Activation of the δ opioid receptor relieves cerebral ischemic injury in rats via EGFR transactivation
Meixuan Chen, Shuo Wu, Bing Shen, Qingquan Fan, Ran Zhang, Yu Zhou, Pingping Zhang, Liecheng Wang *, Lesha Zhang *
A B S T R A C T
Delta opioids are thought to relieve ischemic injury and have tissue-protective properties. However, the detailed mechanisms of delta opioids have not been well identified. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), have been shown to mediate downstream signals of δ opioid receptor (δOR) activation through the metalloproteinase (MMP)-dependent EGF-like growth factor (HB-EGF) excretion pathway, which is called transactivation. In this study, to investigate the role of EGFR in δOR-induced anti-ischemic effects in the brain, we applied the middle cerebral artery occlusion (MCAO) model followed by reperfusion to mimic ischemic stroke injury in rats. Pre-treatment with the δOR agonist [D-ala2, D-leu5] enkephalin (DADLE) improved the neurologic deficits and the decreased infarct volume caused by cerebral ischemia/reperfusion injury, which were blocked by the EGFR inhibitor AG1478 and the MMP inhibitor GM6001, respectively. Further results indicated that DADLE activated EGFR, Akt and ERK1/2 and upregulated EGFR expression in the hippocampus in a time-dependent manner, which were inhibited by AG1478 and GM6001. The enzyme-linked immunosorbent assay (ELISA) results showed that δOR activation led to an increase in HB-EGF release, but HB-EGF in tissue was downregulated at the mRNA and protein levels. Moreover, this protective action caused by δOR agonists may involve attenuated hippocampal cellular apoptosis. Overall, these results demonstrate that MMP-mediated transactivation of EGFR is essential for δOR agonist-induced MCAO/reperfusion injury relief. These findings provide a potential molecular mechanism for the neuroprotective property of δOR and may add new insight into mitigating or preventing injury.
Keywords: δOR EGFR
HB-EGF
MCAO
Cerebral ischemia/reperfusion
1. Introduction
Stroke is one of the leading causes of death worldwide [1], and 87% of stroke cases result from cerebral ischemia injury [2]. δ opioids have been shown to possess a tissue-protective property in ischemia [3–5]. The preconditioning or perfusion of the δ opioid receptor (δOR) agonist [D-ala2, D-leU5] enkephalin (DADLE) can remarkably protect organs, such as the heart and lung, from ischemia-reperfusion injury [6,7]. It has been reported that DADLE may induce a hibernation-like state that protects the brain against experimental stroke [8], regulate neural regeneration after ischemia and improve the recovery of spatial and cognitive function [9], and enhance the anti-anoxic ability [10]. However, direct evidence suggesting the neuroprotective ability of DADLE against brain ischemia in vivo is insufficient, and the specific molecular mechanism is not fully known. δOR, a GPCR, has been shown to activate the downstream signals ERK1/2 or Akt by transactivating receptor tyrosine kinase (RTKs) [11–14]. GPCRs, such as the histamine receptor H3R, can activate ERK1/2 through the PKC/PLD/metalloproteinase (MMP)/epidermal growth factor receptor (EGFR) transactivation pathway to exhibit a neuroprotective effect on cultured mouse cortical neurons under hypoxic conditions [15]. Previous studies have reported that ERK1/2 and Akt have pro-survival effects on DADLE-induced protection of rabbit hearts after left coronary artery occlusion and reperfusion, which may be dependent on the transactivation of EGFR [7]. In vitro evidence indicates that δOR activation leads to metalloproteinase-dependent cleavage of heparin binding EGF-like growth factor (HB-EGF) from the membrane, and HB-EGF as a ligand diffusely interacts with EGFR to activate the intracellular signaling pathway [16], which is different from the classical Gαi/o-dependent pathway of δOR [17]. Additionally, HB- EGF may contribute to recovery from cerebral injury through direct neuroprotective effects [18] and is expressed in the hippocampus [19].
It has been reported that administration of HB-EGF after local cerebral ischemia inhibits the migration of neuronal precursors to the infarct area and modifies neurogenesis [20]. Specific knockout of HB-EGF in the forebrain of rats leads to exacerbated ischemia and reperfusion injury [19]. These findings lead us to hypothesize that δORs, which possibly transactivate EGFR, may relieve cerebral ischemia/reperfusion injury through the release of HB-EGF. In this study, we administered DADLE through an intracerebroventricular injection middle cerebral artery occlusion (MCAO)/reperfusion model rats, analyzed the neurologic and histological outcomes, and detected the molecular mechanism to verify the above-mentioned hypothesis.
