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Abstract

Objective:

Methamphetamine (MA) abuse induces neurotoxicity and causes neuronal cell apoptosis. Gastrodin is a traditional Chinese herbal medicine used for the treatment of nerve injuries, spinal cord injuries, and some central nervous system diseases as well. The present study investigated the neuroprotective effects of gastrodin against MA-induced neurotoxicity in neuronal cells and its potential protective mechanism.

Methods:

The primary cortex neuronal culture was divided into four groups (control group, MA group, MA + gastrodin group, and MA + gastrodin + small interfering RNA group). The neurotoxicity of MA was assessed by detecting apoptotic cells by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay and cell viability by cell counting kit 8 (CCK-8) method, the Tuj1-positive cells and the average axonal length were detected by immunofluorescence, and the expressions of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), cAMP-response element-binding (CREB), and brain-derived neurotrophic factor (BDNF) proteins were detected by Western blot.

Results:

The results of CCK-8 assay showed that 0.5 mM MA was an optimal concentration that induced neurotoxicity (p < 0.01). Pretreatment with 25 mg/L gastrodin exerted maximum protective effects on neuronal cells. The expression levels of cAMP, PKA, phosphorylated PKA, CREB, phosphorylated CREB, and BDNF proteins were decreased in the MA group, and pretreatment with gastrodin upregulated the expression levels of these proteins (p < 0.01). The expressions of PKA and CREB proteins showed no significant changes in the control group, MA group, and gastrodin group. Compared the MA + gastrodin + small interfering RNA group with MA + gastrodin group, the Tuj1-positive cells and the average axonal length were decreased significantly, while the number of apoptotic cells was increased (p < 0.05).

Conclusion:

Gastrodin has neuroprotective effects against MA-induced neurotoxicity, which exerts neuroprotective effects via regulation of cAMP/PKA/CREB signaling pathway and upregulates the expression of BDNF.

Keywords

Methamphetamine gastrodin cyclic adenosine monophosphate protein kinase A cAMP-response element-binding protein brain-derived neurotrophic factor

Introduction

Methamphetamine (MA) is a neural stimulant with high abuse potential¹ and inhibits the uptake of dopamine and norepinephrine in the presynaptic terminal. The MA abuse exerts a significant impact on the nervous system of adolescents as well as adults. Recent studies have claimed that adolescents who took MA led to side effects in the neural system.^2,3 Researchers have confirmed that MA induces oxidative stress, inflammation, and apoptosis in the cerebral regions such as hippocampus,^4
–6 but the underlying molecular mechanisms and signaling pathways involved in MA abuse remained unclear.

Rhizoma Gastrodiae (Tianma) is a famous traditional Chinese herbal medicine and the dried rhizome of Gastrodia elata Blume has been used for the treatment of headache, dizziness, spasm, epilepsy, stroke, amnesia, and other disorders for over centuries.⁷ Gastrodin, a phenolic glycoside, is the main bioactive constituent of Rhizoma Gastrodiae and is used for treating nerve injuries.⁸ In addition, gastrodin is used in the treatment of central nervous system (CNS) diseases including epilepsy, Alzheimer’s disease, Parkinson’s disease, affective disorders, cerebral ischemia/reperfusion, and cognitive impairment as well.⁹

There were no reports to date that focused on whether gastrodin had any neuronal protective effects on MA-induced neurological disorders in subjects or animal models.

Preclinical research has shown that gastrodin can decrease oxidative stress, alcohol cravings, and behaviors in animal models.^10,11

Previous studies have reported that few nerve damage treatment drugs induced neuroprotection against some neurodegenerative disorders via promoting cyclic adenosine monophosphate (cAMP)-response element-binding (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathway.¹² CREB acts on DNA and prompts the production of BDNF, playing an important role in the brain development and neurogenesis.¹³ Besides these, cAMP and protein kinase A (PKA) also mediated gastrodin-induced neuroprotection via CREB/BDNF signaling pathways.¹⁴

Hence, the present study was designed to assess the role of gastrodin and CREB/BDNF pathway in clarifying the neuroprotective effect against MA-induced neurotoxicity.

The present study aimed to explore whether gastrodin has neuroprotective effects on neurotoxicity induced by MA and whether the CREB/BDNF pathway was involved in the molecular mechanism of neuroprotection of gastrodin. The study provided a novel strategy for the treatment of neurotoxicity induced by MA abuse.

