Ginsenoside Rg1 attenuates neurogenesis disorder and neuronal apoptosis in the rat hippocampus after spinal cord injury may involve BDNF/ERK signaling

doi:10.21203/rs.3.rs-1713893/v2

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Ginsenoside Rg1 attenuates neurogenesis disorder and neuronal apoptosis in the rat hippocampus after spinal cord injury may involve BDNF/ERK signaling

https://doi.org/10.21203/rs.3.rs-1713893/v2

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Objective: Our previous results have demonstrated that spinal cord injury(SCI) can induce hippocampus injury and depression in rodents. Ginsenoside Rg1 is efficient to prevent neurodegenerative disorders. This study aim to investigate the effects of ginsenoside Rg1 in hippocampus after SCI.

Method:We used a rat compression SCI model. Western blotting and morphological assays were used to investigate protective effects of Ginsenoside Rg1 in the hippocampus.

Results: The results demonstrated that BDNF/ERK signaling is altered in the hippocampus at 5 weeks after SCI. SCI attenuates neurogenesis and enhances the expression of cleaved caspase-3 in the hippocampus. Moreover, ginsenoside Rg1 can attenuate the expression of cleaved caspase-3 and improve the number of neurogenesis and the expression of BDNF/ERK signaling in the rat hippocampus. The results thus suggest that SCI affects BDNF/ERK signaling, and ginsenoside Rg1 can attenuate the damage of hippocampus after SCI.

Conclusion:We speculate that the protective effects of ginsenoside Rg1 in hippocampal pathophysiological after SCI may involve BDNF/ERK signaling. Ginsenoside Rg1 may serve as a therapeutic pharmaceutical product in the futurewhen seeking to counter SCI-induced hippocampal damage.

Spinal cord injury

hippocampus

neurogenesis

neurotrophin brain-derived neurotrophic factor(BDNF)

extracellular signal-regulated kinases(ERK)

Ginsenoside Rg1

Spinal cord injury (SCI) leads to motor and sensory deficits and to autonomic deregulation, causing permanent disability. Interestingly, cognitive deficits and depression occur in subjects with chronic SCI without head injury ^1-5, which might influence rehabilitation. SCI induces distal lesions in the thalamus, hippocampus, prefrontal cortex ^3,6-8. Nevertheless, the mechanisms of SCI-brain injury are unclear. The hippocampus is related to high-level neural activities such as emotion, learning, and memory and plays a role in SCI-induced neuronal apoptosis, neurogenesis⁹, and depressive-like behaviors^10-11. However, little is known about the exact mechanism of hippocampal injury after SCI.

Extracellular signal-regulated kinases (ERK) 1/2, a member of the mitogen-activated protein kinase (MAPK) subfamily, is activated by physiological extracellular stimuli, such as neurotransmitters, neurotrophic factors, and growth factors. Activated ERK1/2 induces diverse cellular responses after translocating to the nucleus. The ERK1/2 pathway is required for neurodegeneration and neuroprotection under pathological conditions ^12-13. Pignataro et al. showed that ERK1/2 phosphorylation is involved in the neuroprotection induced by remote ischemic post-conditioning¹⁴. Our previous results suggested that SCI can decrease ERK1/2 phosphorylation signaling in hippocampus and induce depression¹⁵.

Neurotrophin brain-derived neurotrophic factor (BDNF) is one of the most widely distributed neurotrophic factors in the CNS. It plays an important role in the pathological progress or functional recovery of neurological trauma and central degenerative diseases^16-18. Increased BDNF levels increase neuronal survival and promote the regeneration and sprouting of damaged neuron axons^19-20. There is a correlation between BDNF and the prevalence and management of a variety of neuropsychiatric disorders, including schizophrenia²¹, Alzheimer disease ^22-23, mood disorders²⁴, and Parkinsonism²⁵. In addition, BDNF activates various intracellular signaling pathways, including ERK^26-27. BDNF may interact with other signaling pathways and co-regulate cellular functions such as survival, differentiation, and metabolism ^28-29. Moreover, it is involved in adult neurogenesis in the hippocampus³⁰. However, the changes of BDNF/ERK signaling in response to SCI in the hippocampus is unclear.

