CaMKII and CaV3.2 T-type calcium channel mediate Connexin-43-dependent inflammation by activating astrocytes in vincristine-induced neuropathic pain

Vincristine (VCR), an alkaloid isolated from vinca, is a commonly used chemotherapeutic drug. However, VCR therapy can lead to dose-dependent peripheral neurotoxicity, mainly manifesting as neuropathic pain, which is one of the dominant reasons for limiting its utility. Experimentally, we discovered that VCR-induced neuropathic pain (VINP) was accompanied by astrocyte activation; the upregulation of phospho-CaMKII (p-CaMKII), CaV3.2, and Connexin-43 (Cx43) expression; and the production and release of inflammatory cytokines and chemokines in the spinal cord. Similar situations were also observed in astrocyte cultures. Interestingly, these alterations were all reversed by intrathecal injection of KN-93 (a CaMKII inhibitor) or l-Ascorbic acid (a CaV3.2 inhibitor). In addition, KN-93 and l-Ascorbic acid inhibited the increase in [Ca2+]i associated with astrocyte activation. We also verified that knocking down or inhibiting Cx43 level via intrathecal injection of Cx43 siRNA or Gap27 (a Cx43 mimetic peptide) relieved pain hypersensitivity and reduced the release of inflammatory factors; however, they did not affect astrocyte activation or p-CaMKII and CaV3.2 expression. Besides, the overexpression of Cx43 through the transfection of the Cx43 plasmid did not affect p-CaMKII and CaV3.2 expressions in vitro. Therefore, CaMKII and CaV3.2 may activate astrocytes by increasing [Ca2+]i, thereby mediating Cx43-dependent inflammation in VINP. Moreover, we demonstrated that the CaMKII signalling pathway was involved in VCR-induced inflammation, apoptosis, and mitochondrial damage. Collectively, our findings show a novel mechanism by which CaMKII and CaV3.2 mediate Cx43-dependent inflammation by activating astrocytes in neuropathic pain induced by VCR.


Introduction
Chemotherapy-induced peripheral neuropathy (CIPN) is a significant health concern that poses severe challenges to cancer patients' physical and mental health, which represents a colossal burden to society and the economy worldwide (Sisignano et al. 2014;Cavaletti et al. 2019). Vincristine (VCR) has long been applied widely to cure various tumours, such as neuroblastomas, lymphomas, and leukaemias (Shimizu et al. 2017;Kantarjian et al. 2004). The pharmacological mechanism of VCR is to disrupt microtubule formation in mitotic spindles, leading to the cessation of cellular mitosis (Gupta et al. 2006). Furthermore, VCR plays anticancer roles by inducing mitochondrial dysfunction and subsequent energy deficiency (Xiao and Bennett 2012). However, VCR treatment can cause dosedependent peripheral neurotoxicity, mainly manifesting as somatosensory and motor dysfunction, which is one of the dominant reasons for limiting its utility (Balayssac et al. 2011;Schouten et al. 2020). Currently, due to an inadequate understanding of the pathogenesis of neuropathic pain caused by VCR, effective prevention and therapeutic strategies are lacking. Existing studies have shown that VCR causes pathological changes in the spinal cord of mice, such as glial cell activation, intracellular ion disorders, inflammation, and apoptosis (Gold and Gebhart 2010;Surmeier et al. 2010;Zhang et al. 2013;Chen et al. 2020).
Neuropathic pain is generally considered as a manifestation of the neuroplasticity of the primary sensory neurons of the central nervous system (CNS) (Zhang et al. 2013). However, in recent decades, increasing researches have confirmed glial cells' critical roles in maintaining neuropathic pain by modulating synaptic transmission (Nedergaard and Verkhratsky 2012). In the CNS, astrocytes are the most abundant type of glial cells. Many studies have revealed that they involve in developing and maintaining neuropathic pain by releasing astroglial mediators to upregulate nociceptive neuron activity in the spinal cord (Gao and Ji 2010). Under spinal cord injury or nerve injury, astrocytes exhibit persistent reactive changes and promote neuropathic pain by producing proteases, inflammatory cytokines, and chemokines (Guo et al. 2007;Zhang et al. 2013;Kawasaki et al. 2008).
The activation of astrocytes requires the participation of various ions, including calcium ions (Ca 2+ ) that regulate a series of physiological responses of neurons via neurotrophic factors and neurotransmitters (Surmeier et al. 2010;Neher and Sakaba 2008). Intracellular Ca 2+ homeostasis imbalance leads to impaired plasticity, synaptic dysfunction, and neuronal degeneration (Nedergaard et al. 2010, Marambaud et al. 2009). Calcium/calmodulin-dependent protein kinase II (CaMKII) is a widely expressed multifunctional serine/ threonine kinase that regulates the transcription of Ca 2+ channels (Naranjo and Mellstrom 2012). Ca 2+ -dependent CaMKII modulates the activity or plasticity of neurons by Ca 2+ signalling in the development of neuropathic allodynia (Song et al. 2010;Kim and Sharma 2004). Voltage-dependent calcium channel 3.2 subunit (Ca V 3.2), a low-threshold calcium channel, is the molecular substrate of neuropathic allodynia in lamina II and III of the spinal cord (François et al. 2015) and a key regulator of dorsal root ganglion (DRG) neuronal excitability (McCallum et al. 2003). Ca V 3.2 antisense oligonucleotide injected into the sheath to reduce the t-type calcium current of DRG neurons was shown to relieve the traumatic response of neuropathic pain in rats (Bourinet et al. 2005). Therefore, we hypothesized that CaMKII and Ca V 3.2 are involved in VCR-induced neuropathic pain (VINP) by activating astrocytes through regulating intracellular free calcium ([Ca 2+ ] i ).
Chemotherapeutic agents have been shown to induce spinal cord neuroinflammation and apoptosis in mice (Chen et al. 2020;Li et al. 2020). Recent studies suggested that decreasing the phosphorylation of CaMKII could inhibit NF-κB signalling pathway activation and diminish lipopolysaccharide-induced neuroinflammation in primary microglial cells (Park et al. 2020). Furthermore, inhibiting the CaMKIIβ expression improved cell viability and reduced the apoptosis rate, which protected DRG cells from ropivacaine hydrochlorideinduced neurotoxicity injury (Wen et al. 2019). The interaction between reactive oxygen species (ROS) and cellular Ca 2+ activates related signalling pathways, inducing neuronal apoptosis (Franklin 2011;Cheng et al. 2012). However, whether VCR causes inflammation and apoptosis through the CaMKII signalling pathway needs further evidence to be collected.
Astrocytes form interconnected networks coupled in the adult CNS through connexin43 (Cx43), which is a major structural component of gap junctions (Wang and Xu 2019;Bennett et al. 2012). There is now evidence that gap junctions are involved in chronic pain induced by tissue inflammation or nervous system damage (Wu et al. 2012;Vicario et al. 2020;Morioka et al. 2019). And neuropathic pain was improved by inhibiting chronic constriction injury (CCI)-induced Cx43 alterations (Vicario et al. 2019). Our previous research revealed that VCR upregulated the expression of Cx43 (Zhou et al. 2020a). However, it is still unclear how Cx43 participates in and maintains neuropathic pain induced by VCR. Therefore, in this study, we aimed to investigate the roles and mechanisms of CaMKII, Ca V 3.2, and Cx43 in VINP.

