Ropivacaine Induce G0 / G1 Phase Cell Cycle Arrest and Apoptosis of PC12 Cells Via Inhibiting Mitochondrial STAT3 Translocation

Lian Zeng Hubei University of Medicine Zhen Zhang Hubei University of Medicine Fuyu Zhang Hubei University of Medicine Chenguang Liu Hubei University of Medicine Jiafeng He Hubei University of Medicine Huaxian Chen Hubei University of Medicine Qingsong Wang Hubei University of Medicine Xudong Ding Hubei University of Medicine Huiyu Luo (  603983267@qq.com ) Hubei University of Medicine https://orcid.org/0000-0003-2126-223X


Introduction
Ropivacaine, as an amide local anesthetic, has been widely used in clinical practice. Because of the longlasting and stable anesthetic effects, ropivacaine is mainly used for pain management and regional anesthesia, which has the characteristic of sensorimotor separation (Martin-Flores M, 2019). However, some studies have shown that ropivacaine has cytotoxicity (Cai XY et al., 2015;CM C et al., 2012), including muscle toxicity, neurotoxicity, and cardiotoxicity. In recent years, increasing attention has been paid to the neurotoxicity because it is directly related to the recovery of patients after surgery (Aps J and Badr N, 2020), but the mechanism of ropivacaine induced neurotoxicity is unclear. In past decades, a growing body of evidence has revealed that apoptosis plays an important role in the ropivacaine induced neurotoxicity (Chen Y et  The DNAzol™ Reagent (Invitrogen™, USA) was used to extracted the genomic DNA according to the manufacturer's instructions. DNA was quanti ed with NanoPhotometer (Implen, Germany). The ratio of mtDNA / nDNA was measured by real-time PCR. The ampli cation of mitochondrial cytochrome c oxidase subunit II (COII, mitochondrial-encoded gene) and β-actin (nuclear-encoded gene) was done with Real-time PCR. The primer used were as follows: COII: 5′-TGAGCCATCCCTTCACTAGG-3′(sense) and 5′-TGAGCCGCAAATTTCA GAG-3′ (anti-sense);β-actin: 5′-CTGCTCTTTCCCAGATGAGG-3′(sense) and 5-CCACAGCA CTGTAGGGGTTT-3′ (anti-sense).
Mitochondrial staining PC12 cells were seeded in a 12-well plate at a density of 1×10 4 cells/well after treated with different concentrations of ropivacaine for 24 h. Then, cells were incubated with Mito-Tracker Red CMXRos (Beyotime, Shanghai, China) according to the instructions. Subsequently, cells were xed in 4% PFA, and Hochest33258 was used to label the nuclei. Finally, the uoresce intensity was observed under a uorescence microscope and the average optical density was measured by ImageJ software.
Mitochondrial membrane potential assay Mitochondrial membrane potential (MMP) 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 and uorescence microscopy were done to measure MMP and the average optical density was determined by ImageJ software.
Mitochondrial separation, extraction of mitochondrial proteins Mitochondria separation was based on the differential centrifugation. According to the manufacturer's instructions, the cells were broken down by the Ultrasonic Crusher (Sonic, USA). Then, the cell debris and large organelles were removed by low-speed differential centrifugation (800 ⋅g). Finally, the mitochondria were harvested by high-speed differential centrifugation (15000 ⋅g). The extraction of mitochondrial proteins was done after lysing mitochondria with lysis buffer containing a protease inhibitor and a phosphatase inhibitor. The mitochondrial proteins were used for Western Blot.
Quantitative real-time PCR PC12 cells were digested with trypsin and seeded into 6-well plates cultured for 2 days. Then, cells were treated with different concentrations of ropivacaine for 24h. Total RNA was extracted with Trizol reagent (Invitrogen, USA), and the concentrations were measured by NanoPhotometer (Implen, Germany). A total of 1µg RNA was reversely transcribed into cDNA according to the instructions of RT reagent kit (Promega, USA). SYBR-based real-time PCR experiments were done to detect the mRNA expression of Bax, Bcl-2, Caspase-3, p53, p21, CDK-4, and CyclinE1 using an ABI 7500 platform. The 2 − ΔΔCT method was used to analyze the data. The primers were used as follow Bax 5'-CTGAGCTGACCTTGGAGC − 3' (forward), 5'-
Detection of cell cycle and apoptosis by ow cytometry PC12 cells were seeded in a 6-well plate at a density of 1×10 6 cells/well after treated with different concentrations of ropivacaine for 24 h, cells were collected and centrifuged at 1000 rpm/min for 5 min at 4℃. After washing with PBS, the cell pellet was xed in 500 µL of 70% cold ethanol for at 2 h. Before ow cytometry, the cells were washed with PBS, and incubated with 100 µL of RNase. The resulting suspension was incubated at 37°C for 30 min. Subsequently, 400 µL propidium iodide was added for nuclear staining at 4°C in dark for 30 min. Cell cycles were analyzed using ow cytometry (BD, San Jose, CA). The apoptosis was detected as follows. PC12 cells in each group were digested with trypsin. Then, cells were centrifuged at 1000 rpm/min for 10 min, and the supernatant was removed. The cells were washed twice with PBS and centrifuged to remove the supernatant. 500 µl of buffer solution was added to cell suspension. Then, 5 µL of Annexin V and 10 µL of propidium iodide was added, followed by incubation at room temperature for 30 min in dark, apoptosis was detected by ow cytometry and apoptosis rate was calculated.

