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Akt signaling pathway
Experimental Gerontology 124 (2019) 110653
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Resveratrol delays 6-hydroxydopamine-induced apoptosis by activating the PI3K/Akt signaling pathway
T
Nanqu Huanga,b,1, Ying Zhanga,1, Mingji Chena, Hai Jinc, Jing Niea, Yong Luod, Shaoyu Zhoua,e, ⁎ Jingshan Shia, Feng Jina, a
Key Laboratory of Basic Pharmacology and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Guizhou, China Drug Clinical Trial Institution, The First People's Hospital of Zunyi & The Third Affiliated Hospital of Zunyi Medical University, Guizhou, China c Institute of Digestive Diseases of Affiliated Hospital, Zunyi Medical University, Guizhou, China d Department of Neurology, The First People's Hospital of Zunyi & The Third Affiliated Hospital of Zunyi Medical University, Guizhou, China e Department of Environmental Health, Indiana University Bloomington, IN, United States b
A R T I C LE I N FO
A B S T R A C T
Section Editor: Thomas Foster
This study aimed to determine whether resveratrol (Res) delays the progression of 6-hydroxydopamine (6OHDA)-induced apoptosis via activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Sprague-Dawley (SD) rats were unilaterally injected with 6-OHDA (8 μg/4 μL) into the substantia nigra of the midbrain. Res (15 and 30 mg/kg) was given orally to the rats for a total of 36 days to examine its protective effects. We first tested whether Res can delay the progression of 6-OHDA-induced damage by measuring weight and performance on behavioral tests (rotarod, open field test and grid test) and further explored whether this effect is related to the activation of the PI3K/Akt signaling pathway using immunohistochemistry (IHC) and Western blotting (WB). Our results showed that the damage induced by 6-OHDA gradually worsened, while Res 30 mg/kg treatment significantly improved motor function and increased body weight. Compared with those in the model group, the number of dopaminergic neurons cells and the expression of PI3K-110α, p-Akt Ser473, and pro-caspase-3 in the Res 30 mg/kg group were significantly increased, and the Bax/Bcl-2 ratio and the level of activated caspase-3 was decreased. The results indicate that Res ameliorates 6-OHDA-induced apoptosis and motor dysfunction via activating the PI3K/Akt signaling pathway, delaying the progression of Parkinson's disease (PD) symptoms in this model.
Keywords: Parkinson's disease Resveratrol Phosphoinositide 3-kinase Protein kinase B 6-Hydroxydopamine
1. Introduction Since its first description by British surgeon James Parkinson approximately 200 years ago, Parkinson's disease (PD) has been extensively studied, and treatments for PD have emerged over the past two centuries (Goetz, 1986; Parkinson, 2002; Przedborski, 2017). However, PD, which is still a threat to public health worldwide, is the second most prevalent neurodegenerative disorder (Cuenca et al., 2018). The causes of PD are not fully understood, but the disease is believed to be associated with genetic, environmental and aging factors (Schneider et al., 2017). Therefore, it is vital to detect PD in its early stage and take effective preventive measures to control its progression. Weight can be used for the early detection of PD because weight loss may indicate progressive deterioration in patients with PD (Umehara et al., 2017; Wills et al., 2016). In addition, the loss of dopaminergic
neurons is a key pathological feature of PD, and the upregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway decreases injury to dopaminergic neurons both in vivo and in vitro (C.R. Wu et al., 2018; Xiromerisiou et al., 2008; Xu et al., 2013; Yang et al., 2018). Increasing the number of dopaminergic neurons is important to slow the progression of PD. Our previous work showed that resveratrol (Res) has a protective effect against 6-hydroxydopamine (6-OHDA)induced PD in rats (Feng et al., 2008). Res, as a phytoalexin, has been found to possess many pharmacological functions, such as inhibiting of neuronal apoptosis and improving energy metabolism (Z. Wu et al., 2018). In the present study, we further explored whether the protective effect of Res against the apoptosis of dopaminergic neurons is related to the regulation of the PI3K/Akt signaling pathway.
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Corresponding author. E-mail address: [email protected] (F. Jin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.exger.2019.110653 Received 13 March 2019; Received in revised form 30 May 2019; Accepted 5 July 2019 Available online 08 July 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.
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2. Materials and methods
injection with 2% sodium pentobarbital at a dose of 50 mg/kg and then placed in a standard stereotaxic device (68038, RWD, Shenzhen, China). The method for the establishment of the model were performed as follows (Fig. 1B): A unilateral midbrain substantia nigra injection of 4 μL of 6-OHDA (2 μg/μL) was carried out to establish a rat model of dopaminergic (DA) neuron injury, and the sham group was injected with an equal volume of normal saline containing 0.2% vitamin C, as described in our previous work (Feng et al., 2008).
2.1. Drugs, reagents and antibodies Reagent grade Res (purity ≥98% by HPLC analysis) was purchased from Zelang Medical Technology (Nanjing, China). 6-OHDA (catalog no. H4381) and L-ascorbic acid (catalog no. A5960) were purchased from Sigma-Aldrich (St. Louis, MO, USA). B-cell lymphoma 2 (Bcl-2, catalog no. D163267), Bcl-2-associated X protein (Bax, catalog no. D120073), activated caspase-3 (catalog no. D260009), and TBS (catalog no. B040126) were purchased from Sangon Biotech (Shanghai, China). Akt (catalog no. 9272S), p-Akt Ser473 (catalog no. 4060P), pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1, catalog no. 3062p), and p-PDK1 (catalog no. 3061S) were purchased from Cell Signaling Technology (Beverly, MA, USA). PI3K p110-alpha (catalog no. 21890-1AP) was purchased from Proteintech Group (Wuhan, China). Goat antirabbit and mouse IgG-HRP (catalog no. M21003) was purchased from Abmart (Shanghai, China). Tyrosine hydroxylase (TH, catalog no. ab41528) and pro-caspase-3 (catalog no. ab179517) were purchased from Abcam (Cambridge, MA, USA). β-Actin (catalog no. AA128), GAPDH (catalog no. AF0006), PMSF (catalog no. ST505), RIPA lysis buffer (catalog no. P0013B), an SDS-PAGE Gel Quick Preparation kit (Catalog No. P0012AC) were purchased from Beyotime Biotechnology (Shanghai, China). Benzylpenicillin sodium for injection (catalog no. H13021633) was purchased from CSPC Zhongnuo Pharmaceutical (Shijiazhuang, China). A two-Step Immunohistochemistry (IHC) Detection kit including HRP-conjugated goat anti-rabbit IgG and H2O2 (catalog no. PV-6001) that was used in IHC was purchased from ZSGBBIO Corporation (Beijing, China). A DAB Substrate kit (catalog no. DA1010), DMSO (catalog no. D8371), 0.01 M PBS (powder, pH 7.2–7.4, catalog no. P1010), phosphatase inhibitor cocktail (catalog no. P1260), trypsin-EDTA solution, 0.25% (with phenol red, catalog no. T1320), penicillin-streptomycin liquid (catalog no. P1400), Tween-20 (catalog no. T8220), Triton x-100 (catalog no. T8200), Tris (catalog no. T8060), glycine (catalog no. G8200), and SDS (catalog no. S8010) were purchased from Solarbio Life Science (Beijing, China). Hydrophobic PVDF (0.45 μm, catalog no. IPVH00010) was purchased from Merck (Darmstadt, Germany). PageRuler™ Prestained Protein Ladder (catalog no. 26616) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). A BCA Protein Assay kit (catalog no. GK5012) was purchased from Generay (Shanghai, China). SDS-PAGE loading buffer (catalog no. ZS306) was purchased from Zoman Biotechnology (Beijing, China). Enhanced chemiluminescence (ECL) reagents (catalog no. E002-100) were purchased from 7seaBiotech (Shanghai, China).