Materials and methods

Animals and materials

Animal use was in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kunming Medical University (KMU), the experimental procedure was approved by the Ethics Committee on Animal Care and Use from KMU. Pregnant Sprague-Dawley (SD) rats used in this study were purchased from the facility of experimental animals of KMU. The MA (purity above 98%) was provided legally by the Yunnan Provincial Public Security Department. The gastrodin was a kind gift from Prof. Di Lu of the Medical Biology Engineering Center of KMU.

Primary cortex neuron culture

Pregnant SD rats weighing 250–300 g and aged 4–6 months were used. Primary cortex neurons were obtained from 1-day-old neonatal SD rats according to the previously reported protocol. Briefly, a pool of neonatal SD rats, regardless of gender difference, were collected and placed in the culture disks after sterilization. The skull was removed after the rat was killed, and the blood vessels and meninges were moved carefully. The cortex region was isolated and excised into approximately 1 mm³ pieces, then digested with 0.05% trypsin (Gibco, Carlsbane, California, USA) at 37°C for 10 min and mixed with 10% bovine serum albumin (BSA; Gibco). The suspension was filtered with a membrane of diameter 0.22 µm and then centrifuged at 1000 r/min for 10 min. The pellets in the bottom were collected and resuspended in the complete culture medium (Hyclone, Logan, Utah, USA), which was composed of DMEM/high glucose, 10% fetal calf serum (Gibco), and 1% penicillin–streptomycin solution (Hyclone). The above steps were repeated twice. Neurons were dispensed and cultured in poly-l-lysine-coated 96-well plates at a density of 5 × 10⁵ cells/mL in DMEM, supplemented with 10% BSA, and 1% penicillin–streptomycin solution in 5% CO₂ at 37°C incubator for 4 h. Subsequently, the complete culture medium was replaced with a neurobasal medium (ThermoFisher Scientific, Waltham, Massachusetts, USA) with 2% B27 (Invitrogen, Carlsbane, California, USA). Half of the culture medium was changed every 3 days.

Transfection of small interfering brain derived neurotrophic factor (BDNF) RNA into cultured cortex neuron

Primary cortex neurons were cultured after reaching 50–80% confluency, followed by transfection procedure. For transplantation of small interfering BDNF (siBDNF) RNA, primary cultured cortex neurons were divided into four groups: control group (neurobasal medium only), MA group, MA + gastrodin group, and MA + gastrodin + siBDNF group. siBDNF was designed and synthesized by RiboBio Company (Guangzhou, China). Transfection was performed using SuperFectin™ II invitro siRNA transfection reagent (Pufei Biotech Co., China) according to the manufacturer’s protocol. In brief, transfection stock buffer and siBDNF were mixed, prepared, and then 3 μL of SuperFectin II reagent was added to the mixture. Mixture of siBDNF (100 nM) was added dropwise to the appropriate wells, respectively. After incubation at 37°C for 24 h, another 1.2 mL of fresh culture medium was added. Red Cy3-5′-fluorescence (RiboBio Co., GuangZhou, China) was observed with a fluorescence microscope (Leica CM1860, Germany) at 72 h after transfection to confirm that the expression of BDNF was downregulated.

TUNEL assay

A terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) kit (In Situ Cell Death Detection Kit, Cat. 12156792910, Roche, Switzerland) was used to determine the apoptosis. Reaction mixture of enzyme solution and labeling solution were added at a ratio of 1:9 (v/v), and the neurons were stored at 4°C overnight in the dark. After washing three times with 0.01 mol/L phosphate buffer saline (PBS), the neurons were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature, and photographs were taken using fluorescence microscopy (CM1860, Leica). In detail, five fields were selected randomly from each section, and three sections from each group were observed. Apoptosis was quantified by determining the percentage of TUNEL/DAPI using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, Maryland, USA).

Immunofluorescence staining

Immunocytochemical analysis of Tuj1 was used as a specific indicator of neurons of MA group, MA + gastrodin group, and MA + gastrodin + siRNA group. Tuj1 immunocytochemical analysis was performed to detect the neural changes. Briefly, neurons were directly permeated in 0.01 mol/LPBS containing 3% goat serum for 30 min at 37°C. Then, the neurons were incubated with appropriate concentrations of Tuj1 primary antibodies and secondary antibodies. The numbers of Tuj1-positive cells were quantified. Then the number of Tuj1-positive cells in different groups was counted by a high content screening system (ThermoFisher Scientific). Data were presented as means ± standard deviation.