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.

Molecular docking
Structure-based virtual screening was employed with molecular docking program Autodock Vina (version 1.1.2). The PyMol (version 2.3) was used to generate the 3D schematic representation of STAT3 protein (PDB ID: 6QHD) which was extracted by in The Research Collaboratory for Structural Bioinformatics Protein Data Bank database. The 2D schematic representation of the interaction between ligand and other amino acid residues was shown by LigPlus (version 2.1). In order to detect whether ropivacaine competitively bind the STAT3 peptide, the ropivacaine molecular structure (PubChem CID: 175804) was obtained from PubChem database. The ligand and the receptor pdb were converted to pdbqt le by AutoDock Tools following the autodock vina tutorial, and the grid size in XYZ was set at 96,66,118, which is large enough to cover all potential pockets in the STAT3 monomer.

Animals and treatment
All procedures performed in studies involving animals were in accordance with the ethical standards of animal ethics committee of Hubei University of Medicine ((IACUC number: 2018DW003), Eight-week-old Sprague Dawley (SD) rats, weighing 200-300g, were purchased from the Experimental Animal Center of Hubei University of Medicine (Hubei, China). The rats were given access to food and water and housed in an environment with relative humidity of 50-60% and 12/12h light/dark cycle for 1 week before surgery. Rats were anesthetized with 10% chloral hydrate (300 mg/kg body weight, Beyotime Biotechnology, Shanghai, China). The animal was established as a previous reported (Y H et al., 2016). A PE-10 catheter was inserted through the L4 / L5 intervertebral space (depth 2 cm) to the L1-L2. The cannulated rats observed for ve days and rats with the neurological injury were not included for further experiment. The remaining rats were randomly divided into four groups (n = 5 per group), including control group (saline group), 0.5% ropivacaine group (0.5% Rop), 1% ropivacaine group (1% Rop) and 2% ropivacaine group (2% Rop). All rats were received 8 injections at 1.5 h intervals for 12h. After injection, rats were observed for 48 h and those with movement disorders were removed from this study.

TUNNEL staining and Immunohistochemistry
The fresh spinal cord tissues were xed in 4% PFA. After being embedded with para n, the para n blocks were cut into 3 mm slices. Then, they were detected with TUNNEL staining according to the manufacturer's instruction to detect the apoptosis in the spinal cord. The protein expression of p-STAT3 (Ser727) in the spinal cord was detected by Immunohistochemistry.

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 was 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.

Ropivacaine decreased viability and reduced the protrusions numbers in PC12 cells
To determine the effects of ropivacaine on PC12 cell viability, CCK-8 assay was done in these cells after treatment with different ropivacaine concentrations (0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM) for 24 h. As shown in Fig. 1A, compared with the control group, ropivacaine suppressed PC12 cell viability in a dosedependent manner. The half lethal concentration of ropivacaine was 3.24 mM. As we know, PC12 cells are a neuron-like cell line, they have long fusiform with protrusions, but after treatment with ropivacaine, the protrusions reduced, and the cells became round, which represented the loss of the neuronal characteristics. Those results indicate that ropivacaine exerted signi cant toxic effects on the PC12 cells by inhibiting cell viability.