2.3. Behavioral experiments All behavioral experiments were carried out between 8:30 a.m. and 5:30 p.m. The animals were transferred to the experimental room at least 1 h before the test to allow them to acclimatize to the test environment. 2.3.1. Rotarod test The rotarod test is a classic method for detecting motor function in rodents (Hamm et al., 1994). The apparatus consisted of a cylindrical arrangement of thin 75-mm diameter steel rods. Before the experiment began, all rats were trained to achieve stable performance. The speed for the training sessions was set at 12 rpm, and the cut-off time was 30 s. The rats in all groups were trained on the rotarod until they stayed on the rod for at least the cut-off time. The animals were allowed to remain stationary for 10 s at 0 rpm, and then the rotational speed was steadily increased to 12 rpm over a 20 s interval until the rats fell off the rod. The time that each animal remained on the rotating bar was recorded. The maximum time was 600 s per trial. The apparatus automatically recorded the time to 0.1 s and stopped recording when the rats fell off the rotating rod. The speed was set at 12 rpm and accelerated at a rate of 3 rpm per min. After the rats were unilaterally injected with 6-OHDA, the rotarod test was performed every 7 days to detect progressive deficits in muscle coordination in the rats. The data are presented the time spent on the rotating rod over the three test trials. 2.3.2. Open field test The open field apparatus contained 4 squares that were 50 cm in length and 50 cm in width, surrounded by a 50 cm high wall, which was painted black. The animals were placed in the center of the apparatus for free exploration, and their behavioral parameters were recorded for 5 min. The apparatus was washed with a 5% ethanol solution before each behavioral test to preclude the possible effects of odors left by the previous subjects. Control and experimental rats were intermixed to minimize the possible influences of the circadian rhythm on rat behavior. We used the TopScan™ behavioral testing system to quantify the distance traveled and the moving speed of the animals.
2.2. Animals and treatment
2.3.3. Grid test The grid apparatus was an independent metallic grid (50 cm × 40 cm, 1 cm × 1 cm per hole) dangling vertically and fixed at a height of 1 m above the ground (Fig. 1C). Wood chips at least 20 cm thick from the cages were prepared in advance and placed on the ground to prevent damage to the rats from falling. In order to prevent the rats from climbing to the top of the grid and hovering, a lid was added to the top of the grid. The rats were placed upright in the center of the grid and were allowed to grasp the mesh with four paws. The time that it took each rat to fall was recorded with a stopwatch, and the cut-off time was 300 s.
A total of 50 male SD rats (210 ± 15 g, 7 weeks old) were obtained from the Experimental Animal Center of the Third Military Medical University (Chongqing, China; specific-pathogen-free grade II; certificate no. SCXK 2012–0005). The rats were housed in specific pathogen free (SPF)-grade animal facilities (certificate no. SCXK 2014-0004) at Zunyi Medical University on a 12 h light/dark cycle at 23 °C ( ± 2 °C) with free access to food and water. The use of animals was in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1978). All procedures in the present study were approved by the Animal Care and Use Committee of Zunyi Medical University. After one week of adaptive feeding, all the rats were divided into the following 5 groups (n = 10 per group) by the random number method: the sham group, the normal group (30 mg/kg Res), the model group (6OHDA), the Res-15 group (6-OHDA +15 mg/kg Res), and the Res-30 group (6-OHDA +30 mg/kg Res). Specific animal experiments were performed as shown in Fig. 1A. Res (15 and 30 mg/kg) was given orally to the rats beginning one day before the establishment of the PD model for a total of 36 days. The rats were anaesthetized by intraperitoneal
2.4. Western blot (WB) and IHC staining and counting Briefly, rat midbrain tissues were homogenized and lysed in RIPA lysis buffer supplemented with the protease inhibitor PMSF and 1× phosphatase inhibitor cocktail. The lysates were treated on ice for 30 min and centrifuged at 14,000 ×g for 15 min. The supernatant was acquired, and the protein levels were quantified by a BCA assay kit. 2
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Fig. 1. Schematic diagrams. (A) A simple schematic diagram of the animal experimental procedure. (B) A simple schematic diagram of the establishment of the animal model. (C) The grid test.
Equal amounts of total protein (30 μg per lane) were loaded. Then, the lysates were separated using 8–12% SDS-PAGE in running buffer (10× running buffer: 30.3 g Tris, 188 g glycine, 10 g SDS, and ddH2O to 1 L; diluted to 1× with ddH2O for use). The gel was run at a low voltage (60 V) to separate the proteins, and a higher voltage (100 V) was used for the stacking gel until the dye ran off the bottom of the gel. PVDF membranes were cut to suitable dimensions. The sponge and filter paper were soaked in transfer buffer (10× transfer buffer: 30.2 g Tris, 150.2 g glycine, and ddH2O to 1 L; diluted to 1× with ddH2O; ratio of water:methanol:10× buffer = 7:2:1), and the PVDF membrane was soaked in methanol. Then, the sandwich was transferred at 4 °C in 1× buffer at 220 mA for 90 min. Following three washes with TBST (15 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20), the membranes were blocked with 5% nonfat milk in TBST at room temperature for 1 h. Following three washes with TBST, the membranes were incubated at 4 °C overnight with the following specific primary antibodies: Bcl-2 (1:1000), Bax (1:1000), activated caspase-3 (1:1000), Akt (1:1000), pAkt Ser473 (1:1000), PDK1 (1:1000), p-PDK1 (1:1000), PI3K-p110α (1:1000), β-actin (1:2000), and GAPDH (1:2000). Following three washes with TBST, the membranes were incubated with secondary antibodies (goat anti-rabbit and mouse IgG-HRP, 1:2000) at room temperature for 2 h. The protein-antibody complexes were visualized using the ECL reagent. The intensity of the bands was quantitated with Quantity One software (Bio-Rad). IHC staining and counting was performed as described previously in detail (Huang et al., 2017) and according to the instructions of the two-step IHC detection kit. Rat brains were cut into 35-μm transverse free-floating sections on a horizontal sliding microtome. The sections were used for immunohistochemical staining with antibodies against TH (a marker of DA neurons; 1: 200).