Animals
Two hundred and sixty adult male ICR mice weighing 20 to 25 g were purchased from Jiesijie Laboratory Animal (Shanghai, China; Production licence No. SCXK (Hu) 2018-0004). Mice were raised on a fixed 12-h/12-h on/off light cycle with controlled room temperature and relative humidity (22-25°C, 60 ± 10%). All animal experiments were performed following the ARRIVE guidelines and complied with the policies of the International Association for the Study of Pain. All experimental procedures complied with the Guidelines for the Care and Use of Laboratory Animals and were approved by China Pharmaceutical University (Nanjing, China; licence No. SYXK (Su) 2016-0011).

Establishment of the VINP model and drug administration protocols
Mice were intraperitoneally injected with VCR (0.1 mg/kg; Shenzhen Main Luck Pharmaceutical Incorporated, China) once daily for 5 consecutive days (Shen et al. 2015).
Cx43-specific small interfering RNA (siRNA) was provided by RiboBio (Guangzhou, China). Cx43 siRNA (50 nM, 10 μl) was dissolved in the transfection reagent riboFETC TM CP and intrathecally injected daily for 2 consecutive days from day 6 after the first-day VCR injection. Missense siRNA was administered intrathecally in the same way as a sham treatment.

Behavioural assessments
In this study, we mainly evaluated the behaviour of mice with mechanical allodynia and heat hyperalgesia in the same quiet behavioural testing room. For the mechanical allodynia assessment, an ascending series of von Frey filaments (0.07-2.0 g, Woodland Hills, Los Angeles, CA) was utilized to determine the mechanical withdrawal threshold for all mice (Dixon 1980). Mice were placed in the transparent plexiglass compartments on an elevated metal grid and acclimated for 30 min before testing. In the resting state, each monofilament was vertically stimulated 5 times in ascending order to a plantar (3 s for each filament). Dixon's up-down method was applied to determine the 50% paw withdrawal threshold (PWT) when the monofilament stimulation led to paw licking or withdrawal in 3 of 5 applications. Heat hyperalgesia was evaluated as infrared stimulation duration and measured using the PL-200 stinging instrument (TechMan, Chengdu, China). The paw withdrawal latency (PWL) of the sham group was ensured at 10 ± 2 s by adjusting the radiant heat (Hu et al. 2017).
Behavioural evaluations were performed from 0 to 21 days after the first intraperitoneal injection of VCR. For the groups treated with KN-93, Gap27, L-Ascorbic acid, or Cx43 siRNA, behaviour was assessed at 2 h after the intrathecal injections.
For the immunofluorescence staining of astrocyte cultures, cells on slides were fixed with 4% paraformaldehyde for 15 min after washing with PBS, and then processed for staining with GFAP antibody (1:200) and Cx43 antibody (Cell Signalling Technology; 3512, 1:100), as indicated above.
Quantitative reverse transcription-polymerase chain reaction According to TRIzol instructions (TaKaRa, Tokyo, Japan), total RNA of spinal cord tissues (10 mg) and cells was isolated. PrimeScript™ Reverse Transcriptase (TaKaRa, Tokyo, Japan) was utilized to generate complementary DNA (cDNA). Subsequently, real-time quantitative PCR (qRT-PCR) was performed using a QuantStudio 3 Real-time PCR System (Applied Biosystems, Foster, CA, USA) with SYBR Green Master Mix (Vazyme, Nanjing, China). In this study, the primer sequences are listed in Table 1. Levels of each sample mRNA were calculated using the delta method from threshold cycle numbers after mRNA normalization to GAPDH.

Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) kits (Dakewe Biotech, Beijing, China) were used to analyse IL-6 and IL-1β levels in serum and cell culture media following the instructions provided by the manufacturers.

Cell viability assay
Cell viability was analysed using CCK-8 (KeyGen Biotechnology, China). Primary astrocytes seeded in the 96-well plate were treated with VCR at different concentrations (0.1 to 100 nM) for 24 h, and then added CCK-8 reagent (10 μl). After 2 h, the absorbance at 450 nm was detected by Multiskan FC microplate reader (Thermo Fisher, USA).

TUNEL assay
The cell apoptosis rate was calculated by TdT-mediated dUTP Nick-End Labeling (TUNEL) in situ cell death detection kit (Beyotime, Shanghai, China) according to the protocols recommended by the manufacturer. TUNEL-positive signals showing green fluorescence were detected by laser scanning confocal microscopy.

Annexin V/PI double staining
According to the manufacturer's protocols, astrocytes were rinsed 3 times with PBS and then double-stained using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, USA) for 15 min at room temperature in the dark. Apoptotic cells were detected by flow cytometer (BD Biosciences, CA, USA).

Measurement of mitochondrial membrane potential (MMP) and total ROS
The MMP △Ψm and total ROS of astrocytes were examined by mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China) and ROS species assay kit (Beyotime, Shanghai, China) respectively, followed by imaging under flow cytometry.

Statistical analysis
All data are presented as the mean ± SEM. Differences between groups were compared using unpaired twotailed Student's t-test or one-way analysis of variance (ANOVA). SPSS 22.0 (IBM, Armonk, USA) and GraphPad Prism software 8.0.1 (GraphPad Software, CA, USA) were applied to analyse the data and graphing, respectively. A value of P < 0.05 was deemed statistically significant.