Ropivacaine arrested PC12 cells in the G0/G1 phase
To explore the effect of ropivacaine on cell death pathway, the cell cycle was detected by ow cytometry in ropivacaine-treated PC12 cells. As shown in Fig. 2A, exposure to different concentrations of ropivacaine for 24 h signi cantly increased the number of cell in the G0/G1 phase and decreased the number of cell in the G2/M phase as compared to the control group, but ropivacaine had no in uence on the S phase. In addition, the expression of genes that regulate the cell cycle of p21, CyclinE1 and CDK4 was detected. After ropivacaine treatment, the mRNA and protein expressions of p21 increased, but the mRNA and protein expression of CyclinE1 and CD4 were inhibited, which indicates that ropivacaine induced PC12 cell death via arresting the cell cycle (Fig. 2C, 2D, 2E).

Ropivacaine induced the apoptosis of PC12 cells and the spinal cord
To determine the effect of ropivacaine on the apoptosis of PC12 cells and the spinal cord, the ow cytometry was used to detected the apoptosis rate of PC12 cells, the results were consistent with our previous study, ropivacaine increased apoptosis in a dose-dependent manner (Fig. 3D). In addition, the mRNA and protein expression of apoptosis-related genes included Bax, Bcl-2 and Caspase 3 were also detected, as shown in Fig. 3A, ropivacaine up-regulated the expression of pro-apoptotic genes and downregulated the anti-apoptotic gene at mRNA and protein levels. To further explore the effect of ropivacaine on the spinal cord, a rat model was established with a subarachnoid catheter. After the treatment with ropivacaine at different concentrations, TUNNEL staining was to detect the apoptosis of the spinal cord.
Compared to the control group, ropivacaine increased the apoptosis rate in the spinal cord in a dosedependent manner (Fig. 3E, 3F).

Ropivacaine induced oxidative stress in PC12 cells
The oxidative stress was assessed in PC12 cells after ropivacaine treatment by measuring SOD, MDA, ROC, and LDH. As shown in Fig. 2A, compared with the control group, 1 mM ropivacaine signi cantly increased ROS in the cytoplasm. After treatment with different ropivacaine concentrations of ropivacaine (0.5 mM, 1 mM, and 2mM), ropivacaine increased the MDA, and LDH increased and suppressed the SOD reduced in a dose-dependent manner. These results suggest ropivacaine induces the oxidative stress damage in the PC12 cells.

Ropivacaine induced mitochondrial dysfunction in PC12 cells
To assess the impact of ropivacaine on mitochondria of PC12 cells, a JC-1 test kit was used to detect the MMP of PC12 cells, and the ow cytometry and uorescence microscopy were done to assess the mitochondrial function. As shown in Fig. 5A, compared with the control group, ropivacaine reduced the amounts of JC-1 polymer in a dose-dependent manner, which indicates the reduction of MMP in PC12 cells after ropivacaine treatment. As shown in Fig. 5B, in the control group, cells had with strong red and green uorescence, but in the 1 mM ropivacaine group, the MMP decreased, and cells had strong green uorescence but low red uorescence. In addition, Mito-Tracker Red CMXRos was used to label mitochondrial with normal function. As shown in Fig. 5D, compared with the control group, 1 mM ropivacaine signi cantly reduced the number of mitochondria with normal function in PC12 cells, the mtDNA / nDNA ratio kit was determined to assess the amounts of mitochondria. The result showed treatment with ropivacaine reduced the mtDNA / nDNA ratio in a dose-dependent manner (Fig. 5F). Those results demonstrate that ropivacaine causes mitochondrial dysfunction in PC12 cells.

