Background
Schizophrenia is a neurodevelopmental disease characterized by positive symptoms, negative symptoms and cognitive symptoms. The mechanisms of schizophrenia have not yet been elucidated. Microglia are neuroprotective, immunocompetent cells of the central nervous system (CNS) that play a pivotal role in neurodevelopmental processes [
1,
2]. Accumulating evidence has shown that abnormal activation of microglia is involved in the pathogenesis of schizophrenia by facilitating the release of proinflammatory cytokines and various free radicals, leading to developmental abnormalities [
3‐
5]. Clinical studies have found that anti-inflammatory drugs that can inhibit the abnormal activation of microglia can alleviate the negative symptoms of schizophrenia. In addition, previous studies have found that atypical antipsychotics, which have been found to be effective in treating negative symptoms, also inhibit the activation of microglia, suggesting that atypical antipsychotics also ameliorate the negative symptoms of schizophrenia by inhibiting the abnormal activation of microglia.
Microglial activation is manifested by changes in cell morphology (retraction of processes and hypertrophy), changes in the expression of specific cell surface markers (OX-42, a complement type III receptor) and the release of some harmful substances (proinflammatory cytokines, free radicals, etc.). There are a wide range of factors that trigger the activation of microglia, such as LPS, interferon alpha (INF-α), and β-amyloid (Aβ). When microglia are stimulated, they are exposed to a series of signals, leading to the expression of related inflammatory factors and thereby regulating the inflammatory response [
1]. Studies have found that some signaling molecules involved in the activation process of microglia are related to mental illness. Studies have found that the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is involved in synaptic plasticity in the hippocampus and related to depression [
6‐
9]. A study involving a model of neuroinflammation found that the atypical antipsychotic risperidone inhibited the increase in p38 mitogen-activated protein kinases (MAPK) expression induced by LPS [
10]. Based on the above results, we believe that these two signaling pathways are likely related to the therapeutic effects of antipsychotics.
Minocycline is a tetracyclic antibiotic that exerts anti-inflammatory and neuroprotective effects by inhibiting the activation of microglia [
11]. Previous study [
12] have shown that the MAPK, and JAK-STAT signaling pathways are involved in the anti-inflammatory effect of minocycline. Clinical evidence has shown that when used as an adjunct therapy, minocycline can alleviate the negative symptoms of schizophrenia better than placebo [
13]. Some in vitro and in vivo studies have found that minocycline and atypical antipsychotics can inhibit the activation of microglia, while typical antipsychotics have little effect on microglial activation [
14,
15]. Our previous study also found that minocycline and risperidone can significantly rescue behavioral deficits and attenuate microglial activation in rat models of schizophrenia [
16,
17]. Haloperidol is a typical antipsychotic used for the treatment of positive symptoms, and its inhibitory effect on microglial activation is significantly weaker than that of risperidone [
18].
Although the “Microglia hypothesis of schizophrenia” has attracted much attention, the molecular mechanism by which atypical antipsychotics inhibit microglia activation is still unclear [
19,
20]. Therefore, the aim of this study was to explore the molecular mechanism of treatment effect of minocycline and antipsychotics on schizophrenia through observation of related molecular signaling pathways in activated microglia. Our research hypothesis is that atypical antipsychotic risperidone, similar to minocycline, inhibits the activation of microglia through the MAPK, and JAK-STAT signaling pathways, thereby exerting a therapeutic effect on the negative symptoms of schizophrenia. The inhibitory effect of typical antipsychotic haloperidol on MAPK, and JAK-STAT signaling pathway and microglia activation is significantly weaker than that of minocycline and risperidone. This study provides evidence for the pathogenesis of negative schizophrenia symptoms and new targets for treatment.
