Xiaoyue Li, Tao Liu, Xuan Mo, Runhua Wang, Xueyan Kong, Robin Shao, Roger S.McIntyre, Kwok-Fai So,Kangguang Lin,8,*

Abstract Strong evidence has accumulated to show a correlation between depression symptoms and inflammatory responses.Moreover, anti-inflammatory treatment has shown partial effectiveness in alleviating depression symptoms.Lycium barbarum polysaccharide (LBP), derived from Goji berries, exhibits notable antioxidative and anti-inflammatory properties.In our recent double-blinded randomized placebo-controlled trial, we found that LBP significantly reduced depressive symptoms in adolescents with subthreshold depression.It is presumed that the antidepressant effect of LBP may be associated with its influence on inflammatory cytokines.In the double-blinded randomized controlled trial, we enrolled 29 adolescents with subthreshold depression and randomly divided them into an LBP group and a placebo group.In the LBP group, adolescents were given 300 mg/d LBP.A 6-week follow up was completed by 24 adolescents,comprising 14 adolescents from the LBP group (15.36 ± 2.06 years, 3 men and 11 women) and 10 adolescents from the placebo group (14.9 ± 1.6 years, 2 men and 8 women).Our results showed that after 6 weeks of treatment, the interleukin-17A level in the LBP group was lower than that in the placebo group.Network analysis showed that LBP reduced the correlations and connectivity between inflammatory factors, which were associated with the improvement in depressive symptoms.These findings suggest that 6-week administration of LBP suppresses the immune response by reducing interleukin-17A level, thereby exerting an antidepressant effect.

Key Words: adolescents; cytokines; efficacy; Goji berry; inflammatory responses; interleukin-17A; Lycium barbarum polysaccharide; randomized controlled study; subthreshold depression

Introduction

The burden of major depressive disorder (MDD) in youth is well documented and associated with a significant risk for suicide (Wesselhoeft et al., 2013;Clayborne et al., 2019).Several lines of evidence indicate that symptoms of psychological distress and rates of depression and anxiety increased in many parts of the world during the coronavirus disease 2019 pandemic (Xiong et al.,2020).Disturbance in the immune and inflammatory system are considered as potentially causative in prevailing pathophysiologic models of depression,and are also associated with select phenomenological characteristics such as cognitive impairment and anhedonia (Lee et al., 2018; Nogo et al., 2021; Peng et al., 2022).Subthreshold depression (StD) is characterized by the presence of significant depressive symptoms that do not meet the diagnostic criteria for MDD (Rodríguez et al., 2012).It is considered to represent an early stage of MDD and significantly increases the risk of an individual progressing to MDD and other psychological disorders (Lee et al., 2019).Limited previous research indicates an increase in proinflammatory cytokines in adults presenting with subthreshold depressive symptoms (Fagundes et al., 2013), but there is still no consensus regarding the association between inflammation and StD in youths.Identifying mechanisms and potential treatments of StD in youths holds promise to preempt and possibly prevent syndromal severity and its associated complications (Noyes et al., 2022).

Although prior evidence has indicated that the levels of cytokines, such as interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α, in peripheral blood significantly change in patients suffering from depression (K?hler et al.,2017), the coregulation relationships between cytokines have not yet been elucidated.Prior studies have also indicated the role of additional cytokines in the context of depression.For instance, the role of IL-17A, predominantly produced by T helper (TH) 17 cells, has garnered increasing attention.Crosssectional studies have detected a marked increase in IL-17A levels in patients with depression (Davami et al., 2016; Beurel and Lowell, 2018).Moreover,preclinical trials have shown that IL-17A administration induces depressionlike behaviors in mouse models (Nadeem et al., 2017; Kim et al., 2021).This expanding body of research underscores the need for a more comprehensive understanding of cytokines in relation to depression.Additionally, given the intricate and varied nature of cytokines in the inflammatory response,along with their potential to exert both pro- and anti-inflammatory effects on different cells, it seems prudent to carry out an extensive analysis of all inflammatory markers and examine the mutual regulation relationships among cytokines.A network-based approach to cytokine analysis could prove advantageous in the formulation and assessment of the effects of anti-inflammatory therapeutics (Song et al., 2022).

