Correlation Analysis between Serum NLRP1 Inflammasome and Depression in Acute Stage of Stroke Open Access

Stroke is an acute cerebrovascular disease that can be classified into two types: hemorrhagic and ischemic. Ischemic stroke, caused by arterial blockage, accounts for the majority of stroke cases (87%), while hemorrhagic stroke, resulting from bleeding, constitutes 13% of stroke types [1, 2]. As a common neurological disease, it often leads to severe neurological sequelae and other complex dysfunction, including post-stroke cognitive impairment and dementia, pain, anxiety, depression, fatigue, and epilepsy [3]. The presence of emotional disorders, particularly depression-related symptoms, during the acute phase of a stroke, has been observed to significantly influence the immediate efficacy of medical interventions. Furthermore, these emotional disturbances are intricately linked to the risk of developing post-stroke depression (PSD) in the subsequent phases of recovery. Patients with acute depression state of stroke mainly show low mood, and some patients may also have self-harm, suicide and other behaviors [4]. Previous studies have shown that depression in the acute stage of stroke not only affects the rehabilitation process of acute stroke patients, but also may endanger the life safety of the patients [5]. Patients with PSD in the acute phase have poor functional recovery after discharge and a high risk of long-term depression [5]. The psychopathological mechanism of acute stroke depression is still unclear. In recent years, immunoinflammation and many cytokines have been considered to be related to the mechanism of PSD. Research has found that the central and peripheral immune-inflammatory responses are immediately activated following acute stroke [6]. Early inflammatory markers in patients, such as high-sensitivity C-reactive protein, ferritin, D- (+)-Neopterin, and glutamate, are elevated, along with pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, IL-6, interferon [IFN]-γ). The pro-inflammatory/anti-inflammatory ratios (TNFα/IL-10, IL-6/IL-10) are also increased, while complement expression is reduced [7]. As research on the role of inflammation in depression has deepened, a multi-protein complex called the inflammasome has attracted great attention for its role in inflammatory modulators [8]. Inflammasome activity is closely associated with many human diseases, including rare genetic syndromes such as autoinflammation, cardiovascular disease, neurodegenerative diseases, and cancer [9]. The NLRP1 inflammasome functions as a polyprotein complex, comprising NLRP1, the adaptor protein apoptosis-associated speckle-like protein containing a CARD domain (ASC), and the effector protein pro-caspase-1. Notably, research indicates that the activation of the NLRP1 inflammasome occurs in brain neurons following instances of brain injury [10]. It has also been reported that the NLRP1 inflammasome is significantly activated in the hippocampus of rats with neuropathic pain induced by chronic systolic injury (CCI). Inhibiting the products of the NLRP1 inflammasome not only alleviates depressive-like behavior in CCI rats but also suppresses the production of mature IL-1β [11]. The inflammasome serves as a critical regulator of the immune inflammatory response, with its activation resulting in significant alterations in inflammatory factor levels. This process can mediate neuroinflammatory cascades, ultimately contributing to neuronal death. Such neuronal loss has profound implications for mental health, as it is increasingly recognized as a key factor in the onset and progression of depression [12]. The present study explored the potential relationship between serum NLRP1 inflammasome components (NLRP1, ASC, Caspase-1), serum inflammatory factors such as IL-1β, IL-18, IL-10, TNF-α, as well as other related factors, and acute phase depression following stroke.

Inclusion criteria (1): first stroke onset, aged between 30 and 80 years (2); all the enrolled patients met the Chinese diagnostic criteria for acute stroke, and the diagnosis was verified by head CT or MRI imaging examination within 7 days of onset (3); able to cooperate in completing the assessments of the National Institute of Health Stroke Scale (NIHSS), Hamilton Depression Scale-24 (HAMD-24), Pittsburgh Sleep Quality Index (PSQI), and the Minimum Mental State Examination (MMSE).

Exclusion criteria (1): patients with severe neurological dysfunction, language impairment, or those with consciousness and cognitive disorders who are unable to cooperate in completing the assessment (2); patients with a history of mental disorders (such as schizophrenia or depression) prior to onset (3); patients with a history of other neurological diseases, such as epilepsy, Parkinson’s disease, dementia, and brain tumors (4); patients with severe liver and kidney function impairment, infectious diseases, malignant tumors, or autoimmune diseases.