Methods
Drugs and reagents
Minocycline hydrochloride, risperidone, haloperidol and LPS were purchased from Sigma Chemical Co. (St. Louis, MO, USA, catalog no. WXBB4793V, R3030, H1512, and 025M4040V, respectively). RPMI 1640 medium, fetal bovine serum, penicillin/streptomycin antibiotics and 0.25% trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY, USA, catalog no. 11875119, 10091148, 15140148, and 25200072, respectively). The measurement of nitric oxide using the Nitrite/Nitrate Assay Kit purchased from Sigma-Aldrich (St. Louis, MO, USA, catalog no. 23479-1KT-F). Rabbit anti-OX42 Polyclonal Antibody was purchased from Absin Bioscience Inc. (Shanghai, China, catalog no. abs137035). Sheep Anti-Rabbit IgG H&L (DyLight® 488) was purchased from Abcam Bioscience Inc. (Cambridge, UK, catalog no. ab96923). Mouse TNF-α, IL-1β ELISA kits were from R&D Systems, Inc. (NE Minneapolis, USA, catalog no. MTA00B, and MLB00C, respectively). Mouse IL-6 ELISA kits purchased from CUSABIO (Fannin St., Houston, USA, catalog no.CSB-E04639m). P38 MAPK (D13E1) XP ®Rabbit mAb, Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP ®Rabbit mAb, iNOS (D6B6S) Rabbit mAb, JNK Rabbit mAb, ERK Rabbit mAb antibody, and α-Tubulin Rabbit mAb antibodies were purchased from CST (Boston, USA, catalog no. 8690, 4511, 13120, 67096, 4695, and 3873, respectively), STAT3 Rabbit mAb and JAK2 Rabbit mAb antibodies were purchased from Novus (Colorado, USA, catalog no. NBP2-61588, and NBP3-15825).
BV-2 cell culture
The BV2 murine microglial cells were gifted from Shanghai Institutes for Biological Sciences (SIBS, Shanghai, China). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a 5% CO2 cell incubator at 37 °C. The cells were subcultured when the cell monolayer reach approximately 80% confluence.
Viability assay
BV-2 cell viability was measured by the CCK-8 assay. In brief, BV-2 cells (100 μL) were seeded in each well of a 96-well culture plate (0.5 × 104 cells/mL). Twenty-four hours later, different concentrations of drug-containing and serum-free medium were added. The cells were treated with different concentrations of minocycline (0.01, 0.1, 1.0, 10, and 100 μmol/L), risperidone (0.1, 1.0, 10, 50 and 100 μmol/L) and haloperidol (0.1, 1.0, 10, 50 and 100 μmol/L); normally cultured cells were established as controls for each experimental group. Cells from each group were plated in 3 wells of a 96-well culture plate and cultured at 37 °C and 5% CO2 for 24 h. Then, 10 μL of CCK-8 reagent was added to each well for 3 h, and the absorbance (OD) at 450 nm was measured using a microplate reader connected to an enzyme labeling instrument. The relative survival rate of the cells was calculated as follows:
$$\begin{array}{*{20}{c}}{{\rm{\% }}\,{\rm{cell}}\,{\rm{viability}}\,{\rm{ = }}\,{\rm{(O}}{{\rm{D}}_{{\rm{experimental}}\,{\rm{group}}}}{\mkern 1mu} {\rm{ - }}{\mkern 1mu} {\rm{O}}{{\rm{D}}_{{\rm{blank}}\,{\rm{group}}}}{\rm{)}}}\\{{\rm{/(O}}{{\rm{D}}_{{\rm{normal}}\,{\rm{group}}}}{\mkern 1mu} {\rm{ - }}{\mkern 1mu} {\rm{O}}{{\rm{D}}_{{\rm{blank}}\,{\rm{group}}}}{\rm{)}}{\mkern 1mu} {\rm{ \times }}{\mkern 1mu} {\rm{100\% }}}\end{array}$$
After selecting the most effective concentrations of each drug, the optimal LPS treatment duration was selected. The experiment was divided into three parts to investigate the effects of minocycline, risperidone and haloperidol on cell viability. In each part of the experiment, the cells were divided into three groups: the normal culture group, LPS treatment group and drug pretreatment plus LPS treatment group. Cells from each group were plated in three wells in each part of the experiment, and the 96-well culture plate was cultured at 37 °C for 24 h in 5% CO2. The cells were treated with the optimal concentrations of the three drugs for 24 h and then treated with 1.0 μg/ml LPS for 12, 24 or 48 h. After incubation for 3 h with 10 μL of CCK-8 reagent, the OD value of each well was measured by an enzyme labeling instrument, and the optimal LPS treatment duration was determined by calculating the relative survival rate of the cells in each well.
Morphological observation
The BV2 cells were plated into 6-well plates at a density of 5 × 104cells per well. After the cells had adhered to the wall overnight, they were randomly divided into 5 groups: (1) the blank control group; (2) the LPS group; (3) the minocycline preconditioning + LPS group; (4) the risperidone preconditioning + LPS group; and (5) the haloperidol preconditioning + LPS group. The cells in each group were photographed under a phase contrast microscope at the end of the treatment period to observe the effects of the drugs on the activation of cells stimulated with LPS. The number of branches and the branch length of each cell were measured using ImageJ.
