The nuclear factor E2-related factor 2 participates in morphine-induced autophagy

doi:10.21203/rs.3.rs-2153863/v1

Download PDF

Research Article

The nuclear factor E2-related factor 2 participates in morphine-induced autophagy

https://doi.org/10.21203/rs.3.rs-2153863/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Morphine is an important potent analgesic which has many side effects such as addiction. Studies have demonstrated that autophagy play an important role during morphine addiction especially in morphine withdrawal. However, the molecular basis of morphine-induced autophagy has been not fully elucidated. The nuclear factor E2-related factor 2 (NRF2) is a well-known transcription factor that plays a critical role in antioxidation and even autophagy, furthermore, previous study showed that it increased in the frontal cortex and striatum of the mice which suffering from morphine withdrawal, indicating that NRF2 may involve in morphine addiction. In the present study, we aim to clarify the role of NRF2 in morphine-induced autophagy in vitro. Our results showed morphine could significantly increase the expression of NRF2 and further induce autophagy in SH-SY5Y cells. In addition, pretreatment of NRF2 inhibitor could reverse the upregulation of NRF2 and autophagy after morphine administration, indicating NRF2 play a key role in morphine-induced autophagy. Interestingly, we found NRF2 was also regulated by autophagy because pretreated with 3-Methyladenine after morphine administration significantly increased the expression of NRF2. In conclusion, our study revealed a critical role of NRF2 in morphine-induced autophagy and the feedback effect of autophagy on NRF2.

addiction

morphine

autophagy

nuclear factor E2-related factor 2

SH-SY5Y

Morphine is treated as an effective medicine for pain treatment for a long time [1], it has been used for treating various types of pain such as postoperative pain, neuropathic pain, and cancer pain in clinical practice [2, 3]. At the same time, it is undeniable that chronic and repeated exposure to morphine also brings many side effects, including tolerance, respiratory depression, immunosuppression, and the most seriously, addiction [4–6]. Nowadays, morphine addiction has become a global problem, however, the exact mechanism of morphine addiction has not been fully understood.

Autophagy is a critical catabolic process to maintain cell homeostasis and it can be activated by various stresses such as nutrient starvation and oxidative stress [7]. During autophagy process, long-lived proteins and damaged cellular components are delivered to the lysosome, then, the contents of the lysosome are degraded into amino acids for recycling. Generally, autophagy can be divided into initiation, phagophore nucleation, autophagosome elongation, and autophagolysosome fusion. During this process, there are many “autophagy-related” proteins governing each of these steps. For instance, the absence of mammalian target of rapamycin (mTOR) mediated ULK1 suppression is believed to regulate lots of autophagy processes such as initiation [8, 9], besides, the LC3II which is a cleaved and lipidated form of MAP1LC3 is proved to be indispensible in autophagosome elongation [10], therefore, mTOR and LC3II are treated as important autophagy biomarkers.

A fine balance of autophagy is necessary for the normal brain function. Study has reported that the dysregulation of autophagy leads to the progression of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases [11]. Furthermore, accumulating evidences indicate that autophagy is closely related to morphine and morphine addiction [12, 13]. For example, previous study have reported that morphine prompts Beclin1 expression and inhibits the interaction between Beclin1 and Bcl-2, besides, knockdown Beclin1 or knockout of autophagy-related 5 (ATG5) could both inhibit morphine-induced autophagy [14]. Morphine is also found to facilitate LPS-induced autophagy, it inhibits autophagolysosomal fusion leading to reduced bacterial clearance and increased bacterial load [15]. In addition, it is worth noting that study came from Su et al. revealed that knockout of ATG5 and ATG7 in dopaminergic neurons impaired the construction of morphine-induced addictive behaviors [13]. Therefore, it could be suggested that autophagy play an important role in morphine addiction, and identifying molecular alterations underlying morphine-induced autophagy is helpful to clarify the mechanism of morphine addiction.

