• Significant Group Differences: Adolescents with first-episode depression and comorbid nonsuicidal self-injury (NSSI) showed distinct demographic, psychological, and biological profiles compared to both depressed patients without NSSI and healthy controls. Key differences included sex distribution, anxiety/depression severity, and levels of folate, complement 3 (C3), and interleukins.

• Identified Correlates of NSSI: Female sex and higher levels of complement C3 were positively associated with the presence of NSSI in depressed adolescents. Conversely, older age and higher folate levels were inversely associated with NSSI.

• Folate as a Potential Protective Factor: Higher folate levels were identified as a protective correlate against NSSI, specifically in the depressed group without self-injury, suggesting a potential role for folate in mitigating self-harming behaviors in adolescent depression.

• Implication of Immune and Inflammatory Pathways: The study highlights the potential involvement of immune system components (specifically C3) and inflammatory factors in the pathophysiology of NSSI comorbid with depression, pointing to possible biological mechanisms underlying this behavior.

After the coronavirus disease 2019 pandemic, the prevalence of depression among children aged 9–15 years has soared from 11.8% to 34.4% [1]. Adolescents in this age group are going through a crucial stage of self-awareness integration and identity formation, and their ability to regulate their emotions is relatively weak. Owing to the lack of emotional stability and inadequate regulation ability, individuals with depression are more prone to atypical symptoms, such as destructive emotions and irritability, and may even resort to extreme behaviors, such as nonsuicidal self-injury (NSSI) or suicide [2]. Individuals with adverse childhood events exhibit greater psychological distress, lower emotional well-being, anhedonia and depression, as well as higher rates of lifetime suicidal ideation and suicide attempts. Studies indicate a close association between adverse childhood events and NSSI in adolescents, with notably increased risks of NSSI across all childhood trauma subtypes [3]. NSSI refers to the intentional and self-initiated act of harming one’s own body tissues without suicidal intent. Currently, the global prevalence of NSSI is on the rise, with an incidence rate ranging from 11.5% to 33.8% [4]. In China, NSSI has been reported in approximately 25% of adolescents [5], seriously hindering their personality development and academic performance. Emotional problems and NSSI are significantly comorbid in adolescents. Research indicates that both depressive disorders and NSSI are key predictors of suicidal behavior in adolescents [6]. Multiple surveys have verified that more than 50% of Chinese adolescents with depression engage in NSSI [7]. When facing adverse childhood events, academic pressure, or peer relationship problems, adolescents with depression often resort to NSSI to regulate their emotions and cognition. Notably, the presence of comorbid NSSI was shown to significantly increase suicide risk among adolescents with depression. Without timely intervention, NSSI may escalate into suicidal behavior, causing serious harm to the physical and mental health of adolescents with depression [8]. Although the aforementioned background highlights the close relationship between depression and NSSI in adolescents and underscores the urgency of further research, the underlying pathological mechanisms of their comorbidity remain poorly understood.

The pathogenesis of depression is closely related to the abnormal upregulation or activation of inflammatory factors, such as cytokines, and the complement system [9, 10, 11]. The immune-inflammatory pathway not only provides a physiological basis for the development of depressive behavior but also reveals the intrinsic connection between inflammatory responses and depressive symptoms [12]. As important cell signaling mediators, cytokines regulate the immune response to internal and external stimuli. This functional class of small proteins includes interleukins (ILs), interferons, chemokines, lymphokines, and tumor necrosis factors [9]. Clinical observations have revealed that IL-6 and white blood cell levels are significantly positively correlated with the severity of depressive symptoms [13, 14]. The underlying mechanism may involve inflammatory responses impairing the function of the frontal lobe, thereby increasing impulsive behavior and suicide risk [15]. Elevated levels of IL-6 are closely associated with somatic symptoms and cognitive dysfunction [16], whereas mice with decreased IL-4 levels exhibited more pronounced depressive-like behaviors in stress tasks [17]. The synergistic effect of anti-inflammatory drugs in the treatment of depression suggests that inflammatory markers may be developed into novel biological targets for assessing the presence of NSSI [18].