Immunofluorescence staining and measurement of mean fluorescence intensity
The protein expression of OX42 (CD11b/c), a microglia-specific marker, in BV-2 cells was assessed by immunofluorescence, and changes in its expression were observed. After the cells in the five groups were digested with 0.25% trypsin, they were inoculated onto slides at a density of 1 × 105 cells/ml and washed with PBS. After that, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Tween in PBS (PBST) and blocked with a 5% BSA working solution. After being diluted 1:100, the first antibody was added to the cells and incubated at 4 °C overnight. The cells in the negative control group were treated with PBS rather than the primary antibody. The next day, the cells were allowed to recover at 37 °C for 45 min, washed with PBS, and then incubated with a sheep anti-rabbit secondary antibody (diluted 1:1000) at room temperature for 30 min. Finally, after washing with PBS, the slides were sealed with glycerin and photographed under a microscope. For relative fluorescence analysis, all settings such as condenser opening, objective, zoom, exposure time, and gain parameters were maintained constant for all samples.
Initially fluorescence images were converted to Lab Stack and then split into single channel images. 32-bit images of single channel were converted into 8-bit images. The gray value of each pixel represents the fluorescence intensity of the point. Mean fluorescence intensity (MFI), mean gray value, was calculated using integrated intensity per unit area. A threshold was then chosen using the ‘Auto threshold’ function of ImageJ. Furthermore, the image was processed with the Analyze Particles algorithm of ImageJ to determine the number of single fluorescence cells computationally. The mean fluorescence intensity of single-cell was quantified in ImageJ using the ROI (region of interest) toolbox.
Measurement of nitric oxide and inflammatory factors production
The BV-2 microglial cells in the logarithmic growth phase were suspended and seeded in 6-well culture plates (10 × 104 cells/mL). The cells were randomly divided into 5 groups. After 24 h, the drugs at the appropriate concentrations, as determined by the CCK-8 assay, were added to the three drug groups, while serum-free medium was added to the blank control and LPS groups. The cells in the five groups were incubated for 24 h. All groups except the blank control group were stimulated with 1 μg/mL LPS for 24 h. The cell supernatant was collected, and the level of NO in the medium was measured with a nitric oxide one-step kit. The levels of IL-1β, IL-6 and TNF-α in the cell supernatant were measured by ELISA kits according to the manufacturer’s instructions.
Western blot analysis
After discarding the culture media of the five groups of BV-2 cells, the cells were washed with PBS twice, and 2 μL of the protease inhibitor PMSF and 200 μL of protein lysate were added. After lysis for 30 min, the cells were centrifuged; the precipitate was discarded, and the supernatant was collected. The proteins were separated by SDS-PAGE (10%) and then transferred onto PVDF membranes. The membranes were incubated with specific antibodies (anti-iNOS, anti-p38, anti-pho-p38, anti-JNK, anti-ERK, anti-JAK-2, anti-STAT3 and anti-α-Tubulin) diluted 1:2000 overnight at 4 °C. The membrane was then washed with TBST, destained, dried, and incubated with a secondary antibody diluted 1:5000 at 37 °C for 2 h. Using α-Tubulin as an internal reference, the relative densities of the bands were analyzed by ImageJ software.
Statistical analysis
Each experiment was repeated more than three times, and the experimental results were expressed as mean ± standard error. The comparison between two groups was compared by one-way analysis of variance (ANOVA) followed by the LSD post-hoc test using SPSS 23.0 software, p < 0.05 was considered to be statistically significant.
Discussion
Negative symptoms have long been challenging in the treatment of schizophrenia. Minocycline and atypical antipsychotics have been reported to be more effective in attenuating negative symptoms [
16,
21], but the specific mechanism underlying their effects is poorly understood. In this study, we first reported that risperidone exerted stronger anti-inflammatory and neuroprotective effects than haloperidol in microglia stimulated with LPS through the MAPK and JAK-STAT signaling pathways, revealing the underlying mechanisms of minocycline and atypical antipsychotics in the treatment of negative schizophrenia symptoms.
Under LPS stimulation, microglia are activated, and their morphology changes from branched to amoeboid. Amoeboid microglia can release inflammatory mediators and proinflammatory cytokines, thus causing neuronal damage. Numerous studies [
18,
22,
23] have found that antipsychotic drugs can inhibit the inflammatory response caused by microglial activation, but few studies have investigated the effects of antipsychotic drugs on the morphological changes attributed to microglial activation. In the present study, we found that minocycline and risperidone inhibited morphological changes and reduced the protein expression of OX-42 in LPS-induced activated microglia, while haloperidol did not rescue cell morphology as effectively as minocycline and risperidone. This further confirmed the inhibitory effect of antipsychotic drugs on the activation of microglia.
