Hydromorphone protects CA1 neurons by activating mTOR pathway

Accepted Manuscript Title: Hydromorphone protects CA1 neurons by activating mTOR pathway Authors: Wenji Xie, Wenqin Xie, Zhenming Kang, Changcheng Jiang, Naizhen Liu PII: DOI: Reference:

S0304-3940(18)30635-9 https://doi.org/10.1016/j.neulet.2018.09.029 NSL 33818

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

10-7-2018 14-9-2018 15-9-2018

Please cite this article as: Xie W, Xie W, Kang Z, Jiang C, Liu N, Hydromorphone protects CA1 neurons by activating mTOR pathway, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.09.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hydromorphone protects CA1 neurons by activating mTOR pathway Wenji Xie, Wenqin Xie*, Zhenming Kang, Changcheng Jiang, Naizhen Liu Department of Anesthesiology, Quanzhou First Hospital, No. 248-252 Dong Road,

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Quanzhou 362000, China

*Corresponding author: Wenqin Xie, Department of Anesthesiology, Quanzhou First

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Hospital, No. 248-252 Dong Road, Quanzhou 362000, China

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E-mail: [email protected]

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Running title: Hydromorphone protect hippocampal CA1 neurons from IR injury

Highlights

Preconditioning with HM increased Latency time,

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Preconditioning with HM decreased apoptosis of hippocampal CA1 neurons



Preconditioning with HM suppressed IR induced oxidative stress



HM increased Bcl-2 and p-mTOR expression levels and decreased Bax expression



Rapa reverses the role of HM in protecting hippocampal CA1 neurons



HM protect hippocampal CA1 neurons from IR injury via mTOR pathway

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

Abstract Hydromorphone has been shown to play protective effect in rat glial cell. However, 1

whether hydromorphone plays important roles in ischemia–reperfusion (IR) injury and the involved signaling pathway remains unclear. In this study, we detected whether HM plays protective effect in IR injury mouse model, further followed by the mechanism exploration. Preconditioning with hydromorphone was performed for

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continuous 4 days at the doe of 2 mg/kg before IR injury induction. Intraperitoneal injection of rapamycin (Rapa) was administrated to examine the role of mTOR in IR

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injury. The mRNA expression level was detected by RT-PCR, and protein expression level was detected by western blot. Latency time and apoptosis of hippocampal CA1

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neurons were detected 72 h after IR injury induction. Preconditioning with

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hydromorphone significantly increased Latency time, decreased apoptosis of

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hippocampal CA1 neurons and suppressed IR induced oxidative stress. Mechanically,

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preconditioning with hydromorphone increased Bcl-2 and p-mTOR expression levels

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and decreased Bax expression levels. Rapa administration reverses the role of hydromorphone in protecting hippocampal CA1 neurons from IR injury.

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Hydromorphone protect hippocampal CA1 neurons from IR injury via activating

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mTOR signaling pathway.

Keywords: Hydromorphone (HM); ischemia–reperfusion (IR) injury; mTOR;

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oxidative stress; Bcl-2.

1. Introduction Ischemia/reperfusion (IR) injury is defined as the tissue damage which is caused 2

by blood supply returns to tissues after ischemia or lack of oxygen. IR injury always leads to myocardial infarction, stroke, and peripheral vascular disease [12, 22]. Through the induction of oxidative stress, the absence of oxygen and nutrients in blood during ischemia result in oxidative damage and the release of inflammatory

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mediators through the induction of oxidative stress [12]. After IR injury in brain, neuronal injury and death often occurs in vulnerable hippocampal CA1 pyramidal

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under the activation of cellular and molecular mechanisms [6]. Global brain I/R injury always developed in patients with systemic hypoperfusion and myocardial infarction,

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is becoming one of the leading reasons of morbidity and mortality [9, 10]. Therefore,

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the strategies to protect neuronal cells from I/R injury are urgently to needed to

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improve clinical outcomes.