General data: A total of 102 patients with acute stroke who were hospitalized for the first time in the Department of Neurology of the First Affiliated Hospital of Dali University from April 2023 to October 2023 were selected. Among them, 8 patients had language disorder, 2 had a history of mental illness, 5 had cognitive impairment, 1 had a history of malignant tumor, 6 refused to participate in cooperation, and 80 met the inclusion criteria. Within 1 week of hospitalization, the Hamilton Depression Scale-24 (HAMD-24) [13] was utilized for psychological assessment and the patients were divided into 31 patients in the acute stage of stroke depression group (HAMD score ≥8)and 49 patients in the acute stage of stroke non-depression group (HAMD score <8). All participants signed written informed consent, and the study protocol was approved by the Ethics Committee of the Clinical School of Medicine of Dali University Hospital in accordance with the Declaration of Helsinki.

Collect clinical data on enrolled patients, including name, gender, age, BMI, smoking history, alcohol consumption history, history of hypertension, diabetes, and coronary heart disease.

The evaluation of neurological deficits, sleep quality, and the severity of cognitive function was conducted by the same trained neuropsychiatrist [1]. The enrolled patients were evaluated by the National Institute of Health Stroke Scale (National Institute of Health) after admission of Health Stroke Scale (NIHSS) [13] score was used to evaluate the neurological impairment of patients. The total score was 0–42 points, ≤15 indicates mild neurological dysfunction, a score of 16–20 indicates moderate neurological dysfunction, and a score of >20 indicates severe neurological dysfunction. The higher the score, the greater the degree of neurological impairment [2]. The Pittsburgh Sleep Quality Index (PSQI) [14] was used to evaluate the sleep of the patients, with a total score of 21 points. The higher the score, the worse the sleep quality. Scores ≤5 were classified as good sleep quality, 6–10 as fair sleep quality, 11–15 as average sleep quality, ≥16 as poor sleep quality [3]. A simple mental state examination (minimum mental state examination, MMSE) [14] was employed to assess the cognitive function of patients, with a maximum score of 30. A score of 27–30 indicates normal cognitive function, while a score of less than 27 suggests cognitive impairment, which excludes the individual from the study criteria.

The Hamilton Depression Scale (HAMD) was administered by the same trained psychiatrist to assess the severity of the patient’s depressive symptoms. The HAMD scale consists of 24 items, with each item scored from 0 to 4, resulting in a total score of 76. The levels of depression are defined as follows: mild depression is indicated by a score of 8–16 (8 ≤ HAMD ≤ 16), moderate depression by a score of 17–23 (17 ≤ HAMD < 24), and severe depression by a score of 24 or higher (HAMD ≥24).

All patients had fasting venous blood collected at 8 a.m. Centrifuge at 3,000 rpm for 15 min, collect the supernatant, and store it at −80°C for further evaluation. The concentrations of serum NLRP1, ASC, Caspase1, TNFα, IL-1β, IL-18, and IL-10 were measured using ELISA kits that include human NACHT, LRR, and PYD domain-containing protein 1 (NLRP1), human apoptosis-associated speck-like protein (ASC), and human caspase-1 (purchased from Huamei Biological Co., CSB-EL015864HU, CSB-EL01911HU, CSB-E13025h), as well as TNFα, IL-1β, IL-18, and IL-10 ELISA kits (purchased from Wuhan Saiweier Biotechnology Co., Ltd, GEH0004-1, GEH0003-1, GEH0002-1, GEH0010-1). The sensitivity of the assays was 18.75 pg/mL for NLRP1, 15.6 pg/mL for ASC, 1.56 ng/mL for Caspase-1, 1.83 pg/mL for TNF-α, 0.46 pg/mL for IL-1β, 8.78 pg/mL for IL-18 and 0.23 pg/mL for IL-10. Mean inter- and intra-assay coefficients of variation were <10%.

Data management and statistical analysis were conducted using SAS 9.4 software (version 9.4 for Windows, SAS Institute, Inc., Cary, NC, USA). Qualitative data were described by n (%), and chi-square test was used for comparison between groups. For normally distributed data, the mean and SD are used for descriptive statistics, and Student’s t test are employed for comparisons between groups. For quantitative data that did not follow normal distribution, the median and interquartile distance M(P25,P75) were used to describe the data, and the rank-sum test was used for inter-group comparison. Fisher exact probability method was used to compare the two groups. Pearson product moment correlation coefficient was used for correlation analysis. Binary logistic regression analysis was used to analyze the risk factors of PSD. Using PSD as the dependent variable (with “yes” as the modeling probability); independent variables include statistically significant from univariate analysis (PSQI, NIHSS score, NLRP1, IL-18, TNF-α). Receiver operating characteristic (ROC) curve was used to analyze the predictive value of related risk factors to PSD. The difference with p ≤ 0.05 was statistically significant.