VCR treatment induces persistent pain hypersensitivity, astrocyte activation, inflammation, and apoptotic
The in vivo experimental process is shown in Fig. 1A. Mice were intraperitoneally injected with VCR daily for 5 consecutive days, and pain behaviour was monitored over time. On 3 days after starting intraperitoneal administration of VCR, the results revealed evident pain hypersensitivity (P < 0.05), which was characterized by mechanical allodynia and heat hyperalgesia (Fig. 1B, C). For mechanical sensitivity, compared with mice treated with saline (sham group), the PWT in response to von Frey hair stimulation in VCR-treated mice (VCR group) was fully decreased at day 7 (1.43 ± 0.08 g vs. 0.35 ± 0.0.07 g; P < 0.05) and persisted at 21 days (1.44 ± 0.11 g vs. 0.84 ± 0.13 g; P < 0.05) (Fig. 1B). For heat sensitivity, the PWL responding to the heat stimulation in the VCR group was reduced wholly compared with that in the sham group at day 7 (11.5 ± 0.30 s vs. 7.3 ± 0.32 s; P < 0.05) and persisted at 21 days (12.8 ± 0.51 s vs. 11.13 ± 0.31 s; P < 0.05) (Fig. 1C). On days 3, 7, 14, and 21, we analysed astrocyte activation and the inflammatory response. Astrocyte reactivity to VCR was evaluated via the spinal dorsal horn GFAP fluorescence intensity. Compared with resting-state GFAP-positive astrocytes in the sham group, numerous GFAP-positive astrocytes in the VCR group showed intense immunoreactivity, and the increase in GFAP-positive astrocytes was most pronounced on day 7 after VCR injection (6.56 ± 0.87-fold that of sham, P < 0.05) (Fig. 1D, E). Simultaneously, the qRT-PCR results revealed that VCR also induced a noticeable increase in the mRNA levels of inflammatory factors (TNF-α, IL-6, IL-1β) and chemokines (CXCL12) in the spinal cord on days 3, 7, 14, and 21 compared to saline treatment ( Fig. 1F-I). To verify whether VCR induces an apoptotic response, TUNEL staining was applied to evaluate the apoptotic response in the spinal cord on day 7. The results confirmed that, compared with rare TUNEL-positive cells in the sham group, VCR induced a distinctly increased number of TUNEL-positive cells (5.08 ± 0.4-fold of sham, P < 0.05) (Fig. 1J, K).
Cx43 plays a critical role in VCR-induced pain hypersensitivity by regulating the release of inflammatory factors in spinal astrocytes Existing research has reported that Cx43 is the major component of intercellular connections in astrocytes (Wu et al. 2012). We detected spinal cord Cx43 protein expression on days 3, 7, 14, and 21 by Western blotting analysis. Compared with saline-treated animals at the same time points, VCR caused profound upregulation of Cx43 level at all the time points tested ( Fig. 2A, B). Next, we examined whether Cx43 played a crucial role in VCR-induced pain hypersensitivity in mice. First, qRT-PCR was used to screen out an antisense siRNA specifically targeting Cx43 mRNA (Fig. 2C). Then, on days 6 and 7, we focally knocked down Cx43 expression in the spinal cord by intrathecal injection of Cx43 siRNA003 (50 nM, 10 μl). As shown in Fig. 2D and E, intrathecal injection of Cx43 siRNA, but not a missense siRNA, significantly improved VCR-induced pain hypersensitivity at 36 h after injection. Western blotting and qRT-PCR analysis suggested that the spinal cord Cx43 protein and mRNA level were markedly decreased after genetic knockdown of Cx43 using Cx43 siRNA ( Fig. 2F-H).
To provide further evidence supporting the role of spinal Cx43 in VCR-induced pain hypersensitivity, we tested the effects of Gap27, a specific inhibitor of Cx43. We first injected Gap27 (144 nM, 10 μl) intrathecally into saline-treated mice to rule out the impact of Gap27 on the mechanical and thermal pain threshold of normal mice (Supplemental 1A, B). Subsequently, we treated mice exhibiting pain hypersensitivity symptoms with Gap27 (14.4, 43.2, 144 nM, 10 μl each) on day 6 and day 7. The results indicated that, compared with the VCR group at the same time point, intrathecal injections of Gap27 dose-dependently reversed mechanical allodynia and heat hyperalgesia at 2 h after treatment (Fig. 3A, B). Western blotting and qRT-PCR analysis suggested that the protein and mRNA levels of Cx43 declined remarkably after the first intrathecal injection of Gap27 for 36 h (Fig. 3C-E). However, as shown in Fig. 3F and G, VCR-induced astrocyte activation was not improved after the downregulation of Cx43 level by Gap27. Then, we tested the effect of Gap27 on inflammatory factors. The ELISA results demonstrated that VCR-induced upregulation of serum IL-1β and IL-6 secretion was strikingly reduced after Gap27 treatment by 38.4% and 24.5%, respectively, (Fig. 3H, I). However, qRT-PCR confirmed that Gap27 did not obviously decrease the mRNA levels of inflammatory factors and chemokines (Fig. 3J). These data confirmed that Cx43 played a critical role in VCR-induced pain hypersensitivity by regulating the release of inflammatory factors. The PWT and C PWL of mice at different time points after VCR (0.1 mg/kg) intraperitoneal injection, compared to sham group at the same point in time, n = 8 mice/group. D, E Confocal images of GFAP (red) and quantitative analysis of GFAP immunofluorescence intensity in the spinal dorsal horn on days 3, 7, 14, and 21 after VCR injection, scale bar = 100 μm, n = 4 mice/group. F-I qRT-PCR analysis of spinal cord TNF-α, IL-1β, IL-6, and CXCL12 mRNA levels, n = 4 mice/group. J, K Immunofluorescence staining for TUNEL (green) and quantitative analysis of TUNEL-positive cells in the spinal dorsal horn on day 7 after VCR injection, scale bar = 100 μm, n = 4 mice/group. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM b CaMKII and Ca V 3.2 are involved in VCR-induced [Ca 2+ ] i upregulation and pain hypersensitivity Currently, the role of CaMKII and Ca V 3.2 in neuropathic pain is mainly studied in neurons (Li et al. 2017;Diniz et al. 2014), but little attention has been paid to the role of astrocytes. Therefore, in this research, confocal images of spinal sections co-stained with CaMKII or Ca V 3.2 and GFAP demonstrated that both CaMKII and Ca V 3.2 were expressed on astrocytes (Fig. 4A). A growing number of studies have found that Ca 2+ metabolism disorders were observed in chemotherapyinduced pain hypersensitivity (Li et al. 2020;Siau and Bennett 2006). We investigated the role of intracellular Ca 2+ in pain hypersensitivity. To determine whether VCR stimulated the [Ca 2+ ] i increase in primary astrocytes, the Ca 2+ indicator Fluo-4 AM was used to measure [Ca 2+ ] i . The results suggested that VCR (3 nM) Fig. 2 Cx43 plays a critical role in VCR-induced pain hypersensitivity. A Cx43 protein expression in the spinal cord, as shown by western blotting, at 3, 7, 14, and 21 days after VCR injection. B Quantitative analysis of Cx43 levels in the spinal cord, n = 3 mice/group. C qRT-PCR analysis of spinal cord Cx43 level after Cx43 siRNA injection, n = 4 mice/group. D, E The PWT and PWL of mice after intrathecal injection Cx43 siRNA003 (50 nM, 10 μl) or Cx43 missense siRNA for 36 h, compared with VCR group, n = 8 mice/group. F, G Western blotting and quantitative analysis of Cx43 protein expression in spinal cord of mice treated with Cx43 siRNA003, n = 3 mice/group. H The mRNA level of spinal cord Cx43 was analysed by qRT-PCR, n = 4 mice/group. Western blotting and qRT-PCR analysis results were presented as a fold of sham group. GAPDH was used as an internal reference. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM following treatment with Gap27 (144 nM, 10 μl), scale bar = 50 μm, n = 4 mice/group. H, I ELISA detected the protein levels of IL-1β and IL-6 in blood circulation, n = 4 mice/group. J The mRNA levels of TNF-α, IL-1β, IL-6, and CXCL12 in the spinal cord following treatment with Gap27 (144 nM, 10 μl), n = 4 mice/group. Western blotting and qRT-PCR analysis results were presented as a fold of sham group. GAPDH was used as the internal reference. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM rapidly stimulated an increase in [Ca 2+ ] i in both normal physiological buffer and Ca 2+ -free physiological buffer, and the increase in [Ca 2+ ] i in Ca 2+ -free physiological buffer was slightly lower than that in normal physiological buffer (Fig. 4B). Therefore, the [Ca 2+ ] i accumulation sources may be ascribed to Ca 2+ release from intracellular stores and Ca 2+ influx from the extracellular fluid, and the former occupied a larger proportion. Earlier, we mentioned that CaMKII and Ca V 3.2 were involved in Ca 2+ regulation. In our study, we detected that the VCR-stimulated [Ca 2+ ] i increase was significantly inhibited by pre-treatment with KN-93 (10 μM), but not by L-Ascorbic acid (300 μM), in Ca 2+ -free physiological buffer for 1 h; however, after adding Ca 2+ into Ca 2+free physiological buffer, [Ca 2+ ] i increased rapidly in the KN-93 group, while the change in the L-Ascorbic acid group was not noticeable (Fig. 4C). An increase in [Ca 2+ ] i was also inhibited after pre-treatment with both KN-93 and L-Ascorbic acid simultaneously in normal physiological buffer (Fig. 4D). These results suggested that VCR caused an increase in [Ca 2+ ] i via promoting CaMKII-sensitive Ca 2+ release from intracellular stores, and Ca 2+ influx regulated by Ca V 3.2 from the extracellular fluid. Therefore, CaMKII and Ca V 3.2 were involved in VCR-induced [Ca 2+ ] i upregulation in astrocyte cultures.
To characterize the roles of CaMKII and Ca V 3.2 in VCR-induced pain hypersensitivity, mice were injected intrathecally with KN-93 (70 nM, 10 μl) and L-Ascorbic acid (30, 100, 300 μM; 10 μl each) on day 6 and day 7 after eliminating the influence of the inhibitors KN-93 and L-Ascorbic acid on the mechanical and thermal pain threshold of mice (Supplemental 1C-F). Behaviour was assessed at 2 h after administration, and the results showed that KN-93 relieved mechanical allodynia and heat hyperalgesia (Fig. 4E, F). Intrathecal injections of L-Ascorbic acid rapidly (2 h) reversed VCR-induced pain hypersensitivity (Fig. 4G, H).