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Hydromorphone (HM), also known as dihydromorphinone, is a centrally acting

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opioid medication for severe acute pain and severe chronic pain [26]. hydromorphone could also be used for relieving cancer pain [5, 29]and neuropathic pain in adults [33].

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However, due to the abuse potential, overdose risk and the side effects such as

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respiratory depression, bronchospasm, and urinary retention, hydromorphone can only be prescribed after other first-line treatments failed [1]. hydromorphone pre- and per-treatment significantly reduced ROS levels of rat glial cells, while selective

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antagonists for the μ, δ, κ opioid receptor partially negated the hydromorphone effect, demonstrating that hydromorphone has protective effects to glial cells via inhibiting ROS levels with the participation of μ, δ, κ opioid receptors [24]. In a case report, pain management with hydromorphone in a 44-year-old women leads to 3

recrudescence of focal stroke symptoms 30 years after the initial stroke events, indicating a potential role of hydromorphone in post-infarction functional neuroplasticity [7]. In this present study, we explored the role of hydromorphone in IR injury. We

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found that preconditioning with hydromorphone significantly increased Latency time, decreased apoptosis of hippocampal CA1 neurons and suppressed IR induced

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oxidative stress. Preconditioning with hydromorphone increased Bcl-2 and p-mTOR expression levels and decreased Bax expression levels. Moreover, Rapa

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administration reverses the role of hydromorphone in protecting hippocampal CA1

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neurons from IR injury. These data suggest that hydromorphone protect hippocampal

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2. Methods & materials

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CA1 neurons from IR injury via activating mTOR signaling pathway.

2.1 Animals and experimental protocol

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Animals were kept in the 21–24°C animal room which has a 12 h light/12 h dark

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cycle. Thirty-two male BALB/c mice weighing 25 to 30 g were divided into four groups randomly. Sham group: 5 days before surgery, normal saline with the same dose was injected subcutaneously. The learning test was performed 1 h before surgery.

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The mice were not performed occlusion of the bilateral common carotidarteries; IR group: normal saline was injected subcutaneously for 5 days. The learning test was performed 1 h before surgery. Then, the mice were performed ischemia for 30 mins; IR+HM group: hydromorphone was injected subcutaneously at 2 mg/kg for 4 days. 4

Then, the mice were performed hydromorphone injection at the dose of 3 mg/kg 4 hours before surgery. The learning test was performed 1 h before surgery. Then, the mice were performed by 30 mins ischemia; I/R+HM+Rapa group: subcutaneous injection of hydromorphone at 2 mg/kg for 4 days, followed by 3 mg/kg

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hydromorphone injection 4 hours before surgery. Then, the mice were administrated by Rapa at 5 mg/kg 3 hours before surgery. The learning test was performed 1 h

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before surgery, followed by 30 mins ischemia. The experimental protocol was showed

in Fig. 1. The dose of hydromorphone was chosen based on published papers [4, 25].

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This study was approved by the ethics committee of Quanzhou First Hospital.

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2.2 Ischemia–reperfusion (IR) induction

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Four hours after the last hydromorphone administration, For IR induction, mice were

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performed intraperitoneally injection using ketamine (50 mg/kg) and xylazine (10

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mg/kg) for anesthetization. Then, the mice were performed a neck incision for the explosion of the whole common carotid arteries, followed by vagus nerve release

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from the surrounding tissues. The arterial clamp was used to occlude bilateral carotid

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arteries for global cerebral ischemia induction, which were removed 30 minutes later. Reperfusion was performed for 24 hours for the determination of protein expression that are associated with enzymatic activity, performed for 72 hours for histological

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evaluation (Supplementary Table S1). During the surgery, mice body temperature was maintained at about 36°C by a rectal probe [37]. 2.3 Neurobehavioral assessments As described previously, passive avoidance test was chosen to determine learning and 5

memory performances, which has been described previously [4]. Briefly, the mice were adapted to the shuttle box conditions in the habituation trial before ischemia. The mice were maintained in light compartment for 5 seconds, followed by opening the guillotine door for mice to pass into dark compartment. After the mice went into

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the next compartment, the guillotine doors were closed for 10 seconds. Then, the mice were allowed to return to the cage. After 30 mins, the acquisition trial was performed.

