VSP-2 attenuates secretion of inflammatory cytokines induced by LPS in BV2 cells by mediating the PPARγ/NF-κB signaling pathway

Abstract Neuroinflammation, characterized by microglial activation and the subsequent secretion of inflammatory cytokines, plays a pivotal role in neurodegenerative diseases and brain injuries, often leading to neuronal damage and death. Alleviating neuroinflammation has thus emerged as a promising strategy to protect neurons and ameliorate neurodegenerative disorders. While peroxisome proliferator-activated receptor gamma (PPARγ) agonists have demonstrated potential therapeutic actions on neuroinflammation, their prolonged use, such as with rosiglitazone, can lead to cardiac risks and lipid differentiation disorders. In this study, we investigated the effects of a newly synthesized PPARγ agonist, VSP-2, on secretion of inflammatory cytokines in BV2 cells. Treatment with VSP-2 significantly reduced the mRNA and protein levels of proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, VSP-2 attenuated the phosphorylation of nuclear factor kappa B (NF-κB) (65 kD) and IκBα, as well as the nuclear translocation of NF-κB (65 kD). Additionally, the use of PPARγ small interfering RNA was able to attenuate the effects of VSP-2 on proinflammatory cytokines and the NF-κB pathway. In conclusion, our findings suggest that VSP-2 effectively suppressed the expressions of IL-1β, IL-6, and TNF-α via the PPARγ/NF-κB signaling pathway. Given its potential therapeutic benefits, VSP-2 may emerge as a promising candidate for the treatment of neurodegenerative diseases or brain injuries associated with neuroinflammation.


Introduction
Neuroinflammation, a common occurrence in numerous neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease (AD) [1][2][3], is a primary contributor to neuronal damage.Microglia, the immune cells, and "defenders" in the brain, play a key role in mediating inflammation in the central nervous system (CNS) [4].In neurodegenerative diseases, microglia become activated and release inflammatory cytokines, facilitating the clearance of harmful factors, such as amyloid plaques in AD [5,6].This mechanism mitigates neuroinflammation during neurodegeneration.Nevertheless, the persistent presence of protein misfolding, aggregation, and activation throughout the progression of these diseases leads to sustained neuroinflammation [6][7][8].Consequently, microglia release increased levels of proinflammatory cytokines, resulting in neuronal damage [8,9].Therefore, the reduction in proinflammatory cytokines in neuroinflammation is considered a viable approach to mitigating brain damage and a strategy for the treatment of neurodegenerative diseases [10,11].
The production of proinflammatory cytokines and neuroinflammation is regulated by numerous signaling pathways [12,13].Evidence is accumulating that the classical nuclear factor kappa B (NF-κB) pathway is chronically active in neuroinflammation, promoting the expression of proinflammatory cytokines [14,15].When exposed to bacterial lipopolysaccharides (LPS) or other cellular stimuli, NF-κB complex, a dimer of p50 and p65, becomes activated and translocated to the cell nucleus, thereby inducing the production of proinflammatory cytokines [16,17].Conversely, suppressing the NF-κB pathway can attenuate the production of proinflammatory cytokines and mitigate the progression of inflammation in CNS [18,19].
Rosiglitazone (Ros), an antidiabetic drug, has demonstrated the potential to ameliorate many neuroinflammatory diseases by downregulating the expression of proinflammatory factors [20,21].This is achieved by activating the peroxisome proliferator-activated receptor gamma (PPARγ), inhibiting NF-κB binding to the promoters of proinflammatory genes [22].However, the long-term use of Ros is associated with high risks of heart failure, myocardial infarction, and weight gain, limiting its application in inflammatory diseases [23][24][25].These observed side effects are believed to be linked to the mechanism by which Ros activates PPARγ [26].To address these limitations, a new PPARγ agonist, activating through an alternative mechanism, could be a superior therapeutic for neuroinflammation.SR1664, a PPARγ inhibitor, binds to a distal region of the PPARγ ligand binding pocket, distinct from the binding domain of Ros [26,27].By leveraging the binding advantages of SR1664 and mitigating the binding disadvantages of Ros, we designed and synthesized VSP-2.The aim of this research is to explore the impact of VSP-2 on the suppression of inflammatory cytokine secretion in BV2 cells, along with its underlying mechanism, particularly involving PPARγ and the NF-κB pathway.

