Protective Effects of Sophoraflavanone G by Inhibiting TNF-α-Induced MMP-9-Mediated Events in Brain Microvascular Endothelial Cells

The regulation of matrix metalloproteinases (MMPs), especially MMP-9, has a critical role in both physiological and pathological events in the central nervous system (CNS). MMP-9 is an indicator of inflammation that triggers several CNS disorders, including neurodegeneration. Tumor necrosis factor-α (TNF-α) has the ability to stimulate the production of different inflammatory factors, including MMP-9, in several conditions. Numerous phytochemicals are hypothesized to mitigate inflammation, including the CNS. Among them, a flavonoid compound, sophoraflavanone G (SG), found in Sophora flavescens has been found to possess several medicinal properties, including anti-bacterial and anti-inflammatory effects. In this study, mouse brain microvascular endothelial cells (bMECs) were used to explore TNF-α-induced MMP-9 signaling. The effects of SG on TNF-α-induced MMP-9 expression and its mechanisms were further evaluated. Our study revealed that the expression of MMP-9 in bMECs was stimulated by TNF-α through the activation of ERK1/2, p38 MAPK, and JNK1/2 via the TNF receptor (TNFR) with a connection to the NF-κB signaling pathway. Moreover, we found that SG can interact with the TNFR. The upregulation of MMP-9 by TNF-α may lead to the disruption of zonula occludens-1 (ZO-1), which can be mitigated by SG administration. These findings provide evidence that SG may possess neuroprotective properties by inhibiting the signaling pathways associated with TNFR-mediated MMP-9 expression and the subsequent disruption of tight junctions in brain microvascular endothelial cells.


Introduction
Cerebrovascular cells, brain microvascular endothelial cells (bMECs), are essential components in the central nervous system (CNS).They have a crucial purpose in preserving the brain's microenvironment and facilitating various processes such as cerebral blood flow regulation, microvascular tone modulation, and maintenance of the integrity of the blood-brain barrier (BBB) [1,2].Nevertheless, the impairment of the vascular endothelium is an initial clinical observation, which is strongly associated with several vascular and cerebral disorders in individuals affected by atherosclerosis, stroke, and neurodegenerative conditions [3][4][5].The disruption and dysfunction of brain microvessels can lead to subsequent damage to neuronal cells within the surrounding microenvironment [3,5].Therefore, maintaining the integrity of the BBB to ameliorate brain disorders is a pivotal aspect in the treatment of several cerebral ailments.The potential for therapeutic intervention exists in preventing or reducing BBB disruption [6,7].Moreover, offering protective strategies for cerebrovascular cells has received increased attention in CNS diseases.
Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases with crucial functions for their pathophysiological roles, especially in extracellular matrix (ECM) turnover [8,9].Among those MMPs, MMP-9 plays an important role in morphogenesis, wound healing, and neurite outgrowth in the CNS [9,10].Overexpression of MMP-9 may have a role in the pathogenesis of brain illnesses resulting from various brain traumas [10,11].Previous studies have demonstrated that inflammatory cytokines and endotoxin could induce MMP-9 expression with their associated activity in brain cells [10,12].MMP-9 can also potentially play a role in the BBB integrity, leading to the onset of neuroinflammation through the influence of several detrimental elements, such as lipopolysaccharides [13].Previous reports have demonstrated that proinflammatory cytokines can induce MMP-9 expression and elicit functional alterations in brain cells, including human cerebral microvascular endothelial cells [14] and rat astrocytes [15,16].These results suggest that proinflammatory cytokines and MMP-9 may play important roles in neural inflammation and brain diseases, arousing our interest in investigating the potential effects of phytochemical compounds on MMP-9-associated events in bMECs.Here, we aim to elucidate the potential of tumor necrosis factor-α (TNF-α), a pivotal proinflammatory cytokine, to promote the MMP-9 expression in bMECs and its implications and underlying mechanism.
Phytochemicals present in natural products have shown potential roles in reducing the risk of several disorders, especially in neurodegenerative diseases [17][18][19].The probable underlying mechanisms may involve the inhibition of oxidative stress and the suppression of the synthesis of different proinflammatory mediators.To date, hundreds of natural products have been widely screened for anti-oxidative and anti-inflammatory properties in in vitro and in vivo experiments.Several natural products can be regarded as potential sources of highly effective molecules with antioxidant and anti-inflammatory properties, explaining their therapeutic and preventive benefits [18,19].Flavonoids belong to natural polyphenolic compounds exerting beneficial effects in cardiovascular and neurological disorders [20].Among these, sophoraflavanone G (SG), a flavonoid compound, has been isolated from Sophora flavescens, which possesses several medicinal properties, including anti-bacterial and anti-inflammatory effects [21,22].Previous reports have indicated that some natural flavonoid compounds exhibit potential neuroprotective agents to treat neurodegenerative disorders [20,23].Although there is extensive research reports that exist to demonstrate the effects of natural flavonoid compounds in various disorders, including brain disorders, there are no studies indicating the effects and mechanisms of SG in the CNS.Hence, we established the paradigm of tumor necrosis factor-alpha (TNF-α)-induced MMP-9 in bMECs to assess the impact of SG on MMP-9-mediated processes, specifically pertaining to the impairment of tight junction integrity.
Drawing on the aforementioned backgrounds and our prior investigations into the mechanisms of brain inflammatory responses triggered by MMP-9 induction [12], the research was conducted to evaluate the effects of SG on TNF-α-induced MMP-9 expression in bMECs.Herein, SG ameliorates the disruption of zonula occludens-1 (ZO-1) arranged integrity by TNF-α-induced MMP-9.Moreover, TNF-α-stimulated activation of TNFR-dependent MAPKs and the NF-κB signaling pathway also been inhibited by SG pretreatment.These findings indicate that the SG may possess neuroprotective properties through its anti-inflammatory properties, which help to prevent the disruption of ZO-1 organized integrity mediated by MMP-9.