2. Materials and methods
2.1. Animal treatment
Sprague Dawley male rats weighing 250–300 g were purchased from the animal center of Anhui Medical University (Hefei, Anhui, China). Rats were housed in 485 mm × 350 mm × 200 mm cages, with two to three rats per cage, and were maintained on a 12 h light/dark cycle with access to food and water ad libitum at room temperature (RT, ~25 ◦C). All studies involving animals are reported in accordance with the ARRIVE guidelines for experiments involving animals [21]. All experiments were approved by the Administration Office of Laboratory Animals of Anhui Medical University and complied with the guidelines of the Institutional Animal Care Unit Committee of Anhui Medical University. All animal experimental procedures were also permitted by the Animal Ethics Committee of Anhui Medical University (License No. LLSC20180209). The total number of animals used was 205. All the rats used for each trials of experiment were randomly divided into each group.
2.2. Intracerebroventricular injection
Rats were anaesthetized with sodium pentobarbital (55 mg/kg, i.p.) in combination with atropine (0.4 mg/kg, i.p.). The depth of anaesthesia was determined by testing for the lack of a pain response to gentle pressure on the hind paws. Then, the animals were placed in a stereotaxic apparatus (RWD Life Science, Shenzhen, China). Guide cannulas were implanted on the left side of the rat brain (AP: − 0.8 mm; ML: +1.5 mm; and DV: − 2.0 mm). A stainless-steel blocker was inserted into each cannula to keep them patent and prevent infection. Rats recovered from surgery for seven days and were separately housed in 370 mm × 270 mm × 170 mm cages, one rat per cage. During these seven days, 80,000 IU penicillin was subcutaneously injected per day per rat to prevent infection. Seven days later, microinfusions were made through a 31- gauge injection cannula (1.8 mm under the tip of the guide cannula) over 5 min at a rate of 2 μL/min and with an additional 1 min for drug diffusion. The δOR agonist DADLE was dissolved in artificial cerebrospinal fluid (ACSF) at a concentration of 5 nmol/10 μL. The EGFR inhibitor AG1478 was dissolved in ACSF at a concentration of 1 or 10 μM, and the MMP inhibitor GM6001 was diluted with ACSF to a final concentration of 0.5 or 1 ng/μL (injection volume 10 μL), and then, one of these inhibitors were microinjected 1 h before agonist injection. Rats in the control group were injected with ACSF at the same time. During the microinjection, the rats were awake and gently restrained by hand. All procedures were performed as described in previous studies [17]. The detailed information of all the agonist and inhibitors used refer to Table S1.
2.3. MCAO and reperfusion model
Cerebral ischemic stroke injury was induced by establishing MCAO and reperfusion [8]. Fifteen minutes after receiving an intracerebroventricular injection of DADLE or ACSF, rats were anaesthetized with sodium pentobarbital (55 mg/kg, i.p.) in combination with atropine (0.4 mg/kg, i.p.). Cerebral blood flow (CBF) was determined in the area of the middle cerebral artery (MCA) in the cerebral hemisphere by laser speckle imaging (PeriCamPSI, PERIMED, Sweden). A customized monofilament based on the weight of the rat was inserted 18 to 20 mm into the internal carotid to occlude the origin of the MCA. The reduction in CBF was 50% on average. Then, the monofilament was removed after 1 h for reperfusion. The temperature was maintained at 37 ◦C during surgery until the animals recovered from anaesthesia. Because the rats were implanted guide cannulas partly damaging the integrity of blood brain barrier plus the injury of cerebral ischemia and reperfusion, moderate death of rats during the surgery of MCAO might not be avoided. By statistics, the total mortality was 21%.
2.4. Evaluation of neurologic deficit scores
To determine the sensory and motor neurologic deficits induced by ischemia/reperfusion injury, neurological evaluations were carried out 72 h after surgery. The animals of each group were scored according to the Garcia criteria, which included six items (maximal deficit score = 3, normal score = 18): spontaneous activity (0–3 points); symmetry in the movement of four limbs (0–3 points); forepaw outstretching (0–3 points); climbing of the wire cage (1–3 points); body proprioception (1–3 points) and response to vibrissae touch (1–3 points) [22]. The total score is the summation of all six individual test scores. The neurologic deficit scores were assessed by examiners blinded to the group assignment of each rat.
