Melatonin Attenuates Ropivacaine-Induced Apoptosis by Inhibiting Excessive Mitophagy Through the Parkin / PINK1 Pathway in PC12 and HT22 Cells

Melatonin, as an endogenous circadian indoleamine, is secreted from the pineal gland and has extensive biological functions, including antioxidant, anti-inammatory, anti-tumor and neuroprotective effects. Its neuroprotective effects make it to be a potential therapeutic for many nerve damages, but its effect on ropivacaine-induced neurotoxicity remains unclear. The aims of our research were to explore the impact and mechanism of melatonin on ropivacaine-induced neurotoxicity. Our results shown that melatonin pretreatment protected the cell viability, morphology, and apoptosis of PC12 and HT22 cells from ropivacaine, it also improved ropivacaine-induced mitochondrial dysfunction and it inhibited an increase of mitophagy levels by ropivacaine. In addition, our study also revealed that the increase of mitophagy levels could reduce the protective effect of melatonin on ropivacaine-induced apoptosis in PC12 and HT22 cells, but the inhibition of mitophagy could enhance this effect of melatonin. To further illustrate the mechanism of melatonin inhibited mitophagy, the expressions of Parkin and PINK1 were detected, and results shown that melatonin inhibited the activation of Parkin / PINK1 pathway. In conclusion, our results indicated that melatonin protected ropivacaine-induced apoptosis through suppressing excessive mitophagy by inhibiting the Parkin / PINK1 pathway. Melatonin may be a useful potential therapeutic agent against ropivacaine-induced neurotoxicity.


Introduction
Melatonin, as an endogenous circadian indoleamine, is secreted from the pineal gland and has extensive biological functions, including antioxidant, anti-in ammatory, anti-tumor and neuroprotective effects [1,2]. The neuroprotective of melatonin have been reported, it plays an important role in the regulation of neurogenesis and may be a potential therapeutic for many nerve damages [3]. Melatonin not only promotes the viability and proliferation of neural stem cells, but also enhances the expression of neuronal markers in the PC12 cells [4,5]. In addition, melatonin plays a protective role in many neurodegenerative diseases [6,7]. Melatonin attenuates nerve impairment in the Parkinson's disease (PD) in vito and vitro experiment [8]. It also has a neuroprotective effect against Alzheimer's disease (AD) and improved the spatial learning and memory de cits in AD by reducing the Aβ production [9]. Apart from protecting brain neurons, melatonin protects against the spinal cord injury and peripheral nerve impairment [3]. In a word, melatonin could be a possible treatment target for many neuronal disorders. But, the effect of melatonin on ropivacaine-induced neurotoxicity remains unclear.
Ropivacaine is widely used for peripheral nerve blocks, epidural anaesthesia, spinal anaesthesia and pain management [10]. However, epidemiological studies shown that ropivacaine induced neurotoxicity at the high dosage and long duration of exposure. The main symptoms are transient neurologic syndrome (TNS), cauda equina syndrome (CES) and delayed sacral nerve disorder [10]. Although most damages are transient, major complications will produce permanent nerve damage. It's important to develop preventive and therapeutic drugs for clinical and basic research toward the neurotoxicity by ropivacaine on basis of understanding the exact mechanism. In recent years, many studies have revealed that there are various pathophysiological processes involved in ropivacaine-induced neurotoxicity, included intracellular Page 3/20 calcium concentration, cell apoptosis, inflammation and autophagy [11][12][13][14]. Wen et al. reported the inhibition of T-type calcium channel could improve ropivacaine-induced cell damage [15]. Wang and Luo et al. found apoptosis was a key point in ropivacaine-induced neurotoxicity, and the Fas / Fasl meditated exogenous apoptosis pathway was involved in the process [16,17]. In addition, Xiong et al. demonstrated ropivacaine up-regulated autophagy levels in neuronal cells and inhibition of autophagy aggravated ropivacaine-induced neurotoxicity [10]. But, in our study, we found that the melatonin pretreatment could inhibit ropivacaine-induced apoptosis of PC12 and HT22 cells via down-regulating the excessive mitophagy, which was a protective mechanism against the neurotoxicity by ropivacaine.
The present study aimed to explore the impact and mechanism of melatonin on ropivacaine-induced neurotoxicity. Our study was the rst to reveal that melatonin protected cell viability, morphology, apoptosis, and mitochondrial dysfunction in PC12 and HT22 cells from ropivacaine, it also reduced the mitophagy levels and down-regulated the expression of Parkin / PINK1 pathway. Subsequently, our study demonstrated that the activation of mitophagy could reduce the protective effect of melatonin on ropivacaine-induced apoptosis, but the inhibition of mitophagy could enhanced this effect of melatonin. Which indicated melatonin attenuates ropivacaine-induced apoptosis via inhibiting the excessive mitophagy.