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The mice were maintained in light compartment for 5 seconds, followed by opening

guillotine door. After the mice went into dark compartment, the doors were closed. At

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the meanwhile, the foot hock (50 Hz, 1 sec, 0.5 mA) was induced immediately.

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Retention trial was performed 24 hours after acquisition trial. The time in light

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compartment before crossing into dark compartment was determined as latency time.

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2.4 TUNEL staining

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The tissues were embedded into paraffin, and cut into 6 mm sections. The situ cell death detection kit was used to perform TUNEL staining according to the

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manufacturer’s instructions (Roche, IN, USA). In the sections, cells with brown

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nuclear cytoplasmic staining were TUNEL-positive. The number of apoptotic cells was quantitatively detected in five random different fields under light microscopy. All TUNEL-positive cells were determined for apoptotic cells. The experimental analyzes

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were done blindly. 2.5 RNA isolation and quantitative real-time PCR Total RNAs were isolated using TRIzol reagent (Invitrogen, CA) according to the manufacturer’s instructions, followed by cDNA synthesis using the PrimeScript RT 6

Reagent Kit (TaKaRa, China). β-actin mRNA expression levels were used for normalization. The relative mRNA expression levels of BCL2 and BAX were detected by 2 –ΔΔCT method. Primers used for detecting mRNA expression levels were listed in the supplementary Table S2.

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2. 6 Western blotting

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The hippocampal tissues were lysed using RIPA buffer, followed by analysis using

BCA protein assay kit. Then, the proteins were electrophoresed into 10% SDS–PAGE

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gel, followed by transfer to polyvinylidene fluoride membranes. The membranes were

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blocked using 5% milk at room temperature for 1 hour. Then, the membranes were

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incubated with anti-BCL2, anti-BAX, anti-p-mTOR or anti- mTOR primary antibody

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at 4°C overnight. Then, corresponding HRP-conjugated secondary antibodies were used for incubation at room temperature for 1 hour. Finally, signals were detected by

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enhanced chemiluminescence. β-Actin (CST) was used as control.

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2. 7 Malondialdehyde (MDA), Nox2 concentrations, superoxide dismutase (SOD) and catalase (CAT) activity

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The hippocampal tissues were put on ice with normal saline 4 hours after reperfusion, followed by frozen at -20°C for 5 mins. Then, the hippocampal tissues were

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centrifuged at 800 ×g for 15 mins. The concentration of MDA production in supernatants was detected using chemical assay kits (Nanjing Jiancheng Biologic Product). The Nox2 concentrations were determined by Nox2 ELISA kit. SOD and CAT activity were detected using chemical assay kits. All the detections were performed following manufacturer’s protocol. 7

2.8 Statistical analysis All data analyzed as mean ± SD are from 3 or more separate experiments. Student’s t-test was used to determine statistical significance.

P < 0.05 was considered

3. Results

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3.1 Hydromorphone treatment promotes memory performance

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significant.

The experimental protocol was showed in Fig. 1, during which the mice were divided

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into four groups: Sham, IR, IR+HM and IR+HM+Rapa.

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To explore the role of hydromorphone treatment on learning and memory

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performance, we performed passive avoidance test on the four groups. As shown in

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Fig. 2, the average latency time in Sham group is ~180 seconds, while significantly

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decreased to ~75 seconds after IR injury (IR group). Hydromorphone treatment (IR+HM group) recovered latency time to ~125 seconds. However, Papa treatment

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(IR+HM+Rapa group) which inhibit mTOR signaling pathway significantly decreased

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latency time to ~ 85 seconds. These data demonstrate that hydromorphone treatment has the protective role to IR injury, and this protective role is involved in mTOR signaling pathway.