2.5. Cerebral infarction volume assessment
After the neurologic evaluation, the infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Briefly, rats were anaesthetized with an overdose and sacrificed. Subsequently, the brain was rapidly removed and frozen at − 20 ◦C for 15 min. The olfactory bulb, cerebellum, and low brain stem were removed, and the rest of the brain was placed in brain matrices made of stainless steel (RWD Life Science, Shenzhen, China) to be sliced into at least seven slices approximately 2 mm thick from rostral to caudal. The coronal sections were stained with PBS containing 2% TTC (T8877, Sigma-Aldrich, USA) at 37 ◦C for 15 min per side in the dark. Photographic images were taken to measure the stained and unstained areas and calculate the percentage of the infarct volume in the whole brain by using image software (Image- Pro Plus 6.0; Media Cybernetics, Maryland, USA). The stained area (red) was defined as normal tissue, and the unstained area (white) was defined as the infarct region.
2.6. RNA extraction and quantitative real-time PCR (qPCR)
The brain was dissected and quickly frozen using liquid nitrogen and stored in a − 80 ◦C freezer until use. Total RNA was extracted from the dorsal hippocampus as described in previous studies [23]. The RNA samples were converted into cDNA with the GoScript™ Reverse Transcription System Kit (A5000, Promega, USA). The relative abundance of each mRNA species was assessed by real-time PCR using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, USA) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System. The primer sequences for hb-egf were 5’-TCATCTGTCTGTCTTCTTGT-3′ (forward) and 5’- ATGCCCAACTTCACTTTCTC-3′ (reverse). The primer sequences for egfr were 5’-CAAGTGCTGGATGATAGA-3′ (forward) and 5’-GAAGTTGGAGTCTGTAGG-3′ (reverse). The primer sequences for β-actin were 5’- CATTGTTACCAACTGGGACGACAT-3′ (forward) and 5’- GCCTCGGTGAGCAGCACA-3′ (reverse). The mRNA levels were normalized against β-actin and are presented as 2-ΔΔCT.
2.7. Enzyme-linked immunosorbent assay (ELISA)
Rats were first anaesthetized using sodium pentobarbital (55 mg/kg, i.p.); then, the meninges overlying the rat’s cisterna magna were exposed, the arachnoid membrane covering the cisterna was punctured using a microneedle, and cerebrospinal fluid (CSF) was collected. After whole CSF samples of rats were gathered, the brain was placed in brain matrices to be sliced into slices approximately 1 mm thick from rostral to caudal. Then, homogenization of the region of the dorsal hippocampus or whole CSF samples was performed. The total protein concentration of each sample was determined by using a BCA assay kit (P0012, Beyotime Biotechnology, China). The protein homogenization supernatant (100 μL) or CSF samples (100 μL) were applied to an HB-EGF antibody-coated 96-well plate and incubated for 2 h at 37 ◦C. Briefly, ELISA for tissue HB- EGF or soluble HB-EGF was carried out using a rat HB-EGF ELISA kit (CSB-EL010154RA, Cusabio, China). The optical density of each well was determined using a microplate reader set to 450 nm. The HB-EGF levels were calculated from a standard curve obtained running recombinant HB-EGF proteins (0, 6.25, 12.5, 25, 50, 100, 200, 400 pg/mL).
2.8. Western blotting
Equal amounts of protein were electrophoresed on 8% SDS-PAGE gels and transferred to PVDF membranes for immunoblotting. The membranes were blocked with a 5% non-fat milk dilution in TBST for 1 h at RT and incubated with primary antibody dilutions kept overnight at 4 ◦C. After incubation with HRP-conjugated goat anti-rabbit IgG (1:5000) or goat anti-mouse IgG (1:10000) (BBI Life Sciences, Shanghai, China), chemiluminescence detection was performed by using the ECL plus Western blotting detection reagent (GE Health, Little Chalfont, UK), and immunoblots were quantified by densitometry using a chemiluminescence gel imaging analysis system (P&Q, Shanghai, China). For repeated immunoblotting, membranes were stripped in Stripping buffer (Beyotime Biotechnology, China) for 20 min. The primary antibodies were as follows: anti-ERK1/2 (mouse, 1:2000, Bioss, Beijing, China), phospho-AKT (Ser473, rabbit, 1:200, Cell Signaling Technology, Boston, MA, USA), Phospho-EGF Receptor (Tyr845, rabbit, 1:100, Cell Signaling Technology, Boston, MA, USA), EGF Receptor (rabbit, 1:200, Cell Signaling Technology, Boston, MA, USA), Phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, rabbit, 1:500, Cell Signaling Technology, Boston, MA, USA), Akt (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA), HB-EGF (H-1, mouse, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH (0411, goat, 1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The detailed information of all the antibodies used refer to Table S2.