Cell culture
The mouse microglia BV2 cells were provided by the laboratory of Yuehan Zhou and purchased from BeNa Culture Collection (China).BV2 cells were cultured in DMEM medium (Thermo Fisher Scientific, USA) with a supplement of 10% FBS and 1% penicillin-streptomycin solution.The cells were maintained in a humidified incubator with an atmosphere of 5% CO 2 /95% air at 37°C.

Cell viability assay
BV2 cells (9,000 cells per well) were seeded into 96-well plates and allowed to adhere approximately for 15-18 h.Subsequently, cells were exposed to nine different concentrations of VSP-2 (0.01-100 μM) for 12 or 24 h.After incubation with MTT solution (0.5%) for 4 h, the supernatant was removed, and formazan crystals within the cells were completely dissolved with DMSO (150 μL).The absorbance was measured at wavelength of 490 nm using a microplate spectrophotometer.

Cell transfection
BV2 cells (9.5 × 10 5 cells per well) were seeded in 6-well plates and allowed to reach 70-80% confluency overnight before transfection.For each well, a mixture containing 100 pmol specific small interfering RNA (siRNA) for PPARγ, 4 μL of Lipo8000 transfection reagent, and 125 μL of Opti-MEM Medium was added to the cells for 6 h.

Statistical analysis
All data were expressed as the mean value ± standard deviation (SD).Statistical comparisons between two groups were performed using t-test, and comparisons among multiple groups were performed using one-way ANOVA analysis, followed by Tukey's test with GraphPad Prism software (v6.0 for Windows, GraphPad Software, USA).P < 0.05 was considered a significant statistical difference.

Effect of VSP-2 on BV2 cells' viability
The MTT assay was performed to investigate the impact of VSP-2 on the viability of BV2 cells.Cells were exposed to varying concentrations of VSP-2 (ranging from 0.01 to 100 μM) or DMSO (0.1%) for durations of 12 or 24 h.The results indicated a reduction in BV2 cells viability upon treatment with 100 μM VSP-2 for 12 h (P < 0.001, Figure 1b) and concentrations ranging from 3 to 100 μM for 24 h (Figure 1c).To mitigate the effects of cell death induced by higher VSP-2 concentrations or prolonged exposure, concentrations of 1, 3, and 10 μM for 12 h were selected for subsequent experimentation, limited to a duration of 12 h.

VSP-2 exerted a concentrationdependent effect on the production of proinflammatory cytokines
Elevated proinflammatory cytokines are hallmarks of neuroinflammation [31].To explore the function of VSP-2 on LPS-induced BV2 cells, we quantified the expression levels of IL-1β, IL-6, and TNF-α using RT-qPCR and Western blot analysis.BV2 cells were pretreated with VSP-2 (1, 3, and 10 μM) or Ros (1 μM) for 12 h, followed by LPS stimulation (0.1 μg/mL) for 24 h.The significantly increased expression of IL-1β, IL-6, and TNF-α mRNA were repressed with different concentrations of VSP-2 or Ros.Notably, the effect of 10 μM VSP-2 on mRNA expression of these proinflammatory cytokines was comparable to that of 1 μM Ros.Furthermore, treatment with 10 μM VSP-2 or 1 μM Ros resulted in IL-1β and IL-6 mRNA levels nearing those of the control group (Figure 2a).Comparable to mRNA expression, the elevated protein levels of IL-1β (17 kD and 31 kD), IL-6, and TNF-α were also reduced by VSP-2 or Ros treatment (Figure 2b and c).The effect of VSP-2 at concentrations ranging from 3 to 10 μM on the protein expression of these proinflammatory cytokines was similar to that of 1 μM Ros.
The data indicated a concentration-dependent inhibitory effect of VSP-2 on the upregulation of proinflammatory cytokines at both mRNA and protein levels.At a concentration of 10 μM, VSP-2 exhibited comparable effects to Ros.