CaMKII and Ca V 3.2 participate in VCR-induced pain hypersensitivity by activating astrocytes and increasing Cx43 expression
To further verify the role of CaMKII in VCR-induced pain hypersensitivity, we examined p-CaMKII protein level in the spinal cord on days 3, 7, 14, and 21 by Western blotting analysis. The data revealed that VCR resulted in an obvious upregulation in p-CaMKII protein level at all the time points tested but did not affect CaMKII expression (Fig. 5A, B). Previous studies have proven that astrocyte activation is associated with Ca 2+ flux in chemotherapy-induced pain hypersensitivity (Sompol and Norris 2018;Zamora et al. 2020). In this study, KN-93 and L-Ascorbic acid inhibited the VCRstimulated [Ca 2+ ] i increase. We wanted to know whether spinal CaMKII and Ca V 3.2 participate in the development of pain hypersensitivity by activating astrocytes. Mice were injected intrathecally with KN-93 (70 nM, 10 μl) or L-Ascorbic acid (300 μM, 10 μl) separately on 2 successive days, and then various biochemical indicators were tested. Confocal images of spinal dorsal horn stained with GFAP demonstrated that the VCR-induced increase in GFAP-positive astrocytes was significantly inhibited in mice injected with KN-93 intrathecally (Fig. 5C, D). Western blotting analysis showed that VCR-induced CaMKII phosphorylation was inhibited by KN-93 (Fig. 5E, F). In addition, KN-93 also decreased the protein and mRNA levels of Ca V 3.2 and Cx43 (Fig.  5E, G-J). Intrathecal injection of L-Ascorbic acid (300 μM, 10 μl) also inhibited the activation of astrocytes (Fig.  6A, B) and reduced the levels of Ca V 3.2, p-CaMKII, and Cx43 ( Fig. 6C-H). However, intrathecal injection of Gap27 did not affect the protein levels of p-CaMKII or Ca V 3.2 ( Fig. 6I-K). These results revealed that CaMKII and Ca V 3.2 might coregulate the expression of Cx43 through astrocyte activation. Moreover, CaMKII and Ca V 3.2 may have a mutual regulatory effect, which is probably related to their abilities to regulate Ca 2+ . Therefore, CaMKII and Ca V 3.2 play essential roles in VCRinduced pain hypersensitivity.
The CaMKII signalling pathway is involved in the inflammation and apoptosis caused by VCR Our previous study observed that VCR could cause an inflammatory response (Zhou et al. 2018). Therefore, we tested the influence of the CaMKII signalling pathway on inflammation in the present experimental setting. First, we examined the expression of NF-κB pathway-related proteins in the spinal cord. Western blotting confirmed that KN-93 suppressed phospho-NF-κB p65 (P-p65) protein level but there was no difference in NF-κB p65 (p65) expression compared with sham group (Fig. 7A-C). Furthermore, KN-93 also inhibited the VCR-induced increase in COX-2 expression (Fig.  7A, D). Subsequently, we tested the changes in inflammation factors and chemokines in the spinal cord after ] i concentration of astrocyte pre-treated with VCR (3 nM) for 1 h in normal physiological buffer or Ca 2+ -free physiological buffer, n = 3 cultures/group. C In a Ca 2+ -free physiological buffer, the [Ca 2+ ] i concentration of astrocyte pre-treated with KN-93 (10 μM) or L-Ascorbic acid (300 μM) for 30 min, exposed to VCR (3nM), and then 1 mM Ca 2+ was added to the cells, n = 3 cultures/group. D In a normal physiological buffer, the [Ca 2+ ] i concentration of astrocyte pre-treated with KN-93 (10 μM) and L-Ascorbic acid (300 μM) for 30 min, exposed to VCR, n = 3 cultures/group. E, F The PWT and PWL of mice after intrathecal injection KN-93 (70 nM, 10 μl), compared with the VCR group at the same point in time, n = 8 mice/group. G, H The PWT and PWL of mice after intrathecal injection L-Ascorbic acid (30, 100, 300 μM, 10 μl), n = 8 mice/group. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM inhibiting CaMKII phosphorylation. The qRT-PCR results suggested that intrathecal injection of KN-93 attenuated the increase in TNF-α, IL-6, IL-1β, and CXCL12 mRNA levels induced by VCR (Fig. 7E).
In this study, we found that VCR caused an apoptotic response in the spinal cord. Therefore, we then verified whether the CaMKII signalling pathway was related to the apoptotic response induced by VCR. TUNEL 50 μm, n = 4 mice/group. E-H Western blotting and quantitative analysis of spinal cord p-CaMKII, Ca V 3.2, and Cx43 protein levels, n = 3 mice/group. I, J qRT-PCR analysis of Ca V 3.2 and Cx43 mRNA levels. All analysis results were presented as a fold of sham group. GAPDH was used as an internal reference. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM Fig. 6 Ca V 3.2 plays roles in VINP by activating astrocytes and increasing Cx43 expression. A, B Confocal images and quantitative analysis of GFAP intensity in the spinal dorsal horn of mice following treatment with L-Ascorbic acid (300 μM, 10 μl), scale bar = 50 μm, n = 4 mice/group. C-F Western blotting and quantitative analysis of p-CaMKII, Ca V 3.2, and Cx43 protein levels in spinal cord following treatment with L-Ascorbic acid (300 μM, 10 μl), n = 3 mice/group. G, H qRT-PCR analysis of spinal cord Ca V 3.2 and Cx43 mRNA levels following treatment with L-Ascorbic acid (300 μM, 10 μl), n = 4 mice/group. I-K Western blotting and quantitative analysis of p-CaMKII and Ca V 3.2 protein levels in the spinal cord of mice after intrathecal injection Gap27 (144 nM, 10 μl), n = 3 mice/group. All analysis results were presented as a fold of sham group. GAPDH was used as an internal reference. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM staining proved that the number of TUNEL-positive cells was significantly decreased by 79% following KN-93 treatment (Fig. 7F, G). Moreover, we tested the expression of apoptosis-associated proteins Cleaved Caspase-3, Bcl-2, and Bax by Western blotting and revealed that KN-93 strikingly reversed the dysregulation of Cleaved Caspase-3, Bcl-2, and Bax ( Fig. 7H-N).
Consistently, qRT-PCR demonstrated that KN-93 could improve the changes in Bcl-2 and Bax mRNA levels ( Fig. 7O, P). These results indicated that the CaMKII signalling pathway participated in VCR-induced inflammation and apoptosis.