Cell culture
The Rat pheochromocytoma cell line (PC12) and Hippocampal neuronal cell line (HT22) were purchased from the Cell Bank of Shanghai Institute of Chinese Academy of Sciences. These cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM, Gibco, USA) containing 10% of fetal bovine serum (FBS, Gibco, Australia), 100 IU/ml of penicillin G sodium, and 100 mg/ml of streptomycin sulfate at 37 °C in an incubator maintained with 5% CO 2 at 37°C.

Cell viability assay
The cytotoxicity of melatonin on PC12 cells and HT22 cells was detected by the cell counting kit-8 (CCK-8, Biosharp, Shanghai, China) assay according to the manufacturer's instruction. PC12 cells and HT22 cells were separately seeded in the 96-well plate at a density of 5×10 3 cells / well and then treated with PBS or various concentrations of melatonin (5-40 μM) in 100 μl of medium. There were six wells in each group. After incubation for 24 h, 10 μl of CCK-8 solution was added to each well, followed by incubation for 2 h at 37 °C. The absorbance was measured in a microplate reader (SpectraMax®iD3, Molecular Devices, USA) at a wavelength of 450 nm. To explore the protective effect of melatonin, cell was pretreated with different concentration of melatonin for 2 h, then adding 1mM ropivacaine to co-culture for different times (24 h, 48 h, 72 h), the cell viability was detected as above.
2.4 Detection of cell apoptosis and necrosis by Hoechst / PI staining Cells were seeded into 6-well plates at a density of 1×10 5 cells / well, and then pre-treated with melatonin for 2 h, 1 mM ropivacaine was added and incubation for 24 h. The medium was removed, 1 ml of phosphate buffer saline (PBS) containing 5 µl of Hoechst33342 and 5 µl of propidium iodide (PI) was added to each well, followed by incubation for 15 min at 4°C. Therefore, cells were washed with PBS, and observed under a fluorescence microscope (Olympus, IX73, Japan). Representative photographs were captured. The red fluorescence positive cells were counted and the percentage of necrotic cells was calculated.

Determination of Caspase-3 activity
Cells were seed in a 6-well plate at a density of 1×10 5 cells / well, when the density reached 60-80%, various concentrations of melatonin (5uM, 10uM, 20uM) were added, after cultured for 2 h, those cells were treated with 1 mM ropivacaine. Then, the caspase-3 activity of PC12 and HT22 cells were detected by Caspase-3 spectrophotometric detection kit (Wanleibio, Shenyang, China) according to the manufacturer's instructions.

Determination of the Mitochondrial reactive oxygen species
Cells were seed in a 12-well plate at a density of 1×10 4 cells / well, when the density reached 60-80%, various concentrations of melatonin (5uM, 10uM, 20uM) were added, after cultured for 2 h, those cells were treated with 1 mM ropivacaine. After the incubation of 24 h, the mitochondrial reactive oxygen species was measured by the H 2 DCFDA ROS assay and the Mito-Tracker Red CMXRos according to the manufacturer's instruction. At last, the Hoechst 33258 was added for 5 min to label the nucleus.