Panax Ginseng C. A. Meyer is a traditional medicine that has been used to prevent against the neurodegenerative disorders^31-34, which is recorded in the Bencao Gangmu (Encyclopedia of Herbs). Ginsenoside Rg1 is one of primary component found in Panax ginseng with plenteous biological activity^35-37. Studies have demonstrated that Ginsenoside Rg1 facilitates neurogenesis and neurotrophin expression^38-39, and is effect to reverse hippocampal synaptic plasticity in Alzheimer’s disease⁴⁰. However, whether ginsenoside Rg1 affects SCI-induced hippocampus injury is still unclear.

The hippocampus receives information and then responds negative effects include depression, cognitive defects, and anxiety. We hypothesized that SCI affects BDNF expression, resulting in intracellular signaling changes, a decreased level of activated ERK, and hippocampus injury, and ginsenoside Rg1 reverses the negative effects of SCI in hippocampus. Therefore, we investigated whether ginsenoside Rg1 reverse the chronic SCI-induced injury in hippocampus involving BDNF/ERK signaling.

Animals and ethics statement

We purchased adult male Sprague-Dawley(SD) rats (body weight 220 ± 20 g) from Laboratory Animal Centre, Jiangxi University of Traditional Chinese Medicine (China). Rats were given enough food and water and housed under stationary conditions: temperature (22 ± 1°C), a humidity of 54–66%, and a light/dark cycle (lights on from 08:00 to 20:00 h). Rats were randomly divided into four groups after 7 days of adaption (n = 10):Sham group(laminectomy), SCI group(laminectomy with SCI), SCI+Ginsenoside Rg1(40 mg/kg) group and sham+Ginsenoside Rg1 group(40 mg/kg). All procedures were approved and implemented in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, revised 1996) and the Animal Ethics Committee of the First Affiliated Hospital of Nanchang University. The experimental process is presented in Figure 1A.

Compression SCI model

Anesthetized rats with sodium pentobarbital (150 mg/kg, i.p.) were performed under aseptic conditions throughout the surgery. Iodophor was applied to sterilize back skin. The laminectomy was performed at the spinal cord (T9) without injuring the dura.The sham animals were subjected to laminectomy without SCI. SCI of other groups were performed using an aneurysm clip (FT220T, Am Aesculap-Platz 78532 Tuttlingen, Germany) with a calibrated force of 70 g ⁴¹. Next, core muscles around vertebra were sutured in layers. An electric blanket was used to help rats recover from anesthesia. To prevent wound and urinary tract infections, penicillin (2.5 mg/kg, subcutaneously) was administered for 3 days postoperatively ⁴². To avoid urinary retention, bladders were kneaded twice daily. The bladder emptying reflex was typically established at 7–14 days. All procedures were approved and implemented in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, revised 1996) and the Animal Ethics Committee of the First Affiliated Hospital of Nanchang University.

Assessment of locomotor activity

Because SCI causes a poor condition on the first day after SCI, spontaneous locomotor recovery was assessed by the Basso, Beattie, Bresnahan (BBB) locomotor rating scale ⁴³ at postoperative day 3 and repeated measurement weekly until 5 weeks. The assessment of locomotor activity was recorded from 0 (no observable movements) to 21 points (normal gait) as previous study⁴³. A locomotor score of > 2 in the SCI rats and an of < 21 points in the sham rats were excluded at postoperative day 3.

Bromodeoxyuridine(BrdU) administration and Ginsenoside Rg1 administration

To assess the effect on cell proliferation and neurogenesis after chronic SCI. From days 25 to 35 post-injury, rats in both groups received once daily intraperitoneal injections of BrdU (50 mg/kg, 10 mg/mL in 0.9% saline) ⁹, ⁴⁴. Ginsenoside Rg1 was purchased by Nanjing Plant Origin Biological Technology Co., Ltd., Nanjing, China. Ginsenoside Rg1 was dissolved in normal saline(10 mg/ml). The dose and route of ginsenoside Rg1 in our experiment were administrated as previous results ⁴⁵. Animals were administered ginsenoside Rg1(intraperitoneally, 40 mg/kg) daily for 35 days after SCI.

Brain tissue sampling

Twenty-four hours after behavioral tests, rats were anesthetized with sodium pentobarbital (150 mg/kg, i.p.), and slowly perfused with 250 mL PBS. For western blotting (n = 4 per group), hippocampus tissue was collected and homogenized (in 150 mM NaCl, 20 mM Tris–HCl, 2 mM EDTA, 1% Triton X-100, 1 mM PMSF, phosphatase inhibitor cocktail; pH 7.4), the supernatant was collected after centrifugation (12,000 rpm, 40 min, 4°C). BCA protein assay kit (Beyotime Biotechnology, Inc., China) was used to assess the protein concentration. The proteins were stored at -80°C until analysis. For morphological assay, brain tissues were collected after perfusion with 4% paraformaldehyde (PFA), transferred to 4% (w/v) PFA in PBS, and treated with 30% sucrose solution until sunk to the bottom. They were next embedded in paraffin (n = 3 per group) or optimal cutting temperature compound (OTC) (n = 3 per group).