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3.2 Hydromorphone treatment inhibits IR induced apoptosis We next detected the role of hydromorphone treatment in IR induced apoptosis of hippocampal CA1 neurons. In the Sham group, almost no TUNEL positive cells in the hippocampal CA1 neurons. After IR injury (IR group), TUNEL positive cells 8

significantly increased, indicating IR injury induced apoptosis in hippocampal CA1 neurons. Hydromorphone treatment (IR+HM group) significantly decreased the proportion of TUNEL positive cells, while inhibition of mTOR signaling pathway with Rapa (IR+HM+Rapa group) greatly increased apoptosis in hippocampal CA1

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neurons (Fig. 3a and 3b). 3.3 Hydromorphone treatment regulates BCL2 and BAX expression levels

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Given that hydromorphone treatment inhibits apoptosis of hippocampal CA1 neurons, we next detected whether hydromorphone treatment has the potential to regulate the

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expression levels of Bcl-2 and Bax, which are the apoptosis related genes (As shown

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in Fig. 4a, Bcl-2 mRNA levels decreased in IR group as compared with Sham group.

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hydromorphone treatment (IR+HM group) increased Bcl-2 mRNA levels, while Rapa

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administration (IR+HM+Rapa group) decreased Bcl-2 mRNA levels. Contrast to

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Bcl-2 expression, Bax mRNA levels increased after IR injury (IR group) as compared with Sham group, decreased after hydromorphone treatment (IR+HM group) and

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increased in IR+HM+Rapa group when compared IR+HM group (Fig. 4b).

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Bcl-2 and Bax protein levels showed a similar trend with the mRNA expression levels after hydromorphone or hydromorphone+Rapa treatment (Fig. 4c). The ratio of Bax to Bcl-2 (Bax/Bcl-2) increased after IR injury (IR group), decreased after

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hydromorphone treatment (IR+HM group). However, Rapa treatment significantly increased Bax/Bcl-2 ratio as compared with IR+HM group (Fig. 4d). 3.4 Hydromorphone treatment inhibits oxidative stress We next explored the influence of hydromorphone treatment on oxidative stress. The 9

levels of MDA (Fig. 5a) and Nox2 (Fig. 5b) increased significantly in IR group as compared with Sham group, and this increase was significantly prevented by hydromorphone treatment (IR+HM group) as compared to IR group. Rapa treatment (IR+HM+Rapa group) greatly increased MDA (Fig. 5a) and Nox2 (Fig. 5b) levels as

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compared with IR+HM group. The SOD (Fig. 5c) and CAT (Fig. 5d) significantly decreased in IR group as

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compared with Sham group. hydromorphone treatment (IR+HM group) significantly increased SOD (Fig. 5c) and CAT (Fig. 5d) levels, while Rapa treatment

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(IR+HM+Rapa group) greatly decreased SOD (Fig. 5c) and CAT (Fig. 5d) levels as

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compared with IR+HM group.

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3.5 Hydromorphone treatment activates mTOR pathway

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We next detected whether the mTOR signaling pathway was involved in the effect of

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hydromorphone treatment in IR injury. As shown in Fig. 6a and 6b, mTOR expression levels showed no great difference in different groups. However, the protein levels of significantly

increased

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p-mTOR

after

IR

injury

(IR

group).

Moreover,

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hydromorphone treatment (IR+HM group) further increased p-mTOR levels as compared with IR group. Rapa treatment (IR+HM+Rapa group) significantly

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decreased p-mTOR levels as compared with IR+HM group.

4. Discussion During

various

clinical

events,

such

as

organ

transplantation,

cardiopulmonary bypass, aortic cross-clamping and coronary angioplasty, IR injury 10

occurs and finally leads to disability and even death, which make IR injury as a major cause of disability and death worldwide [15]. Oxidative stress increased, pro-inflammatory cytokines released, and subsequently cell damage increased after ischemia. Reduced supplies of tissue glucose and oxygen in ischemic condition result

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in low ATP levels in cells. The increased oxidative stress leads to oxidative damage, which further caused cellular injury. Moreover, reperfusion after ischemia results in

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robust release of intracellular Ca2+, neutrophils recruitment, free radicals generation

and inflammatory responses, which further promote necrotic or apoptotic ell death [2].