The nuclear factor E2-related factor 2 (NRF2) plays a key role in cellular defenses against oxidative stress [16]. The expression of NRF2 is controlled by Kelch ECH-associated protein 1 (Keap1). Under normal physiological conditions, NRF2 is degradated through ubiquitin proteasome system which makes it keep at a low level [17], however, when the cell suffers from oxidative stress, NRF2 is released from Keap1 and transferred from the cytoplasm to the nucleus, leading to the accumulation of NRF2 in the nucleus, activating the expression of antioxidant enzymes. Previous study has demonstrated that NRF2 increased in the frontal cortex and striatum of morphine-treated mice which may play a key role in regulating morphine withdrawal symptom [18]. Besides, it is reported that NRF2 participates in the process of autophagy. NRF2 activation is an upstream regulator of autophagy which protects against neurodegeneration in the PD models [19]. Additionally, Daniela Perroni Frias et al. reported that NRF2 might also regulate the expression of autophagy-related genes such as ATG5, p62, and MAP1LC3 [20]. However, whether NRF2 engages in the morphine-caused neuronal cell autophagy remains unclear.

In this study, we aim to clarify the role of NRF2 in morphine-induced autophagy in vitro. To this end, SH-SY5Y cells were exposed to different concentrations of morphine, then, western blotting was used to measure the expression of LC3II/LC3I and p-mTOR/mTOR, and furthermore, the LC3 puncta, as well as the autophagosome were also tested by immunofluorescence and electron microscopy separately to examine morphine-induced autophagy. We also used ML385 (an inhibitor of NRF2) to explore the role of NRF2 in morphine-induced autophagy. Our results indicated that morphine can induce autophagy in neuronal cell and morphine-induced autophagy in immortalized neuronal cell line is NRF2 dependent.

2.1. Regents

The following antibodies and chemicals were used in this study: rabbit anti-LC3 (Cell Signaling Technology, 43566), rabbit anti-NRF2 (Cell Signaling Technology, 12721), rabbit anti Phospho-mTOR (Cell Signaling Technology, 5536), rabbit anti-mTOR (Cell Signaling Technology, 2983), mouse anti-β-Actin (Cell Signaling Technology, 3700), goat anti-mouse IgG-HRP (absin, abs20039), goat anti-rabbit IgG-HRP (absin, abs20040), ML385 (MedChemExpress, HY-100523 ), chloroquine (MedChemExpress, HY-17589A), 3-Methyladenine (MedChemExpress, HY-19312 ), rapamycin (MedChemExpress, HY-10219), morphine (The Third Research Institute of Ministry of Public Security, Shanghai, China, CAS NO. 57-27-2, Purity 99%).

2.2. Cell culture and treatment

SH-SY5Y cells were incubated in Dulbecco’s modified Eagle’s medium (Gibco, C11995500BT) containing 10% foetal bovine serum (Biological Industries, 04-001-1C), and 1% penicillin-streptomycin solution (Hyclone, SV30010), at 37°C with 5% CO₂. SH-SY5Y cells were treated with 0 to 1 mM morphine for 12 h. 10 µM ML385 was added into the culture medium of SH-SY5Y cells to inhibit NRF2 expression.

2.3. Immunofluorescence staining

SH-SY5Y cells were treated with various concentrations of morphine for 12 h. Then, the cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS for 5 min. After that, cells were permeabilized with 0.5% TritionX-100 for 15 min and washed 3 times with PBS, then they were blocked with 1% Bovine Serum Albumin for 30 min at room temperature. The samples were incubated with primary antibodies against LC3 (1:6400) overnight at 4°C. After washing the samples with PBS for 3 times, the samples were incubated with secondary antibodies (FITC goat anti-rabbit IgG, 1:100; AS011, ABclonal) for 2 h at room temperature. Next, DAPI dihydrochloride (DIYIBIO, DY20101) was used for nucleus staining. Finally, the images were recorded with a Zen microscope (ZEN 3.2, ZEISS).