Western blotting analysis

Equal amounts of protein in hippocampal samples from per group (20 µg) were subjected to 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Millipore, Bedford, MA). Then 5% nonfat milk was used to blocked membranes for 2 h and incubated at 4°C for 12 h with primary antibodies: anti-ERK1/2 (1:1000, Abcam Co., UK), anti-phospho-ERK1/2 (1:1000, Abcam Co., UK), anti-BDNF (1:1000, Abcam Co., UK) and anti-β-actin (1:8000, Santa Cruz Biotechnology, Santa Cruz, CA). After extensive washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, Santa Cruz Biotechnology) for 2 h at room temperature. Proteins were detected using an enhanced chemiluminescence detection kit (GE Healthcare, Buckinghamshire, UK) and band densities were quantified using Image-J software (NIH, Scion Corporation, Frederick, MD). Data were generated from four rats per group.

Immunohistochemical staining

Paraffin embedded tissues were cut into coronal sections (5 μm thick) using a microtome (RM2235, Leica Biosystems, Germany), deparaffinized, and rehydrated in a graded series of ethanol to PBS. Antigen retrieval was conducted in citrate buffer (10 mM, pH 6.0) at 99°C for 40 min and blocked with endogenous catalase in 3% hydrogen peroxide. Next, sections were blocked in 5% bovine serum albumin (BSA) at room temperature for 30 min and incubated overnight with anti-phospho-ERK1/2 (1:100, Abcam Co., UK) and anti-BDNF (1:100, Abcam Co., UK) primary antibodies. The sections were rinsed with PBS and incubated with biotinylated secondary antibody and streptavidin-peroxidase conjugate at 37°C for 30 min. Finally, sections were stained with 3.3′-diaminobenzidine tetrahydrochloride (DAB), counterstained with hematoxylin. The hippocampus were visualized and analyzed using a light microscope (Olympus bx51, Japan) in at least six representative images per rat (n = 3 per group). The staining intensity of BDNF and p-ERK was quantified by densitometry using Image J software (NIH).

Immunofluorescence staining

A frozen thermostat (Leica Biosystems, Germany) was used to cut brain tissue into 40 μm coronal sections, the OTC samples were stored in 4°C antifreeze (30% ethylene glycol, 10% 2 M PBS). The sections were blocked in 5% BSA for 2 h and incubated overnight at 4°C with the following primary antibodies diluted in PBS: anti-caspase 3 (1:200; Abcam Co., UK), anti-Bromodeoxyuridine(Brdu; 1/500, Wuhan servicebio technology CO.,LTD, China) . and goat anti-doublecortin (DCX; 1/200, Wuhan servicebio technology CO.,LTD, China). After washing in 0.05 M PBS, the sections were immersed in Alexa-488-conjugated secondary antibody (goat anti-rabbit IgG; rabbit anti-goat IgG, 1:200, Sigma-Aldrich) for 2 h at room temperature in a dark room. The sections were washed in PBS and mounted in 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, CA) for neuronal nuclear staining. Next, the sections were covered with a coverslip and observed under a fluorescence microscope (´400; Olympus). Fluorescence images were acquired using a confocal scanning microscope (LSM780, Carl Zeiss, Germany). Proliferation and neurogenesis in the hippocampus were analyzed using a light fluorescence microscope (Zeiss AxioImager, Germany) in at least six representative images per rat (n = 3 per group). The number of BrdU+ or DCX+ cells and the immunofluorescence intensity of cleaved caapase-3 were analyzed using Image J software (NIH).

Statistical analysis

Data are means ± standard errors of the mean (SEM). Western blot of the target proteins was repeated three times. All data were tested for normality and homogeneity of variance, and repeated measures two-way analysis of variance (ANOVA) was used to compare BBB scores between the groups of rats over 5 weeks. One-way ANOVA was applied to assess differences between groups, and Tukey’s post hoc test was used to the pairwise comparisons. Using SPSS software (version 25.0; SPSS Inc., Chicago, IL, USA) to analysis data, and using GraphPad Prism software (version 8.0; GraphPad Software Inc., San Diego, CA, USA) to plot data.A p-value < 0.05 was considered indicative of statistical significance.