Cx43 regulates the release of inflammatory factors in primary astrocytes
To check the cytotoxicity of VCR, cell viability was measured in cultured primary astrocytes by a CCK-8 kit.
After 24 h of treatment with VCR at a concentration that was increased from 0.1 to 100 nM, we found that VCR concentrations within 3 nM were safe for cells (Fig. 8A). Then, we performed immunohistochemical staining, Western blotting, and qRT-PCR to confirm the effect of VCR on Cx43 expression. Confocal laser images showed that VCR treatment (3 nM, 24 h) evoked a pronounced upregulation in the fluorescence intensity of astrocytes Cx43 and this was reversed by Gap27 (30 μM) (Fig. 8B, C). In agreement with in vivo studies, the Western blotting analysis proved that the increased in Cx43 expression induced by VCR was reduced by 41.2% in astrocyte cultures treated with Gap27 (30 μM) for 24 h (Fig. 8D, E). For the inflammatory response, the ELISA results suggested that inhibiting Cx43 could reduce the release of IL-6 and IL-1β in the cell supernatant (Fig. 8F, G). However, Gap27 did not decrease the increase in inflammatory factors and chemokine mRNA levels induced by VCR in astrocyte cultures (Fig. 8H). In addition, Cx43 siRNA was also used to treat astrocyte cultures. Following Cx43 siRNA treatment (100 nM, 36 h), the expression of Cx43 was downregulated by 55.1% in astrocyte cultures compared with missense siRNA treatment (Fig. 8I, J). Moreover, treatment with Cx43 siRNA also decreased the release of IL-6 and IL-1β in the cell supernatant (Fig. 8K, L).