2.4. Western Blotting

Cells were lysed in RIPA lysis buffer (Beyotime, P0013B) on ice for 5 min, which contained protease inhibitor and phosphatase inhibitor. Then the protein was quantified using a bicinchoninic acid kit (Beyotime, P0012). The cell lysates were separated by SDS-PAGE for immunoblotting. After that the protein was transferred to PVDF membrane and blocked by 5% non-fat milk in PBST. The membrane was incubated with primary antibodies overnight at 4°C. Followed by incubation with secondary antibodies for 2 h at room temperature. The epitopes were visualized using an ECL western blotting detection kit (Millipore, WBKLS0100).

2.5. Transmission electron microscopy

SH-SY5Y cells were collected and fixed with 2.5% glutaraldehyde at 4°C overnight. Transmission electron microscopy was performed by the Microscopy Core Facility at Xi'an Jiao Tong University.

2.6. Statistical analysis

All results were repeated in at least three independent experiments; Data were represented as means ± SEM. The data were evaluated statistically by one-way analysis of variance (ANOVA). Significance was determined as P < 0.05.

3.1. Morphine could induce autophagy in SH-SY5Y cells

During autophagy process, LC3 exists in two forms: LC3I (the cytoplasmic form, 18 kDa) and LC3II (the cleaved and lipidated form, 16 kDa). To explore the relationship between autophagy and morphine, SH-SY5Y cells were treated with various doses of morphine (0, 0.5, and 1 mM) at different time points (0, 6, and 12 h). We found that the LC3II/LC3I ratio increased after morphine treatment whereas the p-mTOR/mTOR ratio decreased (Fig. 1A to E). Those results indicated that morphine might promote SH-SY5Y cell autophagy. We next investigated whether morphine-induced autophagy was widespread in other neuronal cells. As shown in Fig. S1A to D, the similar results were also found in PC12 (a rat undifferentiated neuronal cell line), HT22 (a mouse hippocampal neuronal cell line), but not BV2 (a mouse microglia cell line) cells, thus, our results demonstrated that morphine induced autophagy in neurons. Morphine-induced autophagy was further validated by immunofluorescence which showed increased puncta of LC3 in morphine-treated cells (Fig. 2A). We also used electron microscopy to observe autophagic vacuole in morphine-treated SH-SY5Y cells. As shown in Fig. 2B, autophagic vacuole could be detected by electron microscopy. Taken together, these results demonstrated that morphine could induce autophagy in neuronal cells.

3.2. NRF2 is highly expressed in morphine-treated cells

We next examined the expression of NRF2 in morphine treated SH-SY5Y cell. As shown in Fig. 3A to C, NRF2 as well as LC3II/LC3I ratio increased remarkably in SH-SY5Y cell after morphine stimulation in a dose-dependent manner, furthermore, the correlation analysis revealed that NRF2 may be involved in morphine-induced autophagy because it was positively correlated with LC3II/LC3I ratio (Fig. 3D). Next, in order to identify the increase of LC3II level was due to the increase of autophagosome formation or a defect in the process of the fusion of autophagosome and lysosome, we used chloroquine (CQ), an autophagy inhibitor, which can inhibit the fusion of autophagosome and lysosome. Western blotting results demonstrated that CQ can significantly increase the level of NRF2 and LC3II/LC3I (Fig. 3E to G). Taken together, these results further validated the relationship between NRF2 and autophagy.

3.3. NRF2 is a potential regulator in morphine-induced autophagy

To further analyze whether morphine-induced autophagy is mediated by NRF2, we use ML385 to determine the effect of NRF2 on morphine-induced autophagy. Western blotting results showed that NRF2 inhibitor remarkably diminished the protein level of NRF2 (Fig. 4A and B) and the ratio of LC3II/LC3I in morphine-treated cells (Fig. 4C and D). Meanwhile, the p-mTOR/mTOR expression rate was also rescued in morphine-exposed cells after ML385 treatment (Fig. 4C and E). We also observed significantly decreased puncta of LC3 in immunofluorescence results after the inhibition of NRF2 (Fig. 4F). These results indicated that NRF2 may be a potential regulator in morphine-induced autophagy.