TNF-α Induces MMP-9 Expression and Its Effect on Brain Microvascular Endothelial Cells (bMECs)
TNF-α is a common proinflammatory cytokine that contributes to various inflammationrelated disorders, including CNS [24].To determine whether TNF-α treatment in bMECs cells could induce MMP-9 expression, bEnd.3 cells were treated with TNF-α (30 ng/mL), and then the conditioned media were collected for gelatin zymography analysis.In Figure 1A, TNF-α-treated bMECs at the concentration of 30 ng/mL showed significant increase in MMP-9 expression in a time-dependent manner, especially between the 16 h and 24 h time points, where MMP-2 and GAPDH were used as internal controls.In order to look into the potential relationship between the transcriptional and translational expression of MMP-9 induced by TNF-α, a RT-PCR analysis was carried out.As shown in Figure 1B, the expression of MMP-9 mRNA was increased in a time-dependent manner upon TNF-α stimulation with a significant upregulation from the time point of 6 h to 24 h, where the expression of β-actin mRNA had no significant change and served as the internal control.These data revealed that the upregulation of MMP-9 by TNF-α treatment in bMECs is mediated through the MMP-9 transcriptional event.

TNF-α Induces MMP-9 Expression and Its Effect on Brain Microvascular Endothelial Cells (bMECs)
TNF-α is a common proinflammatory cytokine that contributes to various inflammation-related disorders, including CNS [24].To determine whether TNF-α treatment in bMECs cells could induce MMP-9 expression, bEnd.3 cells were treated with TNF-α (30 ng/mL), and then the conditioned media were collected for gelatin zymography analysis.In Figure 1A, TNF-α-treated bMECs at the concentration of 30 ng/mL showed significant increase in MMP-9 expression in a time-dependent manner, especially between the 16 h and 24 h time points, where MMP-2 and GAPDH were used as internal controls.In order to look into the potential relationship between the transcriptional and translational expression of MMP-9 induced by TNF-α, a RT-PCR analysis was carried out.As shown in Figure 1B, the expression of MMP-9 mRNA was increased in a time-dependent manner upon TNF-α stimulation with a significant upregulation from the time point of 6 h to 24 h, where the expression of β-actin mRNA had no significant change and served as the internal control.These data revealed that the upregulation of MMP-9 by TNF-α treatment in bMECs is mediated through the MMP-9 transcriptional event.
Figure 1.TNF-α induces MMP-9 expression and its effect on brain microvascular endothelial cells (bMECs).(A,B) TNF-α increases the expression of MMP-9 in a time-dependent manner.After treating with TNF-α (30 ng/mL) in bEnd.3 cells for the indicated time intervals, the MMP-9 expression and activity were analyzed by gelatin zymography, Western blot, and RT-PCR (GAPDH and β-actin as the internal controls).The intensity of zymographic and gene expression represent relative fold differences of protein and gene levels on the basis of densitometer quantitation.(C,D) MMP-9 is involved in TNF-α-induced disruption of ZO-1 arranged integrity.After growing on coverslips for 24 h, bEnd.3 cells were pretreated with MMP-9 inhibitor (9i, 1 µM) for 1 h and then incubated with TNF-α (30 ng/mL) for 24 h.After treatment, the conditioned media were collected for gelatin zymography (D), and the cells were fixed for immunofluorescence staining (C, ZO-1).The intensity of the zymographic represents relative fold differences of protein levels on the basis of densitometer quantitation.Data are shown as the mean ± SEM (n = 3).* p < 0.05; ** p < 0.01, as compared to untreated control.# p < 0.05, as compared to TNF-α treatment only.The image represents one of three individual experiments.(A,B) TNF-α increases the expression of MMP-9 in a time-dependent manner.After treating with TNF-α (30 ng/mL) in bEnd.3 cells for the indicated time intervals, the MMP-9 expression and activity were analyzed by gelatin zymography, Western blot, and RT-PCR (GAPDH and β-actin as the internal controls).The intensity of zymographic and gene expression represent relative fold differences of protein and gene levels on the basis of densitometer quantitation.(C,D) MMP-9 is involved in TNF-α-induced disruption of ZO-1 arranged integrity.After growing on coverslips for 24 h, bEnd.3 cells were pretreated with MMP-9 inhibitor (9i, 1 µM) for 1 h and then incubated with TNF-α (30 ng/mL) for 24 h.After treatment, the conditioned media were collected for gelatin zymography (D), and the cells were fixed for immunofluorescence staining (C, ZO-1).The intensity of the zymographic represents relative fold differences of protein levels on the basis of densitometer quantitation.Data are shown as the mean ± SEM (n = 3).* p < 0.05; ** p < 0.01, as compared to untreated control.# p < 0.05, as compared to TNF-α treatment only.The image represents one of three individual experiments.
The upregulation of MMP-9 has been demonstrated with a main contribution to several brain injuries and various CNS disorders [14].Therefore, the effects of TNF-αinduced MMP-9 expression on the functions of bMECs for BBB formation were further investigated, specifically focusing on the maintenance of tight junction protein ZO-1 in its arrangement and integrity.As shown in Figure 1C, the images demonstrated that ZO-1 was completely associated with the plasma membrane (control); however, this integrity was disrupted following TNF-α stimulation (30 ng/mL) for 24 h (Figure 1C, middle).Moreover, the pretreatment of the MMP-9 inhibitor (9i, 1 µM) effectively suppressed the detrimental effects of TNF-α on the structural integrity of ZO-1 (9i/TNF-α), indicating that MMP-9 was indeed involved in this phenomenon.After gelatin zymography analysis to check MMP-9 expression in this particular scenario, TNF-α-induced MMP-9 expression and activity were markedly attenuated upon pretreatment of MMP-9 inhibitor (MMP9i, 1 µM; Figure 1D).These data suggested that TNF-α induces the disruption of ZO-1 arranged integrity in bMECs via the MMP-9 pathway.