CaMKII and Ca V 3.2 regulate the expression of Cx43 in vitro
To further test the changes in CaMKII, Ca V 3.2, Cx43, and their associations, we pre-treated astrocyte cultures with CaMKII, Ca V 3.2, and the Cx43 inhibitors KN-93 (10 μM), L-Ascorbic acid (300 μM), and Gap27 (30 μM) for 6 h and then treated them with VCR for 24 h. The Western blotting analysis results demonstrated that both KN-93 and L-Ascorbic acid inhibited CaMKII phosphorylation and downregulated the protein expression of Ca V 3.2 and Cx43 ( Fig. 9A-D, G-J). qRT-PCR verified that KN-93 and L-Ascorbic acid reduced the mRNA levels of Ca V 3.2 and Cx43 (Fig. 9E, F, K, L). In order to eliminate the contingency of the experiment, we used KN-62 (10 μM) and zinc chloride (ZnCl 2 , 30 μM) for the experiment, and the Western blotting analysis results were consistent with KN-93 and L-Ascorbic acid ( Fig. 9M-P). However, Gap27 only inhibited Cx43 protein and mRNA levels ( Fig. 3C-E), but did not affect CaMKII phosphorylation and Ca V 3.2 protein expression ( Fig. 9Q-S). To further determine whether Cx43 was downstream of CaMKII and Ca V 3.2, we treated astrocyte cultures with the Cx43 plasmid (3 μg/ml) to induce Cx43 overexpression. The results of Western blotting analysis showed that the upregulation of Cx43 expression did not affect CaMKII phosphorylation or Ca V 3.2 protein level (Fig. 9T, U). These results indicated that VCR was capable of activating the CaMKII signalling pathway and increasing Ca V 3.2 expression. Thus, CaMKII and Ca V 3.2 may interact and jointly regulate the expression of Cx43 in astrocyte cultures. Fig. 7 The CaMKII signalling pathway is involved in inflammation and apoptosis caused by VCR. A-D Western blotting and quantitative analysis of P-p65, p65, and COX-2 protein levels in the spinal cord of mice treated with KN-93 (70 nM, 10 μl), n = 3 mice/group. E The mRNA levels of TNF-α, IL-1β, IL-6, and CXCL12 in spinal cord after intrathecal injection KN-93 (70 nM, 10 μl), n = 4 mice/group. F, G Immunofluorescence staining for TUNEL (green) and quantitative analysis of TUNEL-positive cells in sections of spinal cord, scale bar = 100 μm, n = 4 mice/group. H-J Western blotting and quantitative analysis of spinal cord C-Caspase-3 and T-Caspase-3 protein levels, n = 3 mice/group. K-N Western blotting and quantitative analysis of Bcl-2 and Bax protein levels in the spinal cord, n = 3 mice/group. O, P qRT-PCR analysis of spinal cord Bcl-2 and Bax mRNA levels, n = 4 mice/group. All analysis results were presented as a fold of sham group. GAPDH was used as an internal reference. ANOVA followed by Tukey's post hoc test. All data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-p65, phosphorylated p65; C-Caspase-3, Cleaved Caspase-3; T-Caspase-3, Total Caspase-3 The CaMKII signalling pathway is associated with inflammation, mitochondrial injury, and apoptosis in astrocyte cultures treated with VCR To clarify the potential mechanism of CaMKII involved in regulating the VCR-induced inflammatory response, the protein levels of P-p65 and p65 were detected via Western blotting in primary astrocytes. Compared with the control group, P-p65 but not p65 protein levels were apparently upregulated in VCR-treated cells and were significantly blocked by KN-93 treatment (Fig. 10A-C). Then, we examined the mRNA levels of TNF-α, IL- Fig. 8 Cx43 regulates the release of inflammatory factors in primary astrocytes. A Cell viability of astrocytes treated with varying concentrations of VCR from 0.1 to 100 nM for 24 h, n = 6 cultures/group. B, C Immunofluorescence images and quantitative analysis of Cx43 intensity in the astrocyte cultures following treatment with Gap27 (30 μM) for 24 h, n = 3 cultures/group. D, E Western blotting and quantitative analysis of Cx43 protein level in the astrocyte cultures, n = 3 cultures/group. F, G The protein levels of cell supernatant IL-1β and IL-6 were measured by ELISA after treatment with Gap27 (30 μM), n = 3 cultures/group. H qRT-PCR analysis of primary astrocytes Cx43 mRNA level following treatment with Gap27 (30 μM) for 24 h, n = 3 cultures/group. I, J Western blotting and quantitative analysis of Cx43 protein level in the astrocyte cultures following treatment with Cx43 siRNA for 36 h. K, L The protein levels of cell supernatant IL-1β and IL-6 were measured by ELISA after treatment with Cx43 siRNA. GAPDH was used as an internal reference. Western blotting and qRT-PCR analysis results were presented as a fold of control group. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All data are represented as means ± SEM Fig. 9 CaMKII and Ca V 3.2 regulate the expression of Cx43 in vitro. A-D Western blotting and quantitative analysis of p-CaMKII, Ca V 3.2, and Cx43 protein levels in astrocyte cultures following treatment with KN-93 (10 μM) for 24 h. E, F qRT-PCR analysis of Ca V 3.2 and Cx43 mRNA levels in cells treated with KN-93 (10 μM). G-J Western blotting and quantitative analysis of p-CaMKII, Ca V 3.2, and Cx43 protein levels in cells after treatment with L-Ascorbic acid (300 μM). K, L qRT-PCR analysis of Cav3.2 and Cx43 mRNA levels in astrocytes following treatment with L-Ascorbic acid (300 μM). M-P Western blotting and quantitative analysis of p-CaMKII, Ca V 3.2, and Cx43 protein levels in astrocyte cultures following treatment with KN-62 (10 μM) and ZnCl 2 (30 μM) for 24 h. Q-S Western blotting and quantitative analysis of p-CaMKII and Cav3.2 protein levels in astrocyte cultures following treatment with Gap27 (30 μM). T, U Western blotting and quantitative analysis of p-CaMKII, Ca V 3.2, and Cx43 protein levels in cells following transfection with Cx43 plasmid (3 μg/ml). GAPDH was used as an internal reference. Western blotting and qRT-PCR analysis results were presented as a fold of control group. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3 cultures/group. All data are represented as means ± SEM 6, IL-1β, COX-2, and CXCL12 by qRT-PCR and found that VCR-induced increases in inflammatory cytokines, chemokines, and COX-2 were inhibited by KN-93 in astrocyte cultures (Fig. 10D).
Mitochondria play crucial roles in inflammation, immunity, autophagy, and cell death. Recent studies have confirmed that VCR can lead to mitochondrial functional damage (Chen et al. 2020). We wanted to know whether the CaMKII signalling pathway participated in mitochondrial damage caused by VCR. To evaluate the effect of CaMKII on VCR-induced mitochondrial damage, flow cytometry was performed to examine the MMP when primary astrocytes were challenged with VCR and KN-93. As shown in Fig. 10E and F, VCR exposure reduced MMP, which was significantly alleviated by KN-93. We also tested the oxidative stress of primary astrocytes, and the data proved that KN-93 largely inhibited ROS production induced by VCR (Fig. 10G, H). For the apoptotic response, the results of flow cytometry revealed that the VCR-triggered increase in apoptosis was remarkably reversed by KN-93 (Fig. 10I, J). Western blotting analysis demonstrated that VCR-treated primary astrocytes significantly increased their protein expression of Cleaved Caspase-3 and Bax and reduced Bcl-2 expression, and these effects were reversed when they were exposed to . Taken together, the evidence above confirmed that the CaMKII signalling pathway was involved in the VCR-induced inflammatory response, mitochondrial damage, and apoptosis.