3.4. Morphine-induced NRF2 increase is partially inhibited by autophagy feedback

In order to determine whether the increase of NRF2 in morphine-treated cells is regulated by autophagy, we used rapamycin and 3-methyladenine (3-MA) to induce or inhibit the autophagy level in morphine-exposure SH-SY5Y cells. As shown in Fig. 5, our results demonstrated that compared with morphine group, pre-treated with 3-MA significantly increased NRF2 expression while pre-treated with rapamycin had no influence on NRF2, suggesting that NRF2 expression was regulated by autophagy. These results were contradicted with our previous data which NRF2 was positively related with LC3II/LC3I ratio. We believed there was a feedback inhibition of NRF2 protein level by autophagy in morphine-treated cells which could balance the expression level of NRF2.

Chronic morphine exposure could induce drug addiction and affect brain functions [21–24]. As we all know, withdrawal is a part of morphine addiction, this uncomfortable syndrome occurs after morphine discontinued and it was also the major reason of relapse. For the past few years, increasing evidence has demonstrated that autophagy participates in morphine withdrawal. Besides, studies also indicate that oxidative stress functions in morphine withdrawal. Given that NRF2 plays a key role in cellular defenses against oxidative stress, it would be interesting to determine the relationship among NRF2, autophagy and morphine.

Morphine is one of the most abundant alkaloids using for analgesia worldwide. Although morphine has strong analgesic activity, morphine addiction has been a very serious social issue [25]. Many efforts have been made to explore the exact molecular basis of morphine addiction. A lot of studies demonstrate that autophagy participated in morphine addiction. Feng et al. have already found the mitochondrial dysfunction was accompanied by opioid withdrawal and melatonin could regulate those process by attenuate autophagy [26]. The results come from Liu et al. also showed that morphine induced autophagy through RACK1 in SH-SY5Y cell [27], all these results were similar to our present study.

Oxidative stress plays a critical role in morphine addiction. Previous studies have reported that some antioxidants such as thymoquinone, vitamin E, and bergenin not only could block morphine-induced oxidative stress, but also could attenuate morphine withdrawal [18, 28, 29]. On the other hand, oxidative stress-induced mitochondrial dysfunction could also contribute to cellular stress responses such as autophagy [30]. As an important component of antioxidant system, NRF2, which has been demonstrated closely related to autophagy, increased in the frontal cortex and striatum of morphine-treated mice [18]. Similar to these results, in the present study, we found NRF2 also increased in the morphine-treated SH-SY5Y cell, at the same time, our results demonstrated that the up-regulated NRF2 further involved in this process because ML385, a NRF2 inhibitor, significantly attenuated morphine-induced autophagy. Collectively, our results and the recent literature indicated that NRF2 may be a promising candidate in regulating morphine addiction.

It should be noted that there are lots of researches reported the major function of NRF2 is antioxidation. As a transcription factor, NRF2 has been proved to regulate some antioxidant enzymes such as HO1 and NQO1, and those enzymes play protecting roles during the occurrence of oxidative stress [31, 32]. Researches also demonstrated that NRF2 could drive oxidative stress- induced autophagy [33, 34], which resembled our present results. On the other hand, in the study about diabetic cardiomyopathy, NRF2 is proved to either ameliorate or worsen this disease [35]. Besides, NRF2-dependent autophagy may be regulated by a feedback, previous study has reported that a Keap1-NRF2-p62 feedback could induce autophagy and further protect intervertebral disc from degeneration [33], however, whether a Keap1-NRF2-p62 feedback is also existed during morphine-induced autophagy need further explore.

