Pinocembrin Protects Human Brain Microvascular Endothelial Cells against Fibrillar Amyloid-β 1−40Injury by Suppressing the MAPK/NF-κB Inflammatory Pathways

Cerebrovascular accumulation of amyloid-β (Aβ) peptides in Alzheimer's disease (AD) may contribute to disease progression through Aβ-induced microvascular endothelial pathogenesis. Pinocembrin has been shown to have therapeutic effects in AD models. These effects correlate with preservation of microvascular function, but the effect on endothelial cells under Aβ-damaged conditions is unclear. The present study focuses on the in vitro protective effect of pinocembrin on fibrillar Aβ 1−40 (fAβ 1−40) injured human brain microvascular endothelial cells (hBMECs) and explores potential mechanisms. The results demonstrate that fAβ 1−40-induced cytotoxicity in hBMECs can be rescued by pinocembrin treatment. Pinocembrin increases cell viability, reduces the release of LDH, and relieves nuclear condensation. The mechanisms of this reversal from Aβ may be associated with the inhibition of inflammatory response, involving inhibition of MAPK activation, downregulation of phosphor-IKK level, relief of IκBα degradation, blockage of NF-κB p65 nuclear translocation, and reduction of the release of proinflammatory cytokines. Pinocembrin does not show obvious effects on regulating the redox imbalance after exposure to fAβ 1−40. Together, the suppression of MAPK and the NF-κB signaling pathways play a significant role in the anti-inflammation of pinocembrin in hBMECs subjected to fAβ 1−40. This may serve as a therapeutic agent for BMEC protection in Alzheimer's-related deficits.


Introduction
Brain microvascular endothelial cells (BMECs) contribute to the formation of the blood-brain barrier (BBB) and are indispensable to the creation and maintenance of brain homeostasis. They are also the early targets of various toxic molecules, such as amyloid-peptides (A ) and reactive oxygen species (ROS), of neurodegeneration [1,2]. Recent findings demonstrate that fibrillar A (fA ) accumulates not only in the brain parenchyma but also at sites in the cerebrovasculature, particularly around arterioles and capillaries of the cerebral cortex and leptomeninges, resulting in cerebral amyloid angiopathy (CAA) [3,4]. CAA is closely correlated with Alzheimer's disease (AD) and affects 80-90% of AD patients [4][5][6]. The pathology of microvascular CAA is associated with cerebrovascular dysfunction, including destruction of the blood-brain barrier (BBB) and enhancement in vessel-associated inflammation [4,[6][7][8].
In AD, cerebrovascular amyloid deposition primarily comprises aggregated A . Cerebrovascular deficiencies involved in CAA are paralleled by A -mediated responses in cultured endothelial cells [9][10][11], indicating that A can elicit cerebrovascular deficits that contribute to disease progression. In particular, A treatment of endothelial monolayers augments monocyte adhesion and subsequent transendothelial migration [12][13][14], reduces endothelial antioxidant efficacy [15][16][17], stimulates inflammatory responses [18], and increases endothelial permeability [11,19,20]. In this way, endothelial damage appears to be associated with the activation of multiple types of signal transduction. A stimulates the production of ROS, which may be important signaling molecules in endothelial pathological events, especially inflammation [21]. ROS may act as second messengers through the activation of many intracellular signaling pathways, such as mitogen-activated protein kinases (MAPKs) and transcription factors, notably nuclear factor-kappa B (NF-B). The MAPKs are a group of serine and threonine kinases. They regulate gene expression by modulating transcription factors such as NF-B [22]. They have also been implicated in inflammatory and cell death pathways on cerebral damage [23,24]. The NF-B signal pathway, which is activated by ROS-dependent mechanisms [25,26], also plays an important role in the gene expression of a large number of proinflammatory cytokines. In this way, the hope of counteracting these deleterious events might involve strategies aimed at interrupting the oxidant and inflammatory cascades.
