According to the International Association for the Study of Pain, chronic pain is characterized as pain that persists intermittently or exceeds a duration of three months [1,2]. Depression, which ranks as the second most prevalent global disease, manifests as enduring emotional disorders lasting for a minimum of two weeks, thereby posing a substantial risk to both the mental and physical well-being of the general population [3]. Notably, chronic pain emerges as a primary catalyst for the onset of depression, given its classification as a stress-related condition. Empirical evidence suggests that, on average, approximately 50% of individuals suffering from chronic pain encounter severe depressive symptoms [4], and their coexistence often worsens the severity of both conditions [5]. Given the frequent coexistence of these two pathological conditions, it is essential to explore the shared potential targets between chronic pain and depression to suggest more efficient therapeutic approaches.

1. Complement pathways

The complement system encompasses more than 40 proteins that are soluble or bound to membranes, and can be found in serum, tissue fluids, and cell surfaces. In its inactive state, it is present in bodily fluids or the central nervous system (CNS), and becomes activated through a cascade of enzymatic reactions, resulting in diverse biological outcomes [6]. Initially, the liver is believed to be the primary origin of complement proteins. However, further investigation has revealed that within the nervous system, complements can also be synthesized by neurons, oligodendrocytes, microglia, and astrocytes [7,8]. There are three distinct activation routes for the complement system: the classical pathway, the lectin pathway, and the alternative pathway. The classical pathway involves the binding of C1q to the antibody-antigen complex, while the lectin pathway is activated when mannose-bound lectin (MBL) encounters pathogenic carbohydrate sequences. The alternative pathway is initiated by the autonomous hydrolysis of C3 into C3d·H2O. These pathways play crucial roles in modulating inflammation and aiding the host defense mechanisms. The C1 protein complex, composed of C1q, C1r, and C1s, is responsible for initiating the classical pathway. Upon activation, C1s cleaves C4 and C2 to further propagate the complement cascade. The lectin pathway is primarily activated by extracellular sugar residues. The pattern recognition complex of the lectin pathway consists of the MBL paired with two protease complexes formed by MBL-associated serine proteases, MASP-1 and MASP-2. Additionally, other collagenous lectins such as the ficolins and collectin-11 can activate the lectin pathway by interacting with MASPs. Upon substrate recognition, the MBL complex undergoes conversion into an active serine protease, which then cleaves C4 and C2 in a manner similar to the classical pathway. This process results in the production of the classical C3 convertase. The resulting cleavage products induce the assembly of C3 convertase C4bC2a, which further cleaves C3 into C3a and C3b. C3a then facilitates the chemotaxis and activation of microglial cells through the C3a receptor (C3aR), while C3b can be further processed into iC3b. Microglial cells recognize and enhance their activation through the binding of iC3b to complement receptor 3 (CR3). Additionally, the generation of C3b can lead to the formation of C5 convertases (C4bC2aC3b and C3bBbC3b). C5 can undergo cleavage to produce C5a and C5b. C5a acts to enhance chemotaxis and activation of glial cells through its interaction with C5aR. Additionally, an alternative pathway is initiated by the spontaneous hydrolysis of C3 to C3 (H2O). All three of these pathways lead to the formation of convertases, which further contribute to the production of key effectors in the complement system, including anaphylatoxins (C4a, C3a, and C5a), membrane attack complexes (MAC), and opsonins (C3b). Anaphylatoxins are responsible for activating pro-inflammatory signals. C5b, on the other hand, combines with C6, C7, C8, and C9 to form the MAC, which penetrates the cell membrane, leading to cell lysis upon target surfaces, playing a role in the innate immune process and mediating the killing and clearance of pathogens [9] (Fig. 1). Additionally, complements are involved in the coordination of diverse host mechanisms, including synaptic restructuring, axon rejuvenation, neuronal injuries, and enduring stress. These mechanisms, which are closely linked to disease and neural functionality, are increasingly recognized as significant factors in the development and advancement of chronic pain and depression [10].

Figure 1. Activation pathways of the complement system. The complement system is activated by the classical, lectin, or alternative pathways. The initiation of the classical pathway involves the C1 protein complex, which consists of C1q, C1r, and C1s. Upon activation, C1s cleaves C4 and C2. In the lectin pathway, activation occurs when mannose-binding lectin (MBL) encounters a pathogenic carbohydrate motif. Subsequently, MBL associates with MASP-1 and MASP-2, leading to the cleavage of C4 and C2. The resulting cleavage products induce the formation of C3 convertase C4bC2a, which subsequently splits C3 into C3a and C3b. C3a has the ability to induce the chemotaxis and activation of microglia via C3aR. Additionally, C3b can undergo cleavage to form iC3b, which is recognized by microglial cells through complement receptor 3 (CR3), thereby promoting activation. Furthermore, the production of C3b can facilitate the generation of C5 convertase (C4bC2aC3b, C3bBbC3b). C5 can also be cleaved into C5a and C5b, with C5a further promoting the chemotaxis and activation of glial cells through C5aR. Another pathway involves the spontaneous hydrolysis of C3 to C3 (H2O). All three pathways result in the generation of convertases, which subsequently stimulate the production of the primary components of the complement system, namely anaphylatoxins (C4a, C3a, and C5a), membrane attack complexes, and opsonins (C3b). Allergic toxins initiate pro-inflammatory signaling, while C5b associates with C6, C7, C8, and C9 to form membrane attack complexes (MAC), ultimately resulting in cell cleavage.