CCK-8 assay

The cell viability of neurons after MA treatment was estimated by cell counting kit 8 (CCK-8; Biosharp, China). Neonatal rat prefrontal cortical neurons were cultured in a 96-well plate for 5 days, and each group has triplicated wells. Then, the neurons were treated with MA at different concentrations for 24 h, which were 0.125, 0.25, 0.5, 1.0, 1.5, and 2.0 mM, respectively, to determine the optimal concentration of MA which could cause obvious cellular neurotoxicity. Subsequently, 100 μL culture medium with 10 μL of CCK-8 solution was added to each well, and then the plate was incubated for 2 h at 37°C. Cell viabilities of different groups were tested by measuring the absorption at 450 nm with a microplate spectrophotometer reader (Bio-Rad Co., Hercules, California, USA) to represent relative cell viability.

Western blot

The cells were homogenized in cold radioimmunoprecipitation assay lysis buffer supplemented with proteinase inhibitor cocktail and phenylmethylsulfonyl fluoride. Lysates were centrifuged at 12,000 × g for 15 min at 4°C and then the protein concentrations were measured by bicinchoninic acid (BCA) assay. Subsequently, aliquots of cellular lysates were denatured. Then the samples were loaded and separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. The protein bands were transferred onto the polyvinylidene difluoride membrane. The membrane was incubated in the blocking buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4, containing 5% nonfat milk) at room temperature for 2 h, and then incubated overnight at 4°C with primary antibodies, including β-actin mouse polyclonal antibody (1:1000, Abcam, Cambridge, Massachusetts, USA), cAMP rabbit polyclonal antibody (1:1000, Abcam), PKA rabbit polyclonal antibody (1:1000, Abcam), phosphorylated PKA (pPKA) rabbit polyclonal antibody (1:1000, Abcam), CREB rabbit polyclonal antibody (1:1000, Abcam), phosphorylated CREB (pCREB) rabbit polyclonal antibody (1:1000, Abcam), and BDNF rabbit polyclonal antibody (1:1000, Millipore, USA), respectively. After overnight incubation, the membrane was washed with TBST for three times, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibody (anti-mouse immunoglobulin G ,IgG, 1:5000 or anti-rabbit IgG 1:5000(Abbkine, Wuhan, Hubei province, China) at room temperature for 2 h. The membranes were washed with TBST for three times and then was developed in an enhanced chemiluminescent kit (Beyotime, China). The images were captured by the Bio-Gel imaging system (Bio-Rad, Hercules, California, USA) equipped with Image Lab software (NIH, Bethesda, Maryland, USA). Densitometry analysis was performed for all proteins. β-Actin was used as an internal control.

Statistical analysis

Data analysis was performed with SPSS v20.0. Data were shown as means ± standard deviation. The effects of gastrodin on MA-administrated neurons were assessed using a one-way analysis of variance. t-Tests were used to analyze the effects of MA-induced neruotoxicity. The value of p < 0.05 was considered as statistically significant.

Results

MA-induced neurotoxicity in cortical neurons

To clarify the effect of MA on neurons, the primary cortical neurons after culturing were incubated with various concentrations of MA for 24 h. The bright-field images showed that the apoptotic cells were increased and the cell viability was decreased in a dose-dependent manner after the administration of MA. An optimal concentration of 0.5 mM MA induced obvious neurotoxicity on primary cortical neurons (Figure 1(a)). The number of neurons treated with different concentrations of MA was quantified (Figure 1(b)). Meanwhile, the length of axon of neurons after the MA administration was quantified (Figure 1(c)). The results demonstrated that MA-induced nerve injury in a concentration-dependent manner on MA-treated neurons. The CCK-8 assay was performed to investigate the cytotoxicity in primary cortical neurons as well. The results showed that 0.5 mM MA was an optimal concentration which induced obvious neurotoxicity on primary cortical neurons (Figure 1(d); *p < 0.05, **p < 0.01). Data for number of neurons, length of axon, and the viability percentage are presented in Table 1.

Figure 1.

MA-induced neurotoxicity in primary cortical neurons: (a) primary cortical neurons were treated with various concentrations of MA for 24 h, (b) number of neurons in different concentrations of MA, (c) length of axons in different concentrations of MA, and (d) cell viability of neurons detected by CCK-8 assay. The results of (b) to (d) showed that 0.5 mM MA was an optimal concentration that could induce obvious neurotoxicity in primary cortical neurons: *p < 0.05 and **p < 0.01. MA: methamphetamine.

Table 1.