Role of NF-κB in TNF-α-Induced MMP-9 Expression in bMECs
The involvement of NF-κB-dependent pathways in MMP-9 expression has been documented in various cell types [12].In order to determine more dynamically whether the TNF-α-induced MMP-9 expression is mediated through NF-κB activation in bMECs, a NF-κB inhibitor (Bay11-7082) was used.Pretreatment with Bay11-7082 (Bay, 1 µM) in bMECs significantly inhibited TNF-α-induced MMP-9 expression (Figure 4A), suggesting that NF-κB was involved in this event.Furthermore, the results of RT-PCR analysis demonstrated that Bay pretreatment attenuated TNF-α-induced MMP-9 mRNA expression (Figure 4B).These results indicated that the transcription factor NF-κB participates in the signaling of TNF-α-induced MMP-9 expression in bMECs.Furthermore, TNF-α stimulated time-dependent phosphorylation of p65 (a NF-κB subunit) in these cells (Figure 4C) with a significant increase within a time frame of 5 to 30 min, where the event was markedly attenuated by Bay pretreatment (1 µM) (Figure 4C).We further confirmed the TNF-αstimulated NF-κB activation by immunofluorescence stain with a p65 subunit antibody to assess the nuclear translocation of p65 NF-κB.As shown in Figure 4D, TNF-α-stimulated translocation of p65 from the cytosol into the nucleus.On the other hand, pretreatment with Bay (1 µM) inhibited TNF-α-stimulated translocation of p65 (Figure 4D).These data indicated that activation of NF-κB, including phosphorylation and translocation of the p65 subunit, is critical for TNF-α-induced MMP-9 expression in bMECs.Moreover, as shown in Figure 4E, pretreatment with U0126 (U0, 1 µM), SB202190 (SB, 30 µM), or R-7050 (R, 1 µM) significantly inhibited TNF-α-stimulated p65 NF-κB phosphorylation at 10 min, suggesting that TNF-α stimulated p65 NF-κB activation (phosphorylation) via TNFR-dependent MAPKs pathways in bMECs.According to the obtained results, we demonstrated that demonstrated that TNF-α induces MMP-9 expression via TNFR-dependent activation of ERK1/2, p38, and JNK1/2 linking to NF-κB cascade in bMECs.Based on the results, we demonstrated that TNF-α induces MMP-9 expression and thereby disrupts the ZO-1 arranged integrity in bMECs.Therefore, we evaluated the effects of the natural flavanone compound SG on the aforementioned phenomenon.To achieve this objective, cells were pretreated with SG (1 µM) for 1 h and following TNF-α stimulation for the indicated times.In Figure 5A, SG pretreatment significantly blocked TNF-α-induced MMP-9 expression in bMECs.To further analyze the effect on MMP-9 transcription level, SG pretreatment attenuated TNF-α-induced MMP-9 mRNA expression in bMECs (Figure 5B).These results indicated that SG might have brain-protective potential by reducing MMP-9 expression induced by TNF-α in bMECs.Next, we determined the effects of SG on the signaling pathway of TNF-α-induced MMP-9 expression, including ERK1/2, p38, JNK1/2 MAPKs, and NF-κB.As shown in Figure 5C, SG pretreatment (1 µM) markedly reduced TNF-α-stimulated phosphorylation of ERK1/2 both in time-and concentration-dependent manners but not p38 and JNK1/2.These results indicated that SG can inhibit TNF-α-induced MMP-9 expression by attenuating ERK1/2 activation in bMECs.