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In central nervous system, IR injury disrupt blood–brain barrier, which further lead to

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increased intracranial pressure, cerebral oedema, irreversible tissue damage, worsened

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sensory, motor or cognitive functioning and death [13]. People have made a lot of

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efforts to decreased IR injury. When using IR injury mouse model, Maedeh Arabian

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and colleagues found that preconditioning with morphine improved memory performance, which resulted from the inhibition of apoptosis and neuronal loss in

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hippocampal CA1 neurons. In addition, morphine treatment increased p-mTOR

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protein levels and SOD activity in the hippocampus, indicating that chronic morphine treatment protects hippocampal CA1 neurons from IR injury via mTOR pathway activation [3]. In the transient global cerebral IR rat model, medetomidine treatment

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inhibited the number of apoptotic neurons, oxidative stress, inflammation and brain water content, demonstrating the protective role of medetomidine in IR injury [38]. Hydromorphone is a potent analogue of morphine, used for pain management clinically [16].

For opioid-naïve patients, hydromorphone is recommended at the 11

dose of 0.1 to 2 mg [30]. However, high dose of hydromorphone has the potential of opioid toxicity and abuse for patient s[14]. So, the dose-substitution policy was used to allow automatically lower dose of hydromorphone to minimize opioid toxicity incidence [30]. However, there is no evidence suggesting the role of hydromorphone

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in IR injury. In this study, we found that hydromorphone significantly improved the performance of ability of memory, decreased the proportion of TUNEL positive cells

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in hippocampal CA1 neurons, indicating that hydromorphone has the protective role in IR injury.

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When there is an imbalance between reactive oxygen species production and

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antioxidant defenses in systemic manifestation, oxidative stress was induced to

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damage all c the cell components, such as proteins, lipids, and DNA[8]. Oxidative

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stress has been shown play crucial roles in the development of cancer [17],

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Parkinson’s disease [20], autism [21], infection [23] and ect. Oxidative stress is also necessary during the development of IR injury. In the mouse model of renal IR injury,

IR

injury

induction

[19].

Tak

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and

colleagues

found

that

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after

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clopidogrel protect mice via inhibiting oxidative stress, which significantly increased

epigallocatechin-3-gallate reduced oxidative stress, inhibited apoptotic cell death, and had a protective role in hepatic IR injury [34]. In this study, we showed that oxidative

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stress significantly increased after IR injury, while decreased after hydromorphone treatment, indicating that hydromorphone protected mice from IR injury. The results in our study are similar with previous research, which shows that endogenous antioxidant system can be restored by many novel therapeutic agents. Therefore, the 12

role of hydromorphone in oxidative stress may be because of restored endogenous antioxidant system, which will be explored in our next study. mTOR is the mammalian target of rapamycin, also known as FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), is the mammalian target of

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rapamycin. Functionally, mTOR is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases [11, 32]. mTOR regulates cell

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growth, cell proliferation, cell survival, protein synthesis and protein transcription [18, 35]. Remote ischemic preconditioning (RIPC) showed protective role in hippocampus

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along with augment of p-mTOR expression, while rapamycin administration (mTOR

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inhibitor) abolished all protective effects of RIPC, which suggested that mTOR

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played an important role in RIPC induced hippocampal protection [37]. Hydrogen

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sulfide (H2S) treatment increased cell vitality and decreased lactate dehydrogenase

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activity in IR injury. The expression levels of p-mTOR were found to be up-regulated after H2S treatment, while rapamycin decreased the protective effect of H2S,

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indicating that H2S plays a protective role against IR injury via the activation of

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mTOR signaling [36]. In this study, we found that inhibition of mTOR by rapamycin decreased memory performance, increased apoptosis and oxidative stress in

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hippocampal CA1 neurons. We found that preconditioning with hydromorphone protects hippocampal CA1

neurons from IR injury by activating the mTOR pathway. However, mTOR inhibition by therapeutic agents also has the protecting role in neurodegenerative disorders by activating protective autophagy [28]. Regarding the protecting role of hydromorphone 13

in IR injury, the molecular targets of hydromorphone may not only mTOR. The new targets of hydromorphone needed to be explored in our future study. It is worthy to note that rapamycin treatment may have systemic influence on mice, not only mTOR signaling pathway alone. Moreover, as antibiotics, rapamycin

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may have influence on gut microbiota [27], which has been showed to involved in various biological process [31]. Therefore, the influence of rapamycin on some other

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signaling pathways should be detected in future.