As a core component of the innate immune system, the complement system not only mediates proinflammatory signaling but also participates in regulating the development of the central nervous system [19]. Multiple clinical studies have confirmed that the serum levels of complement 3 (C3) and C4 are significantly elevated in patients with depression [15], and C3 expression in the prefrontal cortex is particularly prominent among patients with suicidal tendencies [20]. Conversely, another study showed that the serum level of C3 in patients with first-episode depression was lower than that in patients with recurrent depression and healthy controls, while the serum levels of C4 did not differ significantly among the three patient groups [10]. Given the close connection between the complement system and the inflammatory response, researchers have further explored the role of inflammatory factors in NSSI, but the existing conclusions are also controversial.

In studies on the integration of NSSI, Korean scholars found that the behavioral impulsivity of patients engaging in NSSI was positively correlated with the level of inflammation [21]. Another study demonstrated that the levels of tumor necrosis factor, IL-1, and IL-6 in adolescents engaging in NSSI significantly decreased after treatment, providing strong evidence for the role of inflammatory factors in NSSI [22]. Contrarily, a controlled study in Germany reported no significant differences in the concentrations of IL-6 and other inflammatory factors between patients engaging in NSSI and healthy individuals [23]. Few studies have examined the inflammatory characteristics of Chinese adolescents with depression and NSSI, as well as the underlying pathological mechanism. Therefore, research on the biological basis of depression and its related behaviors (such as NSSI) must take into account the complexity of multi-system interactions, among which metabolic and immune disorders are two important aspects in addition to inflammation.

Neurobiological research indicates that the active form of folate, 5-methyltetrahydrofolate, influences the synthesis of neurotransmitters (dopamine and serotonin) by regulating one-carbon metabolism, with S-adenosylmethionine serving as a key methyl donor. This suggests that folate metabolism disorders may be implicated in the pathophysiology of depression [24]. Multiple controlled studies have confirmed that serum folate levels are significantly lower in patients with depression than in healthy individuals, establishing folate deficiency as an independent risk factor for depression [25]. However, the antidepressant efficacy of folate supplements remains controversial, and their clinical translational value warrants more rigorous causal evidence [26]. Additionally, patients with depression exhibit highly heterogeneous changes in humoral immunity, with multiple studies reporting inconsistent changes in immunoglobulin (Ig) levels (elevated, decreased, or unchanged) [27, 28]. This heterogeneity is a reflection of the complex underlying mechanisms involving multifactorial interactions among psychological, neural, immune, and environmental factors.

In summary, despite evidence substantiating the association of depressive disorders with abnormal folate metabolism, the complement system, Ig levels, and inflammatory factors, the pathological mechanism underlying the comorbidity of depressive disorders with NSSI remains unclear. This study adopts a case-control design to systematically analyze dynamic changes in the levels of immune-neuroregulatory substances in adolescent patients with depression with or without NSSI. The results of this study will shed light on the association between biomarker profiles and NSSI, inform the construction of a risk warning model, and facilitate the early identification of personalized interventions for suicide prevention in adolescents with NSSI comorbid with depression.

This study analyzed the incidence of depressive disorder with comorbid NSSI by sex and age. The results showed that female sex was an independent risk factor for depressive disorder with comorbid NSSI. This finding is consistent with the large-sample study by Sun et al. [31], which reported a 3.4-fold higher incidence of NSSI among female adolescents than male adolescents. The possible mechanism underlying this sex disparity may be that women are more likely to adopt externalized coping strategies for negative emotions. Their prominent emotional expression characteristics and higher pain tolerance thresholds may collectively promote NSSI to become a preferred maladaptive strategy for emotion regulation [32]. Multiple logistic regression analysis identified age as a protective factor for NSSI. This is highly consistent with the age-wise trajectory of NSSI development: the behavior often begins at the age of 12–14 years, peaks at 15–17 years, and gradually subsides in early adulthood [33]. The neurodevelopmental perspective provides a plausible explanation for this phenomenon: the asynchronous development of the prefrontal-limbic system during adolescence leads to an imbalance in emotional regulation, manifested by insufficient top-down control from the orbitofrontal cortex and hyperresponsiveness of the amygdala [34]. This neuroplastic window period may form the biological basis for the high incidence of NSSI in adolescents, while the subsequent maturation of these neural circuits may contribute to the observed decline in NSSI behaviors in adulthood.