Sophoraflavanone G, a Natural Flavanone Compound, Inhibits TNF-α-Induced MMP-9
Expression by Attenuating TNFR/MAPK (ERK1/2)/NF-κB Cascade Based on the results, we demonstrated that TNF-α induces MMP-9 expression and thereby disrupts the ZO-1 arranged integrity in bMECs.Therefore, we evaluated the effects of the natural flavanone compound SG on the aforementioned phenomenon.To achieve this objective, cells were pretreated with SG (1 µM) for 1 h and following TNF-α teraction modes between SG and TNFR protein by DS molecular docking.The molecular docking result indicated that SG can interact with the key protein of TNFR (Figure S1).All these data demonstrate that TNFR/MAPKs/NF-κB cascade plays a regulative role in TNFα-induced MMP-9 expression in bMECs.As expected, pretreatment with SG (1 µM) also significantly repressed the response (Figure 5F), demonstrating that SG may play the role of an inhibitor in TNF-α-induced MMP-9 expression by blocking NF-κB activation in bMECs.The effect of SG on TNF-α-stimulated p65 NF-κB phosphorylation, translocation, and transcriptional activity were further explored in bMECs.The results showed that pretreatment with SG (1 µM) significantly blocked TNF-α-stimulated p65 NF-κB phosphorylation in time-and concentration-dependent manners (Figure 5D).Additionally, we also confirmed that pretreatment with SG (1 µM) markedly inhibited TNF-α-stimulated translocation of p65 NF-κB into the nucleus by immunofluorescence stain (Figure 5E).Moreover, previous studies have demonstrated that there is a regulative NF-κB binding site in the MMP-9 promoter region [15].Thus, we next elucidated whether TNF-α can induce NF-κB transcriptional activity and the effect of SG on this event; a pGL4.32 plasmid with a κB response element was used.As shown in Figure 5F, TNF-α markedly induced the transcriptional activity of κB at 6 h (~3 fold), which was significantly diminished by preincubating with R-7050 (R-7, 1 µM), U0126 (U0, 1 µM), SB202190 (SB, 30 µM), SP600125 (SP, 10 µM), or Bay11-7082 (Bay, 1 µM), suggesting that TNF-α induces NF-κB activation via the TNFR/MAPKs-dependent pathway in bMECs.Here, we further investigated the interaction modes between SG and TNFR protein by DS molecular docking.The molecular docking result indicated that SG can interact with the key protein of TNFR (Figure S1).All these data demonstrate that TNFR/MAPKs/NF-κB cascade plays a regulative role in TNF-α-induced MMP-9 expression in bMECs.As expected, pretreatment with SG (1 µM) also significantly repressed the response (Figure 5F), demonstrating that SG may play the role of an inhibitor in TNF-α-induced MMP-9 expression by blocking NF-κB activation in bMECs.