Discussion
Several studies have verified that astrocytes play a vital role in developing and maintaining chronic pain in the CNS (Sáez and Green 2018;Orellana et al. 2013). The activation of astrocytes requires the participation of Ca 2+ (Surmeier et al. 2010;Neher and Sakaba 2008). Therefore, we focused on CaMKII and Ca V 3.2 t-type calcium channel related to calcium ion regulation. Moreover, astrocytes form interconnected networks coupled through Cx43 . Intriguingly, in the current study, our data suggested that CaMKII and Ca V 3.2 activate astrocytes by upregulating [Ca 2+ ] i , thereby modulating Cx43-dependent inflammatory factor release in the spinal cord of mice treated with VCR. Moreover, CaMKII was also involved in VCR-induced inflammation and apoptosis.
Chemotherapy-induced peripheral nerve damage could result in astrocyte activation in the spinal dorsal horn (Zhou et al. 2020a). Astrocyte activation is characterized by hypertrophy accompanied by an increase in GFAP, whose expression may be associated with proinflammatory cytokines (Sticozzi et al. 2020). Moreover, astrocyte activation promoted the development of CIPN by secreting regulatory factors, such as pro-/antiinflammatory factors, chemokines, and ATP (Dosch et al. 2019;Shen et al. 2015). In our research, we confirmed that VCR could persistently activate astrocytes and increase the production of inflammatory factors (TNF-α, IL-6, IL-1β), COX-2, and chemokines (CXCL12) from day 3 to day 21 after the first injection of VCR. In addition, VCR also led to an apoptotic response in the spinal cord of mice.
Accumulating evidence suggests that astrocyte activation is related to calcium imbalance (Sompol and Norris 2018;Zamora et al. 2020). CaMKII is a widely expressed multifunctional serine/threonine kinase and plays a critical role in linking astrocyte activation and calcium dysregulation in age-related neurodegenerative diseases (Sompol and Norris 2018). Additionally, CaMKII has been verified to participate in the survival and apoptosis of neuronal cells through Ca 2+ signalling (Chen et al. 2010). CaMKII acts as a general integrator of Ca 2+ signalling. We surmised that VCR likely activates astrocytes by promoting the phosphorylation of Ca 2+ -dependent CaMKII and increasing [Ca 2+ ] i in astrocytes. Indeed, our study showed that VCR not only caused [Ca 2+ ] i disturbance in astrocytes but also increased the phosphorylation level of CaMKII. Blocking CaMKII phosphorylation effectively reversed the VCRinduced increase in [Ca 2+ ] i and pain hypersensitivity by treatment with KN-93. Ca V 3.2, a low-threshold calcium channel, has been reported to be involved in regulating DRG neuronal excitability (McCallum et al. 2003) and is a molecular substrate for dorsal horn neuropathic pain (François et al. 2015). In this study, we discovered that Ca V 3.2 controlled extracellular calcium entry into the cytoplasm of astrocytes treated with VCR. Similarly, after using L-Ascorbic acid to inhibit Ca V 3.2 activity, the VCR-induced [Ca 2+ ] i imbalance and pain hypersensitivity were also reversed. Moreover, inhibiting CaMKII phosphorylation and Ca V 3.2 activity could significantly reduce astrocyte activation and downregulate Cx43 level. Thus, we believe that CaMKII and Ca V 3.2 likely activate astrocytes by upregulating [Ca 2+ ] i in VINP. Existing evidence has revealed that Fig. 10 The CaMKII signalling pathway is associated with inflammation, mitochondrial injury, and apoptosis in astrocyte cultures treated with VCR. A-C Western blotting and quantitative analysis of P-p65 and p65 protein levels in astrocyte cultures following treatment with KN-93 (10 μM) for 24 h. D The mRNA levels of TNF-α, IL-1β, IL-6, COX-2, and CXCL12 in astrocyte cultures. E, F JC-1 fluorescent probe was used to analyse the MMP of astrocyte cultures. G, H The analysis of the ROS assay in astrocyte cultures by flowcytometry. I Representative flow cytometry analysis of Annexin V and PI staining. J Quantitative analysis of cells apoptosis proportion. K-M Western blotting and quantitative analysis of C-Caspase-3 and T-Caspase-3 protein levels in astrocyte cultures. N-Q Western blotting and quantitative analysis of Bcl-2 and Bax protein levels in astrocytes. GAPDH was used as an internal reference. Western blotting and qRT-PCR analysis results were presented as a fold of control group. ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3 cultures/group. All data are represented as means ± SEM CaMKII regulates Ca V 3.2 expression to enhance neuronal excitability (Welsby et al. 2003). Indeed, we observed that inhibiting CaMKII phosphorylation by KN-93 could significantly reduce the spinal cord protein and mRNA levels of Ca V 3.2 in this research. However, a reduction in spinal cord p-CaMKII protein expression was found after L-Ascorbic acid administration. In order to eliminate the contingency of the experiment, we used another Ca V 3.2 inhibitor ZnCl 2 (30 μM) for the experiment. The Western blotting results revealed ZnCl 2 largely abolished the VCR-induced enhancement of CaMKII phosphorylation. Therefore, CaMKII and Ca V 3.2 may interact in VINP. It was worth noting that L-Ascorbic acid also downregulated Ca V 3.2 expression. To our knowledge, the inhibitory effect of L-Ascorbic acid on Ca V 3.2 channel expression has not been reported. Combined with the experimental results of this study, we conjecture that the inhibition of Ca V 3.2 expression by L-Ascorbic acid may be an indirect effect. Both Ca 2+ and CaMKII phosphorylation process may be involved in the decrease of Ca V 3.2 expression by L-Ascorbic acid. In addition, L-Ascorbic acid, as a potent antioxidant, has been tested to treat CINP (Maschio et al. 2018). Studies have shown that L-Ascorbic acid plays a role in the treatment of peripheral neuropathic pain by reducing the generation of ROS and inhibiting the phosphorylation of p38 MAPK in spinal cord and DRGs of mice (Lu et al. 2011). Blockage of oxidative pathways with L-Ascorbic acid ameliorates paclitaxelinduced mechanical and thermal hypersensitivity (Miao et al. 2019). Furthermore, L-Ascorbic acid can enhance the therapeutic effect of gabapentin on neuropathic pain (Li et al. 2016), and has effective prevention and treatment effects on cisplatin-induced nerve pain (Guindon et al. 2014), chronic ischemic pain (PMID: 24363848), and neuralgia caused by herpes zoster (Chen et al. 2011;Chen et al. 2009).