2. Complement system in chronic pain and depression

Research has demonstrated that the activation of complements, whether in the peripheral or CNS, contributes to the onset and progression of chronic pain. Tong et al. [11] have examined the correlation between plasma levels of complement C5a and pain in individuals diagnosed with neuromyelitis optica spectrum disorders (NMOSD). They have revealed that patients with NMOSD had significantly elevated levels of plasma C5a. In comparison to pain-free patients, these individuals experience increased levels of anxiety and a decreased quality of life. The concentration of plasma C5a emerges as an independent factor influencing pain in NMOSD patients. Togha et al. [12] further identified that an increase in plasma complement C3 protein levels could serve as a biological marker during pain flare-ups in individuals with chronic migraines. Additionally, bioinformatics has highlighted the importance of the complement system in neuropathic pain (NP). Yi et al. [13] employed the restarted random walk algorithm to identify the complement C3 as a significant gene associated with NP in rats subjected to spinal nerve ligation (SNL). Similarly, Wang et al. [14] investigated the hub genes in rats with spared nerve injury (SNI) that are closely associated with the development of NP, indicating the crucial involvement of both complement C1q and C3 genes in the initiation of NP [14]. Zoster sine herpete is one of the atypical clinical manifestations of herpes zoster, which stems from infection and reactivation of the varicella-zoster virus in the cranial nerve, spinal nerve, viscera, or autonomic nerve [15]. Peng et al. [16] conducted a significant clinical investigation employing least absolute shrinkage and selection operator regression analysis to develop a predictive model for assessing the efficacy of pulsed radiofrequency (PRF) in managing Zoster-associated pain. Their findings revealed a significant correlation between lower levels of complement C4 in peripheral blood and poor PRF outcomes, which holds potential implications for tailoring personalized treatment approaches in the future. Furthermore, the activation of the complement system also contributes to the onset of depression. Numerous clinical studies have demonstrated that individuals diagnosed with major depressive disorder (MDD) exhibit significantly elevated levels of various complement proteins, including C1, C1q, C3, and C3a, in their plasma compared to healthy individuals. This observation suggests a crucial involvement of the complement system in the pathophysiological mechanisms underlying MDD [17–19]. Furthermore, there is a notable increase in mRNA levels of complement C3 in the prefrontal cortex (PFC) of individuals who have died by suicide and were diagnosed with depression [20]. Additionally, mice lacking the complement pathway C3/C3aR display heightened anxiety and fear-like behaviors in comparison to their normal counterparts [21,22]. The aforementioned studies indicate a significant correlation between the complement system and the pathogenesis of chronic pain and depression. Consequently, it is plausible to hypothesize that targeting the complement system may hold promise for the treatment of chronic pain and concurrent depression.

Within the mechanisms of comorbidity between chronic pain and depression, neuroglial cells play a pivotal role in the functioning of the complement system. In three neuropathological pain models, specifically SNI, SNL, and chronic constriction injury (CCI), there is a significant upregulation of complement C1q, C3, C4, C5, and C5a within the microglial cells located in the dorsal horn of the spinal cord. When mice are administered intrathecal injections of C5a, they display an increased sensitivity to cold pain, whereas mice lacking C5 exhibit a reduced sensitivity to pain [23]. Typically, astrocytes express complement C3, while microglial cells express the complement C3a receptor C3aR [24]. In the context of chronic stress-induced depressive mice, the involvement of the C3/C3aR complement pathway is implicated in the abnormal synaptic pruning, which arises from the interaction between astrocytes and microglia [25]. Suppression of the C3a signaling has been found to alleviate the depressive behaviors induced by chronic stress in mice[20]. Prior investigations have suggested that the complement system within neuroglial cells may serve as a crucial connection in the comorbidity mechanism of chronic pain and depression [6,26]. Consequently, this article primarily investigates the molecular mechanisms of the complement system as a prospective therapeutic target for depression and chronic pain.

3. The role of complement system-mediated glial cell synaptic pruning in the comorbidity of chronic pain and depression

1) Synaptic pruning by microglia and astrocytes

Synapses serve as the fundamental components facilitating neuronal communication and memory retention. The process of synaptic pruning, which involves the elimination of superfluous synaptic contacts, plays a critical role in the CNS, ensuring the proper development of neural circuits during the course of neural maturation and sustaining synaptic stability in adulthood. The establishment of accurate synaptic connections is of utmost importance for the optimal functioning of the brain. In the brain, there exists an activity-dependent mechanism for synaptic pruning: neurons selectively stabilize active synapses while getting rid of inactive ones, ensuring optimal synaptic connections throughout brain development [27]. Dysregulation of synaptic pruning is believed to lead to the onset and progression of various psychiatric disorders, such as depression and neurodegenerative diseases including Alzheimer's disease (AD) [28]. Additionally, abnormal synaptic pruning is also considered a mechanism contributing to CNS sensitization and the onset of chronic pain [29].