Discussion
MMPs have a significant role in various biological processes across diverse tissues, including major CNS disorders such as stroke, neurodegenerative diseases, and malignant glioma.Among MMPs, MMP-9 expression and activation play a critical role not only in tissue remodeling but also in the pathogenesis of brain diseases [10,11,27], such as being a risk factor in the integrity of the BBB [28].The BBB disruption is a prominent characteristic observed in CNS illnesses that involve neuroinflammation.Cerebrovascular cells, namely bMECs, play a crucial role in preserving the microenvironment and functional integrity of the BBB [1].The brain can be protected from BBB disruption, cell death, and enhanced neuroinflammation through the inhibition of MMP activity using pharmacological inhibitors or gene knock-out techniques [28,29].Some proinflammatory cytokines such as TNF-α and IL-1β have been shown to induce the expression of MMP-9 following brain injury, including BBB damage [30,31].Our previous data have demonstrated that cytokine IL-1β-induced MMP-9 expression in brain astrocytes may cause astrocytic motility [16].The findings of these researches indicate that the increase in MMP-9 expression caused by proinflammatory mediators could have a significant impact on brain injury and BBB disruption.These results could provide a potential therapeutic approach for addressing brain inflammatory response even for neurodegenerative conditions.The research from the pharmacological studies and knockout mice has demonstrated that MMP-9 and its upstream signaling pathways may be therapeutic targets in brain injury and inflammatory disorders.In this study, we first demonstrated that TNF-α can induce MMP-9 expression with the detailed underlying mechanisms and their effects in bMECs.Next, we evaluated whether SG, the natural flavanone product, possesses brain-protective effects on TNF-α-induced MMP-9-related events and its underlying mechanism.All these data suggested that in bMECs, SG inhibited TNF-α-induced MMP-9-mediated disruption of ZO-1 arranged integrity through blocking the TNFR-dependent MAPKs (ERK1/2) cascade leading to activation of the NF-κB pathway.
In our study, we first found that TNF-α can upregulate MMP-9 expression even in transcriptional and translation events in bMECs.Next, we demonstrated that TNF-α-mediated upregulation of MMP-9 can affect the main tight junction protein ZO-1 arranged integrity, which is closely associated with the degree of BBB destruction, and it becomes a symbol of the BBB dysfunction [6,32].Our results further showed that TNF-α-induced disruption of ZO-1 integrity mediated by MMP-9 in bMECs was inhibited by TNFR antagonist R-7050, including expression of mRNA, protein, and disruption of ZO-1 integrity.These data suggest that the TNF/TNFR system plays a critical role in MMP-9-dependent disruption of ZO-1 integrity in BBB breakdown, where the observation is consistent with previous results [7,33].To explore the protective natural compound for the event, a natural flavanone compound SG was evaluated.The results showed that SG (1 µM) attenuated TNF-α-induced MMP-9 expression in bMECs both in transcriptional and translational levels (Figure 5A,B).Here, we also found that SG can interact with the key protein of TNFR by DS molecular docking (Figure S1).Moreover, pretreatment of SG also affected TNF-α-induced disruption of ZO-1 arranged integrity (Figure 6A), suggesting that SG may have a brain-protective effect by blocking TNF-α-induced events such as BBB dysfunction.These results are the first finding that SG can reduce the disruption of ZO-1 integrity through the MMP-9 pathway in TNF-α-challenged bMECs.