between the means were analyzed by Bonferroni's post hoc t-test with corrections. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Effect of Res on the body weight of 6-OHDA-exposed rats Initially, there was no significant difference in the body weights of the rats in each group. The body weights of the rats in each group continued to increase for 30 days. However, compared with the sham rats, the 6-OHDA rats showed a lower weight gain. At the same time, compared with those of the 6-OHDA rats, the body weights of the Res 30 mg/kg rats increased significantly (Fig. 2A). 3.2. Effect of Res on behavioral changes in 6-OHDA-exposed PD model rats 3.2.1. Rotarod test During the 1st, 2nd and 3rd weeks after model establishment, rat motor function was detected by rotarod. The rats showed motor dysfunction during the first week. During the second week, the motor function of the high-dose Res rats significantly improved. By the third week, the motor function of the 6-OHDA group further decreased, indicating that the damage induced by 6-OHDA gradually worsened, paralleling the progressive worsening of PD (Wills et al., 2016). A high dose of Res significantly improved 6-OHDA-induced motor dysfunction in rats (Fig. 2B). The motor function of the low-dose Res group decreased during the third week, indicating that the protective effect of Res 15 mg/kg was not enough to block the progressive damage caused by 6-OHDA.
2.5. Statistical analysis 3.2.2. Open field test Both the distance traveled (Fig. 2C) and the speed (Fig. 2C). D) of the model group were lower than those of the sham group. At the same time, the spontaneous motor ability of the high-dose Res group
The data in this study are expressed as the mean ± SEM. Statistical significance was assessed by one-way ANOVA using SPSS 22.0. When ANOVA indicated significant differences, pairwise comparisons 3
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Fig. 2. Weight and behavioral results. (A) Body weight results; ⁎P < 0.05 vs the model group, #P < 0.05 vs the sham group. (B) Rotarod results; ⁎P < 0.05 vs the sham group, #P < 0.05 vs the model group; (C) Distance traveled in the open field test. (D) Velocity in the open field test. (E) Grid test results (mean ± SEM, n = 9–10).
analysis with an anti-TH antibody. As shown in Figs. 3B, 6-OHDA caused an approximately 50% decrease in TH-positive nigral neurons compared with the number in the sham group. Meanwhile, compared with no treatment, Res increased the number of TH-positive cells in the SNc of the rats. Similarly, the results of the WB assay regarding TH protein expression were consistent with the IHC results (Fig. 3C).
significantly improved. 3.2.3. Grid test The rats in the model group fell earlier than the rats in the sham group, and their motor function was significantly weakened. A high dose of Res significantly increased the motor function of the model rats, allowing the rats to stay on the grid for longer (Fig. 2E).
3.4. Effect of Res on 6-OHDA-induced apoptosis in the midbrain 3.3. Effect of Res on 6-OHDA-induced DA neuronal loss in the substantia nigra pars compacta (SNc) region
Bax was significantly upregulated in the midbrain of the model group compared with the sham group (Fig. 4B). Bcl-2 was significantly decreased (Fig. 4C), and there was a significant increase in the Bax/Bcl2 ratio (Fig. 4D). Pro-caspase-3 (Fig. 4E) was decreased and activated
After the behavior tests, the rats were sacrificed. The brains were sectioned, and dopaminergic neurons were quantified by IHC and WB 4
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Fig. 3. Protective effects of Res against the 6-OHDA-induced loss of dopaminergic neurons in the rat substantia nigra. (A) Representative pictures of TH-positive cells in the rat substantia nigra (scale bar is 200 μm, solid circle: SNc); a: sham, b: normal (Res 30 mg/kg), c: model (6-OHDA 8 μg), d: 6-OHDA + Res 15 mg/kg, e: 6OHDA + Res 30 mg/kg. (B) Graph summarizing the number of TH-positive cells in the rat SNc. (C) The protein expression of TH (mean ± SEM, n = 4–5).
(Tsai et al., 2001). Compared with that in the sham group, the level of p-PDK1 in the model group decreased, while the level of p-PDK1 in the Res group increased compared with that in the model group. However, these differences were not statistically significant (Fig. 5C). The level of p-Akt Ser473 in the model group was significantly lower than that in the sham group, while the p-Akt Ser473 levels in the high-dose Res group were significantly higher than those in the model group (Fig. 5D).
caspase-3 (Fig. 4F) was increased. These results suggest that 6-OHDA induces apoptosis in midbrain neurons in rats, whereas a high dose of Res can significantly decrease Bax, activated caspase-3, increase Bcl-2, and Pro-caspase-3 expression.
3.5. Effect of Res on 6-OHDA-induced PI3K/Akt signaling pathway changes in the midbrain
4. Discussion
To investigate whether the PI3K/Akt signaling pathway is involved in the protective effects of Res against 6-OHDA-induced neuronal injury, we detected some of the proteins involved in this pathway. The results showed that PI3K-110α was decreased in the model group compared with the sham group, but the difference was not significant. However, it is interesting to note that a high dose of Res significantly increased the expression of PI3K-110α (Fig. 5B). PDK1 mainly phosphorylates the Thr308 site of the Akt protein, resulting in Akt activation
At present, the most frequently used neurotoxins for modeling PD are 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-OHDA (Lane et al., 2012). MPTP-lesioned monkeys best mimic the symptomatology of PD seen in human patients, while rats appear to be resistant to the effects of MPTP (Mokry, 1995). For that reason, 6-OHDA is used to damage the substantia nigra in rodents. Performing 5
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Fig. 4. Effect of Res on the protein level of the 6-OHDA-induced expression of apoptosis-related proteins in the midbrain in rats. (A) Representative bands of Bax, Bcl2, pro-caspase-3 and activated caspase-3 in the midbrain of the different groups. (B) The protein level of Bax. (C) The protein level of Bcl-2. (D) Bax/Bcl-2 ratio. (E) The protein level of pro-caspase-3. (F) The protein level of activated caspase-3 (mean ± SEM, n = 4).