Microglial cells, as a subset of resident mononuclear phagocytes in the CNS, serve as the principal immune cells. They are extensively distributed throughout the brain and spinal cord, contributing to the maintenance of normal brain functions through the release of inflammatory cytokines, phagocytosis of apoptotic cells, synaptic pruning, regulation of synaptic plasticity, and formation of neural networks [30]. In the brain, astrocytes represent the predominant neuroglial cell type and play a vital role in directing synapse formation, plasticity, and restructuring to regulate neuronal functions [31]. Studies have shown that during brain development, the synaptic pruning directed by microglial and astrocytic cells is crucial in establishing the right neural connections. These cells remove weaker synapses to form the appropriate neural pathways, however, in pathological states, glial cells can engulf an excessive number of synapses, resulting in abnormal synaptic loss [32]. The phagocytic and chemotactic functions of microglial cells play a crucial role in synaptic pruning. Studies have shown that mice lacking the microglial phagocytic triggering receptor expressed on myeloid cells-2 (TREM2) [33] or the chemotactic factor C-X3-C Motif Chemokine Receptor 1 (CX3CR1) demonstrate an excessive presence of synapses in their brains [34]. Additionally, astrocytes significantly contribute to the process of synaptic pruning. Research findings have demonstrated that astrocytes utilize multiple EGF like domains 10 (MEGF10) and tyrosine-protein kinase Mer (MERTK) during the development of the retinal newborn system to facilitate synaptic elimination. However, when MEGF10 or MERTK is knocked out in astrocytes of mice, there is a notable decrease in their capacity to engulf surplus synapses by approximately 30% or 50%. This suggests that astrocytes actively contribute to the promotion of synaptic pruning by participating in the elimination process [35]. Additionally, in the adult hippocampal CA1 region, astrocytes play a more prominent role than microglia in eliminating both excitatory and inhibitory synapses through MEGF10-mediated continuous engulfment of excitatory synapses [36]. Hence, through phagocytic molecular mechanisms, various types of glial cells selectively remove synapses, adjusting their numbers.

2) Complement system participated in glial cell-mediated synaptic pruning

In the brain development process, the complement system acts as an intermediary for glial cell phagocytosis and plays a role in synaptic pruning [37]. The central mechanism for synaptic pruning in the developing mammalian brain is now being attributed to the complement-dependent synaptic pruning by glial cells [28]. In 2007, a study initially demonstrated that synaptic pruning during development is mediated by complement C3 [38]. Further investigations have since indicated that the process of complement-mediated synaptic pruning involves the engulfment of weaker synapses labeled with C3 complement by microglial complement receptor 3 (CR3). The classical complement cascade pathway, facilitated by microglia, plays a crucial role in the non-autonomous mechanism of glial cell-mediated synaptic pruning during developmental synapse pruning [37]. Furthermore, the SRPX2 protein, which is expressed in neurons, functions as a complement inhibitor and has the ability to bind directly to C1q, thereby inhibiting its activity and regulating the process of complement-dependent synapse elimination. And mice with a knockout of SPRX2 [39], as well as mice lacking the microglial phagocytosis inhibitory factor CD47 [40], both exhibited a reduction in synaptic density. These findings provide compelling evidence that complement-dependent synaptic pruning, mediated by microglial cells, constitutes a fundamental mechanism in brain development. Moreover, the modulation of synaptic pruning is directly influenced by astrocytes and microglia through complement crosstalk. This is due to the presence of complement C3 in astrocytes and its cleavage fragment C3a receptor C3aR in microglia, which enhances the release of C3 and activates the microglia, thereby exacerbating abnormal synaptic pruning [41]. Additionally, in mice models of AD, astrocytes eliminate synapses in a C1q-dependent manner, resulting in pathological synaptic loss. Furthermore, astrocyte phagocytosis can compensate for the dysfunction of microglial phagocytosis [32].

Complement C1q serves as a pivotal mediator connecting microglia and synapses, thereby initiating the process of synaptic phagocytosis [42]. In mice and adult individuals, complement C1q is predominantly expressed within the brain and microglia [43]. In the CNS, C1q has been demonstrated to be upregulated as an initial response to injury and functions as the initiating protein for the classical complement cascade, leading to the generation of MAC, anaphylatoxins, and opsonins [44]. C1q plays a role in guiding synaptic pruning through glial cells. Studies show that under high-resolution microscopy, C1q co-labels with pre-synaptic or post-synaptic proteins [38]. Proteomic studies found that synapses marked with C1q have elevated levels of apoptotic indicators casepase-3 and membrane protein-5 in comparison to synapses labeled negatively for C1q, indicating that synaptic pruning associated with C1q exhibit mechanisms akin to apoptosis [45]. Synaptic pruning by complement C1q are typically associated with complement C3, its split fragments C3a and C3b, and the presence of the complement receptor CR3 on microglia. C1q, predominantly secreted by microglia, binds to target synapses and facilitates the cleavage of complement C3 on the synaptic surface into C3a and C3b. C3b is recognized as the "eat-me" signal, while its breakdown product, iC3b, is recognized by microglia's CR3, leading to microglia-mediated synaptic pruning in the affected brain region [46]. In mouse models of AD, an increase in C1q levels has been observed to be associated with synaptic specificity. The inhibition of C1q or the removal of C3 or CR3 has been found to result in a decrease in the number of microglia involved in phagocytosis, synaptic loss, and improvements in learning and memory-related tasks [47]. In the context of the developmental visual system, the synaptic pruning is regulated by components of the classical complement cascade. This process involves the activation of C1q, the synaptic pruning through complement fragment C3b, and the interaction with complement receptor CR3 present on microglia, which directly engulfs synapses expressing C3b [10].