MAPKs act as a pivotal inflammatory event through activation of MAPK-dependent cascades in different cell types [34,35].In our observation of the signaling of TNF-α-induced MMP-9 expression in bMECs, MAPKs also serve as second messengers to modulate inflammatory responses.In several experimental models with CNS inflammation and injury, the activity of MAPKs is dysregulated [34,35].Several studies also indicate that proinflammatory factors induced MMP-9 expression in various cells via the MAPKs-dependent pathways [36].In brain astrocytes, our previous studies have demonstrated that activation of ERK1/2, p38, and JNK1/2 are required for MMP-9 upregulation by proinflammatory cytokines [15,16].Here, our data showed that the MAPKs participated in MMP-9 ex-pression upon TNF-α stimulation in bMECs via the TNFR-mediated pathway (Figure 3).The findings are similar with the report showing that IL-1β induces MMP-9 expression via ROS-dependent MAPKs in rat brain astrocytes [16].Moreover, SG markedly inhibited TNF-α-stimulated phosphorylation of ERK1/2 (Figure 5C) but not p38 MAPK and JNK1/2 in these cells.All these results indicated that SG can reduce TNF-α-induced MMP-9 expression through blocking the ERK1/2-dependent cascade in bMECs.The SG data are consistent with the previous reports showing that SG has anti-inflammatory activity by blocking the MAPKs-mediated pathway in LPS-activated mouse macrophages [37] and BV2 microglia [38] and Streptococcus mutans surface antigen I/II-challenged RAW 264.7 macrophages [39].These results suggested that MAPKs (ERK1/2) may also be effective and novel therapeutic targets for CNS-related diseases.
The progressive increase of MAPKs activity during the action of several proinflammatory factors not only causes damage signaling transduction but also modulates the gene expression pattern through functional alterations of transcription factors.The transcription factor NF-κB plays a critical role in the modulation of functional gene expressions in physiological and pathological events, such as inflammation, cell proliferation, and apoptosis [12,40].The induction of inflammatory mediators, such as MMP-9, in brain cells during brain inflammation is a result of numerous stimuli.This induction occurs through the activation of NF-κB in a MAPKs-mediated way [12].Our previous study demonstrated that NF-κB contributes to the upregulation of several genes, including MMP-9, by proinflammatory cytokines through a MAPKs-mediated manner in brain astrocytes [12].These results indicate that NF-κB is critical for upregulation of several inflammatory genes such as MMP-9 in the CNS inflammation [12,41].Therefore, in the present investigation, we focused on the effects of SG on TNF-α-stimulated NF-κB activation in bMECs.The transcription factor NF-κB participated in TNF-α-induced MMP-9 expression (Figure 4).Moreover, the TNFR-dependent activation of MAPKs (i.e., ERK1/2, p38, and JNK1/2) cascades was involved in TNF-α-stimulated NF-κB activation (Figure 4E).We further demonstrated that TNF-α-stimulated activation of p65/NF-κB, including phosphorylation, nuclear translocation, and transcriptional activity, which were significantly suppressed by SG (Figure 5D,F).These results demonstrated that SG can reduce TNF-α-induced MMP-9 expression via inhibiting NF-κB activation in bMECs.These results are consistent with the idea that SG inhibits the expression and production of inflammatory mediators by blocking the ERK/MAPK-mediated pathway and inhibiting the activation of NF-κB in macrophages [37,39].Finally, our data showed that increased MMP-9 expression by TNF-α stimulation was via TNFR-mediated MAPKs signals leading to activation of NF-κB, resulting in the disruption of ZO-1 integrity (representing BBB damage) in bMECs (Figure 6A).In this study, we also found that SG can alleviate this MMP-9-mediated event by inhibiting a series of signaling in bMECs, as mentioned in the text.However, there still exists an obstacle in clinical use due to the difficulties in flavonoid administration.Because of low bioavailability, SG needs to be formulated using some smart drug release and/or delivery technologies, like other flavonoid compounds [42][43][44].Therefore, the optimal delivery system for SG needs to be further verified, especially in the CNS system.