2.9. TdT-mediated dUTP nick-end labelling (TUNEL)
At 72 h after MCAO/reperfusion, rats were deeply anaesthetized and perfused with 4% paraformaldehyde. Then, the brains were removed and kept in a 30% sucrose solution for five days to dehydrate until they sunk. TUNEL was performed on 30 μm thick cryostat sections of brains of using a TUNEL BrightGreen apoptosis detection kit (A112, Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. Nuclei were stained with a 4′-6-diamidino-2-phenylindole (DAPI) staining solution (Beyotime Biotechnology, China). Finally, the fluorescence signals were visualized with an Axio Observer 3 microscope (Carl Zeiss AG, Germany).
2.10. Data analysis and statistics
All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± SEM. Phosphoproteins, such as ERK and Akt, are quantified as the ratio to total protein. Statistical significance was determined by using Student’s unpaired t-test when only two groups were compared or one-way analysis of variance (ANOVA) followed by Newman–Keuls comparison test when more than two groups were compared in accordance with the experimental design. Statistical significance was defined as p < 0.05. Two-way ANOVA analysis was used to do a longitudinal analysis among time.
3. Results
3.1. The δOR agonist DADLE protects against MCAO-induced acute brain ischemia/reperfusion injury and improves neurologic deficits via transactivation of EGFR
To mimic ischemic stroke injury, we applied the MCAO model followed by reperfusion in rats. For animals that exhibit individual variations, the infract volume cannot be controlled by reducing the difference between the respective weights of rats and matching the monofilaments. To stabilize the variations of injuries in each animal, we tested the blood flow of rat brains during surgery. Because guide cannulas were implanted on the left side of the rat brain, the monitoring area focused on the right side of the brain. As shown in Fig. 1, the blood flow of the targeted area was reduced by almost half compared with that before surgery, showing a significant difference.
According to the schedule presented in Fig. 2A, ischemic animals were pre-treated with ACSF, the EGFR inhibitor AG1478 (1 or 10 μM) or the MMP inhibitor GM6001 (0.5 or 1 ng/μL) 1 h before injection of ACSF or 5 nmol DADLE. The MCAO model was established 15 min after injection of ACSF or DADLE, and then, the blood flow of the cerebral ischemic area was recovered by reperfusion 1 h later. Seventy-two hours after reperfusion, the cerebral infarction volume assessed by TTC staining indicated that DADLE at a dosage of 5 nmol could markedly shrink the ischemic infarct area by over half of the area of the ACSF group. In contrast, both AG1478 and GM6001 reversed this protective effect in a dose-dependent manner (Fig. 2B,C). Specifically, 10 μM AG1478 and 1 ng/μL GM6001 blocked the ischemic injury relief caused by DADLE. This result indicated that the improvement of cerebral ischemia/reperfusion injury induced by DADLE involves the EGFR transactivation pathway.
Correspondingly, behavioural tests were also performed in rats of each group. Consistently, the behavioural observation data 72 h after MCAO showed that 5 nmol DADLE obviously improved neurologic deficits compared to the ACSF group (p < 0.001). However, both AG1478 and GM6001 decreased the total neurologic deficit scores in a dose-dependent manner. This observation of sensory and motor abnormalities in DADLE-treated ischaemic animals pre-treated with AG1478 and GM6001 suggests that DADLE exerts its protection via MMP- mediated EGFR transactive mechanisms (Fig. 2D,E).
3.2. DADLE decreases cellular apoptosis in the hippocampal brain region
Because previous studies have reported that δOR agonists exert protective effects by reducing cellular apoptosis [24–26], we speculated that δOR may improve cerebral ischemia/reperfusion injury via anti- apoptosis effects. Therefore, we detected the cellular apoptosis level by performing TUNEL assays on brain slices of ACSF- or DADLE-treated MCAO/reperfusion model rats. As shown in Fig. 3, TUNEL-positive cells were obviously visible in the CA1 and CA3 regions of the dorsal hippocampus of MCAO/reperfusion model rats. In contrast, the number of TUNEL-positive cells in the DADLE group was downregulated (Fig. 3A, line 2). Statistical analysis of the TUNEL-positive fluorescence intensity in the ACSF- and DADLE-treated groups showed that DADLE significantly reduced the level of cellular apoptosis (Fig. 3B). This result indicated that δOR may be involved in the protection of ischemia/ reperfusion injury in the hippocampus by attenuating cellular apoptosis.