3) C1q/C3-CR3 in chronic pain and depression comorbidity

The C1q/C3-CR3 co-mediated microglial synaptic pruning of the complement cascade pathway is a crucial factor in the pathogenesis of chronic pain and depression. Wang et al. [48] utilized the chronic restraint stress (CRS) methodology to establish a mouse model of depression. In the amygdala of the depressive mouse model, there was a significant increase in neuroinflammation and the expression of complements C1q and C3. This led to the activation of microglial cells, resulting in a decrease in synaptic content in the amygdala, as evidenced by reduced levels of Syn and PSD95 proteins. The aforementioned alterations resulted in the manifestation of behaviors resembling depression in mice. Notably, the absence of the C1q gene impeded the synaptic loss and depressive behaviors induced by CRS in mice, thereby illuminating the significant involvement of C1q/C3-mediated microglial activation and synaptic pruning in the mechanisms underlying the onset of depression [48]. This was observed specifically in the hippocampal CA1 region, leading to ameliorated depressive symptoms in mice with Parkinson's disease. Moreover, within the context of the Parkinson's depression model in mice induced by reserpine, the administration of botulinum neurotoxin A (BonT/A) resulted in a notable decrease in the expression levels of complement C1q and C3 proteins in the hippocampus of the depressed mice. This reduction was accompanied by a downregulation of microglial CR3 mRNA levels. Following the treatment, the markers Iba1 and CD68 in hippocampal microglia exhibited a significant decrease, suggesting a decrease in microglial activation. This treatment resulted in the restoration of synaptic density, as evidenced by the increased co-localization of the excitatory presynaptic vesicle protein vesicular glutamate transporter-2 (VGLut2) with PSD-95, and the decreased fluorescence co-localization of VGlut2 with Iba-1 and CD68, indicating a reduction in microglial phagocytic activity. Furthermore, the expression of pro-inflammatory factors, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in microglial cells decreased, indicating the inhibitory effect of BonT/A on the C1q-C3/CR3-mediated complement cascade signaling pathway in the hippocampal CA1 region and subsequent alleviation of depression in Parkinson's mice [49]. In mouse models of visceral chronic pain induced by chronic stress, there was a significant increase in the expression levels of complement C1q and integrin alpha M (ITGAM) mRNA (which encodes the CD11b subunit of CR3) within amygdala microglial cells. Additionally, a heightened co-localization of C1q with the microglial activation marker Iba1 was detected through enhanced immunofluorescence. The utilization of Iba-1 to label the postsynaptic protein PSD95 demonstrated a significant increase in synaptic modifications by microglial cells. However, the use of CR3 antagonists could mitigate this synaptic pruning and ameliorate visceral hypersensitivity and pain [50]. In essence, the complement cascade pathway C1q/C3-CR3 likely plays a role in the intertwined pathogenic mechanisms of chronic pain and depression. This occurs by mediating synaptic pruning primarily within the amygdala and hippocampal microglial cells (refer to Fig. 1 and Table 1 for a summarized overview).

Table 1 The possible mechanisms of chronic pain and depression comorbidity mediated by complement cascade C1q/C3-CR3 and C3/C3a-C3aR

Complement cascade	Molecular mechanisms	Method/experimental models	Type of disease
C1q	C1q is the hub gene of NP [14]	SNI model, random walk with restart (RWR) methodology [14]	NP
C1q	The knockout of C1q led to a significant increase in the mRNA expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the PFC of mice [68]	The LH model of depression [68]	Depression
C3	C3 is the hub gene of NP [13,14]	SNI model, RWR [13,14]	NP
C3	The expression of PFC C3 increased significantly in depressed suicide patients. Chronic stress leads to increased C3 expression in PFC [20]	CUMS mice [20]	Depression
C1q/C3	Neuroinflammation was observed in the glial cells, of the amygdala, C1q and C3 activation, Syn and PSD-95 levels decreased [48]	CRS mice [48]	Chronic pain and depression
C1q/C3-CR3	BoNT/A treatment decreased the levels of complement C3\CR3 and C1q, decreased the colocalization of iba-1 and CD68, decrease the mRNA expression of CX3CR1 in microglia cells, recovered the density of PSD-95, decreased the mRNA levels of TNF-α and IL-1β [49]	PD mouse model, Mice were administered reserpine (3 μg/mL in the drinking water) for 10 wk. BoNT/A (10 U·kg-1·d-1) was injected into the cheek for 3 consecutive days [49]	Chronic pain and depression
C1q/C3-CR3	The mRNA levels of amygdala complement C1q and ITGAM exhibited a significant increase in microglia within the CeA. Iba-1 and PSD95 colocalization levels increased [50]	Fischer-344 rats, micropellets containing either corticosterone (CORT) were bilaterally implanted onto the CeA using stereotaxic techniques [50]	Chronic pain and depression
C3/C3a-C3aR	Treatment with hUC-MSCs and a C3aR antagonist resulted in an increase in protein levels of PSD-95 and AMPA, suppressing TNF-β and IL-10. CD16+/Iba1+ in the hippocampus and the level of C3a protein in GFAP+ cells were inhibited by hUC-MSCs [53]	CUMS mice, hUC-MSCs were administered intravenously to CUMS mice once a week for a duration of 4 wk [53]	Chronic pain and depression
C3/C3a-C3aR	LPS resulted in the activation of the C3/C3aR in PFC increased polarization of microglia, hUC-MSCs can reduced the C3aR and STAT3, C3aR antagonism treatment restored PSD-95 and SYN levels and improved microglia morphology IL-1R blockers block activation of the pNF-κB/C3 pathway in neurotoxic A1 astrocytes [25]	CUMS mice, hUC-MSCs mice was injected with LPS (0.83 mg/kg, i.p.), The animals received intraperitoneal injections of either saline or C3aR blockade once a day for 4 wk [25]	Chronic pain and depression
C3/C3a-C3aR	Gypenoside XVII: resulted in a decrease in the number of Iba1 positive cells and an upregulation of C3 levels, the downregulation of pSTAT3, the levels of IL-1β, IL-6 and TNF-α in the PFC, reduction of VGIut2 within the microglial and PSD95 [54]	CUMS mice, Gypenoside XVII was dissolved in a solution of 0.9% saline containing 0.3% carboxymethyl cellulose and administered orally once daily for a duration of 4 consecutive wk [54]	Chronic pain and depression
C3/C3a-C3aR	The downregulation of C3aR was observed to inhibit the activation, of A1 astrocytes induced by LPS, resulting in a decrease in the expression of C3aR, C3, and GFAP. These proteins are known to be involved in the transition from acute to chronic pain [57]	The induction of A1 astrocytes was achieved through intraperitoneal injection of LPS [57]	Chronic pain and depression