Cell Cultures and Treatments
Mouse brain microvascular endothelial cells (bMECs: bEnd.3) were cultured and used throughout this study as described previously [45].Cells were grown in DMEM/F-12 containing 10% FBS and antibiotics (100 U/mL penicillin G, 100 µg/mL streptomycin, and 250 ng/mL fungizone) at 37 • C in a humidified 5% CO 2 atmosphere.Confluence cells were released with 0.05% (w/v) trypsin/0.53mM EDTA for 5 min at 37 • C. The cell suspension (2 × 10 5 cells/mL) was plated onto 6-well culture plates (2 mL/well) or 10 cm culture dishes (10 mL/dish) for the measurement of protein or RNA expression, respectively.Culture medium was changed after 24 h and then every 3 days.Experiments were performed with cells from passages 5 to 13.When the inhibitors were used, cells were pretreated with the inhibitor for 1 h before exposure to TNF-α (30 ng/mL).Treatment of bMECs with these inhibitors alone had no significant effect on cell viability determined by an XTT assay.

MMP Gelatin Zymography
Growth-arrested cells were incubated with TNF-α for the indicated time intervals.After treatment, the cultured media were collected and analyzed by gelatin zymography [15].Gelatinolytic activity was manifested as horizontal white bands on a blue background.Because cleaved MMPs were not reliably detectable, only pro-form zymogens were quantified.

Western Blot Analysis
Growth-arrested cells were incubated with TNF-α at 37 • C for the indicated time intervals.The cells were washed with ice-cold PBS, scraped, and collected by centrifugation at 45,000× g for 1 h at 4 • C to yield the whole cell extract, as previously described [15].Samples were analyzed by Western blot, transferred to nitrocellulose membrane, and then incubated overnight using an anti-phospho-ERK1/2, phospho-p38, phospho-JNK1/2, phospho-p65 NF-κB, or GAPDH antibody.Membranes were washed four times with TTBS for 5 min each, incubated with a 1:2000 dilution of anti-rabbit horseradish peroxidase antibody for 1 h.The immunoreactive bands were detected by ECL reagents and captured by a UVP BioSpectrum 500 Imaging System (Upland, CA, USA).The image densitometry analysis was quantified by UN-SCAN-IT gel 6.1 software (Orem, UT, USA).

Immunofluorescence Stain
Growth-arrested cells were treated with 30 ng/mL TNF-α for the indicated time intervals, washed twice with ice-cold PBS, fixed with 4% (w/v) paraformaldehyde in PBS for 30 min, and then permeabilized with 0.3% Triton X-100 in PBS for 15 min.The stain was performed by incubating with 10% normal goat serum in PBS for 30 min followed by incubating with an anti-p65 NF-κB or ZO-1 antibody (1:200 dilution) for 1 h in PBS with 1% BSA, washing thrice with PBS, incubating for 1 h with a FITC conjugated goat anti-rabbit antibody (1:200 dilution) in PBS with 1% BSA, washing thrice with PBS, and finally mounting with aqueous mounting medium.The images were observed under a fluorescence microscope (Axiovert 200 M, Carl Zeiss, Göttingen, Germany).

Promoter-Luciferase Reporter Gene Assay
The κB binding sites were cloned to the pGL4.32 (luc2P/NF-κB-RE/Hygro) vector containing the luciferase reporter system.All plasmids were prepared by using QIAGEN plasmid DNA preparation kits.These constructs were transfected into bEnd.3cells by using a Lipofectamine reagent according to the instructions of the manufacturer.The transfection efficiency (~60%) was determined by transfection with enhanced GFP.After incubation with TNF-α, cells were collected and disrupted by sonication in lysis buffer (25 mM Tris, pH 7.8, 2 mM EDTA, 1% Triton X-100, and 10% glycerol).After centrifugation, aliquots of the supernatants were tested for promoter activity using a Dual-Luciferase ® Reporter Assay System (Promega, Madison, WI, USA).Firefly luciferase activities were standardized for Renilla luciferase activity.