In the present study, we found that 3-methyladenine which is an autophagy inhibitor could increase the expression of NRF2. The above evidence seems to give us a hint that NRF2 could either induce autophagy or inhibit by autophagy. Furthermore, the results come from Liu et al. also showed that autophagy feedback also can be detected in morphine-induced RACK1 up-regulation in SH-SY5Y cell [27], which was similar to our results. They believe although autophagy could inhibit the expression of RACK1, the function is insufficient to resist the RACK1 up-regulation induced by morphine. We think this explanation also applies to our present results.

In summary, we believe that NRF2 is an important regulator in morphine-induced autophagy. Besides, autophagy, in turn, produces a negative effect on the increase of NRF2 in morphine-exposed cells. However, the detailed mechanism how NRF2 was upregulated in the morphine-treated cells remains unclear. It is possible that upregulated expression of NRF2 was mediated by the mu opioid receptors because it has been reported that morphine-induced autophagy is mediated by the mu opioid receptor [14]. Also, it would be interesting to elucidate the potential signaling pathway of NRF2 in regulating morphine-induced autophagy. In addition, although our study has experimentally demonstrated the mechanisms of morphine-induced autophagy in cultured cells, the role of autophagy and NRF2 in morphine addiction needs further validation in animal models. Taken together, identify the exact molecular signaling mechanisms underlying the regulation of autophagy by NRF2 and how NRF2 has effect on brain function during morphine treatment, will contribute to clinical application of morphine or therapeutic interventions of morphine abuse.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (82171880).