Taken together, we found that preconditioning with hydromorphone significantly

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increased Latency time, decreased apoptosis of hippocampal CA1 neurons and

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suppressed IR induced oxidative stress. Mechanically, preconditioning with

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hydromorphone increased Bcl-2 and p-mTOR expression levels and decreased Bax

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expression levels. Inhibition of mTOR by rapamycin administration attenuates the

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role of hydromorphone in protecting hippocampal CA1 neurons from IR injury. These

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data indicate the potential role of hydromorphone for clinical treatment of IR injury.

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5. Conclusion

Hydromorphone protect hippocampal CA1 neurons from IR injury via activating mTOR signaling pathway. These data indicate the potential role of hydromorphone

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for clinical treatment of IR injury.

Acknowledgements None. 14

Disclosure of potential conflicts of interest The authors declare that they have no conflict of interest.

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Figure legends

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PLoS One 11 (2016) e0151620.

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Figure 1. Experimental protocol.

Figure 2. Latency time in the passive avoidance test in the different groups. Data are

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presented as mean ± SD. *p ＜ 0.05, **p ＜ 0.01 compared to sham group. #p ＜ 0.05 compared to IR group. &p ＜ 0.05 between the comparison of IR+HM and IR+HM+Rapa groups.

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Figure 3. Effects of preconditioning with hydromorphone on ischemia-induced apoptosis of NM. a) Representative immunohistochemical images of TdT-mediated dUTP nick end-labeling (TUNEL, 400×) in ischemic hippocampal CA1 neurons. b) TUNEL-positive cell counts after reperfusion. Data are presented as mean ± SD. *p

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＜ 0.05, ***p ＜ 0.001 compared to sham group. #p ＜ 0.05 compared to IR group.

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&p ＜ 0.05 between the comparison of IR+HM and IR+HM+Rapa groups.

Figure 4. Preconditioning with hydromorphone suppressed ischemia-induced

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apoptosis of hippocampal CA1 neurons. a-b) The expression of bcl-2, bax was

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measured by Real time PCR. c) Western blotting was used to assay the protein

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expressions, β-actin was used as a loading control and relative expressions of

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bax/bcl-2 from the western blotting (d). Data are presented as mean ± SD. *p ＜

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0.05, **p ＜ 0.01 and ***p ＜ 0.001 compared to sham group. #p ＜ 0.05 ##p ＜ 0.01 and ###p ＜ 0.001 compared to IR group. &p ＜ 0.05 and &&p ＜ 0.01

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between the comparison of IR+HM and IR+HM+Rapa groups.

Figure 5. Preconditioning with hydromorphone of hippocampal CA1 neurons. MDA and Nox2 levels along with, and SOD and CAT activity in hippocampal tissue isolated

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from each group. Data are presented as mean ± SD. *p ＜ 0.05, **p ＜ 0.01 and ***p ＜ 0.001 compared to sham group. #p ＜ 0.05 ##p ＜ 0.01 compared to IR group. &p ＜ 0.05 and &&p ＜ 0.01 between the comparison of IR+HM and IR+HM+Rapa groups. 18

Figure 6. Preconditioning with hydromorphone protects ischemia-reperfusion injury on hippocampal CA1 neurons by activating the mTOR pathway. a) Western blotting was used to assay the protein expressions of p-mTOR and mTOR, β-actin was used as

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a loading control and relative expressions from the western blotting (b). Data are presented as mean ± SD. **p ＜ 0.01 compared to sham group. #p ＜ 0.05

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compared to IR group. &p ＜ 0.05 between the comparison of IR+HM and

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IR+HM+Rapa groups.

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