VSP-2 blocked LPS-induced activation of NF-κB pathway
Evidence indicates that NF-κB pathway has been activated during neuroinflammation [32].To assess whether treatment with VSP-2 can attenuate the activation of the NF-κB pathway, we examined the protein expression of phosphorylated NF-κB (65 kD), total NF-κB (65 kD), phosphorylated IκBα, and total IκBα in BV2 cells using Western Blot analysis.Our findings reveal that both increasing concentrations of VSP-2 and a constant concentration of 1 μM Ros suppressed phosphorylation of IκBα and NF-κB.At concentrations of 3 and 10 μM, these treatments effectively abrogated LPS-induced phosphorylation, returning levels to those observed in the control group (Figure 3a-c).In contrast, the protein level of IκBα remained relatively unchanged across all groups following LPS stimulation (Figure 3d).Once activated outside the nucleus, NF-κB complex translocate into the nucleus to regulate gene expression [33,34].To assess the impact of VSP-2 on NF-κB pathway, we employed immunofluorescence to detect the NF-κB nuclear translocation.Our findings indicate that NF-κB nuclear translocation induced by LPS stimulation was progressively suppressed by increasing concentrations of VSP-2 (Figure 4).Notably, at a concentration of 10 μM, VSP-2 exerted a similar inhibitory effect to 1 μM Ros.These results suggested that VSP-2 blocked the activation of the NF-κB pathway triggered by LPS.Furthermore, at a concentration of 10 μM, the inhibitory effect of VSP-2 is comparable to that of 1 μM Ros.

PPARγ siRNA reversed the function of VSP-2 on the expression of proinflammatory cytokines
To confirm the function of PPARγ in downregulation of proinflammatory cytokines mRNA and protein by VSP-2, we transfected BV2 cells with control siRNA or PPARγ siRNA (siPPARγ) for 6 h.Total mRNA or protein was harvested for RT-qPCR or Western blot analysis 36 or 48 h after transfection respectively.As shown in Figure 5a, siPPARγ 3 exhibited the highest silencing efficiency.Consistent with this, PPARγ protein levels were significantly reduced following transfection with siPPARγ 3 (Figure 5b and c).Therefore, siPPARγ 3 was chosen for subsequent experiments.
After transfection with siPPARγ 3 (hereinafter referred to as siPPARγ), BV2 cells were stimulated with 0.1 μg/mL LPS and subsequently treated with 10 μM VSP-2.The mRNA levels of IL-1β, IL-6, and TNF-α were downregulated by VSP-2 treatment.However, this suppressive effect of VSP-2 on the mRNA expression of the proinflammatory cytokines was reversed by the knockdown of PPARγ using siPPARγ (Figure 6a).Furthermore, the protein levels of these cytokines, which were reduced by VSP-2, were also reversed by siPPARγ treatment (Figure 6b and c).These findings suggested that suppressive action of VSP-2 on the expression of proinflammatory cytokines was mediated through PPARγ.

PPARγ siRNA reversed the effect of VSP-2 on NF-κB pathway
To elucidate the mechanism by which PPARγ mediates the blockade of NF-κB signaling pathway by VSP-2, we quantified the protein levels of p-IκBα, p-NF-κB (65 kD), IκBα, NF-κB (65 kD), and nuclear NF-κB (65 kD) by Western blot analysis.Treatment with VSP-2 led to a decrease in the phosphorylation of both NF-κB (65 kD) and IκBα.However, the inhibitory effect of VSP-2 on phosphorylation of these proteins was reversed by knockdown of PPARγ (Figure 7a and b).
Concurrently, an increase in nuclear NF-κB was observed upon transfection with siPPARγ, suggesting that the regulatory action of VSP-2 on nuclear NF-κB was reversed by PPARγ knockdown (Figure 7c and d).Collectively, these findings suggested that VSP-2 blocked the NF-κB pathway through PPARγ.