Statistical Analysis
All data were estimated using the GraphPad Prism Program (GraphPad, San Diego, CA, USA, https://www.graphpad.com/,accessed on 1 December 2023).Quantitative data were analyzed by one-way ANOVA followed by Tukey's honestly significant difference tests between individual groups.Data were expressed as mean ± SEM.A value of p < 0.05 was considered significant.

Conclusions
Based on our observations and findings, Figure 6B depicts a brain-protective model of SG on TNF-α-mediated disruption of ZO-1 integrity in bMECs.Herein, SG pretreatment markedly inhibits the activation of related signaling molecules in TNF-α-induced MMP-9 expression, including ERK1/2-dependent activation of the NF-κB pathway.These results are the first evidence indicating that SG can inhibit MMP-9-mediated events in bMECs.These findings concerning the natural flavanone compound SG reduces TNF-αinduced ERK1/2-mediated NF-κB and MMP-9-dependent disruption of ZO-1 integrity in bMECs, suggesting that SG may be a promising natural candidate for relieving brain inflammatory events.

Figure 1 .
Figure 1.TNF-α induces MMP-9 expression and its effect on brain microvascular endothelial cells (bMECs).(A,B) TNF-α increases the expression of MMP-9 in a time-dependent manner.After treating with TNF-α (30 ng/mL) in bEnd.3 cells for the indicated time intervals, the MMP-9 expression and activity were analyzed by gelatin zymography, Western blot, and RT-PCR (GAPDH and β-actin as the internal controls).The intensity of zymographic and gene expression represent relative fold differences of protein and gene levels on the basis of densitometer quantitation.(C,D) MMP-9 is involved in TNF-α-induced disruption of ZO-1 arranged integrity.After growing on coverslips for 24 h, bEnd.3 cells were pretreated with MMP-9 inhibitor (9i, 1 µM) for 1 h and then incubated with TNF-α (30 ng/mL) for 24 h.After treatment, the conditioned media were collected for gelatin zymography (D), and the cells were fixed for immunofluorescence staining (C, ZO-1).The intensity of the zymographic represents relative fold differences of protein levels on the basis of densitometer quantitation.Data are shown as the mean ± SEM (n = 3).* p < 0.05; ** p < 0.01, as compared to untreated control.# p < 0.05, as compared to TNF-α treatment only.The image represents one of three individual experiments.

Figure 2 .
Figure 2. TNF-α induces MMP-9 expression via TNF receptor-dependent pathway.(A,B) Cells were pretreated with a TNF receptor antagonist R-7050 (1 µM) following TNF-α (30 ng/mL) incubation for the indicated times.After the stimulation, the MMP-9 expression and activity were analyzed by gelatin zymography, Western blot, and RT-PCR (GAPDH and β-actin as the internal controls).The intensity of the zymographic quantitated by densitometry and gene expression was expressed as fold of untreated control.Data are expressed as the mean ± SEM (n = 3).* p < 0.05, # p < 0.05, as compared to untreated control and TNF-α treatment only, respectively.(C) After stimulation, the cells were fixed and labeled with anti-ZO-1 antibody and a FITC-conjugated secondary antibody, and then the cell images were observed by immunofluorescence stain (ZO-1).The image represents one of three individual experiments.

Figure 5 .
Figure 5. Effects of SG on TNF-α-induced MMP-9 expression in bMECs.(A,B) After SG pretreatment (1 µM) and TNF-α (30 ng/mL) stimulation in cells for the indicated time intervals, samples were collected and assayed for MMP-9 expression by gelatin zymography with GAPDH protein expression as control by Western blot (A) and RT-PCR (B).(C,D) Cells pretreated with SG (0.1 or 1 µM) for 1 h and stimulated with TNF-α (30 ng/mL) were harvested, and the cell lysates were assessed for