In vivo [
10,
24] and in vitro [
15,
18] studies have confirmed that minocycline and atypical antipsychotics, such as risperidone, can inhibit the production of proinflammatory cytokines in activated microglia, which is consistent with the findings of our study. The anti-inflammatory effect of typical antipsychotics in vivo has been reported. Two studies [
25,
26] found that flupentixol and trifluperidol reduced the release of IL-1β, IL-2 and TNF-α by activated microglia, and chlorpromazine and loxapine [
27] have also been reported to reduce the release of IL-1β and IL-2 by activated microglia. A recent study using single-cell RNA-sequencing has provided further evidence that pathways associated with microglial activation and inflammation play a role in the response to antipsychotics. The study also found that long-term exposure to atypical antipsychotic drugs, such as olanzapine, resulted in significantly more differentially expressed genes in mouse striatal microglia compared to typical antipsychotic drugs like haloperidol [
28]. However, research on whether haloperidol can inhibit the release of proinflammatory cytokines by activated microglia is limited. Another recent study found that both haloperidol and risperidone reduce the pro-inflammatory action of BV2 cells [
29].In the present study, we explored the anti-inflammatory effect of haloperidol in vitro and found that it had a slight anti-inflammatory effect, as it inhibited the production of IL-6 but not IL-1β and TNF-α. These data are consistent with the findings of a previous study [
18] showing that risperidone significantly inhibited inflammatory responses following interferon-gamma-induced microglial activation.
NO has been reported to be related to the etiology of schizophrenia. iNOS is the key enzyme responsible for NO production and can be found in neuronal cells [
30]. Clinical [
31] and postmortem histochemical [
32] studies have found that NO and iNOS in the brains and plasma of schizophrenia patients are expressed at higher levels than in those in the brains and plasma of healthy controls. In a neurodevelopmental model of schizophrenia, the use of minocycline was found to reduce iNOS expression in the prefrontal cortex and caudate-putamen while also preventing morphometric abnormalities in the third ventricle [
33]. Both in vivo [
10] and in vitro [
18,
34] studies have found that atypical antipsychotics, such as risperidone and olanzapine, can inhibit the production of NO and the expression of iNOS caused by the activation of microglia. However, whether typical antipsychotics have similar effects remains unclear. In the present study, both risperidone and haloperidol inhibited the production of NO but had no significant effect on iNOS expression. The lack of effect of risperidone on the expression of iNOS may have been related to the concentration of drug administered, as the concentrations of antipsychotics used in this study were selected based on their effects on cell viability, and a concentration gradient was not used. Researchers have differing opinions on whether haloperidol can inhibit the production of NO in activated microglia [
18,
34]. The results of this study suggest that haloperidol does inhibit NO production in activated microglia but that the effect of haloperidol is weaker than that of risperidone. Whether haloperidol can inhibit NO production remains to be verified by further experiments.
The MAPK signaling pathway plays an important role in regulating neuroplasticity and inflammation and is closely related to negative effects caused by the activation of microglia and the secretion of cytokines [
35‐
37]. A study involving a model of mild neuroinflammation [
10] reported that risperidone suppressed the increase in p38-MAPK expression in the prefrontal cortex. In the present study, minocycline and risperidone inhibited the LPS-induced increase of JNK, ERK and dephosphorylation of p38, which are involved in the MAPK signaling pathway, while haloperidol suppressed the activation of only ERK signaling and dephosphorylation of p38. The above results indicate that minocycline and risperidone exert anti-inflammatory effects through the same MAPK pathway, while haloperidol affects only part of the pathway.
The JAK-STAT signaling pathway, which mediates the signals that induce the early secretion of inflammatory cytokines in activated microglia and may also be closely related to the gene expression of late inflammatory mediators [
38], is involved in neuroimmune regulation. Previous study [
39] has found that the activation of microglia induced by ganglioside and interferon is involved in the immune inflammatory response in the central nervous system through the JAK-STAT signaling pathway. The results herein demonstrated that minocycline suppressed the activation of JAK2 and STAT3 induced by LPS, while risperidone and haloperidol suppressed the activation of only STAT3. These results suggest that the JAK-STAT signaling pathway is involved in the anti-inflammatory effects of minocycline, risperidone and haloperidol.
The limitations of our study include the use of in-vitro conditions and immortalized BV-2 microglial cells instead of primary microglial cells. While BV-2 microglial cells are commonly used for microglial studies due to their similarity to primary microglia, further research using primary cultures and in vivo settings is necessary to validate our findings.
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