clinical symptom of PD (Zesiewicz and Hauser, 2001). Because weight loss may predict the exacerbation of PD (Umehara et al., 2017; Wills et al., 2016), we detected the progressive development of PD through changes in behavior and weight. The results showed that the exogenous toxic substance 6-OHDA can cause progressive damage in rats. Compared with that of the rats in the 6-OHDA group, the body weight of the rats in 30 mg/kg Res group increased significantly. In addition, Res at a dose of 30 mg/kg significantly alleviated 6-OHDA-induced motor dysfunction. To confirm whether Res delays the progression of PD, we executed two other behavioral experiments to verify the protective effects of Res. In both the open field and grid tests, Res showed neuroprotective effects. Based on these results, we further explored the potential mechanisms of Res. The number of dopaminergic neurons is closely related to motor function (Li et al., 2018). In this study, WB and IHC were used to detect
behavioral tests on animals with nigrostriatal lesions represents a valuable noninvasive method for assessing the influence of the damaged DA system on locomotor activity. This model produces well-defined and stable behavioral deficits, but the success rate of the model is closely related to the researcher's familiarity with the technique and requires much practice. We conducted two preliminary experiments before this, but they were abandoned due to failure of the model. For our third experiment, we collected and analyzed data. The model was established by a skilled graduate student, and the experiment was carried out successfully. In this study, we validated our hypothesis that Res could effectively delay the progression of PD and further explored the potential underlying mechanisms. Due to the degeneration of dopaminergic neurons in the SNc, the ability of the brain to control motor coordination decreases. This eventually leads to motor dysfunction, which is the main
6
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Fig. 5. Effect of Res on the PI3K/Akt signaling pathway in the midbrain of rats exposed to 6-OHDA. (A) Representative bands of PI3K-110α, p-PDK1, and p-Akt. (B) The protein level of PI3K-110α. (C) The protein phosphorylation level of p-PDK1. (D) The protein phosphorylation level of p-Akt Ser473 (mean ± SEM, n = 4).
Fig. 6. A schematic diagram showing how Res delays 6-OHDA-induced apoptosis by activating the PI3K/Akt signaling pathway.
expression of activated caspase-3. The results indicate that Res may have neuroprotective effects against dopaminergic neuron damage, possibly by modifying the apoptotic pathway. The PI3K/Akt signaling pathway participates in the development, survival and function of neurons and plays an important role in the occurrence and progression of PD (Ding et al., 2016; Xiromerisiou et al., 2008; Yang et al., 2018; Zhong, 2016). PI3K p110α plays an important protective role in the process of oxidative stress-induced apoptosis (Matheny and Adamo, 2009). 6-OHDA induces oxidative stress and apoptosis, which is suitable for simulating oxidative stress-induced PI3K/Akt inhibition (Liu et al., 2015; Schober, 2004; C.R. Wu et al., 2018; Xu et al., 2013). The results showed that, compared with that in the sham group, the expression of PI3K-110α in the model group exhibited a downward trend, but the difference was not statistically significant. However, it is interesting that Res at the dose of 30 mg/kg significantly increased the expression of PI3K-110α. The level of p-Akt Ser473 in the model group was significantly lower than that in the sham group, while the p-Akt Ser473 levels in the Res treatment groups were
TH expression in rats, which directly and indirectly reflects the number of dopaminergic neurons. TH is the rate-limiting enzyme in the synthesis of catecholaminergic neurotransmitters and catalyzes the conversion of tyrosine to L-DOPA. It is not only a target gene of gene therapy for Parkinson's disease but also a specific marker of dopaminergic neurons (Dunkley et al., 2005). The results showed that 6-OHDA induced an approximately 50% decrease in the number of TH-positive nigral neurons compared with the number in the sham group. Meanwhile, compared with no treatment, Res increased the number of THpositive cells in the SNc area of the rats. Similarly, the results the WB assay for TH expression were consistent with the IHC results. These results showed that Res can reduce the loss of dopaminergic neurons in 6-OHDA-exposed rats. To verify whether the protective effect is caused by anti-apoptotic effects, we detected changes in apoptosis-related proteins. The results showed that 6-OHDA induced the apoptosis of midbrain neurons in rats, whereas Res at a dose of 30 mg/kg significantly decreased the protein expression of Bax, increased the protein expression of Bcl-2 and pro-caspase-3, and decreased the protein 7
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significantly higher than those in the model group. We hypothesize that Thr308 is not the major phosphorylation site for Akt activation by Res (Fig. 6).
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5. Conclusion We propose that Res may play a protective effect by activating PI3K110α and p-Akt Ser473, which in turn inhibit the initiation and function of apoptosis-related proteins. Res protects dopaminergic neurons from 6-OHDA-induced apoptosis via activating the PI3K/Akt signaling pathway, ultimately delaying the progression of PD. Funding This work was supported by the National Natural Science Foundation of China (81660599, 81460548), The Postgraduate Education Foundation of Guizhou Province (KYJJ2017008), The Zunyi Medical University Funds (2013F-686, F-738), The Open Funds of Key Laboratory of Basic Pharmacology of Ministry of Education of China (JCYL-K-012), The 15851 Talent Elite Project, The Funds of Department of Education of Guizhou Province ([2016]038), and Shijingshan's Tutor Studio of Pharmacology [GZS-2016(07)]. Declaration of Competing Interest All authors declare no conflict of interest. References Cuenca, L., Gil-Martinez, A.L., Cano-Fernandez, L., Sanchez-Rodrigo, C., Estrada, C., Fernandez-Villalba, E., Herrero, M.T., 2019. Parkinson's disease: a short story of 200 years. Histol. Histopathol. 34, 573–591. Ding, G., Zhao, J., Jiang, D., 2016. Allicin inhibits oxidative stress-induced mitochondrial dysfunction and apoptosis by promoting PI3K/AKT and CREB/ERK signaling in osteoblast cells. Exp. Ther. Med. 11, 2553–2560. Dunkley, P.R., Bobrovskaya, L., Graham, M.E., von Nagy-Felsobuki, E.I., Dickson, P.W., 2005. Tyrosine hydroxylase phosphorylation: regulation and consequences. J. Neurochem. 91, 1025–1043. Feng, J., Wu, Q., Lu, Y.F., Gong, Q.H., Shi, J.S., 2008. Neuroprotective effect of resveratrol on 6-OHDA-induced Parkinson's disease in rats. Eur. J. Pharmacol. 600, 78–82. Goetz, C.G., 1986. Charcot on Parkinson's disease. Mov. Disord. 1, 27–32. Hamm, R.J., Pike, B.R., O'Dell, D.M., Lyeth, B.G., Jenkins, L.W., 1994. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. J. Neurotrauma 11, 187–196. Huang, C., Zhu, L., Li, H., Shi, F.G., Wang, G.Q., Wei, Y.Z., Liu, J., Zhang, F., 2017.