Pinocembrin (5,7-dihydroxyflavanone, Figure 1) is a flavonoid abundant in propolis. It can be extracted as a pure compound and has been shown to be effective in the protection of brain injury from ischemic and A impairment. In 2008, it was approved by the State Food and Drug Administration of China for the treatment of stroke. Pinocembrin was shown to protect against ischemic injury and reduce the area of cerebral infarction in ischemia models via neurovascular protection by decreasing the severity of oxidative damage and inhibiting inflammatory responses [27][28][29]. It was also demonstrated that pinocembrin protected the BBB from ischemic injury by mitigating ultrastructural damage, reducing permeability, improving microvascular blood flow, and protecting BMECs from oxygen-glucose deprivation/reoxygenation-induced toxicity [30]. Recently, pinocembrin was found to alleviate cognitive deficits in intracerebroventricular A -injected and A -precursor protein (APP)/presenilin 1 (PS1) double transgenic AD mouse models [31,32]. It has also been shown to attenuate cerebral degeneration in AD by inhibiting inflammatory pathways mediated by the receptor for advanced glycation end products and to be involved in the preservation of the microvascular function, maintenance of the BBB integrity, and reduction of inflammatory mediator levels [31,32]. Relative studies have demonstrated beneficial effects of pinocembrin in endothelial cells, such as the improvement of the biological functions of endothelial progenitor cells and the suppression of vascular endothelial growth factor-induced angiogenesis in the mouse aortic ring [33,34]. Although previous studies have suggested that pinocembrin may show protection in endothelial cells from insults, no preexisting study has reported the direct effect of pinocembrin on brain microvascular endothelial cells mediated by A . Therefore, current in vitro studies are yet to be conducted to investigate the effect of pinocembrin on human BMECs (hBMECs) in the fA 1-40 damaged condition and explore its mechanism during inflammatory processes.

Material and Methods
2.1. Cell Culture and Treatment. hBMECs were purchased from ScienCell Research Laboratories (ScienCell Research Laboratories, Carlsbad, CA, USA). The hBMECs were cultured in endothelial cell complete medium (ScienCell Research Laboratories, Carlsbad, CA, USA) in a 37 ∘ C incubator with 5% CO 2 , according to the supplier's recommendations. Experiments were conducted within cell passages 4-6. At these passages, cells displayed a cobblestone appearance, which is morphologically normal for endothelial cells. All treatments were performed after the hBMECs were 60-70% confluent.
Synthetic A 1-40 was purchased from Sangon Biotech Company (Shanghai, China) and dissolved in water to make a stock solution of 0.1 mM to foster the fibrillization state, as previously reported [35,36]. Pinocembrin (purity >

Lactate Dehydrogenase (LDH) Release
Assay. LDH released from hBMEC-compromised membranes was determined using a CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, cells were grown to 70% confluence in 96-well culture plates and were exposed to fA 1-40 , pinocembrin, or both for the indicated lengths of time. Then 50 L culture supernatants were transferred to a separate assay plate, leaving behind the cells that were used for Hoechst 33343 and ROS detections. Then 50 L of CytoTox-ONE Reagent was added to each well, and the contents of the plates were mixed for 30 s. The LDH assay was allowed to proceed at room temperature for 10 min prior to an addition of 25 L/well stop solution containing 10% sodium dodecyl sulfate. The contents of the wells were mixed by shaking the plates for 10 s prior to measurement of resorufin fluorescence (560 nm excitation/590 nm emission).

2.4.
Hoechst 33342 and DCFH 2 -DA Staining Assay. Nuclear change and intracellular ROS level were measured in hBMECs using Hoechst 33342 (Dojindo Laboratory, Kumamoto, Japan) and DCFH 2 -DA (2 ,7 -dihydrodichlorofluorescein diacetate, Sigma Chemical Co., St. Louis, MO, USA) staining, respectively. Nuclei were labeled with 5 g/mL of Hoechst 33342 at 37 ∘ C for 10 min after the fA 1-40 injury or pinocembrin treatment. ROS was measured based on the oxidation of DCFH 2 -DA to 2 ,7dichlorofluorescein, and DCFH 2 -DA was added to the culture plates at a final concentration of 5 M at 37 ∘ C for 40 min. The intensity of fluorescence was detected and analyzed by a Cellomics ArrayScan V TI HCS Reader (Cellomics Inc., Pittsburgh, PA, USA) provided with the Morphology Explorer BioApplication software. The images were acquired using the 386/23 nm excitation/460/40 nm emission and 485/20 nm excitation/535/50 nm emission filters, respectively. Nuclear change and ROS level were quantified by the value of average fluorescent intensity [31].