3.3. Stimulation of δORs with DADLE activates EGFR, ERK1/2 and Akt and upregulates EGFR expression in a time-dependent manner
Because it is notable that the cellular apoptosis in the ischemic penumbra within the region of dorsal hippocampus could be reduced by DADLE pretreatment, and we found that the down-stream signals varied most robust in dorsal hippocampus during the preliminary experiment searching the target brain region (data not shown), next we detected δOR-mediated EGFR activation in the dorsal hippocampus. Autophosphorylation of EGFR tyrosine residues leads to the activation of EGFR. In particular, tyrosine 845, which is located in the activation segment of the kinase domain containing trans-phosphorylation sites (e.g., mitogen- activated protein kinase, Akt protein kinase) [27], has been reported to be phosphorylated in the transactivation pathway of EGFR [28,29]. As shown in Fig. 4B, treatment with DADLE (5 nmol) led to a marked increase in EGFR Y845 phosphorylation 24 h after MCAO/reperfusion on the ischemic side of the hippocampus region. However, this effect was not observed 2 h or 72 h after MCAO/reperfusion (Fig. 4A, C). Interestingly, the protein expression level of EGFR increased after treatment with DADLE 24 and 72 h after MCAO/reperfusion on both the ischemic and contralateral sides of the hippocampus region (Fig. 4B). To further verify the change in EGFR at the transcription level, we used qPCR to analyze the quantity of EGFR mRNA. The results showed that the quantity of EGFR mRNA in the ischemic hippocampus region was significantly increased in the DADLE-treated group compared to the ACSF-treated group 24 h after MCAO/reperfusion (Fig. 4D). In order to understand the role of time and agonist administration as sources of variation among the EGFR’s phosphorylation or expression changing, we used two-way ANOVA test to analyze between the ischemic side of ACSF group and DADLE group (Fig. 4E,F). Results showed that for phospho-EGFR the time factor accounted for approximately 27.58% of the total variance (F(2, 18) = 5.89, p = 0.0107), the agonist administration factor accounted for 10.75% of the total variance (F(1, 18) = 2.96, p = 0.1027), the interaction accounted for 10.9% of the total variance (F(2, 18) = 3.5, p = 0.0519).Whereas for protein expression level changing of EGFR, the time factor accounted for approximately 35.89% of the total variance (F(2, 18) = 27.5, p < 0.0001), the agonist administration factor accounted for 22.12% of the total variance (F(1, 18) = 33.89, p < 0.0001), the interaction accounted for 10.9% of the total variance (F(2, 18) = 13.83, p = 0.0002). Overall, these results demonstrate that δOR activates EGFR and upregulates EGFR expression in a time-dependent manner.
Because δOR can activate the pro-survival kinases ERK1/2 and Akt by transactivating RTKs [30–32], we investigated whether δOR could activate ERK1/2 and Akt in the ischemic hippocampus. As shown in Fig. 5C, phosphorylation of Akt (Ser473) in the DADLE-injected group reperfused for 24 h was increased compared to that in the ACSF-treated group. Similar results were obtained by immunoblotting using an antibody against phospho-ERK1/2 (Fig. 5D). Nevertheless, 2 h after MCAO/ reperfusion, the phosphorylation levels of Akt and ERK1/2 showed no difference between the DADLE-injected group and ACSF-injected group (Fig. 5A,B). Additionally, 72 h after MCAO/reperfusion, although DADLE induced slight Akt and ERK1/2 activation, there was still no significant difference (Fig. 5E,F). When compared the Akt or ERK1/2 expression changing folds of DADLE-treated group along with the time after MCAO, both underwent an increasing from 2 h to 24 h followed with a decline from 24 h to 72 h (Fig. 5G,H). To access the role of time in this figure legend, the reader is referred to the web version of this article.) and agonist administration as sources of variation, we analyzed their expression changing folds between the ischemic side of ACSF group and DADLE group by two-way ANOVA test. Results showed that for phospho-Akt the time factor accounted for approximately 29.76% of the total variance (F(2, 17) = 5.35, p = 0.0158), the agonist administration factor accounted for 10.75% of the total variance (F(1, 17) = 3.87, p = 0.0658), the interaction accounted for 10.9% of the total variance (F(2, 17) = 1.96, p = 0.1714).While for phospho-ERK1/2, the time factor accounted for approximately 17.77% of the total variance (F(2, 17) = 3.45, p = 0.0551), the agonist administration factor accounted for 15.77% of the total variance (F(1, 17) = 6.13, p = 0.0241), the interaction accounted for 10.9% of the total variance (F(2, 17) = 4.06, p = 0.0361). These results indicate that pre-administration of DADLE results in an increase in phosphorylation of both Akt and ERK1/2 in a time- dependent manner.