Ropivacaine inhibited STAT3 activation and mitoSTAT3 translocation
To investigate the effect of ropivacaine on the STAT3 signaling pathway in PC12 cells and the spinal cord, STAT3 phosphorylation and upstream transduction proteins expression were evaluated using Western Blot, and the distribution of phosphorylated STAT3 in subcellular compartments was assessed by Western Blot and Immuno uorescence staining, As shown in Fig. 6A, ropivacaine inhibited the serine727 residue phosphorylation of STAT3 and down-regulated IL-6 expression in PC12 cells. In addition, ropivacaine decreased p-STAT3 (ser727) expression in mitochondrial fraction, but increased it in the cytoplasmic fraction (Fig. 6B). Which indicated that ropivacaine inhibited the mitochondrial STAT3 translocation. The result of Immuno uorescence was consistent with Western Blot, ropivacaine reduced the co-localization between p-STAT3 (ser727) with mitochondria in a dose-dependent manner (Fig. 6D). To further explore the effect of ropivacaine on p-STAT3 (ser727) expression in the spinal cord, as expected, ropivacaine inhibited the p-STAT3 (Ser727) expression of the spinal cord in a dose-dependent manner (Fig. 6E). Finally, to explore the potential relationship between STAT3 and ropivacaine, Autodock-Vina was to verify the interaction between them. As shown in Fig. 6F, the pocket was composed of Asn257, Ala377, Glu324, Ile258, Gln326, Pro333, Pro336, Asp 334, Ala250, Cys251 and Pro256. They interacted through hydrophobic bond to affect the activation of STAT3, and further con rming the inhibitory effect of ropivacaine on STAT3 activation.