Discussion
Neuroinflammation encompasses the inflammatory response of the CNS, which is triggered by internal or external stimuli [2,35].Persistent neuroinflammation can lead to neuronal damage and even affect the entire brain.Therefore, alleviating neuroinflammation may help to protect neurons and potentially impact the entire brain, emphasizing the need for therapeutic strategies to mitigate this inflammatory response.In the present study, we successfully synthesized VSP-2 and demonstrated its ability to alleviate production of neuroinflammatory cytokines in BV2 cells by downregulating the expression of proinflammatory cytokines.Furthermore, our findings suggest that VSP-2 decreases production of neuroinflammatory cytokines of BV2 cells by suppressing NF-κB pathway and activating PPARγ.Drawing upon the structural advantages of SR1664 and the structural disadvantages of Ros, we designed VSP-2, which was synthesized through a series of reactions including amide bond formation, ester hydrolysis, and carbon-nitrogen coupling.Ros is known as a full agonist for PPARγ and typically binds to helix 12 (H12) of the PPARγ ligand binding domain, leading to the formation of  an AF-2 motif that regulates gene transcription [26,36,37].In contrast, SR1664 acts as a PPARγ inhibitor binding to PPARγ without directly interacting with H12 [26].Given the structural similarities between VSP-2 and SR1664, we hypothesize that VSP-2 binds to PPARγ in a manner that is comparable to SR1664.However, this hypothesis remains to be further validated through subsequent studies.
Despite potential differences in the mechanisms of PPARγ activation between Ros and VSP-2, VSP-2 demonstrates a comparable ability to attenuate the increase in inflammatory cytokines following microglial activation.In our study, BV2 cells were stimulated with LPS to induce similar microglial response in neuroinflammatory reaction, leading to elevated expressions of IL-1β, IL-6, and TNF-α.Administration of VSP-2 or Ros resulted in reduced mRNA and protein levels of the proinflammatory factors.These findings suggest that, in the inflammatory response of neurodegenerative diseases, VSP-2 may also possess the ability to reduce inflammation, similar to Ros.
The mechanism underlying VSP-2's ability to suppress inflammatory cytokines is analogous to Ros, involving the NF-κB signaling pathway.Evidence indicates that the hyperactivation of the NF-κB pathway plays a pivotal role in the inflammatory response, facilitating the release of proinflammatory cytokines [38,39].In the normal state, the NF-κB complex, composed of a dimer of p50 (NF-κB1) and p65 (NF-κB 3 or RelA), remains bound to the inhibitory proteins of κB (IκBs) [40].Upon exposure to bacterial LPS or other noxious cellular stimuli, IκB kinase (IKK) is activated, resulting in phosphorylation of the IκB proteins, such as IκBα [41].Consequently, phosphorylated IκBs are isolated from the NF-κB complex and degraded by the proteasome [16].This degradation process triggers the activation of the NF-κB complex, leading to its translocation to the nucleus and subsequent binding to the specific NF-κB DNA-binding sites, thereby promoting the production of proinflammatory cytokines [15,17].Consistent with previous studies, we observed elevated levels of p-IκBα and p-NF-κB (65 kD) following LPS stimulation, indicating activation of the NF-κB pathway.Notably, treatment with VSP-2 or Ros resulted in decreased protein levels and NF-κB nuclear translocation, suggesting that VSP-2 may exert inhibitory effects on the NF-κB pathway comparable to Ros.Interestingly, we did not observe the degradation of IκBα following LPS exposure.This observation could be attributed to NF-κB feedback mechanisms leading to upregulated IκBα expression [42], or alternatively, VSP-2 may have a greater impact on NF-κB nuclear translocation.
The inhibitory effects of VSP-2 on NF-κB signaling pathway may be linked to PPARγ, a nuclear receptor involved in fat differentiation, glucose metabolism, and inflammatory responses [22,43].PPARγ and its agonists have been reported to modulate neuroinflammation and are used as a nonspecific class of drugs in AD mouse models [44].These agonists can stimulate PPARγ, promoting its binding to the p65 subunit in the nucleus, thereby inhibiting the NF-κB complex binding to the promoters of proinflammatory genes [45,46].In our previous work, we developed VSP-2 as a PPARγ agonist, distinct from Ros in its mode of activation.Notably, both VSP-2 and Ros share similar abilities in reducing inflammatory cytokines and suppressing the NF-κB signaling pathway.To further investigate the role of PPARγ in VSP-2's mechanism, we knocked down the PPARγ using a siPPARγ.Our results suggest that the effects of VSP-2 on proinflammatory factor expression and NF-κB signaling pathway were abolished after PPARγ knockdown.This finding verifies that the effect of VSP-2 on reducing proinflammatory factors and inhibiting NF-κB signaling pathway is PPARγ-dependent.
In the present study, we discovered that VSP-2 effectively reduces the release of inflammatory factors by activating PPARγ and inhibiting the NF-κB pathway, a mechanism similar to that of Ros.This suggests that VSP-2 has the potential to emerge as a viable alternative to Ros in the treatment of neuroinflammation associated with neurodegenerative disorders.However, our study has limitations.First, our focus has been primarily on the anti-inflammatory effects of VSP-2, yet confirmation of its agonistic activity on PPARγ is still outstanding.This could be addressed by performing molecular docking method or TR-FRET assay, as well as examining mRNA expression of CD36, a target gene of PPARγ.Second, although we have demonstrated VSP-2's impact on NF-κB signaling pathway, further research is needed to ascertain whether it indeed triggers the binding of PPARγ to the p65 subunit in the nucleus.Finally, although VSP-2 attenuates inflammatory factors, further studies are required to assess its neuroprotective potential against neuronal damage.