Determination of the Mitochondrial membrane potential assay
The Mitochondrial membrane potential (ΔΨm) assay was based on JC-1 staining (5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide). JC-1 has two forms including monomers and polymers, the emission spectra of which are different. When the mitochondrial membrane potential reduces, the JC-1 polymer with red uorescence decreases, and so JC-1 monomer with green uorescence was in the cytoplasm. Following the instructions of JC-1 assay kits, the ow cytometry was to measure ΔΨm.

Immuno uorescence staining
The cells mitochondria were labeled by Mito-Tracker Red CMXRos according to the manufacturer's instructions. Cells were xed in 4% PFA and permeabilized with 0.2% Tween X-100. After being blocked with normal goat serum, cells were incubated with rabbit anti-rat p-STAT3 (1:200) overnight at 4℃, and the second antibody (1:1000) was incubated for 1 h at 37℃ in dark. Hochest33258 was added followed by incubation for 5 min to label nuclei. The uorescence microscope (Olympus, Japan) was used to observe the cells. The uorescence intensity was analyzed with ImageJ software.

Statistical analysis
Statistical analysis was performed using SPSS version 22.0 software. The quantitative data were expressed as mean ± standard deviation (SD). The comparisons between two groups were done with Student's t-test. The comparison among groups were performed by one-way ANOVA. A value of P < 0.05 was considered statistically different.

Melatonin pretreatment blocked ropivacaine-induced decrease in cell viability
Cell viability was rstly assessed in the presence of melatonin, The CCK-8 assay showed that PC12 and HT22 cell viability did not change after melatonin at concentrations of 5-40 μM (Fig.1A). Our previous study revealed that 1 mM ropivacaine could induce a signi cant decrease in cell viability. However, melatonin pretreatment blocked ropivacaine-induced decrease in PC12 cells and HT22 cells in a dosedependent manner. Meanwhile, we compared the cell viability of drug combinational treatment at different times intervals (24h, 48h, 72h), the protective effect of melatonin gradually diminished with the extension of exposure times ( Figure 1B, 1C). Furthermore, cell exposed to ropivacaine showed apoptotic bodies and were fewer in number compared with control, but melatonin pretreatment improved the morphological changes and cell numbers decrease by ropivacaine in PC12 cells and HT22 cells ( Figure   1D), those results further validated the protective potential of melatonin.

Melatonin pretreatment blocked ropivacaine-induced apoptosis
To investigate the prevention of melatonin against ropivacaine-induced apoptosis, the levels of apoptosis was detected by Western Blot and DAPI staining. PC12 cells and HT22 cells exposed to ropivacaine showed obviously apoptotic changes, such as increasing the expression of pro-apoptotic proteins BAX, Cleaved-caspase-3 and decreasing the expression of anti-apoptotic protein BCL-2 (Figure 2A, 2B).
And the numbers of apoptotic and necrotic cells were increased ( Figure 2C). Furthermore, caspase-3 cleavage and activity were also examined. Caspase-3 activity and cleavage were notably elevated after ropivacaine treatment ( Figure 2F, 2G). However, melatonin pretreatment protected cells from ropivacaineinduced apoptosis in a dose-dependent manner, included decreasing the ratio of BAX / BCL-2, uorescence intensity and Caspase-3 activity and cleavage. The above results con rmed that melatonin pretreatment suppressed ropivacaine induced apoptosis in the endogenous apoptotic pathway.

Melatonin pretreatment improved ropivacaine-induced mitochondrial dysfunction.
To further demonstrated the protective effects of melatonin on ropivacaine-induced neurotoxicity, the mitochondrial function of PC12 and HT22 cells were accessed by the mitochondrial reactive oxygen (mitoROS) and mitochondrial membrane potential (ΔΨm) before and after the treatment with ropivacaine and melatonin. As shown in Figure 3A, the overlap between ROS and MitoTracker-labeled mitochondria was increased after ropivacaine treatment, but melatonin reversed this effect and decreased the generation of Mito-ROS in a dose-dependent manner. Furthermore, the ΔΨm detection kit (JC-1) was measured by the ow cytometry. Compared with control group, ropivacaine decreased the cell numbers in the red uorescent channel, so that reduced the ΔΨm of PC12 and HT22 cells, but melatonin pretreatment improved this effect and increased the ΔΨm in a dose-dependent manner. Those results indicated that melatonin pretreatment improved ropivacaine-induced mitochondrial dysfunction.