Intracellular Superoxide Dehydrogenase (SOD) and Glutathione Peroxidase (GSH-Px) Assay.
After the fA 1-40 injury and pinocembrin treatment, hBMECs were collected and crushed by sonication (60 W at 0.5 s intervals for 10 min). The cell lysate was centrifuged at 10,000 g for 15 min, and the supernatant was used to measure the activities of SOD and GSH-Px using a WST-1 based SOD inhibition kit (Dojindo Laboratory, Kumamoto, Japan) and a GSH-Px detection kit (Jiancheng Bioengineering, Nanjing, China), respectively. The solutions in each well were added according to the manufacturer's protocols. The absorbance of the endpoint reactions was measured using a SpectraMax Plus microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA). The relative SOD inhibition of each sample was calculated using the following equation: where A1, A2, A3, and As were the absorbance at 440 nm for the uninhibited test, blank sample, blank reagent, and sample, respectively [37]. GSH-Px activity was determined by quantifying the rate of oxidation of reduced GSH to oxidized GSH by H 2 O 2 and catalyzed by GSH-Px. One unit of GSH-Px was defined as the amount that could reduce the level of GSH at 412 nm by 1 M in 1 min per mg of protein.
Briefly, images were acquired in independent channels with fixed exposure times. Based on the Hoechst nuclear staining, a nuclear region mask was created and used to quantify nuclear protein distribution. By expanding the nuclear region mask, a concentric ring was generated and used as an approximation of the cytosolic compartment. Cytosolic and nuclear staining intensities were normalized to total nuclear region and cytosolic ring area, allowing for the quantification of protein translocation between the nucleus and cytosol for phosphor-ERK1/2, phosphor-p38, phosphor-MK2, phosphor-SAPK/JNK, and NF-B p65. The capacity of translocation of the four proteins was illustrated by the value of Mean CircRingAvgIntenDiff. For the c-Jun detection, nuclear fluorescence intensity was acquired and calculated as the value of protein expression.

ELISA Assay for Tumor Necrosis Factor
(TNF-), Interleukin-1 (IL-1 ), and Interleukin-6 (IL-6). Group divisions and treatments were as described above. The culture medium was collected and centrifuged for 10 min at 4 ∘ C to eliminate the cell debris. The proinflammatory cytokines of TNF-, IL-1 , and IL-6 in culture medium were measured by ELISA assays. Quantitative levels were measured according to the manufacturer's instructions (Jiameinuosi Biotech, Beijing, China). The optical density was measured at 450 nm, and values were calculated with reference to standard curves.
2.9. Statistical Analysis. All data are represented as the mean ± the standard error of the mean (SEM). Comparisons were performed using one-way analysis of variance (ANOVA), and multiple comparisons were performed using post-hoc least significant difference comparisons. A value of <0.05 was considered statistically significant.

Pinocembrin Protects hBMECs from
1-40 -Induced Cytotoxicity. In the present study, the direct protective effects of pinocembrin on hBMECs against fA 1-40 -induced toxicity were examined in three cytotoxicity assays. In the MTS assay, cell viability was found to be significantly decreased in the presence of 20 M fA 1-40 in hBMECs (Figure 2 The cytoprotective effects of pinocembrin were confirmed in nuclear changes by Hoechst 33342 staining as well. Control hBMECs were uniformly stained with faint blue fluorescence. In contrast, hBMECs treated with fA  showed denser nuclei with more intense fluorescence indicating nuclear shrinkage or condensation which is one of the early signs of damage ( < 0. found to significantly increase the viability of cells, decrease the level of LDH, and relieve the injury of nucleus injured by fA  . Pinocembrin detected at the same concentrations did not show effects in these cytotoxicity assays. Due to the dose-dependent manner in which pinocembrin is found to act, concentrations ranging from 3.0 M to 30.0 M are selected for further investigation in hBMECs in the presence to fA 1-40 .