3.4. Transactivation of EGFR is required for δOR-mediated EGFR, ERK1/ 2, and Akt activation
To determine whether MMP-mediated transactivation of EGFR is required for δOR-induced EGFR, Akt and ERK1/2 activation, we next examined the effect of the EGFR inhibitor AG1478 (10 μM) and the MMP inhibitor GM6001 (1 ng/μL) on DADLE-induced phosphorylation of EGFR, Akt and ERK1/2 on the ischemic side of the hippocampus region of the rat brain. As shown in Figs. 6, 24 h after MCAO/reperfusion, compared to the DADLE group, pre-treatment with AG1478 significantly reduced phosphorylation of Y845 of EGFR, as well as the expression of EGFR (Fig. 6A), which evidently abolished the activation of Akt and ERK1/2 induced by stimulation with δOR (Fig. 6B,C). The MMP inhibitor GM6001 had a similar blocking effect on the DADLE-induced increases in phosphorylation and upregulation of EGFR (Fig. 6D), Akt activation (Fig. 6E) and ERK1/2 activation (Fig. 6F). Taken together, these results indicate that MMP-mediated transactivation of EGFR is essential for phosphorylation and upregulation of EGFR and downstream signal pathway activation in response to δOR activation.
3.5. Stimulation of δORs with DADLE induces an increase in soluble HB- EGF but a reduction of the expression of tissue HB-EGF
It is thought that the release of HB-EGF is initiated by MMP-mediated cleavage of membrane-bound HB-EGF before EGFR is transactivated [11]. As a result, activated HB-EGF is the crucial soluble mediator in the extracellular environment after δOR stimulation and is the ligand of EGFR. HB-EGF has been reported to participate in the recovery of cerebral injury through direct neuroprotective effects [18]. Consequently, to understand the role of HB-EGF in the participation of δOR stimulation, we further detected hippocampal HB-EGF in ACSF- and DADLE- treated MCAO/reperfusion rats (Fig. 7). Compared with the ACSF group, the level of HB-EGF mRNA decreased in the DADLE group (Fig. 7A). This result is also consistent with the result obtained from western blotting (Fig. 7B), which showed a significant decrease in the HB-EGF protein level when DADLE was administered. Additionally, the MMP inhibitor GM6001 could obviously reverse this reduction, while the EGFR inhibitor AG1478 could partly influence this effect (Fig. 7B).
Next, we explored the distributive changes of HB-EGF in tissue and CSF together, our data reveal that δOR activation upregulates the MMP- triggered by δOR activation using ELISA. The results showed that there dependent HB-EGF/EGFR transactivation pathway in MCAO/reperfuwas a slight decrease in the concentration of HB-EGF in tissue, which sion injury relief (Fig. 8). was recorded from a sample of the dorsal hippocampal homogenate (Fig. 7C). The concentration of soluble-HB-EGF in CSF increased after DADLE treatment, indicating that δOR mediated HB-EGF release. Taken
4. Discussion
Increasing evidence has shown that the δOR agonist DADLE can produce a neuroprotective effect against hypoxia- or ischemia-induced brain damage [8,33,34]. However, this effect is different from the classical analgesic function and addictive effect of δOR [35]. δOR can activate the MAPK and PI3K/Akt transduction pathways through multiple mechanisms, including G-protein dependent and independent processes [36]. In our previous study, we verified that δOR transactivates RTKs in a G-protein independent manner [17]. The present study extends our previous findings by demonstrating that transactivation of the EGFR pathway is involved in δOR agonist-induced cerebral ischemic relief in vivo. Although there is evidence indicating that DADLE exerts a protective effect on the heart during reperfusion via EGFR transactivation [7], no research shows that a similar signaling pathway under δOR activation exists in the central nervous system. Moreover, this is the first time δOR was found to upregulate the expression level of EGFR in the ischemic hippocampus region of the brain.