The study also uncovered significant differences in SAS/SDS scores among the three groups (NSSI group $>$ non-NSSI group $>$ control group), and univariate logistic regression confirmed that the SAS/SDS score was an independent risk factor for depressive disorder with comorbid NSSI. This finding is in line with the longitudinal study by Zhu et al. [35], which showed that the persistence of depressive and anxiety symptoms significantly increases the risk of NSSI in adolescents, while symptom relief can effectively reduce the incidence of self-harm behavior. Kim and Yu [36] reported that individuals with high anxiety achieve immediate emotional regulation by engaging in NSSI, which is aligned with the negative emotion accumulation–release model proposed by Baker et al. [37]. When the emotional load in the form of depression and anxiety exceeds an individual’s tolerance threshold, NSSI may become the preferred strategy to relieve psychological distress [37].

In terms of biological indicators, levels of IL-6, IL-4, and C3 were identified as significant positive correlates of the NSSI group. However, after adjustment in a multivariate model, only the C3 level remained a statistically significant indicator. Moreover, the levels of C3 and IL-6 significantly differed across the three groups (NSSI group $>$ non-NSSI group $>$ control group). After adjustment in the multivariate model, IL-6 and IL-4 levels did not differ significantly among the three groups. This might be due to the fact that the patients’ disease courses and severity were different when the data were collected, and the time of data collection after the occurrence of emotional events was also different. Secondly, it might be due to the insufficient sample size. The sample size of this study was adequate for univariate analysis, but when multiple variables such as IL-4 and IL-6 were simultaneously included, the model complexity increased, and the statistical power decreased. Beyond analyzing the potential causes for the lack of significant differences in the levels of IL-6 and IL-4 in the model, conducting an in-depth exploration of the theoretical pathophysiological mechanisms of these inflammatory factors will contribute to a more comprehensive understanding of the possible roles they play in NSSI.

Studies have shown that elevated levels of inflammatory cytokines can exacerbate depressive symptoms and increase the risk of NSSI through two possible mechanisms [38]. First, inflammation activation can alter the metabolism of key neurotransmitters in the brain, affecting frontal lobe function and diminishing the ability to inhibit responses, thereby leading to an increase in impulsive behavior and NSSI [21]. Second, the activation of inflammatory cytokines can promote the release of endogenous opioid peptides [39], reducing pain sensitivity. Notably, IL-4 can induce macrophages to release endogenous opioid peptides [40], and this mechanism may underpin the formation and development of NSSI [41]. A study by Li et al. [14] discovered that the peripheral IL-6 levels in their 185 study participants increased during the stress period and decreased as the stress period ended, closely mirroring the individual differences in depression susceptibility. Another study compared adolescents who had experienced at least five NSSI events over the past year with a healthy control group, reporting no differences in the levels of IL-6 or C-reactive protein [23]. In future research designs, the exact time of each emotional event (or the time when the researcher induces emotional stimuli) and the subsequent time of blood sample collection should be clearly recorded to more clearly reveal the independent association between emotional events and cytokine levels.

Abnormalities in the complement pathway have been detected in many neurological and neuropsychiatric disorders [42, 43]. In the pathophysiology of these diseases, the complement pathway is inappropriately reactivated, leading to synaptic defects, disruption of neuronal connections, and cognitive impairment [44]. The C3 protein is considered a hub. Research has found that oligodendrocytes can express C3 under stress conditions, thereby participating in the neuroinflammatory cascade reaction and inducing depression [45]. Mechanism studies have shown that in mouse models, synaptic pruning by microglia can be reduced by blocking the HMGB1 (high mobility group box 1)/C1q (complement component 1q)/C3 pathway, thereby improving depressive-like behaviors [46]. Another study found that by knocking down C3 in the hippocampus, the downstream C3aR (complement C3a receptor 1)-GSK3$\beta$ (glycogen synthase kinase 3 beta) pathway can be inhibited, restoring the activation level of glial cells under inflammatory conditions and alleviating anxiety and depression [47]. In terms of clinical research findings, the levels of C3 and C3a in the plasma of patients with depression are elevated, with higher levels in female patients than in male patients [48]. A study has reported significantly elevated C3 levels in the cerebral cortices of mice exposed to chronic unpredictable stress and mice exhibiting depression-like or suicide-related behaviors [49]. In our study, the serum level of C3 in the NSSI group was elevated, consistent with some previous studies [15, 50]. These results suggest that complement C3 may be a specific biological marker for depression with comorbid NSSI.