Melatonin pretreatment inhibited ropivacaine-induced autophagy.
To explore the effects of melatonin and ropivacaine on the autophagy levels, the expression of molecules involved in autophagy were determined by Western-blot. As shown in Figure 4, ropivacaine elevated LC3 II / LC3 I ratio and decreased the expression of p62 and Beclin1 protein levels in PC12 and HT22 cells. But melatonin pretreatment reduced the LC3 II / LC3 I ratio and upregulated p62 and Beclin1 levels. The above results indicated that melatonin pretreatment inhibited ropivacaine-induced an increase of autophagy levels. To further demonstrated the protective effects of melatonin on autophagy, the overlap between LC3 distribution and Mito Tracker-labeled mitochondrial was also detected by the uorescence microscope before and after the treatment with ropivacaine and melatonin. Compared with control cells, ropivacaine increased the numbers of autophagic vacuoles engul ng mitochondria in PC12 and HT22 cells, but this effect was inhibited by melatonin pretreatment (Figure 4C, 4D).
3.5 Inhibiting autophagy enhanced the protective effect of melatonin on ropivacaine-induced apoptosis.
To further illustrated the relationship between melatonin inhibiting apoptosis and downregulating autophagy levels, rapamycin (an autophagy activator) and 3-methyladenine (3-MA, an autophagy inhibitor) were added to the medium before the treatment with ropivacaine and melatonin. And the apoptosis and autophagy index proteins were detected by Western-blot, as shown in Figure 5A, ropivacaine increased LC3 II / LC3 I ratio and decreased p62 levels, but this effect was reversed by melatonin pretreatment. And after the treatment with rapamycin, the inhibition of melatonin on ropivacaine-induced autophagy was downregulated. In addition, cells were pretreated with 3-MA, the inhibition of melatonin on ropivacaine-induced autophagy was upregulated. At the same time, the apoptosis-related proteins BAX and BCL-2 were also tested, when the autophagy levels in PC12 and HT22 cells were activated by rapamycin, the protective effect of melatonin on ropivacaine-induce apoptosis was inhibited, in addition, 3-MA pretreatment strengthened the effect of melatonin on ropivacaineinduced apoptosis. Those results indicated that the protective effect of melatonin on ropivacaine-induced apoptosis was based on its inhibition of excessive autophagy. To further demonstrated above results, the overlap between LC3 distribution and Mito Tracker-labeled mitochondrial was also detected after the treatment with rapamycin and 3-MA, results were consistent with western-blot, rapamycin pretreatment reduced the inhibitory effect of melatonin on autophagy, but 3-MA increased the effect of melatonin ( Figure 5B). Finally, the protein expression of Cleaved-caspase-3 was detected by immuno uorescence, ropivacaine increased Cleaved-caspase-3 levels, and melatonin pretreatment inhibited it, but this effect was reversed because of rapamycin and was strengthened by 3-MA ( Figure 5C).

Melatonin pretreatment inhibited the activation of Parkin / PINK1 pathway
The result from Figure 5B indicated that melatonin inhibited ropivacaine-induced an increase of mitophagy, to further explore the mechanism of mitophagy in ropivacaine-induced neurotoxicity, the expression of mitophagy-key protein, including PINK1, Parkin, Tomm20 and COXIV were detected by Western-blot, as shown in Figure 6A, 6B, compared to the control group, ropivacaine down-regulated Tomm20 and COXIV levels and upregulated the expression of PINK1 and Parkin in PC12 and HT22 cells.
Subsequently, compared to the ropivacaine group, melatonin pretreatment obviously decreased the expression of PINK1 and Parkin and increase Tomm20 and COXIV levels. Those results demonstrated that melatonin inhibited ropivacaine-induced the activation of Parkin / PINK1 pathway.

