseases, such as diabetic kidney disease [9], diabetic neuropathy [17], pulmonary fibrosis [18], and cancer [8]. An accumulation of IGFBP5 was found in hippocampal pyramidal neurons and amyloid plaques in brains of Alzheimer patients [19], which suggested IGFBP5 as a risk factor for Alzheimer disease. Here, we demonstrated the high expression of IGFBP5 in the 6-OHDA-toxicant model of PD. Elevated IGFBP5 expression may serve as a potential biomarker for the clinical diagnosis of PD.

IGFBP5 has long been known as an IGF-binding protein that inhibits the activity of IGF [5]. Interestingly, the biological functions of IGFBP5 independent of IGF have gained significant interest. For instance, IGFBP5 is involved in prostate cancer cell radiosensitivity and G2/M phase arrest [20]. IGFBP5 is capable of inducing cell senescence, which may be due to its regulation of hypercoagulation and cell inflammation [21]. Repressing IGFBP5 expression in PC-12 cells and the striatum of rats relieved methamphetamine-induced neurotoxicity and neuronal apoptosis [11]. In addition, IGFBP5 contributes to dentinogenesis and tumor cell survival via regulating MAPK and PI3K/AKT signaling pathways [5], indicating IGFBP5 as a multifunctional protein. In this study, we demonstrated that silencing of IGFBP5 relieved 6-OHDA-induced neurotoxicity. Silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. However, cell culture in this study is missing the brain environment involved in the progressive nature of PD, including neuronal atrophy, microglial activation, astrocytosis, interaction between the central nervous system and the immune system, etc. Further studies should address this concern to reveal the complexity of IGFBP5 in the pathogenesis of PD.

The Hedgehog signaling pathway is an evolutionarily conserved molecular cascade that plays a significant regulatory role in human embryonic development as well as organism homeostasis [22]. In this study, KEGG analysis revealed that Hedgehog signaling may be a downstream signaling pathway of IGFBP5, which is involved in IGFBP5-mediated neurotoxic regulation. The hedgehog family comprises three mammalian proteins, sonic hedgehog, Indian hedgehog, and desert hedgehog. Sonic hedgehog is the best studied in the nervous system [23] and has marked roles in hippocampal neurogenesis [24], neuron-astrocyte communication [25], and neuronal apoptosis and survival [26, 27]. Importantly, sonic hedgehog is effective against the toxic actions of MPP+ and 6-OHDA and prevents neuron death via regulating the expression of neurotropic genes, such as BDNF, GDNF, and BMP7 [28‒31]. A number of animal studies have suggested the beneficial effects of sonic hedgehog in treating PD [30, 32, 33]. We studied the role of IGFBP5 in regulating sonic hedgehog signaling pathway in vitro. Silencing of IGFBP5 significantly increased the expression of key sonic hedgehog signaling proteins, including SHH, SMO, and Gli1, in 6-OHDA-treated neurons. In addition, by treating neurons with a direct inhibitor of the sonic hedgehog signaling pathway (cyclopamine), the sonic hedgehog signaling pathway was confirmed to be a downstream signaling pathway involved in IGFBP5-mediated neuroregulation.

IGFBP5 was found to be highly expressed in neurons exposed to 6-OHDA, a neurotoxin, which is topically used to mimic PD models. Silencing of IGFBP5 relieved 6-OHDA-induced neuron death possibly due to its regulation of the sonic hedgehog signaling pathway.

Animal studies were approved by the Animal Care and Use Ethics Committee of Shaanxi Provincial People’s Hospital and performed in accordance with the standards of Animal Care and Use and the Animal Welfare Act. This study did not involve human samples and did not require consent to participate.

The authors declare that they have no conflicts of interest.

No funds, grants, or other support was received.

Conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing: Shenglong Guo, Qi Lei, Qian Yang, and Ruili Chen; administrative support and provision of study materials or patients: Qian Yang and Ruili Chen. Final approval of the manuscript: all authors.

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

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