NP: neuropathic pain, SNI: spared nerve injury, TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β, PFC: prefrontal cortex, LH: learned helplessness, CUMS: chronic unpredictable mild stress, PSD-95: postsynaptic density protein 95, CRS: chronic restraint stress, SYN: synaptophysin, BoNT/A: botulinum neurotoxin A, CX3CR1: C-X3-C motif chemokine receptor 1, PD: Parkinson’s disease, ITGAM: integrin, alpha M, CeA: central nucleus of amygdala, hUC-MSCs: human umbilical cord mesenchymal stem cells, LPS: lipopolysaccharide, STAT3: signal transducer and activator of transcription 3, NF-κB: nuclear factor-kappa B, VGlut2: vesicular glutamate transporter-2.

4) C3/C3a-C3aR in chronic pain and depression comorbidity

Complement component C3, which plays a crucial role in the classical, alternative, and lectin pathways of complement activation, occupies a central position in the activation process, rendering it a pivotal focus for therapeutic interventions [51]. C3a, an activation fragment produced upon C3 activation via the three complement pathways, can be released by astrocytes and bind to the G-protein coupled transmembrane receptor C3aR on microglia, thereby exerting an influence on the functionality of these microglial cells. Consequently, it is plausible that the C3/C3a-C3aR pathway facilitates interactions between astrocytes and microglia [52]. Multiple studies have demonstrated that the activation of C3/C3a-C3aR signaling plays a significant role in the development and advancement of chronic pain and depression in mice exposed to chronic unpredictable mild stress (CUMS) or lipopolysaccharide (LPS)-induced depression. In an attempt to investigate this phenomenon, Li et al. [53] conducted an experiment wherein human umbilical cord mesenchymal stem cells (hUC-MSCs), known for their immunomodulatory and anti-inflammatory properties, were administered to mice subjected to the CUMS model. The findings of this study revealed that the administration of either hUC-MSCs or C3aR antagonists effectively ameliorated depression and anxiety-like behaviors induced by CUMS, and reduced the M1 polarization of microglia and pro-inflammatory cytokine levels (TNF-α and IL-10) in hippocampal neurons, and the activation of the complement pathway was due to the activation of C3a in astrocytes, which further activated the C3aR receptor on microglia, promoting its M1 polarization process [53]. Zhang et al. [25] conducted a study on mice with depression induced by CUMS or LPS. They found that the C3/C3aR pathway showed an early upregulation in the PFC, suggesting its potential as an early indicator of depression onset. Additionally, blocking the C3/C3aR signal not only alleviated depressive-like behaviors in mice but also suppressed the translocation of microglial signal transducer and activator of transcription 3 (STAT3) and the expression of inflammatory cytokines in the PFC. This blockade facilitated synaptic phagocytosis, highlighting its therapeutic potential in depression treatment. Furthermore, the administration of an IL-1R antagonist resulted in a noteworthy reduction in the NF-κB pathway and C3 levels within the PFC of mice subjected to CUMS. This blockade also impeded the activation of the IL-1R/pNF-κB/C3 pathway in neurotoxic A1 astrocytes [25]. Gynostemma pentaphyllum, a commonly employed herbal medicine, has been scientifically validated to possess potent anti-inflammatory properties through its saponin extract. Cheng et al. [54] conducted a study that demonstrated the ability of Gypenoside XVII, an active component of Gynostemma pentaphyllum, to inhibit the C3/C3aR/STAT3/cytokine signal pathway in the PFC of mice. This inhibition resulted in a decrease in microglia activation and a reduction in excessive synaptic pruning in the PFC of mice subjected to CUMS, leading to antidepressant effects. These findings suggest that the activation of abnormal synaptic pruning in depression, mediated by astrocytes-microglia, is facilitated through the C3/C3a-C3aR complement signaling pathway. Moreover, astrocytes-microglia are also implicated in the development of chronic pain through the C3/C3a-C3aR complement pathway. Previous studies have demonstrated abnormal activation of complement C3 in the dorsal horn of the spinal cord in rats with CCI, which contributes to the initiation of NP and the development of pain hypersensitivity [55]. In a subsequent investigation conducted by Mou et al. [56], it was observed that C3/C3aR levels were elevated in the dorsal horn of rats in the CCI model. Furthermore, the administration of C3 or C3aR antagonists via intrathecal route resulted in a reduction in M1 polarization in microglia and alleviated symptoms of NP. Zhu et al. [57] conducted a study in which they utilized thoracotomy and intraperitoneal injection of LPS to induce the expression of neurotoxic A1 astrocytes in rats. Through intrathecal injection, they antagonized the C3aR in microglia and observed that the downregulation of C3aR inhibited the activation of LPS-induced neurotoxic A1 astrocytes and M1 polarization of microglia. Additionally, this downregulation increased the presence of A2 astrocytes and resulted in a reduction in both the mechanical pain threshold and the incidence of chronic pain in mice. In light of these findings, it is evident that the C3/C3a-C3aR complement pathway is central to the molecular crosstalk and synaptic pruning between astrocytes and microglia. This interaction likely plays a vital role in the combined mechanisms underpinning chronic pain and depression (Fig. 2 and Table 1).