This study included 80 patients with acute stroke who met the inclusion and exclusion criteria, comprising 66 cases of ischemic stroke and 14 cases of hemorrhagic stroke. Through univariate analysis, no statistically significant differences were found between the two groups in terms of age, BMI, gender, stroke type, and vascular risk factors (p > 0.05). See Table 1 

From Table 2, it can be observed that the Pittsburgh Sleep Quality Index (PSQI) scores in the severe depression group during the acute phase of stroke were higher than those in the mild depression group (p = 0.002). Additionally, the neurological function deficit scores (NIHSS) in the severe depression group during the acute phase of stroke were also higher than those in the mild depression group (p = 0.008). This indicates that PSQI and NIHSS scores are related to the severity of depression during the acute phase of stroke.

Correlation analysis was conducted using Pearson’s correlation coefficient. A correlation heatmap is a common visualization method used to display the correlations between different variables. The deeper the color, the greater the Pearson correlation coefficient, indicating a stronger correlation. According to the correlation heatmap between variables (Fig. 1), it can be observed that the NIHSS score and NLRP1 levels have a strong correlation with the HAMD score, while the PSQI score and the inflammatory factors IL-18 and TNF-α show a certain degree of correlation with the HAMD score.

From Tables 1 and 2, it can be observed that the serum NLRP1 levels in the acute phase of stroke are significantly higher in the depression group compared to the non-depression group (p < 0.001). Furthermore, in the acute phase of stroke, the NLRP1 levels in the moderate to severe depression group are significantly higher than those in the mild depression group (p = 0.001, Table 2). The expression level of NLRP1 was positively correlated with HAMD score (r = 0.476, p < 0.001; see Fig. 2a). R² = 0.226 suggests that the expression level of NLRP1 could explain 22.6% of the change in HAMD score. This means that NLRP1 expression level has a certain degree of explanatory power to HAMD score, but it also suggests that changes in this score are also influenced by other factors. The levels of serum Caspase-1 and ASC showed no significant statistical difference between the two groups (Table 1) and had no apparent correlation with HAMD scores (see Fig. 2b, c).

As can be seen from Table 1, serum IL-18 in the depression group at acute stroke stage was significantly higher than that in the non-depression group at acute stroke stage (p < 0.001). The level of TNF-α was significantly higher in the acute stage depression group than in the non-depression group (p < 0.001). The expression levels of IL-18 and TNF-α were positively correlated with HAMD score (p = 0.017; p = 0.020 see Fig. 2d, e). There was no significant difference in the expression levels of serum IL-1β and IL-10 between the depression group and the non-depression group at the acute stage of stroke (Table 1). There was no significant correlation between IL-1β and IL-10 level and HAMD score (see Fig. 2f, g).

The PSQI, NIHSS scores, and HAMD scores are all positively correlated, indicating that the severity of sleep disorders and neurological impairment is associated with a higher degree of depression. A lower R² value indicates that PSQI and NIHSS scores have some explanatory power for HAMD scores, but also suggests that changes in HAMD scores are influenced by a variety of factors (r = 0.351, r² = 0.123, p = 0.001; r = 0.403, r² = 0.162, p < 0.001, see Fig. 2h, i).

NIHSS score, NLRP1, and IL-18 all had p < 0.05. This indicates that NIHSS score, NLRP1, and IL-18 levels are independent risk factors for PSD (see Table 3).

The analysis of the model’s ROC curve revealed that the area under the ROC curve (AUC) for the model is 0.870, indicating a good predictive value. The AUC for the NIIHSS score is 0.618 (p = 0.036); the AUC for NLRP1 is 0.831 (p < 0.01); and the AUC for IL-18 is 0.737 (p < 0.01). All AUC values are greater than 0.5, suggesting that prediction of depression in the acute phase of stroke involves multiple factors. Inflammatory factors (such as NLRP1 and IL-18) are closely related to the occurrence of depression, while single variables like the NIHSS score have limited predictive value for depression. When inflammatory responses are adequately intervened and neurological function is improved, it is possible to effectively reduce the incidence of depression during the acute phase of stroke and enhance patient prognosis (see Table 4).