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Resveratrol delays 6-hydroxydopamine-induced apoptosis by activating the PI3K/Akt signaling pathway
T
Nanqu Huanga,b,1, Ying Zhanga,1, Mingji Chena, Hai Jinc, Jing Niea, Yong Luod, Shaoyu Zhoua,e, ⁎ Jingshan Shia, Feng Jina, a
Key Laboratory of Basic Pharmacology and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Guizhou, China Drug Clinical Trial Institution, The First People's Hospital of Zunyi & The Third Affiliated Hospital of Zunyi Medical University, Guizhou, China c Institute of Digestive Diseases of Affiliated Hospital, Zunyi Medical University, Guizhou, China d Department of Neurology, The First People's Hospital of Zunyi & The Third Affiliated Hospital of Zunyi Medical University, Guizhou, China e Department of Environmental Health, Indiana University Bloomington, IN, United States b
A R T I C LE I N FO
A B S T R A C T
Section Editor: Thomas Foster
This study aimed to determine whether resveratrol (Res) delays the progression of 6-hydroxydopamine (6OHDA)-induced apoptosis via activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Sprague-Dawley (SD) rats were unilaterally injected with 6-OHDA (8 μg/4 μL) into the substantia nigra of the midbrain. Res (15 and 30 mg/kg) was given orally to the rats for a total of 36 days to examine its protective effects. We first tested whether Res can delay the progression of 6-OHDA-induced damage by measuring weight and performance on behavioral tests (rotarod, open field test and grid test) and further explored whether this effect is related to the activation of the PI3K/Akt signaling pathway using immunohistochemistry (IHC) and Western blotting (WB). Our results showed that the damage induced by 6-OHDA gradually worsened, while Res 30 mg/kg treatment significantly improved motor function and increased body weight. Compared with those in the model group, the number of dopaminergic neurons cells and the expression of PI3K-110α, p-Akt Ser473, and pro-caspase-3 in the Res 30 mg/kg group were significantly increased, and the Bax/Bcl-2 ratio and the level of activated caspase-3 was decreased. The results indicate that Res ameliorates 6-OHDA-induced apoptosis and motor dysfunction via activating the PI3K/Akt signaling pathway, delaying the progression of Parkinson's disease (PD) symptoms in this model.
Keywords: Parkinson's disease Resveratrol Phosphoinositide 3-kinase Protein kinase B 6-Hydroxydopamine
1. Introduction Since its first description by British surgeon James Parkinson approximately 200 years ago, Parkinson's disease (PD) has been extensively studied, and treatments for PD have emerged over the past two centuries (Goetz, 1986; Parkinson, 2002; Przedborski, 2017). However, PD, which is still a threat to public health worldwide, is the second most prevalent neurodegenerative disorder (Cuenca et al., 2018). The causes of PD are not fully understood, but the disease is believed to be associated with genetic, environmental and aging factors (Schneider et al., 2017). Therefore, it is vital to detect PD in its early stage and take effective preventive measures to control its progression. Weight can be used for the early detection of PD because weight loss may indicate progressive deterioration in patients with PD (Umehara et al., 2017; Wills et al., 2016). In addition, the loss of dopaminergic
neurons is a key pathological feature of PD, and the upregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway decreases injury to dopaminergic neurons both in vivo and in vitro (C.R. Wu et al., 2018; Xiromerisiou et al., 2008; Xu et al., 2013; Yang et al., 2018). Increasing the number of dopaminergic neurons is important to slow the progression of PD. Our previous work showed that resveratrol (Res) has a protective effect against 6-hydroxydopamine (6-OHDA)induced PD in rats (Feng et al., 2008). Res, as a phytoalexin, has been found to possess many pharmacological functions, such as inhibiting of neuronal apoptosis and improving energy metabolism (Z. Wu et al., 2018). In the present study, we further explored whether the protective effect of Res against the apoptosis of dopaminergic neurons is related to the regulation of the PI3K/Akt signaling pathway.
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Corresponding author. E-mail address: [email protected] (F. Jin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.exger.2019.110653 Received 13 March 2019; Received in revised form 30 May 2019; Accepted 5 July 2019 Available online 08 July 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.
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2. Materials and methods
injection with 2% sodium pentobarbital at a dose of 50 mg/kg and then placed in a standard stereotaxic device (68038, RWD, Shenzhen, China). The method for the establishment of the model were performed as follows (Fig. 1B): A unilateral midbrain substantia nigra injection of 4 μL of 6-OHDA (2 μg/μL) was carried out to establish a rat model of dopaminergic (DA) neuron injury, and the sham group was injected with an equal volume of normal saline containing 0.2% vitamin C, as described in our previous work (Feng et al., 2008).
2.1. Drugs, reagents and antibodies Reagent grade Res (purity ≥98% by HPLC analysis) was purchased from Zelang Medical Technology (Nanjing, China). 6-OHDA (catalog no. H4381) and L-ascorbic acid (catalog no. A5960) were purchased from Sigma-Aldrich (St. Louis, MO, USA). B-cell lymphoma 2 (Bcl-2, catalog no. D163267), Bcl-2-associated X protein (Bax, catalog no. D120073), activated caspase-3 (catalog no. D260009), and TBS (catalog no. B040126) were purchased from Sangon Biotech (Shanghai, China). Akt (catalog no. 9272S), p-Akt Ser473 (catalog no. 4060P), pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1, catalog no. 3062p), and p-PDK1 (catalog no. 3061S) were purchased from Cell Signaling Technology (Beverly, MA, USA). PI3K p110-alpha (catalog no. 21890-1AP) was purchased from Proteintech Group (Wuhan, China). Goat antirabbit and mouse IgG-HRP (catalog no. M21003) was purchased from Abmart (Shanghai, China). Tyrosine hydroxylase (TH, catalog no. ab41528) and pro-caspase-3 (catalog no. ab179517) were purchased from Abcam (Cambridge, MA, USA). β-Actin (catalog no. AA128), GAPDH (catalog no. AF0006), PMSF (catalog no. ST505), RIPA lysis buffer (catalog no. P0013B), an SDS-PAGE Gel Quick Preparation kit (Catalog No. P0012AC) were purchased from Beyotime Biotechnology (Shanghai, China). Benzylpenicillin sodium for injection (catalog no. H13021633) was purchased from CSPC Zhongnuo Pharmaceutical (Shijiazhuang, China). A two-Step Immunohistochemistry (IHC) Detection kit including HRP-conjugated goat anti-rabbit IgG and H2O2 (catalog no. PV-6001) that was used in IHC was purchased from ZSGBBIO Corporation (Beijing, China). A DAB Substrate kit (catalog no. DA1010), DMSO (catalog no. D8371), 0.01 M PBS (powder, pH 7.2–7.4, catalog no. P1010), phosphatase inhibitor cocktail (catalog no. P1260), trypsin-EDTA solution, 0.25% (with phenol red, catalog no. T1320), penicillin-streptomycin liquid (catalog no. P1400), Tween-20 (catalog no. T8220), Triton x-100 (catalog no. T8200), Tris (catalog no. T8060), glycine (catalog no. G8200), and SDS (catalog no. S8010) were purchased from Solarbio Life Science (Beijing, China). Hydrophobic PVDF (0.45 μm, catalog no. IPVH00010) was purchased from Merck (Darmstadt, Germany). PageRuler™ Prestained Protein Ladder (catalog no. 26616) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). A BCA Protein Assay kit (catalog no. GK5012) was purchased from Generay (Shanghai, China). SDS-PAGE loading buffer (catalog no. ZS306) was purchased from Zoman Biotechnology (Beijing, China). Enhanced chemiluminescence (ECL) reagents (catalog no. E002-100) were purchased from 7seaBiotech (Shanghai, China).