Recovery of hindlimb locomotor function after SCI

Rats without SCI maintained a score of 21 points. The SCI group exhibited significant impairment of hindlimb locomotor function (p<0.001, Figure. 1B). The increase in hindlimb function from day 7 to day 14 was greater than during other periods. The score was moderate for up to 5 weeks, indicating severe SCI ⁴³. Compared with SCI group, hindlimb locomotor function was increased in ginsenoside Rg1 treatment group(p<0.001, Figure. 1B).

Ginsenoside Rg1 improves cell proliferation and neurogenesis in the dentate gyrus(DG)

The protective response of ginsenoside Rg1 in cell proliferation and neurogenesis were assessed by counting BrdU+ and DCX+ cells at 35 days post-injury. Compared to the sham group, the SCI group showed a significant decline in BrdU+ cells (t = 10.32, df = 10, p < 0.001) (Figure. 2A,B). In addition, SCI significantly attenuated the number of DCX+ cells in the hippocampal DG region (t = 5.51, df = 10, p < 0.001) (Figure. 2A,C). However, compared to the SCI group, ginsenoside Rg1 can improve the number of DCX+ cells(t=7.673, df=10, p < 0.001 ) and BrdU+ cells(t=8.258, df=10, p < 0.001)(Figure.2) after SCI. Therefore, chronic SCI suppressed proliferation and neurogenesis in the hippocampal DG region, ginsenoside Rg1 can reverse these changes induced by SCI.

Ginsenoside Rg1 attenuates the immunofluorescence intensity of cleaved caspase-3 in hippocampus

Increased expression of cleaved-caspase3 is a biomarker of mitochondrial apoptosis. Compared with sham group, the immunofluorescence intensity of cleaved caspase-3 was significant increased in SCI group in hippocampus (DG, t=18.65, df=10, p < 0.001; CA1, t=5.919, df=10, p < 0.001) . However, compared to the SCI group, ginsenoside Rg1 can attenuate the immunofluorescence intensity of cleaved caspase-3 in DG(t=6.233, df=10, p < 0.001)(Figure. 3) and CA1(t=18.27, df=10, p < 0.001)(Figure. 4), indicating that ginsenoside Rg1 may prevent neuronal apoptosis in the hippocampus after SCI.

Changes of BDNF/ERK signaling in hippocampus

Compared to the sham group, the western blotting results showed that SCI decreases the expression of BDNF (t = 2.837, df = 6, p < 0.05) and phosphorylated ERK1/2 (p-ERK1/2) (t = 10.90, df = 6, p<0.001) (Fig. 4B), in agree with the immunohistochemical results. Indicating SCI changes BDNF/ERK signaling in the hippocampus. However, the western blotting results(Figure. 5) and the immunohistochemical results(Figure. 6) showed that ginsenoside Rg1 activates the expression of BDNF and p-ERK1/2 in the hippocampus after SCI.

SCI impairs sensorimotor ability, bladder/bowel function, sexual function, and emotion. Prior studies focused on the physiopathology of the injured spinal cord. Clinical studies have shown that SCI can lead to anxiety and depression ^46-47. SCI impacts neurogenesis, hippocampus-dependent learning and memory or emotions ^48-50. However, the molecular details of neurogenesis remain unclear. In addition, there is little data regarding the injury mechanisms of hippocampus induced by SCI and the effects of these mechanisms with ginsenoside Rg1. In this study, we used an SCI model to investigate the pathomechanism of hippocampal remodeling and neurogenesis(Figure. 7). The results suggest that chronic SCI not only changes BDNF/ERK signaling in the hippocampus but also leads to neuroapoptosis and neurogenesis disorders. In addition, SCI caused significant impairment of locomotion of the posterior limbs up to 5 weeks postoperatively. Therefore, SCI can change BDNF/ERK signaling, which may be involve in remodeling and neurogenesis in the hippocampus. More importantly, our results showed that chronic treatment with ginsenoside Rg1 can reverse these changes in neuroapoptosis, neurogenesis disorders and BDNF/ERK signaling in hippocampus after SCI. We speculate that the reversed effects of ginsenoside Rg1 in hippocampal pathophysiological after SCI may involve BDNF/ERK signaling.