Lycium barbarumpolysaccharide (LBP), extracted from the Goji berry, has various pharmacological properties including attenuating neuroinflammation and anti-aging effects (Xiao et al., 2022).Previous preclinical research including ours in mice has demonstrated that LBP has antidepressant effects(Zhao et al., 2019; Fu et al., 2021).Recently, we launched a clinical trial to determine whether LBP provides an antidepressant benefit to adolescents with StD.Interim analysis of the results indicated a significant improvement in depressive symptoms in adolescents treated with LBP compared with placebo over 6 weeks (Li et al., 2022).However, the precise antidepressant mechanism of LBP remains uncertain.Previous research has shown that LBP inhibits the release of inflammatory cytokines, such as TNF-α and IL-6 (Liu et al., 2023).Typically, these factors affect the differentiation of Th17 cells,leading to abnormal secretion of IL-17A (Beurel and Lowell, 2018).

Informed by the inflammation-based theory of depression and the antiinflammatory properties of LBP, we hypothesized that the antidepressant effect of LBP is likely connected to its effect on inflammatory cytokines,particularly IL-17A.In the present study, we evaluated the cytokine response to LBP in youths with StD, with a particular focus on the role of IL-17A.Additionally, we undertook a broader investigation to determine the effect of LBP on various cytokines and their interplay.We aimed to identify potential moderators or correlates across select inflammatory markers for treatment in adolescents with StD.

Methods

Study design and participants

This study sample was obtained from a 6-week double-blind randomized placebo-controlled trial, registered on ClinicalTrials.gov (Identifier ID:NCT04032795) on July 25, 2019, that aimed to evaluate the efficacy of LBP in youths with StD.A detailed description of the study design and procedure of assessment for the trial is available in a previous publication (Li et al.,2022).Briefly, eligible youths aged 12 to 18 years who were identified as having StD were recruited from the Fifth Affiliated Hospital of Guangzhou Medical University (Guangzhou, China).Participants were randomly divided with an allocation ratio of 1:1 into two groups: one receiving a daily dose of 300 mg LBP (tablet) and the other receiving a placebo (microcrystalline cellulose and starch blend), both administered for 6 weeks.Both the LBP and the placebo were supplied by Tianren Ningxia Wolfberry Biotechnology Co.,Ltd.(Zhongwei, China).The randomization list was created using computer software (Excel 2013; Microsoft Office, Seattle, WA, USA) and kept securely in the possession of an independent investigator (KL) at the Affiliated Brain Hospital of Guangzhou Medical University.Both assessors and participants were unaware of the treatment assignments, as the LBP and placebo tablets were identical in both their visual appearance and taste.The primary outcome involved changes in depression severity, measured by the Hamilton Depression Scale (HAMD), a 24-item scale with responses ranging from 0 to 4(Bech et al., 1975).The study was approved by the Medical Ethics Committee of the Fifth Affiliated Hospital of Guangzhou Medical University (Guangzhou,China, approval No.L2019-08) on April 4, 2019.All of the participants and their guardians provided written informed consent.The trial complied with theDeclaration of Helsinkiand was performed in accordance with the CONsolidated Standards Of Reporting Trials (CONSORT) Statement (Additional

file 1; Schulz et al., 2010).

Inclusion and exclusion criteria

Participants were considered as having StD if they recorded a score of 15–25 on the Beck Depression Rating Scale (Beck et al., 1996), yet did not meet the formal criteria for MDD.Eligible participants were confirmed by face-toface psychiatric interviews.The exclusion criteria were: 1) diagnosis of MDD or any other psychiatric disorders according to the Diagnostic and Statistical Manual of Mental Disorders, 5thedition criteria (First et al., 2016), through conducting The Schedule for Affective Disorders and Schizophrenia for Schoolaged Children-Present and Lifetime versions (Ambrosini, 2000); 2) diagnosis of severe physical disease; 3) had consumed Chinese herbs containing Goji berries in the past 3 months; and 4) had been taking hormone or endocrine medications.

Cytokine measurements

Peripheral blood samples were collected from the upper limb veins of the participants at the Fifth Affiliated Hospital of Guangzhou Medical University,China.Collections were made between 8:00 a.m.and 10:00 a.m.at two time points: baseline and 6 weeks after the LBP intervention.Venous blood (5mL) was drawn into additive-free vacutainer tubes, and then centrifuged to generate sera.These samples were then preserved at –80°C for subsequent analysis.Concentrations of 33 cytokines, including IL-17A,IL-6, and IL-1β, were determined using a bead-based, human ProcartaPlex immunoassay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.Briefly, antibody-coated beads were incubated with either premixed standards or sample supernatants for 120 minutes at room temperature at a rotational speed of 500 r/min using a 96-well plate mixer (Olabo, Jinan, Shandong province).After incubation, premixed detection antibodies were added and incubated under the same conditions for 30 minutes.After washing, Phycoerythrin-conjugated streptavidin was added and incubated under the same conditions.Finally, the washed beads were resuspended in Bio-Plex cytokine assay buffer (Bio-Rad, San Francisco,CA, USA) and analyzed on the Bio-Plex 200 system with a low PMT setting.Fluorescence was measured with a Bioplex200 system (Bio-Rad) and analyzed with ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific) (Maisonnasse et al., 2020; Ma et al., 2021).