2.3. Behavioral experiments All behavioral experiments were carried out between 8:30 a.m. and 5:30 p.m. The animals were transferred to the experimental room at least 1 h before the test to allow them to acclimatize to the test environment. 2.3.1. Rotarod test The rotarod test is a classic method for detecting motor function in rodents (Hamm et al., 1994). The apparatus consisted of a cylindrical arrangement of thin 75-mm diameter steel rods. Before the experiment began, all rats were trained to achieve stable performance. The speed for the training sessions was set at 12 rpm, and the cut-off time was 30 s. The rats in all groups were trained on the rotarod until they stayed on the rod for at least the cut-off time. The animals were allowed to remain stationary for 10 s at 0 rpm, and then the rotational speed was steadily increased to 12 rpm over a 20 s interval until the rats fell off the rod. The time that each animal remained on the rotating bar was recorded. The maximum time was 600 s per trial. The apparatus automatically recorded the time to 0.1 s and stopped recording when the rats fell off the rotating rod. The speed was set at 12 rpm and accelerated at a rate of 3 rpm per min. After the rats were unilaterally injected with 6-OHDA, the rotarod test was performed every 7 days to detect progressive deficits in muscle coordination in the rats. The data are presented the time spent on the rotating rod over the three test trials. 2.3.2. Open field test The open field apparatus contained 4 squares that were 50 cm in length and 50 cm in width, surrounded by a 50 cm high wall, which was painted black. The animals were placed in the center of the apparatus for free exploration, and their behavioral parameters were recorded for 5 min. The apparatus was washed with a 5% ethanol solution before each behavioral test to preclude the possible effects of odors left by the previous subjects. Control and experimental rats were intermixed to minimize the possible influences of the circadian rhythm on rat behavior. We used the TopScan™ behavioral testing system to quantify the distance traveled and the moving speed of the animals.
2.2. Animals and treatment
2.3.3. Grid test The grid apparatus was an independent metallic grid (50 cm × 40 cm, 1 cm × 1 cm per hole) dangling vertically and fixed at a height of 1 m above the ground (Fig. 1C). Wood chips at least 20 cm thick from the cages were prepared in advance and placed on the ground to prevent damage to the rats from falling. In order to prevent the rats from climbing to the top of the grid and hovering, a lid was added to the top of the grid. The rats were placed upright in the center of the grid and were allowed to grasp the mesh with four paws. The time that it took each rat to fall was recorded with a stopwatch, and the cut-off time was 300 s.
A total of 50 male SD rats (210 ± 15 g, 7 weeks old) were obtained from the Experimental Animal Center of the Third Military Medical University (Chongqing, China; specific-pathogen-free grade II; certificate no. SCXK 2012–0005). The rats were housed in specific pathogen free (SPF)-grade animal facilities (certificate no. SCXK 2014-0004) at Zunyi Medical University on a 12 h light/dark cycle at 23 °C ( ± 2 °C) with free access to food and water. The use of animals was in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1978). All procedures in the present study were approved by the Animal Care and Use Committee of Zunyi Medical University. After one week of adaptive feeding, all the rats were divided into the following 5 groups (n = 10 per group) by the random number method: the sham group, the normal group (30 mg/kg Res), the model group (6OHDA), the Res-15 group (6-OHDA +15 mg/kg Res), and the Res-30 group (6-OHDA +30 mg/kg Res). Specific animal experiments were performed as shown in Fig. 1A. Res (15 and 30 mg/kg) was given orally to the rats beginning one day before the establishment of the PD model for a total of 36 days. The rats were anaesthetized by intraperitoneal
2.4. Western blot (WB) and IHC staining and counting Briefly, rat midbrain tissues were homogenized and lysed in RIPA lysis buffer supplemented with the protease inhibitor PMSF and 1× phosphatase inhibitor cocktail. The lysates were treated on ice for 30 min and centrifuged at 14,000 ×g for 15 min. The supernatant was acquired, and the protein levels were quantified by a BCA assay kit. 2
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Fig. 1. Schematic diagrams. (A) A simple schematic diagram of the animal experimental procedure. (B) A simple schematic diagram of the establishment of the animal model. (C) The grid test.
Equal amounts of total protein (30 μg per lane) were loaded. Then, the lysates were separated using 8–12% SDS-PAGE in running buffer (10× running buffer: 30.3 g Tris, 188 g glycine, 10 g SDS, and ddH2O to 1 L; diluted to 1× with ddH2O for use). The gel was run at a low voltage (60 V) to separate the proteins, and a higher voltage (100 V) was used for the stacking gel until the dye ran off the bottom of the gel. PVDF membranes were cut to suitable dimensions. The sponge and filter paper were soaked in transfer buffer (10× transfer buffer: 30.2 g Tris, 150.2 g glycine, and ddH2O to 1 L; diluted to 1× with ddH2O; ratio of water:methanol:10× buffer = 7:2:1), and the PVDF membrane was soaked in methanol. Then, the sandwich was transferred at 4 °C in 1× buffer at 220 mA for 90 min. Following three washes with TBST (15 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20), the membranes were blocked with 5% nonfat milk in TBST at room temperature for 1 h. Following three washes with TBST, the membranes were incubated at 4 °C overnight with the following specific primary antibodies: Bcl-2 (1:1000), Bax (1:1000), activated caspase-3 (1:1000), Akt (1:1000), pAkt Ser473 (1:1000), PDK1 (1:1000), p-PDK1 (1:1000), PI3K-p110α (1:1000), β-actin (1:2000), and GAPDH (1:2000). Following three washes with TBST, the membranes were incubated with secondary antibodies (goat anti-rabbit and mouse IgG-HRP, 1:2000) at room temperature for 2 h. The protein-antibody complexes were visualized using the ECL reagent. The intensity of the bands was quantitated with Quantity One software (Bio-Rad). IHC staining and counting was performed as described previously in detail (Huang et al., 2017) and according to the instructions of the two-step IHC detection kit. Rat brains were cut into 35-μm transverse free-floating sections on a horizontal sliding microtome. The sections were used for immunohistochemical staining with antibodies against TH (a marker of DA neurons; 1: 200).