Wu et al. reported that SCI disrupts neurogenesis in the hippocampus⁶. SCI decreases the expression of NMDA, GABAA, and M1 receptors, which was accompanied by fluctuations of the theta rhythm and spatial memory function in the hippocampus and an effect on hippocampal neurogenesis⁹. However, the injured neurogenesis of hippocampus after SCI was remain debated. Steffen et al. reported that neurogenesis was unaltered in neurogenic regions (DG and SVZ) of the brain after SCI⁴⁴. Our results are in agreement with previous reports ^6,9, and suggest that SCI decreases the number of DCX+ and BrdU+ cells in the DG, which implies induction of neurogenesis disorder. The discrepant results may be a result of injuries at different levels (cervical cord or thoracic cord), different degrees of injury (mild, moderate or severe), and/or different detection times (acute or chronic)⁵¹. SCI may affect axon pathways communicating with the hippocampus. Li S ea al. reported that ginsenoside Rg1 can improve neuron proliferation of dentate cells in gerbils performed to transient global ischemia and proliferation ability of hippocampal progenitor cells^38,52. In our study, the results showed that ginsenoside Rg1 can improve the number of DCX+ and BrdU+ cells in the DG after SCI.

Neural stem cells persist in the adult mammalian brain, and may engage in neurogenesis ^53-54. The neurogenic areas are distributed principally in the subependymal zone and the hippocampal DG. Neurogenesis plays an important role in learning and memory. BDNF binds to the tyrosine kinase tropomyosin-related kinase B (TrkB) receptor and activates intracellular downstream signaling ⁵⁵, including that of the ERK pathway ²⁶, possibly contributing to differentiation of immature neurons ^56-57 and neuroapoptosis ^58-60. BDNF promotes neural differentiation and cell survival ^61-62. The BDNF/TrkB system enhances the neurogenesis-promoting role of BDNF in the hippocampus of organotypic mice⁶³. BDNF influences both neurogenesis and the migration of novel neurons ^62, ⁶⁴. To further investigate the effects of ginsenoside Rg1 linked to the potential neuroprotective ability in SCI-induced hippocampus injury associated with the BDNF/ERK signaling. We examined the expression of BDNF/ERK and found that the preventive effects of ginsenoside Rg1 in this SCI model were also accompanied with changes in BDNF/ERK protein expression in hippocampus. Here, we found that chronic SCI reduced hippocampal BDNF expression, consistent with the finding of a previous study on acute SCI ⁶⁵. Immunofluorescence staining showed that the cleaved caspase-3 levels increased significantly in hippocampus after SCI; these are markers of apoptosis. More interesting, chronic dose with ginsenoside Rg1 in SCI rats significantly increased hippocampal BDNF/ERK protein and decreased the cleaved caspase-3 levels. Ginsenoside Rg1 has been reported neuroprotective effects within the central nervous system. Therefore, we speculate that BDNF/ERK signaling may involve the hippocampal changes after SCI. Moreover, further work showed that ginsenoside Rg1 owns neuroprotective ability in hippocampus changes induced by SCI.

However, these results can not clearly explain the complex changes in hippocampus induced by SCI. This study had several limitations. First, there was no explanation for the suppressed BDNF/ERK signaling. Second, we did not identify other factors affecting the positive effects of ginsenoside Rg1 in BDNF/ERK signaling; these may weaken both neurogenesis and neuroapoptosis.

We used a compression SCI model to investigate hippocampus remodeling after SCI and the neuroprotective effects of ginsenoside Rg1. Ginsenoside Rg1 not only increased BDNF/ERK signaling but also suppressed neurogenesis disorder in the hippocampus after SCI. Attenuation of BDNF/ERK signaling may increase the risk of hippocampal remodeling. However, ginsenoside Rg1 may play positive role in injured hippocampus after SCI. Our study provides a new insight in protective effects of ginsenoside Rg1 against neurogenesis disorder and neuronal apoptosis. Ginsenoside Rg1 may be a valuable therapeutic drug in SCI-induced hippocampal damage.

Funding

The Scientific Research initial foundation of Jiangxi Provincial People's Hospital(NO.13)

Disclosure Statement

The authors report no conflict of interest.

Availability of data and material

Please contact the authors for data requests.

The English in this document has been checked by Textcheck at least two professional editors, both native speakers of English.

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No competing interests reported.

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Ginsenoside Rg1 attenuates neurogenesis disorder and neuronal apoptosis in the rat hippocampus after spinal cord injury may involve BDNF/ERK signaling

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