Experimental results of neurons.^a

Group	Number of neurons	Length of axon (μm)	CCK-8 assay (OD)
Control	37.33 ± 0.58^b	38.33 ± 0.76^b	0.96 ± 0.18^b
0.125 mM	33.00 ± 1.00^b	35.50 ± 0.50^b	0.65 ± 0.08^b
0.25 mM	34.33 ± 1.15^b	31.50 ± 0.5^b	0.652 ± 0.14^b
0.5 mM	21.00 ± 1.00	25.43 ± 0.43	0.42 ± 0.09
1.0 mM	10.66 ± 0.55^c	3.33 ± 0.54^c	0.20 ± 0.01^c
2.0 mM	9.66 ± 0.57^c	1.66 ± 0.57^c	0.16 ± 0.03^c

OD: optical density; MA: methamphetamine.

^a Values are represented as mean ± standard deviation; n = 3. Number of neurons— ^b p < 0.01: compared 0.5 mM MA group with the control group, 0.125 mM MA group, 0.25 mM MA group, respectively; ^c p < 0.01: compared 0.5 mM MA group with 1.0 mM MA group, 2.0 mM MA group, respectively. Length of axon— ^b p < 0.01: compared 0.5 mM MA group with the control group, 0.125 mM MA group, 0.25 mM MA group, respectively; ^c p < 0.01: compared 0.5 mM MA group with 1.0 mM MA group, 2.0 mM MA group, respectively. Average of OD of CCK-8 assay— ^b p < 0.01: compared 0.5 mM MA group the with control group, 0.125 mM MA group, 0.25 mM MA group, respectively; ^c p < 0.01: compared 0.5 mM MA group with 1.0 mM MA group, 2.0 mM MA group, respectively.

Different concentrations of gastrodin exerted neuroprotective effects on MA-induced neurotoxicity in primary cortical neurons

Four hours before MA administration, the cultured neurons were pretreated with different concentrations of gastrodin. The concentration series were 12.5, 25, 50, and 100 mg/L. The number of neurons incubated with 0.5 mM of MA and with different concentrations of gastrodin was quantified. The results showed that 25 mg/L gastrodin could obviously ameliorate neurological damage caused by MA (Figure 2(a)). The results showed that 25 mg/L was the optimal concentration of gastrodin which could obviously ameliorate neurological damage caused by MA (Figure 2(b)). Meanwhile, the length of axons of neurons was also quantified. The data showed that the average axon length was the longest in the 25 mg/L gastrodin group (Figure 2(c)). The cell viability was measured by CCK-8 assay. The results showed that 25 mg/L gastrodin administration could significantly alleviate neurological damage induced by MA (Figure 2(d)).

Figure 2.

(a) Pretreatment of primary cortical neurons with different concentrations of gastrodin and then treated with 0.5 mM MA for 24 h, (b) number of neurons after treating with different concentrations of gastrodin, (c) length of axons after treating with different concentrations of gastrodin, and (d) cell viability detected by CCK-8 assay. The results of (b) to (d) showed that 25 mg/L gastrodin was an optimal concentration which could exert obvious neuroprotective effects on primary cortical neurons: *p < 0.05 and **p < 0.01. MA: methamphetamine.

The expression of BDNF, cAMP, PKA, pPKA, CREB, and pCREB in neurons treated with MA and gastrodin

The expression of BDNF was decreased in MA-administrated neurons during 24 h. Meanwhile, the crucial proteins of CREB/BDNF pathway were downregulation in neurons of MA-induced neurotoxicity group. The cAMP and PKA proteins were decreased in the MA group, while the cAMP and PKA proteins were increased in the MA + gastrodin group (**p < 0.01, Figure 3(a) and (b)). The expression of pPKA protein was decreased in the MA group, while the pPKA protein was increased in the MA + gastrodin group (**p < 0.01, Figure 3(c)). Meanwhile, the expression of CREB protein showed no significant change in the control group, MA group, and MA + gastrodin group (p > 0.05, Figure 3(d)). The expression of pCREB protein was decreased in the MA group, while pCREB protein was increased in the MA + gastrodin group (**p < 0.01, Figure 3(e)). The BDNF protein was decreased in the MA group, and BDNF protein was increased in the MA + gastrodin group (**p < 0.01, Figure 3(f)).

Figure 3.