Figure 2. Potential mechanisms by glial cell synaptic pruning mediated by the complement system in chronic pain and depression comorbidity. Complement cascade C1q/C3-CR3 pathway. The amygdala and hippocampus exhibit heightened activation of complement C1q and C3 in response to stimuli associated with chronic pain and depression. This activation facilitates the differentiation of microglial cells into a pro-inflammatory M1 phenotype. Complement C3 undergoes lysis, leading to the generation of C3b and iC3b. This iC3b acts as a marker for synapses, facilitating their identification by the CR3 receptor on pro-inflammatory M1 microglial cells, potentially resulting in synaptic pruning. Notably, there is a reduction in the levels of synaptic proteins SYP and PSD95, as well as a decrease in the secretion of the VGlut2. The BonT/A can counteract these changes by inhibiting the activation of C1q and C3, thereby reducing synaptic pruning in microglia. Complement cascade C3/C3a-C3aR signals pathway. In the presence of LPS or chronic stress, neurotoxic A1 astrocytes in the prefrontal cortex become activated, resulting in an increase in complement C3 and C3a levels. Specifically, C3a targets the C3aR receptor on microglial cells, promoting their polarization towards an M1 phenotype. This activation further stimulates the STAT3 and NF-κB pathways in microglial cells. This amplifies synaptic pruning in microglia and hUC-MSCs, IL-1R, and Gynostemma inhibit and improve these processes. PSD95: postsynaptic density protein 95, VGlut2: vesicular glutamate transporter-2, BoNT/A: botulinum neurotoxin A, LPS: lipopolysaccharide, STAT3: signal transducer and activator of transcription 3, NF-κB: nuclear factor-kappa B, hUC-MSCs: human umbilical cord mesenchymal stem cells.

4. The complement system mediates glial neuroinflammation and plays a role in the chronic pain and depression comorbidity

1) Relationship between neuroinflammation and chronic pain and depression

Neuroinflammation refers to the immune response occurring in the CNS that is initiated by microglia and astrocytes. This response is characterized by the activation of resident glial cells, including microglia and astrocytes, the release of cytokines and chemokines, and the activation and infiltration of leukocytes [58]. Several studies have suggested that the cerebral cortex and its subdivisions, such as the PFC, anterior cingulate cortex, amygdala, hippocampus, and median raphe nucleus, play a crucial role in the co-occurrence of pain and depression. The coexistence of chronic pain and depression is associated with central inflammation, specifically involving TNF-α, L-1β, and IL-6, as well as variations in peripheral cortisol levels [59]. Additionally, persistent neuroinflammation plays a vital role in the initiation and perpetuation of concurrent chronic pain and depression [60]. Consequently, targeted interventions aimed at reducing neuroinflammation have the potential to enhance the management of comorbid depression and chronic pain [61].

2) Complement system-mediated glial cell induced neuroinflammation

In the CNS, the activation of complement plays a crucial role in inducing inflammatory responses within immune effector cells, thereby protecting neurons from potential hazards and toxins [62]. The classical complement pathway, which encompasses essential components such as C1q, as well as the resultant activation byproducts C3a and C5a and their corresponding receptors, serves as the foundation for the complement-driven inflammatory and noninflammatory processes within the brain [63]. Additionally, the activation of glial cells serves as a significant mediator of complement-induced inflammation in the CNS. In response to inflammatory challenges, neurons within the CNS produce various components, including C1r, C1s, C4, C2, and C3, which contribute to the formation of the C1 complex (consisting of C1q, C1r2, and C1s2). Subsequently, C3 is cleaved into C3b/iC3b, which activates microglial cells through the CR3 receptor, leading to synaptic alterations. Following C3 cleavage, C5a is generated along with C3b and C3a, which further promote inflammation-associated microglial and astrocytic cells to increase neurotoxicity, ultimately resulting in the impairment of neuronal function and eventual neuronal death [64]. The findings of this study indicate that in the LPS-induced mouse model of neuroinflammation, there is an upregulation of complement C1q levels in the mouse hippocampus. This upregulation is accompanied by an increase in synaptic phagocytosis by microglial cells, resulting in a higher loss of synapses and subsequent cognitive impairment in the mice. However, it was observed that neutralizing C1q signaling can prevent these changes, suggesting that activated microglial cells and the complement cascade C1q signaling may play a role in the synaptic loss and cognitive impairment observed in the LPS-induced neuroinflammation mouse model [65]. In another study involving neurons and glial cells stimulated by LPS, the levels of C3 were also significantly increased, which can enhance LPS-induced neuroinflammation and neurodegeneration through the Mac1/NOX2 pathway [66]. To conclude, the activation of glial cells via the complement cascade is crucial in the pathogenesis of central neuroinflammation.

The interaction between C1q and microglia, as a key activator of the classical complement pathway, has been demonstrated to effectively suppress neuroinflammation by inhibiting the secretion of pro-inflammatory cytokines [67]. This inhibitory effect of C1q on neuroinflammation is also implicated in the development of depression and chronic pain. In a study conducted by Madeshiya et al. [68], a mouse model of depression was established using the learned helplessness paradigm. Their findings revealed that the knockout of C1q intensified the learned helplessness behaviors induced by electric foot shocks in mice. Furthermore, there was a significant increase in the mRNA levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the mouse PFC, along with an increase in M1-polarized microglia, suggesting that the absence of C1q worsened depressive-like behaviors in mice and is associated with induced neuroinflammation and M1 polarization of microglia. Moreover, studies that employed in vivo, nucleus-specific calcium signaling perturbation strategies, supplemented with gene mapping, bioinformatics, and functional analyses, identified that C1q promotes activity-dependent spinal remodeling and persistent pain hypersensitization in a manner related to inflammatory hypersensitivity reactions [69].