Statistical analysis

This study is an extension of our previous interim analysis (Li et al., 2022),with a specific emphasis on IL-17A-related outcomes.It is worth noting that we employed a subset of the original dataset in this study.Betweengroup comparisons on baseline demographics and clinical characteristics were analyzed using independent samplest-tests or non-parametric tests(Mann-WhineyUtest and Chi-squared test) according to types of variables and data distribution.Baseline cytokine levels were normalized (base 10 logtransformed) to increase the data normality.The change level of individual cytokines in each group was computed by subtracting participants’ week 6 values from their baseline values, in order to eliminate the influence of baseline cytokine concentrations.To better understand the global regulation of systemic cytokine response by interventions, we applied network-level analysis of cytokine data.We then constructed a network in which one node represented a cytokine and one edge was weighted using absolute Pearson correlation coefficients between cytokines.The MetScape (version 3.1.3)application in Cytoscape 3.8.0 (University of California, San Diego, CA, USA)was used to build the network map.We evaluated the network connectivity and the importance of each node in the network based on degree and eigenvector centrality score using the CytoNCA (version 2.1.6) application in Cytoscape 3.8.0.Wilcoxon tests, conducted with 1000 resamples, were used to assess the differences in network connectivity and centrality between the placebo and LBP groups.Statistical analyses were conducted using R software 4.1.2 (R Development Core Team, Vienna, Austria) andP-value significance was set at 0.05.

Results

Participant characteristics

Between May 15, 2019 and October 30, 2020, 29 participants were randomly assigned to either the LBP or placebo group.Figure 1 illustrates the plan for group allocation and the schedule for blood sample collection.During the trial interval, five participants (four in the placebo group and one in the LBP group) dropped out during the follow-up period.Twenty-four valid peripheral blood samples (LBP = 14 and Placebo = 10) were collected and included in the analysis.

Figure 1 ｜Scheme for group assignment and timeline for blood sample collection.LBP: Lycium barbarum polysaccharide.

Demographic characteristics are shown in Table 1.The two groups were matched on age, sex, and education level.HAMD was used to assess the severity of depression, and the groups were also matched on total HAMD scores (P> 0.05).The baseline levels of cytokines were generally balanced between the groups (P> 0.05); the LBP group had a higher level of IL-10 than the placebo group at baseline (t= -2.25,P= 0.04; Table 1).

Cytokine response

We next studied the effect of interventions on cytokine response in participants with StD.As shown in Table 2, 33 cytokines were measured.Ten cytokines were identified as not conforming to a normal distribution through the histogram test method: IL-2, IL-5, IL-6, IL-10, IL-15, IL-18, IL-31,interferon-α, TNF-α, and granulocyte-macrophage colony-stimulating factor.Statistical analysis for these cytokines was conducted using Mann-Whitney tests.Notably, there was a significant difference in the changes in IL-17A level between the two groups, which suggested that LBP downregulated the peripheral level of IL-17A (t= -2.25,P= 0.04).No significant differences were found between the two groups for the other cytokines (P> 0.05).Additionally,there was no significant correlation between the changes in IL-17A and the total score changes on the HAMD (P> 0.05).

Network-level analysis of cytokines

Figure 2A shows correlations between changes in cytokine levels for the two groups.The density distribution curves, which represented the distribution of the correlation coefficient matrix, were significantly different between groups (D= 0.33,P< 0.01; Figure 2B).Next, network maps were constructed,wherein darker black lines indicate a stronger correlation (Pearson correlationr> 0.5,P< 0.05; Figure 2C).The overall cytokine network connectivity (degree)in the LBP group was significantly different from that in the placebo group(Z= 3.34,P< 0.01; Figure 2D), suggesting that LBP downregulated systemic cytokine levels and diminished connectivity between cytokines.Centrality analysis of the cytokines network did not detect significant differences between groups (Z= -0.73,P= 0.76).

To understand the relationship between the cytokines network and depressive symptoms, we analyzed the correlation between relative change in HAMD score and cytokine levels in the LBP-treated group from baseline to 6 weeks.As shown in Figure 3, the change in HAMD score was strongly correlated with IL-1α (r= 0.64,P= 0.01), IL-6 (r= 0.6,P= 0.02), IL-21 (r= 0.76,P= 0.02),TNF-α (r= 0.78,P< 0.01), interferon-α (r= 0.72,P< 0.01), IL-1β (r= 0.59,P= 0.03), IL-1RA (r= 0.57,P= 0.03), and IL-22 (r= 0.71,P< 0.01), suggesting a connection between modulation of the cytokine network and HAMD score.