The expression of cAMP, PKA, pPKA, CREB, pCREB, and BDNF proteins were detected with Western blot: (a) expression of cAMP protein: **p < 0.01, (b) expression of PKA protein: **p < 0.01, (c) expression of pPKA protein: **p < 0.01, (d) expression of CREB protein: **p < 0.01, (e) expression of pCREB protein: **p < 0.01, and (f) expression of BDNF protein: **p < 0.01. cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; pPKA: phosphorylated PKA; CREB: cAMP-response element-binding; pCREB: phosphorylated CREB; BDNF: brain-derived neurotrophic factor.

Gastrodin exerts a neuroprotective effect on MA-induced neurotoxicity by regulating CREB/BDNF signaling pathway

Tuj1-positive cells were observed in the control group, MA group, gastrodin group, and gastrodin + siBDNF group (Figure 4(a)). The number of Tuj1-positive cells was significantly increased in the MA + gastrodin group when comparing with the MA group. However, after the transfection of siBDNF, the number of Tuj1-positive cells was significantly decreased (**p < 0.01, Figure 4(b)). Compared with the MA group, the average axonal length of neurons was increased in the MA + gastrodin group. After the transfection of siBDNF, the average axonal length was significantly decreased (**p < 0.01, Figure 4(c)).

Figure 4.

Gastrodin could exert a neuroprotective effect on MA-induced neuronaldamage via upregulating the expression of BDNF: (a) Tuj1-positive neurons in the control group, MA group, MA + gastrodin group, and gastrodin + siBDNF group, (b) number of Tuj1-positive neurons in different groups: **p < 0.01, and (c) average axonal length of neurons in different groups: **p < 0.01. MA: methamphetamine; BDNF: brain-derived neurotrophic factor.

TUNEL-positive cells were counted in all four experimental groups (Figure 5(a)). Compared with the MA group, the number of apoptotic cells was decreased in the MA + gastrodin group (**p < 0.01). After transfection of siBDNF, the number of apoptotic cells was significantly increased in the gastrodin + siBDNF group when comparing with the MA + gastrodin group (**p < 0.01, Figure 5(b)).

Figure 5.

Gastrodin exerted a neuroprotective effect on MA-induced apoptosis of neuron by upregulating the expression of BDNF: (a) TUNEL staining of neurons in the control group, MA group, MA + gastrodin group, and gastrodin + siBDNF group and (b) number of apoptotic cells in different groups: **p < 0.01. MA: methamphetamine; BDNF: brain-derived neurotrophic factor.

Totally, the results showed that the expressions of cAMP, PKA, pPKA, CREB, pCREB, and BDNF proteins were obviously changed after gastrodin being an intervention factor with the MA-administered neurons, the neuroprotective effect of gastrodin was exerted on MA-induced neurotoxicity by upregulating the expressions of cAMP, PKA, CREB, and BDNF proteins.

Discussion

The present study investigated whether gastrodin has a preventive effect on MA-induced neurotoxicity. The experiments were conducted to demonstrate the hypothesis that gastrodin might enhance the function of CREB/BDNF signaling pathway, which contributes to the neuroprotective effects against MA-induced neurotoxicity. The present study data indicated that 0.5 mM MA could induce obvious neuronal death, and pretreatment of 25 mg/L gastrodin could reverse neuronal injury caused by MA to a greater extent. Gastrodin could exert neuroprotective effects by modulating the expression of crucial components of CREB/BDNF signaling pathway (the pathway shown as Figure 6). Our data provided a novel potential clinical application of gastrodin on MA-induced neurotoxicity.

Figure 6.

A schematic diagram of MA and gastrodin act on neuron via CREB/BDNF signaling pathway. MA: methamphetamine; CREB: cyclic adenosine monophosphate-response element binding; BDNF: brain-derived neurotrophic factor.

MA could induce neuronal injury. MA-induced necrosis in rat cortical neurons in vitro via a time- and dose-dependent manner, and necroptosis might be an important and newly identified method for cortical neuronal death by single high-dose MA administration.¹⁵ It is apparent that MA exposure was associated with significant effects on neural adaptation and innate immunity. And alterations in lymphocyte activity and quantity, changes of cytokine signaling, impairment of phagocytic functions, glial activation, and gliosis have been reported were concerned to the MA abuse or dependence. These drug-induced changes in immune response, particularly within the CNS, played a critical role in the addictive process of MA dependence, as well as for other substance-related disorders.¹⁶ According to a previous study, MA could accelerate cellular senescence and activated transcription of genes involved in cell-cycle control and inflammation by stimulating the production of sphingolipid messenger ceramide.¹⁷ In the present study, the MA-induced neurotoxicity cortical neuron model was established and the cellular neurotoxicity induced by MA was gradually accelerated with increased concentration of MA. Meanwhile, 25 mg/L gastrodin markedly attenuated the neurotoxicity. In previous studies, researchers have found that gastrodin could prevent steroid-induced osteonecrosis of the femoral head (ONFH) by anti-apoptosis,¹⁸ and gastrodin was demonstrated which had protective effects against N-Methyl-D-Aspartate (NMDA) toxicity on cultured hippocampal slices.¹⁹ In the present study, we confirmed that 25 mg/L gastrodin was the optimal concentration that could attenuate the neurotoxicity of MA in cortical neurons.