Pinocembrin Cannot Remarkably Regulate the Redox Imbalance of hBMECs against
1-40 -Induced Toxicity. A exerts toxicity against the endothelial cells of the brain via enhanced ROS production and redox imbalance [37,39]. In this study, fA 1-40 increased endogenous ROS generation in hBMECs by about 2.95-fold ( < 0.001, Figures 3(a) and 3(b)). fA 1-40 also reduced endothelial antioxidant efficacy through decreasing GSH-Px and SOD activities, two markers of oxidative stress, to 43.36% and 58.82%, respectively ( < 0.001, Figures 3(c) and 3(d)). These effects indicated a severe redox imbalance in this endothelial cell model. However, pinocembrin neither decreased the ROS generation nor increased GSH-Px or SOD activity in the present model at the concentrations evaluated here, suggesting that pinocembrin cannot exert sufficient effects on amelioration of the antioxidative ability of hBMECs subjected to fA 1-40induced toxicity.

Pinocembrin Inhibits the MAPK Pathways in hBMECs against
1-40 -Induced Toxicity. MAPKs are regulated by ROS in endothelial cells to express the proinflammatory phenotype through the phosphorylation activation and the subsequent nuclear transduction [40][41][42]. Here, in control hBMECs, basal levels of phosphor-p38 and phosphor-MK2 were significantly confined to the cytosolic and nuclear compartment, showing a low and a high Mean CircRingAvgIntenDiff value, respectively. Depending on stimulus of fA 1-40 , phosphor-p38 and phosphor-MK2 translocation were activated, as illustrated by the significant increase and a marked decrease in Mean CircRingAvgIntenDiff values, respectively ( < 0.001, Figures 4(a) and 4(b)). Pinocembrin treatment significantly inhibited the p38 MAPK pathway. The translocation of cytosolic phosphor-p38 to the nucleus and nuclear phosphor-MK2 to the cytoplasm was significantly inhibited at concentrations of 3.0 M, 10.0 M, and 30.0 M in a dose-dependent manner ( < 0.05, < 0.01, < 0.001).
The basal level of phosphor-SAPK/JNK was confined to the cytosolic compartment, shown as a low Mean CircRingAvgIntenDiff value in control hBMECs. Similarly, its downstream phosphor-c-Jun was seen in low average fluorescence intensity in the nucleus. After fA 1-40 treatment, phosphor-SAPK/JNK translocation was promoted by a significant increase in Mean CircRingAvgIntenDiff values ( < 0.001, Figures 4(a) and 4(c)). The level of phosphor-c-Jun increased by 3.6-fold, as indicated by average intensity of fluorescence in the nucleus ( < 0.001, Figures 4(a) and 4(d)). Pinocembrin treatment was found to significantly inhibit the SAPK/JNK pathway. The

Discussion
As an extension of previous research, the present study clarified the beneficial effects of pinocembrin on AD-associated microvascular endothelial pathology. The present findings indicate that pinocembrin can protect hBMECs from fA 1-40 -induced toxicity. In these effects, pinocembrin increases cell viability, reduces the amount of LDH release, relieves nuclear condensation, inhibits the MAPK pathways, relieves I B degradation, blocks NF-B p65 nuclear translocation, and reduces the levels of extracellular proinflammatory cytokines. In addition, pinocembrin can inhibit phosphor-IKK activation modestly. However, pinocembrin does not show remarkable effects on the regulation of redox imbalances. Pinocembrin's ability to protect microvascular endothelial cells from fA 1-40 mainly contributes to antiinflammation.