PSD is a common emotional disorder following an acute stroke [15]. PSD can occur during the acute phase after a stroke (less than 1 month), the intermediate phase (1–6 months), and the recovery phase (more than 6 months), with incidence rates of 33%, 33%, and 34%, respectively [16]. In the acute stage of stroke, if the depressive state is not recognized early or given intervention, it is very likely to develop into PSD in the later stage [17]. Therefore, early identification, accurate diagnosis and timely treatment are of great clinical significance [16]. In this study, the proportion of patients with depression during the acute phase of stroke reached 38.75%, further highlighting its high prevalence and clinical significance.

Despite extensive research conducted in recent years on the factors influencing depression during the acute phase of stroke, the current evidence remains insufficient. Some studies indicate that there is a positive correlation between age and the incidence of depression [18]. Some studies indicate that women are more likely to be depressed than men [19]. Moreover, there may be complex interactions between cardiovascular risk factors (such as smoking, alcohol consumption, hypertension, diabetes, and coronary heart disease) and depression [18].This study shows that due to limitations in sample size and specific disease course characteristics, no statistically significant differences were found between the two groups regarding age, BMI, gender, stroke type, and vascular risk factors (smoking, alcohol consumption, hypertension, diabetes, coronary heart disease). However, this does not negate the possibility that the aforementioned factors may be unrelated to the occurrence of depression in the acute phase of stroke. Future research should expand the sample size for further investigation.

Sleep disorders are a common issue among stroke patients. These disorders not only affect the rehabilitation of stroke patients but are also closely associated with depression during the acute phase [20]. Previous research by Dong has shown that patients with PSD have significantly higher scores in sleep quality (PSQI) and neurological deficit (NIHSS) compared to non-depressed patients [21]. The severity of stroke has been consistently determined to be significantly associated with PSD [22]. As the results of this study indicate, high PSQI and NIHSS scores are significant risk factors for depression during the acute phase, while also suggesting their predictive value for early identification of depression. By analyzing the PSQI and NIHSS scores, along with related inflammatory factors in patients with acute stroke, this research aims to explore the potential biological mechanisms leading to PSD.

Following an acute stroke, both central and peripheral inflammatory responses are activated [23]. In recent years, inflammation and various cytokines have been recognized as key factors related to the mechanisms of PSD, becoming a focal point of research [24]. The NLRP1 inflammasome is the first member of the NLR family and is highly expressed in the brain [25‒27]. The activation of the NLRP1 inflammasome can induce the cleavage of pro-caspase-1 and the pore-forming protein gasdermin-D, as well as the maturation of pro-IL-18 and pro-IL-1β, ultimately leading to neuronal apoptosis [26, 28‒30]. This represents a form of programmed pro-inflammatory neuronal death that is distinct from apoptosis. The NLRP1 inflammasome promotes the production of pro-inflammatory cytokines, resulting in neuronal cell death and behavioral dysfunction [26]. In this study, we compared the levels of the NLRP1 inflammasome and its component proteins (ASC and caspase-1) in the serum of patients with acute stroke depression and those without depression during the acute phase of stroke. Our results show a positive correlation between serum NLRP1 levels and HAMD scores (Hamilton Depression Rating Scale). Specifically, in the acute phase of stroke, patients with moderate to severe depression exhibit significantly higher NLRP1 expression levels compared to those with mild depression. This finding suggests that measuring the levels of NLRP1 in peripheral blood may provide a valuable biological indicator for predicting the severity of PSD. However, there was no significant association between serum ASC and caspase-1 levels in the two groups, possibly due to the lower protein content of ASC and caspase-1 compared to NLRP1, which may have masked differences in detection. Although ASC and caspase-1 proteins did not show statistical significance in this study, this does not negate the possibility of peripheral inflammasome activation. Furthermore, anti-inflammatory treatment in some patients with DDD has been shown to effectively alleviate depressive symptoms [31], which further support the evidence for the activation of inflammasomes in a subset of DDD patients. Given the limited sample size of moderate to severe depressed patients in the acute phase of stroke in this study, it is necessary for future research to expand the sample size to more comprehensively validate the relationship between inflammasome levels and the severity of depression.