Sacerdote P (2006) Opioids and the immune system. Palliat Med 20(1):s9–15
Bruel BM, Burton AW (2016) Intrathecal Therapy for Cancer-Related Pain. Pain Med 17(12):2404–2421
Cooper TE et al (2017) Morphine for chronic neuropathic pain in adults. Cochrane Database Syst Rev 5:CD011669
Xiao X et al (2021) NADPH-Oxidase 2 Promotes Autophagy in Spinal Neurons During the Development of Morphine Tolerance. Neurochem Res 46(8):2089–2096
Qiu S et al (2015) Chronic morphine-induced microRNA-124 promotes microglial immunosuppression by modulating P65 and TRAF6. J Immunol 194(3):1021–1030
Kenny PJ (2011) Common cellular and molecular mechanisms in obesity and drug addiction. Nat Rev Neurosci 12(11):638–651
Zhang W, Feng C, Jiang H (2021) Novel target for treating Alzheimer's Diseases: Crosstalk between the Nrf2 pathway and autophagy. Ageing Res Rev 65:101207
Zhu Z et al (2019) Balancing mTOR Signaling and Autophagy in the Treatment of Parkinson's Disease.Int J Mol Sci, 20(3)
Al-Bari MAA, Xu P (2020) Molecular regulation of autophagy machinery by mTOR-dependent and -independent pathways. Ann N Y Acad Sci 1467(1):3–20
Rodríguez AE et al (2016) BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism. Autophagy 12(2):287–296
Kiriyama Y, Nochi H (2015) The Function of Autophagy in Neurodegenerative Diseases. Int J Mol Sci 16(11):26797–26812
Su LY et al (2021) Molecular Mechanism of Neuroprotective Effect of Melatonin on Morphine Addiction and Analgesic Tolerance: an Update. Mol Neurobiol 58(9):4628–4638
Su LY et al (2017) Atg5- and Atg7-dependent autophagy in dopaminergic neurons regulates cellular and behavioral responses to morphine. Autophagy 13(9):1496–1511
Zhao L et al (2010) Morphine induces Beclin 1- and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus. Autophagy 6(3):386–394
Wan J et al (2015) Morphine potentiates LPS-induced autophagy initiation but inhibits autophagosomal maturation through distinct TLR4-dependent and independent pathways. Acta Physiol (Oxf) 214(2):189–199
Ma Q (2013) Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol 53:401–426
Dhamodharan U et al (2018) Association of single-nucleotide polymorphisms of the KEAP1 gene with the risk of various human diseases and its functional impact using in silico analysis. Pharmacol Res 137:205–218
Yun J et al (2015) Bergenin decreases the morphine-induced physical dependence via antioxidative activity in mice. Arch Pharm Res 38(6):1248–1254
Guo Q et al (2021) Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models.Cells, 10(8)
Frias DP et al (2020) Nrf2 positively regulates autophagy antioxidant response in human bronchial epithelial cells exposed to diesel exhaust particles. Sci Rep 10(1):3704
Torkaman-Boutorabi A et al (2019) Morphine Exposure and Enhanced Depression-like Behaviour Confronting Chronic Stress in Adult Male Offspring Rat. Basic Clin Neurosci 10(4):323–332
Ide S et al (2021) Caenorhabditis Elegans Exhibits Morphine Addiction-like Behavior via the Opioid-like Receptor NPR-17. Front Pharmacol 12:802701
Wang T, Chen X, Zeng K (2022) Molecular mechanism and candidate biomarkers of morphine for analgesia and addiction effects. Ann Transl Med 10(2):89
Gillespie A et al (2022) microRNA expression levels in the nucleus accumbens correlate with morphine-taking but not morphine-seeking behaviour in male rats. Eur J Neurosci 55(7):1742–1755
Kim J et al (2016) Brain Reward Circuits in Morphine Addiction. Mol Cells 39(9):645–653
Feng YM et al (2013) Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin. Autophagy 9(9):1395–1406
Liu LT et al (2020) Morphine-induced RACK1-dependent autophagy in immortalized neuronal cell lines. Br J Pharmacol 177(7):1609–1621
Abdel-Zaher AO et al (2013) Inhibition of brain oxidative stress and inducible nitric oxide synthase expression by thymoquinone attenuates the development of morphine tolerance and dependence in mice. Eur J Pharmacol 702(1–3):62–70
Pinelli A et al (2009) Morphine or its withdrawal affects plasma malondialdehyde, vitamin E levels and absence or presence of abstinence signs in rats. J Pharm Pharmacol 61(4):487–491
Nunnari J, Suomalainen A (2012) Mitochondria: in sickness and in health. Cell 148(6):1145–1159
Wang Y et al (2019) HO-1 reduces heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative stress. Aging 11(15):5535–5547
Feng Y et al (2019) Methane Alleviates Acetaminophen-Induced Liver Injury by Inhibiting Inflammation, Oxidative Stress, Endoplasmic Reticulum Stress, and Apoptosis through the Nrf2/HO-1/NQO1 Signaling Pathway. Oxid Med Cell Longev, 2019: p. 7067619
Tang Z et al (2019) Nrf2 drives oxidative stress-induced autophagy in nucleus pulposus cells via a Keap1/Nrf2/p62 feedback loop to protect intervertebral disc from degeneration. Cell Death Dis 10(7):510
Ichimura Y et al (2013) Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. Mol Cell 51(5):618–631
Zang H et al (2020) Autophagy Inhibition Enables Nrf2 to Exaggerate the Progression of Diabetic Cardiomyopathy in Mice. Diabetes 69(12):2720–2734

No competing interests reported.

SupplementaryMaterial.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

The nuclear factor E2-related factor 2 participates in morphine-induced autophagy

Status:

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1. Regents

2.2. Cell culture and treatment

2.3. Immunofluorescence staining

2.4. Western Blotting

2.5. Transmission electron microscopy

2.6. Statistical analysis

3. Results

3.1. Morphine could induce autophagy in SH-SY5Y cells

3.2. NRF2 is highly expressed in morphine-treated cells

3.3. NRF2 is a potential regulator in morphine-induced autophagy

3.4. Morphine-induced NRF2 increase is partially inhibited by autophagy feedback

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1