Neighbouring astrocytes can communicate directly through intercellular gap junctions (Orellana et al. 2013). In addition, gap junction proteins also form unopposed hemichannels, which provide channels for cells to exchange molecules such as ATP to coordinate activities between the cytoplasmic and extracellular compartments (Vicario et al. 2017;Dosch et al. 2019;Retamal et al. 2007). Cx43 is a significant component of intercellular connections in astrocytes. Increased expression of spinal cord Cx43 was reported following paclitaxel-induced and oxaliplatin-induced peripheral neuropathy, CCI, and bone cancer pain (Dai et al. 2020;Yang et al. 2018;Tonkin et al. 2018). Astrocytic Cx43 also enhances spinal cord synaptic transmission and maintains neuropathic pain in the late-phase via releasing chemokines (Chen et al. 2014). However, a previous research suggested that the downregulation of spinal Cx43 expression by intrathecal treatment with Cx43-targeting siRNA increased IL-6 and COX-2 expression and induced hind paw mechanical hypersensitivity (Morioka et al. 2018). In our present study, we found that VCR could induce continuous upregulation of the spinal cord Cx43 level and increase the release of IL-1β and IL-6 in blood circulation. Cx43 expression was significantly suppressed by intrathecal injection of the Cx43 inhibitor Gap27 or siRNA, which reduced the release of IL-6 and IL-1β and relieved VCR-induced pain hypersensitivity. However, Gap27 did not inhibit VCR-induced spinal cord astrocyte activation and the production of inflammatory cytokines and chemokines. Gap27 also did not reverse the increase in p-CaMKII and Ca V 3.2 expression induced by VCR. Likewise, astrocytes treated with Gap27 or Cx43 siRNA also decreased Cx43 expression and inflammatory factors release. In addition, we treated astrocyte cultures with a Cx43 plasmid to induce Cx43 overexpression, and the results revealed that the upregulation of Cx43 expression also did not affect CaMKII phosphorylation or Ca V 3.2 levels. Therefore, CaMKII and Ca V 3.2 mediate Cx43-dependent inflammation by activating astrocytes in VINP.
In this study, we discovered that VCR (3 nM) stimulated an increase in [Ca 2+ ] i in both normal physiological buffer and Ca 2+ -free physiological buffer. Hence, the [Ca 2+ ] i accumulation sources may include extracellular fluid influx and intracellular store release, and intracellular store release provided a larger proportion of [Ca 2+ ] i . Subsequently, we pretreated astrocytes with KN-93 and L-Ascorbic acid for 1 h in Ca 2+ -free physiological buffer. The results suggested that KN-93, not L-Ascorbic acid, efficiently inhibited the release of Ca 2+ from intracellular stores. Therefore, we believe that the CaMKII signalling pathway is essential for VINP. Previous studies have shown that astrocyte and microglia activation, oxidative stress, neuronal damage, and apoptosis are several important mechanisms of CINP (Siddiqui et al. 2021). Recent reports have revealed that reducing the p-CaMKII inhibits NF-κB signalling pathway activation and diminishes lipopolysaccharideinduced neuroinflammation in primary microglial cells (Park et al. 2020). During our research, we noticed that when KN-93-pretreated primary astrocytes were treated with VCR for 24 h, the P-p65 level was prominently decreased compared to that of vehicle-pretreated cells. In vivo experiments also showed similar results. Therefore, CaMKII may participate in VCR-induced neuroinflammation by inhibiting NF-κB signalling pathway activation. For VCR-induced apoptosis, the question is how CaMKII governs this process. A growing body of researches has investigated that the interaction between ROS and cellular Ca 2+ activates related signalling pathways, leading to neuronal apoptosis (Cheng et al. 2012;Circu and Aw 2010). Moreover, mitochondria play crucial roles in cellular Ca 2+ and redox homeostasis and apoptosis induction (Koopman et al. 2010;Cheng et al. 2012). Oxidation can damage mitochondrial function, resulting in hindered energy regulation (Zsurka and Kunz 2015). In our experiments, we observed VCR led to mitochondrial damage by increasing mitochondrial ROS production and changing MMP in astrocyte cultures. These disorders were reversed by KN-93. Thus, we tentatively conclude that CaMKII may participate in VCR-induced apoptosis by regulating the crosstalk between Ca 2+ signalling and mitochondrial ROS. Undoubtedly, more researches are necessary to further delve into this problem.
In conclusion, we provide evidence that CaMKII, Ca V 3.2, and Cx43 play vital roles in neuropathic pain and reveal that CaMKII and Ca V 3.2 facilitate Cx43mediated inflammatory factor release in spinal cord astrocytes to underlie the development of peripheral neuropathic allodynia. To the best of our knowledge, our study is the first report that CaMKII and Ca V 3.2 mediate Cx43-dependent inflammation by activating astrocytes in VINP. Furthermore, we confirmed that CaMKII was also involved in VCR-induced inflammation and apoptosis. These findings may have considerable benefits for the future treatment of the CIPN.