Discussion

Participants included in the present study are a subsample of the clinical trial,which demonstrated that LBP-treated youths with StD achieved accelerated recovery of depressive symptoms and a higher remission rate compared with those who received placebo, and that LBP was well tolerated (Li et al., 2022).Despite the small sample size in the present study, our major finding was that youths treated with LBP had an increased reduction in peripheral IL-17A compared with those treated with placebo.Moreover, those treated with LBP had a diminished correlation connectivity of systemic cytokine expression.

IL-17A, commonly known as IL-17, is a proinflammatory cytokine and the most studied member of the IL-17 family (McGeachy et al., 2019).It is secreted mainly by Th17 cells but also by granulocytes, monocytes, and astrocytes(Beurel and Lowell, 2018).The Th17 cell population is mainly located in the lamina propria of the small gut and is responsible for immune surveillance(Ghosh et al., 2022).Physiologically, Th17 cells and IL-17A are almost absent in other organs and not detectable in blood (Ivanov et al., 2009; Shen et al., 2009).Elevated levels of Th17 cells and IL-17A in the central nervous system have been associated with increased neuroinflammation (Beurel and Lowell, 2018; Ghosh et al., 2022), as seen in experimental autoimmune encephalomyelitis (Wu and Wan, 2020).Despite increasing evidence implicating IL-17A in depression, the pathological role of IL-17A in depression is still unclear (Ghosh et al., 2022).It has been suggested that IL-17A impairs the brain–blood barrier integrity and activates astrocytes and microglia to induce the production of inflammatory mediators (such as intercellular adhesion molecule 1, prostaglandin E2, matrix metalloproteinases, other cytokines, and antimicrobial peptides) to mediate neuroinflammation, which further damages synaptic function in the hippocampus, amygdala, and prefrontal cortex (Zhu and Qian, 2012; Zimmermann et al., 2013; Kim et al.,2021).Induction and release of those mediators also appear to amplify IL-17A production through a positive feedback loop, thereby spreading inflammatory damage (Benedettiand Miossec, 2014).

Anti-IL-17 treatment may reduce neuroinflammatory response and mediate antidepressant effects.In the present study, we found that LBP-treated youths with StD had an improvement in depressive symptoms accompanied by downregulation of IL-17A.This result is in accordance with a previous study,which reported that escitalopram reduced the peripheral level of IL-17 during therapy (Munzer et al., 2013).In addition, Kim et al.(2021) recently reported that administering anti-IL-17/IL-17A antibodies improved depressive-like behavior in mouse models.Of note, a longitudinal study that evaluated various cytokines during antidepressant therapy reported that peripheral IL-17A levels increased significantly in the non-responder group compared with the responder group, implying that IL-17A may be a useful marker for nonresponsiveness to antidepressant treatment (Nothdurfter et al., 2021).Given that, IL-17A could be a potential therapeutic target and treatment-responsive marker for depression.

Figure 3 ｜The relationship between antidepressant effect and systemic cytokine changes in LBP treatment.The network map displays the relationships (Pearson correlation r > 0.5, P < 0.05)between the relative change of HAMD score and cytokine levels from baseline to 6 weeks.Red nodes and lines represent direct correlations.CCL: Chemokine (C-C motif)ligand; CXCL: chemokine (C-X-C motif) ligand; GM-CSF: granulocyte-macrophage colony-stimulating factor; HAMD: Hamilton Depression Rating Scale; IFN: interferon; IL:interleukin; LBP: Lycium barbarum polysaccharide; SDF: stromal cell-derived factor; TNF:tumor necrosis factor.