The cleavage products of the complement system, namely C3a and C5a, have been observed in various systems as mediators that elicit neuroinflammation. By means of their seven-transmembrane coupled receptors, C3aR and C5aR as well as C3a and C5a have the ability to stimulate glial cells to generate pro-inflammatory cytokines and participate in a range of diseases [70]. In general, microglial cells express C3aR and C5aR on their surface. Activation of these receptors can augment the chemotaxis of both immune and glial cells, leading to the production and release of inflammatory factors in a Ca2+-dependent manner [71]. The binding of C5a to C5aR has the potential to enhance the secretion of pro-inflammatory cytokines and intensify the activation of pro-inflammatory microglial cells via the p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways [72].

3) C3-C3aR axis and chronic pain and depression comorbidity

The involvement of the C3a-C3aR and C5a-C5aR axis in chronic pain and depression can be attributed to their ability to induce central neuroinflammation. Prior research has demonstrated that C3a possesses the ability to enhance the calcium current in dorsal root spinal neurons and the intracellular calcium flow induced by capsaicin. Consequently, this mechanism serves to activate and sensitize pain receptors during the pain perception process [23]. Furthermore, investigations have indicated that the neuropeptide TLQP-21 Trifluoroacetate can elicit hyperalgesia and NP by activating the C3aR1 receptors located on the microglial cells of the dorsal spinal cord [73]. The findings from research conducted on the CUMS model demonstrate that the augmentation of the C3a-C3aR signaling pathway has the potential to induce M1 polarization of microglial cells, leading to heightened levels of pro-inflammatory cytokines, such as TNF-α and IL-1β, within the hippocampus. Consequently, this can result in the manifestation of anxiety and depressive-like behaviors in mice [53]. Additionally, another investigation proposes that within the same CUMS model, the inhibition of C3aR effectively diminishes the expression of NOD-like receptor protein 3 inflammasomes and the subsequent inflammatory response in the hippocampus. As a result, this intervention exhibits an improvement in depressive-like behaviors observed in mice [74]. Crider et al. [20] propose that in mice with a chronic stress model, monocytes expressing C3a-C3aR migrate into the PFC, leading to increased levels of the inflammatory cytokine IL-1β in the PFC. However, when C3aR is knocked out, the recruitment of PFC monocytes is significantly reduced, resulting in decreased IL-1β levels in the PFC and improved depressive-like behaviors. These findings suggest that C3a-C3aR may play a role in the development of chronic pain and depression by promoting neuroinflammation.

4) C5-C5aR axis and chronic pain and depression comorbidity

The activation of C5a-C5aR has been extensively investigated in the context of chronic pain. C5a, when bound to C5aR, exhibits potent inflammatory properties by inducing the production of chemotactic factors and pro-inflammatory effects. This subsequently leads to the upregulation of inflammatory cytokines such as IL-6, TNF-α, and PGE-2, while simultaneously reducing the secretion of anti-inflammatory factors, resulting in the manifestation of hyperalgesia [75]. Moreover, C5a has the ability to mediate NP at sites of nerve injury, within dorsal root ganglia (DRGs), and in the spinal cord. Specifically, at injury sites, C5a recruits macrophages and T cells through C5aR activation, thereby contributing to the development of neural pain. Additionally, C5a in DRGs may directly sensitize primary sensory neurons. In the spinal cord, C5aR is primarily expressed in neural glial cells, and these neural glial cells can be stimulated by C5a, leading to their activation [26]. Recent clinical studies have indicated a significant correlation between levels of C5a and C5 in both plasma and cerebrospinal fluid, and the manifestation of depression [11,76,77]. Consequently, it can be postulated that an elevation in plasma complement C5a concentration may serve as an indicator of a persistent inflammatory process, potentially resulting in compromised integrity of the blood-brain barrier (BBB). Subsequently, the infiltration of the peripheral complement system into the CNS may occur, thereby triggering the activation of central immune-inflammatory agents and exacerbating symptoms associated with depression [78]. Therefore, the complement C5a in plasma may penetrate brain tissues a few hours after BBB leakage, inducing neuroinflammation, which affects the CNS [79]. To conclude, the complement pathways C3a-C3aR and C5a-C5aR may participate in the comorbidity mechanisms of chronic pain and depression through inducing central neuroinflammation (Fig. 3 and Table 2 for summary).

Table 2 The possible mechanisms of chronic pain and depression comorbidity mediated by complement cascade C3a-C3aR and C5a-C5aR

Complement cascade	Molecular mechanisms	Method/experimental models	Type of disease
C5/C5aR	Peripheral complement component C5 and C5aR is upregulated in spinal microglia after peripheral nerve injury [23]	SNI, CCI and SNL model [23]	Neuropathic pain
C5a-C5aR	The levels of C5a and C5 in plasma and cerebrospinal fluid are significantly increased in patients with major depression [11,77,78]		Depression
C3a-C3aR	C3a can enhance calcium current and capsaicin-induced calcium inflow in dorsal root neurons of spinal cord [23]. TLQP-21 activates the C3aR1 receptor on the dorsal horn microglia of the spinal cord, which induced heat hyperalgesia and contributed to nerve injury-induced [73]	SNI, CCI and SNL model [23] SNI model [73]	Chronic pain and depression
C3a-C3aR	C3a-C3aR signaling pathway promotes M1 polarization of microglia and increases levels of hippocampal pro-inflammatory cytokines TNF-α and IL-1β [53]. C3aR blockade decreased the expression and inflammatory response of NLRP3 inflammasome in the hippocampus [74] C3a-C3aR monocytes infiltrated the prefrontal cortex, and the levels of inflammatory cytokine IL-1β in the PFC increased [20]	CUMS mice [20,53,74]	Chronic pain and depression
C5a-C5aR	C5a can induce upregulation of pro-inflammatory factors such as IL-6, TNF-α depending on C5aR [75]. C5a mediates neuropathic pain by recruiting macrophages and T cells through C5aR. C5a in DRGs may cause direct sensitization of primary sensory neurons [26]	Transgenic MAFIA mice drug-inducible macrophage depletion [76]	Chronic pain and depression

SNI: spared nerve injury, CCI: chronic constriction injury, SNL: spinal nerve ligation, CUMS: chronic unpredictable mild stress, TLQP-21: TLQP-21 Trifluoroacetate, TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1, NLRP3: NOD-like receptor protein 3, PFC: prefrontal cortex, DRG: dorsal root ganglia, MAFIA: macrophage Fas-induced apoptosis.