Figure 5 :
Figure 5: siPPARγ 3 downregulated PPARγ mRNA and protein expression.Control siRNA or siPPARγ was applied to BV2 cells for 6 h.Total mRNA or protein was obtained 36 or 48 h after transfection, respectively.(a) siPPARγ 3 exhibited greater silencing efficiency than siPPARγ 1 and 2. (b and c) PPARγ protein level was decreased after transfection with siPPARγ 3. Identical letters indicate non-significant differences (P > 0.05), while different letters indicate significant differences (P < 0.05), n = 3.

Figure 6 :
Figure 6: Knockdown of PPARγ reversed the suppressive effect of VSP-2 on the expression of proinflammatory cytokines.BV2 cells were transfected with siPPARγ for 6 h, then stimulated with LPS for 24 h, followed by treatment with 10 μM VSP-2 for an additional 12 h.(a) Knockdown of PPARγ abolished the downregulation of proinflammatory cytokines mRNA by VSP-2.(b and c) Knockdown of PPARγ reversed the inhibitory effect of VSP-2 on the protein levels of proinflammatory factors.Identical letters indicate non-significant differences (P > 0.05), while different letters indicate significant differences (P < 0.05), * P < 0.05, *** P < 0.001, n = 3.

Figure 7 :
Figure 7: Knockdown of PPARγ reversed the inhibitory effect VSP-2 on the NF-κB signaling pathway.BV2 cells were transfected with siPPARγ for 6 h, followed by LPS stimulation for 24 h and subsequent treatment with 10 μM VSP-2 for 12 h.(a and b) Knockdown of PPARγ attenuated the inhibitory effect of VSP-2 on phosphorylation of NF-κB and IκBα.(c and d) Knockdown of PPARγ led to an increase in nuclear NF-κB.Identical letters indicate nonsignificant differences (P > 0.05), while different letters indicate significant differences (P < 0.05), n = 3.