The microvascular endothelial cells of the brain form a highly specialized endothelial tissue that serves as the BBB. These cells appear to be a primary target and an important responsive component of cerebral inflammation in AD [1,2]. Cerebrovascular A deposition plays a role in the progression of AD. Fibrillar A accumulates at sites in the cerebrovasculature, particularly around arterioles and capillaries of the cerebral cortex and leptomeninges [3,4]. This phenomenon is present in more than 80% of AD patients, and cerebrovascular amyloidosis is causally involved in the development of neurodegeneration in this disease [42].
Given experimental data reporting the contradictory findings of A toxicity in endothelial cell culture [9,10,43], the present work first involved confirmation of whether fA 1-40 treatment causes any significant cell death in the present experimental paradigm. In line with the decreased viability of hBMECs in the presence of fA 1-40 , the increased release of LDH and the injury of nuclei were observed in the same manner. These results demonstrate that fibrillar A 1-40 is directly toxic to hBMECs.
Herein, the effective administration conditions for pinocembrin were screened using both control and fA 1-40injured hBMECs in the first step. Pinocembrin at the determined optimal concentrations of 3.0 M, 10.0 M, and 30.0 M is found to significantly increase the viability of cells injured by fA  . Results of the decrease of LDH release and the relief of nuclear injury also indicate the protective effects of pinocembrin on this process. It is here confirmed that there are no differences among the pinocembrin treatments in control cells, indicating that pinocembrin has no toxic effect under basal conditions.
Oxidative stress is implicated in AD pathology. Excessive generation of ROS within endothelial cells in response to A  leads to oxidative stress and cellular injury [39]. In vitro studies have revealed that oxidative stress results in dysfunction of the endothelial cell, destroying the integrity of the vascular barrier and leading to increased endothelial permeability, mitochondrial dysfunction, generation of cytokines, chronic inflammatory processes, and amyloid deposition in blood vessels, which are involved with the imbalance of endothelial transductions during the pathogenesis of Alzheimer's deficits [11,19,[44][45][46][47][48]. A strategy involving neuroprotective properties is here recommended for reduction of the severity of oxidative injury and maintaining the integrity of the BBB for the treatment of brain damage.
Pinocembrin is a flavonoid so that it might be thought to be effective in quenching free radicals. Here, pinocembrin merely produced a slight increase in the effectiveness of the antioxidant defense system of hBMECs when subjected to fA  . Pinocembrin was reported to possess a limited antioxidative effect in ischemia models [28,29], but it is not found to produce sufficient effects on the regulation of the redox imbalance under the conditions that are rich in A [31,32]. In general, the cytoprotective capacity of flavonoids against different insults has been mainly attributed to their antioxidant potency [49,50]. Nonetheless, cytoprotection of flavonoids was no more defined to be correlated with the antioxidation potency [51]. The report that the hydroxy substitutions in the A-ring (C5 and C7) and in position C3 (C-ring) of the flavones would be necessary to afford neuroprotection indicated that the structural requirements for cytoprotection are different from those that afford antioxidant capacity [52]. Additionally, many flavonoids show an important pharmacological effect on modulating the activities of protein kinases, lipid kinases, and enzymes of mitochondrial respiratory chain independent of their antioxidant capacity [53][54][55]. Therefore, although antioxidation is not involved in the major mechanisms that prevent A -mediated toxicity, pinocembrin may act synergistically with other crucial mechanisms for the treatment in our experimental model.
The MAP kinase family is correlated with activation of intracellular signal events during the pathological process of AD. To date, at least three major MAPK cascades have been described that involve the activation of ERK, SAPK/JNK, and p38 MAPK in the brain. The ERK cascade is mostly responsive to mitogenic and differentiation stimuli, whereas the JNK and p38 MAPK pathways are preferentially activated by proinflammatory cytokines and extracellular stress [56] and contribute to the regulation of synaptic function, the BBB permeability, inflammatory response, and apoptotic process [57][58][59]. One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by ROS. In case MAPK-signaling pathways are activated, several inflammatory cytokines are further overproduced and released [56].