Cytokines are secreted or membrane-presenting small protein molecules that can mediate a wide range of cellular functions, including development, differentiation, growth, and survival [8]. They can exert pro-inflammatory or anti-inflammatory effects. In the context of psychoneuroimmunology, the most studied cytokines are pro-inflammatory interleukin (IL)-6, TNF, IL-1β, and IFN, as well as the anti-inflammatory IL-10 [32]. Research reports indicate that the primary lymphoid organs in patients with PSD is typically activated after a stroke, leading to the production of increased pro-inflammatory cytokines [33]. Cytokines such as IL-1β, IL-6, IL-8, and TNFα have increased secretion that can lead to neuronal degeneration and apoptosis [33]. Meanwhile, IL-6, IFNγ, and TNFα can induce abnormal degradation of tryptophan as a result of the accumulation of the end product quinolinic acid in glial cells [32]. The excessive excitation of N-methyl-d-aspartate receptors promotes glutamate excitotoxicity and antagonizes 5-hydroxytryptamine, reduced synaptic plasticity and neuronal survival, thereby contributing to depression [33]. IL-10, as an inhibitory cytokine, is primarily produced by Th2 cells. It suppresses the production of pro-inflammatory cytokines, inhibits immune responses, and plays a protective role for the nervous system [18]. The levels of anti-inflammatory cytokines, including transforming growth factor-β and IL-10, are often elevated in patients with major depressive disorder, raising questions about their role in depression [29] and their potential impact on cellular immune responses [32]. In this study, we measured the levels of cytokines such as IL-1β, IL-10, IL-18, and TNF-α in the serum samples of patients. We found that IL-18 and TNF-α levels were significantly higher in the PSD group compared to the non-depressed group, and these levels were positively correlated with the severity of depression. Further analysis revealed that IL-18 is an independent risk factor for the occurrence of depression in the acute phase of stroke and has good predictive value for the development of depression during this period. In contrast, there were no significant differences in IL-1β and IL-10 levels between the two groups. These findings are contradictory to certain previous studies [30], which may be attributed to differences in the cytokine components studied, variations in disease progression, and potential confounding factors such as comorbidities and the effects of medications (non-steroidal anti-inflammatory drugs, statins). In clinical research, case selection often takes into account the presence of confounding factors to minimize bias in the results.

In summary, this study indicates that the activation of central inflammasomes in the central nervous system may induce the onset of depression by mediating the production of inflammatory factors. However, considering the limitations of cross-sectional studies in inferring causality, further research is needed to confirm the causal relationship between inflammasome activation and the development of depression. The results of this study indicate that patients with acute phase depression following a stroke have higher levels of the NLRP1 inflammasome in their peripheral blood compared to healthy individuals. Additionally, there is an observed increase in certain pro-inflammatory and anti-inflammatory cytokines. However, the specific signaling pathways involved in the production of inflammatory components remain unclear. The activation of peripheral inflammasomes and their mediation of cytokine production require further extensive research for validation. Nonetheless, it is undeniable that peripheral inflammation has a certain impact on the occurrence and development of depression during the acute phase of stroke in some individuals. Given the roles of peripheral and central inflammation in depression, future targeted anti-inflammatory treatments may represent a new candidate for interventions in acute phase depression following a stroke.

All participants signed written informed consent, and the study protocol was approved by the Ethics Committee of the Clinical School of Medicine of Dali University Hospital in accordance with the Declaration of Helsinki (DEY20230123001).

The authors declare that there is no conflict of interest.

Funding 404, this study was supported by Joint special fund project for Basic research of local undergraduate 405 universities in Yunnan Province (202101AO070200) and Dali University longitudinal research 406 project (KYBS2023035).

Song Li and Kun Geng: writing, resources, and data analysis. Lin Yang, Yaling Zhang, Xiaoyang Tao, and Jierui Cai: resources and data analysis. Lingyang Li, Zeming Luo, and Birendra Mahato: research design. Yonglei Liu, Xiaoling Yin, Hiu Cai, Jishuai Zhao, and Heyan Cheng: resources. Lixia Wang: research design, writing, data analysis, and validation.

Song Li and Kun Geng made the same contributions to this article as co-first authors.

This document contains all data generated or analyzed during this study. All data are provided in the body. Patient data selected patients with acute stroke diagnosed for the first time in the Department of Neurology of the First Affiliated Hospital of Dali University between April 2023 and October 2023. For further inquiries, contact the corresponding author directly.