Discussion
In the current study, we revealed that ropivacaine was toxic to PC12 and HT22 cells, included impairing cell viability and morphology. But melatonin pretreatment alleviated this nerve damages. Then, we also demonstrated that apoptosis was the underlying mechanism of ropivacaine-induced neurotoxicity, but it was inhibited by melatonin via down-regulating BAX / BCL-2 ratio and Cleaved-caspase-3 levels. Subsequently, our study found that ropivacaine increased mitochondrial reactive oxygen species (mitoROS) levels and decreased mitochondrial membrane potential (ΔΨm) , but this effect was reversed by melatonin. Furthermore, our results indicated melatonin signi cantly suppressed the increase of autophagy levels from ropivacaine in PC12 and HT22 cells, included decreasing the LC3 II / LC3 I ratio, the expression of Beclin1, and increasing p62 levels. And we also found that rapamycin pretreatment reduced the inhibition of melatonin on autophagy levels, but 3-MA pretreatment enhanced this effect of melatonin. In addition, when autophagy levels were once activated by rapamycin, the protective effect of melatonin on ropivacaine-induced apoptosis was reduced. On the contrary, 3-MA enhanced this effect of melatonin by the inhibition of autophagy. Finally, our results indicated that melatonin down-regulated PINK1 and Parkin (mitophagy index proteins) levels but increased the expression of COX IV and Tom 20 (mitochondrial index proteins), which demonstrated that melatonin inhibited Parkin / PINK1 pathway mediated mitophagy.
Ropivacaine, as a representative of amide local anesthetics (LAs), is widely used in clinic for peripheral nerve blocks, epidural anaesthesia, spinal anaesthesia and pain management [18]. Although there is a report that ropivacaine has the least neurotoxicity among the other LAs including lidocaine and bupivacaine [19,20], the permanent nerve damage from ropivacaine is concerned by many doctors and there lacks some effective preventive and therapeutic measures. However, the molecular mechanism of ropivacaine-induced neurotoxicity remains unclear, but the effect is likely muti-factorial, there are several possibilities have been proposed, including intercellular calcium overload [21][22][23], neuronal mitochondrial dysfunction [24,25], neuronal apoptosis [26,27] and autophagy [10]. Some studies have reported that Ttype calcium channels and its regulatory proteins were intimately involved in ropivacaine-induced neurotoxicity, and the inhibition of those could improve the neurotoxicity from ropivacaine [28]. Apart from the intercellular calcium overload, Niu et al. found that ropivacaine damaged the mitochondrial biogenesis of neuronal cells by reducing the mitochondrial mass and impairing the mitochondrial respiratory rate [24]. Our results also demonstrated that ropivacaine made the mitochondrial dysfunction of PC12 and HT22 cells by up-regulating mitoROS levels and reduced the MMP. In addition to the above factors, many studies demonstrated that neuronal apoptosis was the key point of ropivacaine-induced neurotoxicity, including our previous studies, we found that ropivacaine up-regulated the expression of Fas and Fasl during the process of neuronal apoptosis [17], the current results were consistent with before, ropivacaine induced the apoptosis of PC12 and HT22 cells via increasing BAX / BCL-2 ratio and Clever-caspase 3 levels, but melatonin pretreatment could decrease it.
Autophagy is a dynamic process in which macromolecules and damaged organelles in cytoplasm are degraded. It stabilizes the intercellular environment in most cells including neurons [29,30]. But in some case, autophagy promotes the cell death [31]. The relationship between autophagy with ropivacaineinduced neurotoxicity have been reported, results found that ropivacaine increased autophagy ux, as re ected by increases in autolysosome formation and LC3-II generation, and decrease in p62 levels [10]. Our study also con rmed that ropivacaine up-regulated the autophagy levels of PC12 and HT22 cells, and we further found that melatonin pretreatment could inhibit ropivacaine-induced neurotoxicity via down-regulating the mitophagy levels. As we know, mitophagy is a special type of selective autophagy, the dysfunction of mitophagy might be responsible for many neuronal disorders, including neurodegenerative diseases, cerebrovascular diseases and some neurotoxicity diseases [32,33].