Immunological processes are complex and multi-regulated.It is necessary to consider not only a single cytokine of the inflammation system, but also its interactions in evaluating immune response (Himmerich et al., 2019).Cytokine signaling has both synergistic and antagonistic effects in regulating the growth and differentiation of immune cells.For example, IL-4 amplifies IL-3 to induce differentiation and activation of mast cells in a synergistic way, but also blocks the secretion of IL-12 (Del Prete, 1992; Georas et al.,2005; Kane et al., 2015).In addition, cytokine cascades play a crucial role in cytokine signaling, where the activation of one cytokine induces an immune cell type to produce another cytokine (Himmerich et al., 2019).For example,if TH0 cells produce IL-6, the consequence is activation of Th17 cells, which secrete proinflammatory cytokines like IL-17, IL-21, and IL-22 (Gaffen et al.,2014).Because of the coregulation and interactions between cytokines, using network analysis to demonstrate the connectivity and centrality of multiple inflammatory mediators is useful in assessing the effect of anti-inflammatory therapeutics (Song et al., 2022).Although our research did not uncover a direct connection between changes in HAMD scores and the downregulation of IL-17A, we postulate that the decline in IL-17A may be associated with LBP’s modulation of the cytokine network, as suggested by the outcomes of our network analysis.In this study, we observed that LBP treatment, when compared with the placebo group, resulted in reduced correlations and connectivity of cytokines among youths with StD, indicating its potential impact on the peripheral inflammatory environment and subsequent alteration of the systemic cytokine network.In addition, our results support the idea that anti-inflammatory agents that block the immune response may have a vital role in the alleviation of depressive symptoms (Dionisie et al.,2021; Lucido et al., 2021).

The pharmacological pathway by which LBP acts on the central nervous system is still unknown; however, it may be related to the microbiota–gut–brain axis.A preclinical study reported that LBP treatment in mice improved lipid metabolism and insulin levels, and significantly decreased peripheral levels of TNF-α and IL-17A (Zhang et al., 2020).The authors suggested that these positive effects of LBP could be attributed to the function of the gut microbiota in upregulating host defense against infection after treatment.In addition, our preclinical study on a mouse model of depression showed that LBP administration improved depressive-like behavior by preventing aberrant neuronal activity and microglial activation in the lateral habenula, which was associated with a reduced neuroinflammatory response (Fu et al., 2021).On the basis of these previous studies, we speculate that LBP may modulate inflammation response through the microbiota-gut-brain axis to produce an antidepressant effect.However, more studies will be needed to elucidate the mechanism of LBP on the inflammatory response and its relationship with the microbiota-gut-brain axis and depression.

Several limitations of this study need to be mentioned, including its post hoc design.First, the strength of this study is hindered by the small sample size and distribution (80% of participants were female) that impede the generalizability of the findings.Second, we initially used an undirected network to examine the pathway of LBP’s influence on inflammatory cytokines.The precise inflammatory pathways and pharmacological mechanisms affected by LBP need further investigation in future research.Third, given the limited sample size in this study and the absence of rigorous multiple corrections, it is necessary to further investigate the exploratory findings regarding the ability of LBP to reduce the network connectivity of inflammatory cytokines.Future studies with larger sample sizes and robust multiple corrections are warranted to validate these findings.Fourth,throughout the 6-week follow up, there were no periodic assessments of participants’ inflammatory conditions, medication usage, and stressful life events, all of which may have potentially confounded the results.Future research should measure and carefully control for those variables.Fifth,peripheral levels of cytokines could be influenced by confounding factors such as age, body mass index, and blood pressure; cytokine concentrations in cerebrospinal fluid might better reflect cytokine signaling in the brain than levels in the peripheral blood and should be applied in the future (Himmerich et al., 2019).

In conclusion, the present study indicates that immune response is downregulated after repeated LBP administration, and that the changes correlate with improvement of depressive symptoms.In addition, our findings suggest that altering the inflammatory pathway may be involved in the antidepressant effect.Future studies with larger sample sizes are required to replicate and validate our findings.

Acknowledgments:We would like to thank all participants and staffs involved in the data collection for this study.We are deeply grateful for the invaluable support provided by the Department of Psychiatry at the Fifth Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) in conducting clinical assessments and collecting blood specimens.

Author contributions:Study conceptualization and design: KL, KFS; data collection and interpretation: XL, XM, TL, RW and XK; Statistical analysis and writing manuscript: XL; manuscript revision: KL, KFS, RS, RSM.KL and KFS verified the underlying data reported in the manuscript.All authors approved the final version of this manuscript.

Conflicts of interest:All authors declare that they have no conflict of interest.Editor note: The corresponding author KFS is the Editor-in-Chief of Neural Regeneration Research.KFS did not participate in the peer review of decisionmaking of the review.

Author statement:This paper has been posted as a preprint on Research Square with doi: https://doi.org/10.21203/rs.3.rs-2031269/v1, which is available from: https://assets.researchsquare.com/files/rs-2031269/v1/33acf3b5-fbdb-4555-8f0f-8aa1f973c50f.pdf?c=1663103604.

Data availability statement:The data supporting this finding in this study,along with the corresponding statistical analysis procedure, are available from the corresponding author upon reasonable request.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional file:

Additional file 1: CONSORT 2010 checklist.