Figure 3. Potential mechanisms in which neuroinflammation is mediated by the complement system is involved in the comorbidity of chronic pain and depression. Complement cascade C3a-C3aR pathway. Under the influence of injury and inflammation, astrocytes have the ability to enhance the production of complement C3a. This molecule then interacts with the receptor C3aR found on microglia, resulting in the promotion of M1 type polarization of microglia in specific regions of the central nervous system, including the dorsal horn of the spinal cord, dorsal root neurons in the spinal cord, hippocampus, and prefrontal cortex. Consequently, this process leads to an upregulation of the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and NLRP3 in the hippocampus, as well as an elevation in the levels of the inflammatory cytokine IL-1β in the prefrontal cortex. These events ultimately induce neuroinflammation, which is closely associated with the development of chronic pain and depression. Complement cascade C5a-C5aR pathway. Response to injury and inflammation, astrocytes have the capacity to elevate the expression of complement C5a. The interaction between C5a and its receptor (C5aR) can subsequently enhance the secretion of pro-inflammatory factors via the activation of p38MAPK and ERK1/2 signaling pathways. This process further facilitates the M1-type polarization of microglia in the spinal dorsal horn and dorsal root ganglion (DRG), thereby inducing neuroinflammation. Ultimately, these events contribute to the development of chronic pain and depression. TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1, NLRP3: NOD-like receptor protein 3.

Based on the complement system, this article reviews the involvement of different complement pathways in glial cell synapse elimination and neuroinflammation in thecomorbidmechanisms of chronic pain and depression,and explores the idea of the complement system as a new potential target.The complement cascades C1q/C3-CR3 and C3/C3a-C3aR signals, as well as the C3a-C3aR and C5a-C5aR pathways, may be potential common therapeutic targets in the mechanisms underlying thecomorbiditiesof chronic pain and depression. However, there exist certain limitations to this review. An overview of prior research indicates that the complement cascade pathways exhibit a stronger correlation with depression than chronic pain, and the current body of clinical and preclinical studies pertaining to these complement signaling pathways in relation to pain remains insufficient. Furthermore, more experiments are needed to validate the role of complement cascade-mediated synaptic pruning in glial cells and the resultingneuroinflammation in the comorbidity mechanisms of chronic pain and depression, ensuring their consistent presence during the disease progression.

Complement cascade-related targets are also expected to have shared therapeutic effects in both depression and chronic pain. Drugs targeting the blockade of the complement cascade C5a/C5aR signaling are under development, such as the monoclonal antibody TNX-558, which can interfere with the interaction between C5a and C5aR. This reduces inflammatory responses without decreasing the activation of the complement system, ensuring immune responses against pathogens [80]. In various rodent models of inflammatory and painful conditions, the non-reversible C5aR antagonist PMX-53 demonstrated notable therapeutic outcomes [81]. Although the short half-life and bioavailability of PMX-53 have hindered its clinical application [82]. The efforts to improve the pharmacokinetic properties of PMX-53 are still going on [83]. In vitro studies revealed that the selective non-competitive inhibitor DF2593A, targeting the C5a receptor, can treat NP and suppress the migration of neutrophils induced by C5a in both human and rodent models [84]. Given the close association of the C5a/C5aR signaling with chronic pain and depression, the prospects of targeted drug treatments for chronic pain and depression are also promising. Additionally, clinical studies have shown that C1q and translocator protein (TSPO) are elevated in depression, TSPO ligand drugs can exert anti-anxiety and anti-depressive effects by promoting endogenous neurosteroid synthesis [85]. Recent studies have shown that TSPO ligands can cause neuronal atrophy and loss by marking C1q, and treatment with TSPO ligands can reduce the levels of C1q in microglial cells [86]. Hence, TSPO ligands may work synergistically with C1q to exhibit anti-inflammatory properties, potentially paving new avenues for the treatment of depression [87].

The complement system is a newer area for the study of comorbidity mechanisms between chronic pain and depression. While many preclinical and clinical studies regarding chronic pain and depression models have suggested changes in complement component expression levels, and suppression of complement signaling can mitigate model pain and depression-like behaviors and change respective molecular biological phenotypes, the role of the complement system in the co-morbidity mechanisms of chronic pain and depression still demands more substantiation. From the perspective of the involvement of the complement system in synaptic modification and neuroinflammation, this review provides the basis and direction for how to better treat the comorbidity of chronic pain and depression by modifying the molecular mechanism of the complement system in the future.

Data sharing is not applicable to this article as no datasets were generated or analyzed for this paper.

No potential conflict of interest relevant to this article was reported.

This study was supported by the National Natural Science Foundation of China (81960263, 82260231), and the Famous Clinical Doctor Program ([2021]002) of the Zunyi Medical University.

Shanshan Tang: Writing/manuscript preparation; Wen Hu: Writing/manuscript preparation; Helin Zou: Writing/manuscript preparation; Qingyang Luo: Writing/manuscript preparation; Wenwen Deng: Study conception; Song Cao: Study conception.

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