Another novelty of the present study is the change in HB-EGF determined by detecting its concentration in CSF and tissue in response to δOR activation. Given that a number of reports have shown that HB-EGF is crucial for cerebral anti-ischemia function [18,20,37], we speculate that EGFR activation by HB-EGF could result in a pro- survival effect against ischemic damage. Nevertheless, although previous studies have shown that MMP can cleave the precursor of HB-EGF, i. e., pro-HB-EGF, which leads to the excretion of HB-EGF [38] and is necessary for the improvement of the tissue-protective property of δOR agonists [7], the release of HB-EGF has not been detected in vivo. Our results show that compared to the ACSF group, the concentration of soluble HB-EGF in CSF increased in the DADLE group, but the concentration of HB-EGF in tissue in the hippocampus decreased in the DADLE group. HB-EGF decreased the mRNA and protein levels after DADLE administration (Fig. 7). The increase in soluble HB-EGF in CSF is thought to be correlated with the decrease in HB-EGF in tissue. The explanation for this correlation may be that the abundance of MMP-cleaved pro-HB- EGF is increased due to δOR activation; then, the level of HB-EGF released into CSF rises, and HB-EGF in the membrane is reduced. Additionally, because HB-EGF is the ligand of EGFR and it can activate EGFR through paracrine or autocrine signaling [39], upregulation of EGFR may give rise to the relative downregulation of HB-EGF via negative feedback.
It is generally acknowledged that MCAO/reperfusion is one of the most commonly used models for cerebrovascular ischemic disease. Based on the neuroprotective mechanism of DADLE in the MCAO/ reperfusion model, which has been shown to induce a hibernation-like state to protect against stroke [8], we chose to pre-treat DADLE before the MCAO/reperfusion model was established. However, although recent research mostly focuses on the preconditioning protective effect of DOR agonists, potential ischemia might hardly be anticipated so that further studies should be aim at the application after stroke in clinic. Relief of cerebral injury after stroke is crucial for the prognosis of patients. Several reports have indicated that the attendance of delta opioid peptide during reperfusion is beneficial. The addition of delta opioid peptide could enhance neuroprotective effects of combination the 34 ◦C hypothermia and stem cell treatment against reperfusion injury after oxygen-glucose deprivation in primary rat neurons [40]. Furthermore, the finding that δOR-induced EGFR upregulation is time-dependent in this study may supply a reference for the timing of administration of DADLE. On one hand, acute therapies make efforts to restore blood flow to the infract penumbra before irreversible tissue injury has occurred [41]. Administration of DOR agonist as early as possible might strive for more time before irreversible injury. On the other hand, we found that the activation of EGFR and HB-EGF releasement induced by DADLE was notable at 24 h after ischemic happening (Fig. 4). While the sensory and motor functional relief as well as cellular apoptosis reduction sustain till 72 h after stroke (Figs. 2 and 3). It is possible for DOR agonists to keep more cellular survival after reperfusion. However, there is a limitation for opioids’ application in clinic of which is the known existing addictive potency [42]. Indeed, the DOR has emerged as a potential target for therapy because of its weak unwanted effects when compared with μ-opioid receptor (MOR) [43,44]. In addition, through modification of ligands’ structure, pharmacists have developed many exogenous ligands in order to reduce the unexpectable addiction and tolerance [45–47]. Meanwhile, pharmacologists have found that the diversity of ligands brings many other biological functions of DOR, such as tissue-protective function in ischemia [48,49], regulating emotion [50,51], promoting cell proliferation and regeneration [52,53], revealing that DOR may play a variety of regulatory roles in vivo. Thus, besides analgesia, the role of DOR in other biological processes needs to be further studied in order to provide new directions in drug discovery by targeting specific signaling pathway.
5. Conclusion
In summary, this study shows that pre-treatment with the δOR agonist DADLE relieves MCAO/reperfusion-induced neural injury through MMP-mediated HB-EGF secretion followed by EGFR activation and upregulation in the hippocampus of rats. The decrease of cellular apoptosis is involved in this protective effect. This investigation explains the molecular mechanism underlying the neuroprotective effect of δOR and may supply a new strategy to control or block the development of ischemic/reperfusion injury.
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