MAPK activation may be secondary to many different extracellular stimuli, but the series of phosphorylation cascade activation events are critical to the responses. Our results showed that fA 1-40 exposure induced activation of ERK1/2, p38 MAPK/MK2, and SAPK/JNK-c-Jun cascades secondary to overproduction of ROS in the hBMECs. This is consistent with the results of the proinflammatory nature of the MAPK pathway [60]. Pinocembrin is found to markedly inhibit the activation of phosphorylated p38 MAPK/MK2 and SAPK/JNK-c-Jun pathways at each tested concentration, and only the high concentration (30 M) was effective in the blockade of phosphor-ERK1/2 activation. These results are essentially consistent with the finding that pinocembrin modulates transduction of these MAPKs in neuronal and endothelial cells as in previous reports as well [31,32]. Besides, many molecular components are involved in apoptosis tightly linked to the presence and activation of MAPK family, one of which is the JNK-mediated cytochrome release contributing to caspase-3 activation and the onset of apoptosis [61,62]. Therefore, these inseparable processes can be inhibited not only by antioxidant treatment but also by MAPK activation inhibition [63][64][65]. Although pinocembrin does not show strong antioxidative effects through the clearance of ROS, coincided with the reversal from the endothelial injury, it is plausible that pinocembrin treatment attenuates fA 1-40induced cytotoxicity, at least in part, through the inhibition of ERK1/2, SAPK/JNK, and p38 cascades. However, in this study, we only determined the effect of pinocembrin on translocation cascades of MAPKs following the phosphorylation. It is worth investigating more precisely in the future the effect of pinocembrin on the phosphorylation levels of MAPK family.
It is evidenced that ROS and MAPK signal pathways are involved in the regulation of NF-B activation in response to stress. As a redox-sensitive transcription factor, NF-B is activated via the activation of I B-kinase complex which then phosphorylates I B on Ser 32 and Ser 36, resulting in its ubiquitination and subsequent proteasomal degradation as well as the release of NF-B, which can translocate into the nucleus to activate the transcription of proinflammatory target genes, such as TNF-, IL-1 , and IL-6 [66,67]. The protective effects of pinocembrin against A -stimulated endothelial responses were also mediated by blocking NF-B signaling pathways. However, different concentrations of pinocembrin were required to inhibit specific inflammatory transduction in these signal pathways. Only 30 M of pinocembrin was found to significantly decrease the levels of phosphorylation of IKK and IKK , which indicates a slight reduction in the concentration of the I B-kinase complex degradation of the NF-B signaling regulated by this compound. Activation of NF-B needs I B to be phosphorylated, which then leads to targeted degradation of I B . The following dissociation of I B causes the translocation of NF-B from the cytoplasm to the nucleus where it binds and triggers the inflammatory gene expression. All concentrations of pinocembrin tested in this study significantly attenuated the degradation of I B and inhibited the nuclear translocation of p65. Although multiple signal molecules are involved in the NF-B pathway, we suggested that pinocembrin could remarkably inhibit the activation of NF-B signal transduction by attenuating the degradation of the inhibitory protein I B and blocking the translocation of NF-B p65.
The level of proinflammatory cytokines also conduces to evaluate endothelial cell injuries. In response to A stimulus, a series of intracellular signaling cascades are initiated which ultimately lead to activation of inflammation and the release of proinflammatory cytokines in endothelial cells [68]. Many studies demonstrated that some of the proinflammatory cytokines, such as TNF-, IL-1 , and IL-6, play a key role in the development and maintenance of inflammation, and this cytokine elevation is associated with neurodegenerative diseases [69]. TNF-, IL-1 , and IL-6 may also serve as biomarkers of the NF-B inflammatory pathway [70]. Considering that the transcription levels of these cytokines are under the control of NF-B, we investigated whether the reduced secretion levels of TNF-, IL-1 , and IL-6 in pinocembrin-treated cells were due to inhibition of NF-B signaling. In line with the above findings regarding NF-B transduction, the levels of TNF-, IL-1 , and IL-6 in culture medium were significantly decreased by pinocembrin at all tested concentrations. Thus, we suggest that pinocembrin has anti-inflammatory effects against fA 1-40 -induced toxicity, and its mechanisms can be routed through the NF-B signaling pathways to inhibit the secretion of TNF-, IL-1 , and IL-6.