Discussion
Ropivacaine, as a representative of the long-acting local anesthetics (LAs), is available for the spinal anesthesia, and its duration is 1.  (Takenami T et al., 2002). Hence, it is important to investigate the mechanism underlying the ropivacaine induced neurotoxicity, which is helpful for the development of preventive measures and will make the spinal anesthesia safer.
The mechanism of ropivacaine induced neurotoxicity is unclear, and some studies reported it is related to cell apoptosis. Our previous study also revealed that ropivacaine increased apoptosis via up-regulating Fas / Fasl expression in PC12 cells (Zeng Y,Wang R,Bian Y,Chen W and Peng L, 2019), which were also con rmed by Wang et al. (S W et al., 2019). As we know, apoptosis may execute in two pathways: the death receptor pathway and the mitochondrial pathway (Zeng Y,Wang R,Bian Y,Chen W and Peng L, 2019). The death receptor pathway is represented by the up-regulation of Fas/Fasl expression, and the mitochondrial pathway is characterized by the mitochondrial dysfunction. In addition to inducing neurotoxicity through the death receptor pathway, ropivacaine has also been proved to cause neuronal damage by impairing the mitochondrial function. Niu et al. reported ropivacaine affected the mitochondrial biogenesis of neuronal cells by reducing the mitochondrial mass and impairing the mitochondrial respiratory rate via suppressing PCG-1a (Z N et al., 2018). In the present study, our ndings con rmed that apoptosis played an important role in the ropivacaine induced neurotoxicity. At rst, the cell viability was detected after treatment with different concentrations of ropivacaine. Results showed that ropivacaine decreased the cell viability in a dose-dependent manner. In addition, ropivacaine reduced the protrusions of PC12 cells and cells became round after ropivacaine treatment, which means the cell death increases. In order to determine the cell death pathway induced by ropivacaine, we analyzed the change of cell cycle and apoptosis of ropivacaine-treated PC12 cells using ow cytometry, and results showed that ropivacaine arrested the PC12 cells in G0/G1 phase and increased cell apoptosis. The mRNA and protein expressions of genes regulating the cell cycle and apoptosis were further detected in cells after ropivacaine treatment. p21 is one of those genes that block the cell cycle progression ( To further clarify the effects of ropivacaine on the cellular oxidative stress and mitochondria, the reactive oxygen species (ROS) generation was assessed in cells before and after ropivacaine treatment. Our results showed that the ROS increased with the increase in the concentration of ropivacaine. Similarly, the contents of MDA and LDH also increased signi cantly, while the level of SOD decreased after ropivacaine treatment. These suggest ropivacaine induces cellular oxidative stress in PC12 cells. Those results were consistent with those reported by Chen et al (Chen Y,Yan L,Zhang Y and Yang X, 2019). Next, the mitochondria function was examined in PC12 cells treated with ropivacaine. Our results con rmed that ropivacaine induced cell mitochondrial dysfunction, which was mainly manifested the decreased of MMP and the ratio of mtDNA / nDNA. Those results were also con rmed by other studies (Y C et al., 2019;Z N,J T,Y R and W F, 2018).
On the basis above ndings, we further investigated the role of STAT3 in the ropivacaine induced apoptosis. STAT3 was rst described as a transcriptional enhancer of acute-phase genes activated by IL-6, which mediated extracellular signals such as cytokines and growth factors through interacting with polypeptide receptors on the cell surface (You L et al., 2015). The canonical STAT3 signaling becomes transcriptionally activated primarily by tyrosine phosphorylation (Tyr705). Activated STAT3 translocate to the nucleus, and binds to sequence-speci c DNA elements for consequent transcription of target genes (Cui P et al., 2020). The canonical STAT3 pathway has been demonstrated to promote tumor development(Fathi N,Rashidi G,Khodadadi A,Shahi S and Shari S, 2018). The non-canonical STAT3 signaling is represented by serine phosphorylation (Ser727), and STAT3 phosphorylated at Ser727 functions to rescue the impaired mitochondrial function after exposure to the stimuli(Srivastava J and DiGiovanni J, 2016). When apoptosis is induced via the mitochondrial pathway, the non-canonical STAT3 signaling is activated to further protect cells against apoptosis. Except for the non-canonical pathway, STAT3 has been shown to regulate the mitochondrial function by transporting to the mitochondria (mitoSTAT3), which is also dependent on serine phosphorylation (Ser727) (Szczepanek K,Chen Q,Derecka M,Salloum FN,Zhang Q,Szelag M,Cichy J,Kukreja RC,Dulak J,Lesnefsky EJ and Larner AC, 2011;Yang R and Rincon M, 2016). As expected, ropivacaine inhibited the activation of STAT3 by down-regulating the expression of p-STAT3 (Ser727) and decreased the expression of upstream genes of STAT3. To further investigate the effect ropivacaine on mitoSTAT3 expression, the p-STAT3 (Ser727) expression was detected in the mitochondrial fraction and cytoplasmic fraction, which was used to determine that the mitoSTAT3 translocation in PC12 cells Results showed that mitoSTAT3 translocation was inhibited by ropivacaine. We also con rmed this by immuno uorescence staining, the co-localization of p-STAT3 (Ser727) in the mitochondria was signi cantly reduced after ropivacaine treatment. The anti-apoptotic effect of mitoSTAT3 has been demonstrated in many studies. Li et al. reported that the STAT3 activation is key for the ischemic postconditioning, and the increased of mitoSTAT3 can protect the cardiomyocytes against mitochondrial dysfunction . Zhang et al. reported Serine 727 (Ser727) phosphorylation of STAT3 plays a role in the regulation of mitochondrial respiration (Zhang G et al., 2018). In addition, mitoSTAT3 translocation can improve the oxidative phosphorylation and inhibit ROS generation during reperfusion. As we know, STAT3 activation depends on the phosphorylation of its hydrophobic domain, our results showed that ropivacaine could bind to the hydrophobic domain of STAT3, and this phosphorylation was inhibited, which con rms above ndings. At last, we compared the levels of apoptosis and p-STAT3 (Ser727) expression in the spinal cord with the treatment of different concentrations of ropivacaine. The results showed that ropivacaine increased the apoptosis of the spinal cord in a dose-dependent manner, and the expression of p-STAT3 (Ser727) decreased signi cantly after ropivacaine treatment. This indicates that there was a negative relationship between p-STAT3 expression and apoptosis in the spinal cord. The above ndings indicate ropivacaine can induce the neurotoxicity by promoting apoptosis via down-regulating STAT3 phosphorylation.

Conclusion
In conclusion, ropivacaine decreased cell viability and reduced protrusions of PC12 cells to induce neurotoxicity. Furthermore, ropivacaine arrested PC12 cells in the G0/G1 cycle phase, and promoted cell apoptosis through the mitochondrial pathway. Moreover, it also induces the Cellular Oxidative Stress and mitochondrial dysfunction in PC12 cells. These effects may be related to the inhibition of STAT3 activation and mitoSTAT3 translocation. These ndings suggest STAT3 may serve as a target in the prevention a treatment of ropivacaine induced neurotoxicity