between the means were analyzed by Bonferroni's post hoc t-test with corrections. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Effect of Res on the body weight of 6-OHDA-exposed rats Initially, there was no significant difference in the body weights of the rats in each group. The body weights of the rats in each group continued to increase for 30 days. However, compared with the sham rats, the 6-OHDA rats showed a lower weight gain. At the same time, compared with those of the 6-OHDA rats, the body weights of the Res 30 mg/kg rats increased significantly (Fig. 2A). 3.2. Effect of Res on behavioral changes in 6-OHDA-exposed PD model rats 3.2.1. Rotarod test During the 1st, 2nd and 3rd weeks after model establishment, rat motor function was detected by rotarod. The rats showed motor dysfunction during the first week. During the second week, the motor function of the high-dose Res rats significantly improved. By the third week, the motor function of the 6-OHDA group further decreased, indicating that the damage induced by 6-OHDA gradually worsened, paralleling the progressive worsening of PD (Wills et al., 2016). A high dose of Res significantly improved 6-OHDA-induced motor dysfunction in rats (Fig. 2B). The motor function of the low-dose Res group decreased during the third week, indicating that the protective effect of Res 15 mg/kg was not enough to block the progressive damage caused by 6-OHDA.
2.5. Statistical analysis 3.2.2. Open field test Both the distance traveled (Fig. 2C) and the speed (Fig. 2C). D) of the model group were lower than those of the sham group. At the same time, the spontaneous motor ability of the high-dose Res group
The data in this study are expressed as the mean ± SEM. Statistical significance was assessed by one-way ANOVA using SPSS 22.0. When ANOVA indicated significant differences, pairwise comparisons 3
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Fig. 2. Weight and behavioral results. (A) Body weight results; ⁎P < 0.05 vs the model group, #P < 0.05 vs the sham group. (B) Rotarod results; ⁎P < 0.05 vs the sham group, #P < 0.05 vs the model group; (C) Distance traveled in the open field test. (D) Velocity in the open field test. (E) Grid test results (mean ± SEM, n = 9–10).
analysis with an anti-TH antibody. As shown in Figs. 3B, 6-OHDA caused an approximately 50% decrease in TH-positive nigral neurons compared with the number in the sham group. Meanwhile, compared with no treatment, Res increased the number of TH-positive cells in the SNc of the rats. Similarly, the results of the WB assay regarding TH protein expression were consistent with the IHC results (Fig. 3C).
significantly improved. 3.2.3. Grid test The rats in the model group fell earlier than the rats in the sham group, and their motor function was significantly weakened. A high dose of Res significantly increased the motor function of the model rats, allowing the rats to stay on the grid for longer (Fig. 2E).
3.4. Effect of Res on 6-OHDA-induced apoptosis in the midbrain 3.3. Effect of Res on 6-OHDA-induced DA neuronal loss in the substantia nigra pars compacta (SNc) region
Bax was significantly upregulated in the midbrain of the model group compared with the sham group (Fig. 4B). Bcl-2 was significantly decreased (Fig. 4C), and there was a significant increase in the Bax/Bcl2 ratio (Fig. 4D). Pro-caspase-3 (Fig. 4E) was decreased and activated
After the behavior tests, the rats were sacrificed. The brains were sectioned, and dopaminergic neurons were quantified by IHC and WB 4
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Fig. 3. Protective effects of Res against the 6-OHDA-induced loss of dopaminergic neurons in the rat substantia nigra. (A) Representative pictures of TH-positive cells in the rat substantia nigra (scale bar is 200 μm, solid circle: SNc); a: sham, b: normal (Res 30 mg/kg), c: model (6-OHDA 8 μg), d: 6-OHDA + Res 15 mg/kg, e: 6OHDA + Res 30 mg/kg. (B) Graph summarizing the number of TH-positive cells in the rat SNc. (C) The protein expression of TH (mean ± SEM, n = 4–5).
(Tsai et al., 2001). Compared with that in the sham group, the level of p-PDK1 in the model group decreased, while the level of p-PDK1 in the Res group increased compared with that in the model group. However, these differences were not statistically significant (Fig. 5C). The level of p-Akt Ser473 in the model group was significantly lower than that in the sham group, while the p-Akt Ser473 levels in the high-dose Res group were significantly higher than those in the model group (Fig. 5D).
caspase-3 (Fig. 4F) was increased. These results suggest that 6-OHDA induces apoptosis in midbrain neurons in rats, whereas a high dose of Res can significantly decrease Bax, activated caspase-3, increase Bcl-2, and Pro-caspase-3 expression.
3.5. Effect of Res on 6-OHDA-induced PI3K/Akt signaling pathway changes in the midbrain
4. Discussion
To investigate whether the PI3K/Akt signaling pathway is involved in the protective effects of Res against 6-OHDA-induced neuronal injury, we detected some of the proteins involved in this pathway. The results showed that PI3K-110α was decreased in the model group compared with the sham group, but the difference was not significant. However, it is interesting to note that a high dose of Res significantly increased the expression of PI3K-110α (Fig. 5B). PDK1 mainly phosphorylates the Thr308 site of the Akt protein, resulting in Akt activation
At present, the most frequently used neurotoxins for modeling PD are 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-OHDA (Lane et al., 2012). MPTP-lesioned monkeys best mimic the symptomatology of PD seen in human patients, while rats appear to be resistant to the effects of MPTP (Mokry, 1995). For that reason, 6-OHDA is used to damage the substantia nigra in rodents. Performing 5
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Fig. 4. Effect of Res on the protein level of the 6-OHDA-induced expression of apoptosis-related proteins in the midbrain in rats. (A) Representative bands of Bax, Bcl2, pro-caspase-3 and activated caspase-3 in the midbrain of the different groups. (B) The protein level of Bax. (C) The protein level of Bcl-2. (D) Bax/Bcl-2 ratio. (E) The protein level of pro-caspase-3. (F) The protein level of activated caspase-3 (mean ± SEM, n = 4).