The mechanism of neuroprotective effect of gastrodin against MA-induced neurotoxicity on neuron was related to the modulation of CREB/BDNF signaling pathway. It was acknowledged that MA could modulate the production of interleukin-6 and tumor necrosis factor α via cAMP/PKA/CREB signaling pathway in lipopolysaccharide-activated microglia.^20
–22 Gastrodin treatment caused vasodilation in vascular smooth muscle cells via the PKA-dependent signaling pathway. The CREB/BDNF pathway has been shown to play a critical role in the memory processes.^23

–28 Therefore, we explored whether CREB/BDNF pathway was involved in the neurotoxicity induced by MA. The expression levels of pCREB and BDNF in primary cortical neurons were detected. The data showed that pCREB was decreased in primary cortical neurons treated with MA and the ratio of pCREB to CREB was decreased as well. Similar pattern changes occurred in BDNF expression. Taken together, these results suggested that the expressions of pCREB and BDNF were reduced in the MA-induced neurotoxicity model,^29,30 except for the altering CREB levels.

The results of expressions of cAMP, PKA, pPKA, CREB, pCREB, and BDNF proteins proved our hypothesis that the neuroprotective action of gastrodin might be related to the regulation of neurotrophic factors in neurons. Gastrodin only reduced the apoptotic neurons but also protected the neurons by regulating the expression of BDNF in MA-treated neuron.

BDNF is a member of the neurotrophic factor family.³¹ Literature has confirmed that the spinal cord injury could inhibit BDNF expression in the neurons, and also demonstrated significant inhibition of apoptosis weeks after sciatic nerve injury and facial nerve injury.³² In our previous study, gastrodin increased the expression of BDNF in the CNS of MA addictive rats. Meanwhile, after interference of the expression of BDNF, the number of neurons and length of axon of MA + gastrodin + siBDNF group was lower than the MA + gastrodin group, which indicating that pretreatment with siBDNF could reverse the protective effects of gastrodin.

In general, our data showed that MA administration could decrease the expressions of cAMP, pPKA, and pCREB proteins. Gastrodin could attenuate the neurotoxicity induced by MA. Gastrodin has neuroprotective effects against MA-induced neurotoxicity, which exerts the regulation of cAMP/PKA/CREB signaling pathway and upregulates the expression of BDNF.

Footnotes

Author contributions

Authors C-L M and L Li contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China [Grant Nos. 81160380, 81560302, and 81760337], the Joint Research Project of Science and Technology, Department of Yunnan Province & Kunming Medical University [No. 2017FE467-003], Program Innovative Research Team in Science and Technology in Yunnan Province [No. 2017HC007], the Science and Technology Innovation Team Project of Kunming Medical University [No. CXTD201604], and Scientific Research Fund of Education Department of Yunnan province [No. 2016YJS051].

ORCID iD

S-J Hong

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Neuroprotective effect of gastrodin in methamphetamine-induced apoptosis through regulating cAMP/PKA/CREB pathway in cortical neuron

Abstract

Objective:

Methods:

Results:

Conclusion:

Keywords

Introduction

Materials and methods

Animals and materials

Primary cortex neuron culture

Transfection of small interfering brain derived neurotrophic factor (BDNF) RNA into cultured cortex neuron

TUNEL assay

Immunofluorescence staining

CCK-8 assay

Western blot

Statistical analysis

Results

MA-induced neurotoxicity in cortical neurons

Different concentrations of gastrodin exerted neuroprotective effects on MA-induced neurotoxicity in primary cortical neurons

The expression of BDNF, cAMP, PKA, pPKA, CREB, and pCREB in neurons treated with MA and gastrodin

Gastrodin exerts a neuroprotective effect on MA-induced neurotoxicity by regulating CREB/BDNF signaling pathway

Discussion

Footnotes

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

References