This study sheds light on the association between folate levels and depressive disorders. Folate levels exhibited a gradient decline in patients with depressive disorders (non-NSSI group control group). Further, multivariate regression analysis confirmed folate level as an independent protective factor for depressive disorders. However, the direct association between folate levels and NSSI was weak, with no significant difference between the NSSI and non-NSSI groups. Folic acid may regulate inflammatory responses through DNA methylation and synthesis processes. Insufficient folate supply may be related to the progression of inflammation, or chronic inflammation may lead to increased folate demand. Supplementation of folate may have anti-inflammatory benefits by reducing CRP (C-reactive protein) levels [24, 51]. Previous studies have shown that folate deficiency can lead to depressive symptoms [24, 52, 53] and supplementation of active folate can synergistically enhance the efficacy of antidepressants [54, 55]. This further indicates the complex potential mechanism of multi-factor interaction in the pathophysiology of depression. However, further research on larger and more diverse populations is needed to verify and expand these findings. Moreover, a recent study conducted in South Korea found a significant association between folate levels and fatal and non-fatal suicide attempts during follow-up, suggesting that folate levels can serve as an auxiliary means for predicting suicidal behavior in patients with depression [56]. Overall, folate status assessment is suggested to be incorporated into clinical practice for treating depression and NSSI. This study found no significant differences in Ig levels among the three groups, implying that humoral immune dysfunction is not the core pathophysiological mechanism of NSSI. However, the potential effects of limited sample size on statistical power must be considered before drawing any strong conclusions.

This comparative study revealed significant differences in clinical profiles and serum biomarkers among depressive adolescents with NSSI, without NSSI, and healthy controls. Female sex and elevated complement C3 levels were identified as significant positive correlates of NSSI comorbidity. Conversely, older age and higher serum folate levels were significant negative correlates of NSSI. A higher folate level was also a protective correlate in depressed adolescents without NSSI. Given the increasing incidence and substantial public health burden of adolescent depression and NSSI, and the current limitations in prevention and treatment, our findings underscore the potential role of immune-inflammatory pathways and metabolic factors in the pathophysiology of NSSI. To advance this field, future research should prioritize multicenter longitudinal studies to elucidate the spatiotemporal dynamics of these biomarkers. The ultimate goal is to develop artificial intelligence-powered, multi-omics prediction models, facilitating a shift in the diagnostic and therapeutic approach for depression with NSSI from a “symptom-driven” paradigm to a more precise “mechanism-targeted” one.

This study has some limitations. First, the cross-sectional design of this study precludes observations of the trends in various factors at different stages of the disease. Second, the sample size was relatively small, preventing the stratification of depression and anxiety by severity and duration. Third, the recruitment of participants from a single center and the local community may introduce selection bias and limit the generalizability of our findings to other regions or healthcare settings. Future studies should recruit a larger and more diverse sample from multiple centers to enhance representativeness and investigate more related factors, especially objective evaluation indicators such as magnetic resonance imaging scans, biochemical tests, and genetic assessments, to further elucidate the mechanisms underlying NSSI behaviors. Such research will eventually result in the formulation of more effective prevention and intervention measures.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

HYX: Data curation, Investigation, Project administration, Resources, Writing-original draft, Writing-review & editing. SJZ: Data curation, Investigation, Methodology, Validation, Supervision, Writing-review & editing. XDD: Validation, Supervision, Writing-review & editing, Reorganize and recalculate the data. JHL: Conceptualization, Formal Analysis, Methodology, Software, Supervision, Writing-review & editing. PF: Data curation, Investigation, Writing-review & editing. YYW: Data curation, Investigation, Writing-review & editing. YZ: Data curation, Investigation, Writing-original draft. YJM: Data curation, Investigation, Writing-review & editing. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Informed consent was obtained from all subjects involved in the study. This study was approved by the Ethics Committee of the Fourth Affiliated Hospital of Zhejiang University School of Medicine (Approval Number: k2022003). The study was conducted according to the Declaration of Helsinki.

We would like to express our deep appreciation to all non-author contributors who supported this study.

This study was supported by The Basic Public Welfare Research Program of Zhejiang Province (Grant No. LGF20H090013).

The authors declare no conflict of interest.