clinical symptom of PD (Zesiewicz and Hauser, 2001). Because weight loss may predict the exacerbation of PD (Umehara et al., 2017; Wills et al., 2016), we detected the progressive development of PD through changes in behavior and weight. The results showed that the exogenous toxic substance 6-OHDA can cause progressive damage in rats. Compared with that of the rats in the 6-OHDA group, the body weight of the rats in 30 mg/kg Res group increased significantly. In addition, Res at a dose of 30 mg/kg significantly alleviated 6-OHDA-induced motor dysfunction. To confirm whether Res delays the progression of PD, we executed two other behavioral experiments to verify the protective effects of Res. In both the open field and grid tests, Res showed neuroprotective effects. Based on these results, we further explored the potential mechanisms of Res. The number of dopaminergic neurons is closely related to motor function (Li et al., 2018). In this study, WB and IHC were used to detect
behavioral tests on animals with nigrostriatal lesions represents a valuable noninvasive method for assessing the influence of the damaged DA system on locomotor activity. This model produces well-defined and stable behavioral deficits, but the success rate of the model is closely related to the researcher's familiarity with the technique and requires much practice. We conducted two preliminary experiments before this, but they were abandoned due to failure of the model. For our third experiment, we collected and analyzed data. The model was established by a skilled graduate student, and the experiment was carried out successfully. In this study, we validated our hypothesis that Res could effectively delay the progression of PD and further explored the potential underlying mechanisms. Due to the degeneration of dopaminergic neurons in the SNc, the ability of the brain to control motor coordination decreases. This eventually leads to motor dysfunction, which is the main
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Fig. 5. Effect of Res on the PI3K/Akt signaling pathway in the midbrain of rats exposed to 6-OHDA. (A) Representative bands of PI3K-110α, p-PDK1, and p-Akt. (B) The protein level of PI3K-110α. (C) The protein phosphorylation level of p-PDK1. (D) The protein phosphorylation level of p-Akt Ser473 (mean ± SEM, n = 4).
Fig. 6. A schematic diagram showing how Res delays 6-OHDA-induced apoptosis by activating the PI3K/Akt signaling pathway.
expression of activated caspase-3. The results indicate that Res may have neuroprotective effects against dopaminergic neuron damage, possibly by modifying the apoptotic pathway. The PI3K/Akt signaling pathway participates in the development, survival and function of neurons and plays an important role in the occurrence and progression of PD (Ding et al., 2016; Xiromerisiou et al., 2008; Yang et al., 2018; Zhong, 2016). PI3K p110α plays an important protective role in the process of oxidative stress-induced apoptosis (Matheny and Adamo, 2009). 6-OHDA induces oxidative stress and apoptosis, which is suitable for simulating oxidative stress-induced PI3K/Akt inhibition (Liu et al., 2015; Schober, 2004; C.R. Wu et al., 2018; Xu et al., 2013). The results showed that, compared with that in the sham group, the expression of PI3K-110α in the model group exhibited a downward trend, but the difference was not statistically significant. However, it is interesting that Res at the dose of 30 mg/kg significantly increased the expression of PI3K-110α. The level of p-Akt Ser473 in the model group was significantly lower than that in the sham group, while the p-Akt Ser473 levels in the Res treatment groups were
TH expression in rats, which directly and indirectly reflects the number of dopaminergic neurons. TH is the rate-limiting enzyme in the synthesis of catecholaminergic neurotransmitters and catalyzes the conversion of tyrosine to L-DOPA. It is not only a target gene of gene therapy for Parkinson's disease but also a specific marker of dopaminergic neurons (Dunkley et al., 2005). The results showed that 6-OHDA induced an approximately 50% decrease in the number of TH-positive nigral neurons compared with the number in the sham group. Meanwhile, compared with no treatment, Res increased the number of THpositive cells in the SNc area of the rats. Similarly, the results the WB assay for TH expression were consistent with the IHC results. These results showed that Res can reduce the loss of dopaminergic neurons in 6-OHDA-exposed rats. To verify whether the protective effect is caused by anti-apoptotic effects, we detected changes in apoptosis-related proteins. The results showed that 6-OHDA induced the apoptosis of midbrain neurons in rats, whereas Res at a dose of 30 mg/kg significantly decreased the protein expression of Bax, increased the protein expression of Bcl-2 and pro-caspase-3, and decreased the protein 7
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significantly higher than those in the model group. We hypothesize that Thr308 is not the major phosphorylation site for Akt activation by Res (Fig. 6).
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5. Conclusion We propose that Res may play a protective effect by activating PI3K110α and p-Akt Ser473, which in turn inhibit the initiation and function of apoptosis-related proteins. Res protects dopaminergic neurons from 6-OHDA-induced apoptosis via activating the PI3K/Akt signaling pathway, ultimately delaying the progression of PD. Funding This work was supported by the National Natural Science Foundation of China (81660599, 81460548), The Postgraduate Education Foundation of Guizhou Province (KYJJ2017008), The Zunyi Medical University Funds (2013F-686, F-738), The Open Funds of Key Laboratory of Basic Pharmacology of Ministry of Education of China (JCYL-K-012), The 15851 Talent Elite Project, The Funds of Department of Education of Guizhou Province ([2016]038), and Shijingshan's Tutor Studio of Pharmacology [GZS-2016(07)]. Declaration of Competing Interest All authors declare no conflict of interest. References Cuenca, L., Gil-Martinez, A.L., Cano-Fernandez, L., Sanchez-Rodrigo, C., Estrada, C., Fernandez-Villalba, E., Herrero, M.T., 2019. Parkinson's disease: a short story of 200 years. Histol. Histopathol. 34, 573–591. Ding, G., Zhao, J., Jiang, D., 2016. Allicin inhibits oxidative stress-induced mitochondrial dysfunction and apoptosis by promoting PI3K/AKT and CREB/ERK signaling in osteoblast cells. Exp. Ther. Med. 11, 2553–2560. Dunkley, P.R., Bobrovskaya, L., Graham, M.E., von Nagy-Felsobuki, E.I., Dickson, P.W., 2005. Tyrosine hydroxylase phosphorylation: regulation and consequences. J. Neurochem. 91, 1025–1043. Feng, J., Wu, Q., Lu, Y.F., Gong, Q.H., Shi, J.S., 2008. Neuroprotective effect of resveratrol on 6-OHDA-induced Parkinson's disease in rats. Eur. J. Pharmacol. 600, 78–82. Goetz, C.G., 1986. Charcot on Parkinson's disease. Mov. Disord. 1, 27–32. Hamm, R.J., Pike, B.R., O'Dell, D.M., Lyeth, B.G., Jenkins, L.W., 1994. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. J. Neurotrauma 11, 187–196. Huang, C., Zhu, L., Li, H., Shi, F.G., Wang, G.Q., Wei, Y.Z., Liu, J., Zhang, F., 2017.
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