Further, MAPK transductions which have been implicated to be key modulators in inflammatory signaling cascades are associated with activation with NF-B in the inflammatory response [71][72][73]. These MAPKs regulate NF-B activation through I B / kinase activation which induces I B degradation [72,73]. Thus, we deduce that the NF-B along with MAPKs may participate in the amplifying loop of the inflammatory responses after hBMECs subjected to fA  , and that the modulations of the three-tiered cascades provide a rationale to evidence the therapeutic effects of pinocembrin against fA 1-40 -mediated toxicity in hBMECs. Previous studies evidenced that the MAPKs especially p38 and JNK have been implicated in the regulation of inflammatory mediators, including the proinflammatory cytokines, which make them potential targets for anti-inflammatory therapeutics [74,75]. Since p38 and JNK activation in the present study was more responsive to the inhibitory effects of each concentration (from 3 M to 30 M) of pinocembrin than ERK that required the high concentration (30 M), it is assumed that the anti-inflammatory effect of pinocembrin against fA 1-40 in hBMECs may depend primarily on the inhibition of the p38 MAPK and JNK activation. As another point of view, the relevant results may provide the explanation that due to the secondary effect in the inflammatory loop pinocembrin showed a slight reduction in the phosphorylation levels of IKK and IKK .
Taking the results of cytotoxicity assays and inflammatory measurements together, pinocembrin is capable of reducing the fA 1-40 damage, increasing cell survival, and decreasing the proinflammatory cytokine levels in a consistent and effective manner, suggesting that pinocembrin might affect the regulation of the balance of cell survival and inflammatory response in cerebral endothelial cells. Previous studies have shown the therapeutic role and mechanism of pinocembrin involved in Alzheimer's-related deficits. It has been demonstrated that it is effective in conferring neurovascular protection through maintenance of neuropil ultrastructure and the reduction of glial activation and levels of inflammatory mediators in the brain [31,32]. Pinocembrin has been shown to promote neurovascular inflammatory pathways against various types of A toxicity [31,32]. In the present study, the direct recovery of hBMECs by pinocembrin was confirmed and the underlying mechanisms were identified. Three possible intracellular signaling pathways were found to be involved in the modulation of inflammatory responses of pinocembrin in endothelial cells ( Figure 6). Firstly, because pinocembrin suppresses activation of several subfamilies of MAPK-signaling cascades induced by A injury in hBMECs in accordance with the inhibition of neurovascular inflammatory pathways, it is here suggested that MAPKs pathway may be one of the mechanisms by which pinocembrin inhibits overproduction of proinflammatory cytokines. Secondly, because modulation of NF-B activation provides a mean of reducing the generation of inflammatory factors against multiple A insults conducted both in this study and in previous research, it is here suggested that pinocembrin also exerts an anti-inflammatory effect through attenuating the degradation of I B and blocking the nuclear translocation of NF-B p65. Thirdly, as the NF-B along with MAPKs may participate in the amplifying loop of the inflammatory responses in our experiments, pinocembrin shows a slight reduction in the phosphorylation levels of IKK as a compensative modulation in the NF-B signaling.

Conclusion
In summary, the present study demonstrates that fA 1-40induced cytotoxicity in hBMECs can be rescued by pinocembrin treatment. The endothelial protective effects against fA 1-40 exhibited by pinocembrin are achieved via antiinflammation. The mechanisms of this reversal from A may be involved in the inhibition of MAPK activation, the decrease in phosphor-IKK activation, the relief of I B degradation, the blockage of NF-B p65 nuclear translocation, and the reduction of proinflammatory cytokine release. Pinocembrin does not show sufficient activity on regulating the redox imbalance. Taken together, the suppression of MAPK and NF-QB signaling pathways might play a significant role in the endothelial protection of pinocembrin. In this way, it may serve as a potential therapeutic agent for BMECs in the prevention of Alzheimer's-related deficits.