Increasing evidences revealed that excessive mitophagy could lead to the death of neurons [34,35], our data demonstrated that the triggering of excessive mitophagy during ropivacaine-induced neurotoxicity was damaging to neurons, and we also found that the activation of mitophagy could inhibit the protective effect of melatonin on ropivacaine-induced neurotoxicity. In addition, the expression of PINK1 and Parkin were signi cantly increase after the treatment with ropivacaine, whereas COX IV and Tom20 levels were reduced, on the contrary, melatonin pretreatment decreased PINK1 and Parkin expression and increased COX IV and Tom20 levels, indicating ropivacaine activated the Parkin / PINK1 mediated mitophagy, but this effect was reversed by melatonin.
Melatonin, as a chief hormone secreted from the pineal gland, regulates numerous biological functions in a variety of cells including neurons [2]. The neuroprotective effects of melatonin have been reported [36,37]. Melatonin regulated the neurogenesis in both in vivo and in vitro models, including it maintains the neuronal characteristic of different neuronal stem cells, and it increases the structure plasticity of nerve bers in the 8-week-old mice [3,38]. In addition, melatonin can decelerate the progress of many neuronal disorders such as depression [39], aging [40] and neurodegenerative diseases [8]. It reduced the depressionlike behavior in male mice and enhanced the e cacy of antidepressant citalopram [38,41]. Similarly, melatonin can improve the spatial memory de cits in aging mouse mode and attenuate neuronal impairment in PD animal modes [42,43]. Apart from protecting the brain neurons, it was reported that melatonin can promote the nerve regeneration after the spinal cord injury and peripheral nerve impairment [44,45]. In recent years, many clinical drugs have been reported to induce the neurotoxicity, including scopolamine (a prescription drug used for the prevention of nausea and vomiting), but melatonin pretreatment reversed the behavioral de cits and reduced the neurotoxicity of scopolamine [46,47]. In our study, we discovered that melatonin pretreatment had protective effects on ropivacaineinduced neurotoxicity, as evidence by increasing cell viability, decreasing apoptosis, improving mitochondrial dysfunction, and reducing mitophagy levels. Although the inhibition of melatonin on mitophagy have been reported by other studies[48-50], our results were the rst to illustrate that melatonin can inhibit the activation of mitophagy by ropivacaine, which may a potential treatment to ropivacaine-induced neurotoxicity.
In conclusion, ropivacaine induced neurotoxicity by decreasing cell viability, promoting neuronal apoptosis and mitochondrial dysfunction and up-regulating the autophagy and mitophagy levels, but those effects were drastically reversed by melatonin pretreatment. And we also demonstrated that the protective effects of melatonin against ropivacaine-induced apoptosis were modulated by suppressing excessive mitophagy via the inhibition of Parkin / PINK1 pathway. The above results indicated that melatonin could be a candidate treatment in LAs-induced neurotoxicity, and Parkin / PINK1 mediated mitophagy presents a potentially target as well.

Declarations Funding
Page 10/20 The Innovative Research Program for Graduates of Hubei University of medicine (NO. YC2020029)

Competing Interests
The authors declare no con icts of interest regarding this study and publication.

Authors' Contributions
LHY and DXD designed the studies, ZL and HJF undertook the cell experiments and the construction of animal experiments, LCG and ZFY undertook the molecular biology testing, ZL, ZFY, ZZ analyzed data and wrote the draft of manuscript.

Availability of data and materials
Data that support the study ndings are available from the corresponding author upon reasonable request.