Pectolinarin Inhibited LPS-stimulated Inammation in Microglial BV 2 Cells via NF-κB Signaling Pathway

Background: Neuro-inammation is regarded as one of the critical pathogenesis in neurodegenerative diseases, which is characterized by the activated microglial cells. Pectolinarin (Pec), a natural avonoid exists in many Chinese herbal medicines, has been reported to have various biological activities. However, the effects and mechanisms on neuro-inammation are not clear. Methods: In this study, the inhibitory effects and mechanisms of Pec on neuro-inammation were investigated in the LPS-stimulated microglial BV 2 cells. BV 2 microglial cells were treated with Pec or vehicle (1% DMSO), followed by LPS. ELISA, RT-PCR, NO assay, and Western blot were performed to examine the effects of Pec on neuro-inammatory responses. Results: We showed that Pec signicantly inhibited the expression of TNF-α and IL-6 in mRNA and protein levels induced by LPS. Moreover, the production of NO, iNOS and COX-2 were suppressed by Pec in LPS-stimulated microglial BV 2 cells. In addition, Pec inhibited LPS-induced inammation via NF-κB signaling pathway, as evidenced by reduction of the phosphorylation of IKK, the degradation of IκBα and the nuclear translocation of p65. Conclusions: Taken together, Pec exhibited anti-inammatory effects in LPS-stimulated microglial BV 2 cells via NF-κB signaling pathway, which might provide therapeutic potential for neuro-inammation and neurodegenerative diseases. NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; IKK, inhibitor of nuclear factor kappa-B kinase; IκBα, inhibitor of NF-κB; CNS, central nervous system; PD, Parkinson’s disease; AD, Alzheimer’s disease; LBP, LPS-binding protein ; TLR4, Toll-like receptor 4; RT-PCR, Real-time quantitative PCR; IL-1β, interleukin-1β; DMEM, Dulbecco’ modied Eagle’s medium; FBS, Fetal bovine serum; PTGS2, prostaglandin-endoperoxide synthase 2; ELISA, enzyme-linked immunosorbent assay; PVDF, polyvinylidene uoride.


Introduction
Neuro-in ammation is a defense mechanism to multiple exogenous stimuli and pathogens in the central nervous system (CNS) [1]. It is regarded as the pathogenesis of several neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD) [2][3][4]. Microglia, the main immune defense cells, constitute 10-15% of the glial cell population in the brain [5,6], which play a vital role in the innate immune response and represent the rst line of defense against invading pathogens and proin ammatory reactions [7,8].
In a resting stage, microglia survey the microenvironment in real-time with their rami ed processes and secrete various neurotrophic factors to help the development and maintenance of neuronal. When the microglia cells were activated, the shape of them could be changed from highly rami ed morphology into ameboid shape. In addition, a series of cellular and molecular events happened. Microglia would secrete a high level of pro-in ammatory factors and cytotoxic mediators, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) [9]. Therefore, regulation of microglial activation might represent a potential therapeutic strategy for neuro-in ammation.
Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, which is a strong stimulator of microglial activation [10]. LPS recognizes and binds with LPS-binding protein Page 3/16 (LBP) and glycosylphosphatidylinositol-anchored protein CD14, interacts with Toll-like receptor 4 (TLR4) and activates downstream signaling pathway. Activated TLR4 activates the NF-κB signal pathway [11], which is the most frequently used to investigate the mechanism of in ammatory responses in microglia.
In response to external stimulation, IκB family members can be phosphorylated and degraded, the NF-κB moved from the cytoplasm into the nucleus, leading to the expression of various pro-in ammatory mediators [12].
Traditionally, Chinese herbal medicine has been widely used to treat various diseases with little side effects, including neurodegenerative diseases [13][14][15][16][17]. Pec is a glycosylated avone which is rst isolated from Linaria vulgaris [18]. Moreover, Pec has widely reported due to its presence in many medicinal plants, such as Cirsium [19]. According to recent studies, Pec has turned out to be good biological activities, including anti-tumor, antioxidant [20], antiviral [21,22], anti-in ammatory [23,24] and anti-cancer effects. As for anti-in ammatory effects, a previous study showed that Pec inhibited the acidinduced writhing in mice in a dose-dependent manner [25]. However, the effects of Pec on neuroin ammation are still largely unknown. In this study, the anti-in ammatory effects of Pec in LPSstimulated murine microglial cell line BV 2 were investigated, and the underlying mechanisms were further elucidated.

Cell culture and treatment
Murine microglial cell line BV 2 was provided by the National Infrastructure of Cell Line Resource (Wuhan University, China). BV 2 cells were cultured in DMEM supplemented with 10% FBS (v/v) and 1% P/S (v/v) in a humidi ed chamber under 37°C and 5% CO 2 atmosphere. In the subsequent experiments, the cells were pretreated with the indicated concentrations of Pec for 2 h prior to the addition of LPS (1µg/mL).

Cell viability
Cell Counting Kit (CCK8, MedChemExpress, China) was used to detect cell viability in 96-well plates. Cells were plated in each well at a density of 1×10 5 cells/mL and treated with Pec for 24 h. After treatment, 10 µL of CCK-8 was added into the cell culture medium and then the plate was incubated for 1 h at 37°C.
The plate was detected for absorbance at 490 nm by a microplate reader and the results were calculated by the following formula: Viability=(A (experiment) -A (blank) )/(A (control) -A (blank) )×100%.

NO assay
Cells were rstly treated with Pec for 2 h and then stimulated by LPS (1µg/mL) for 24 h. Then, the cell supernatant was added in a new 96-well plate, mixed with equal volumes of Griess reagent I and II (Beyotime, Shanghai, China) and then detected the absorbance at a wavelength of 540 nm within 10 min. Sodium nitrite was used as a standard in the assay.

Enzyme-linked immunosorbent assay (ELISA)
The BV 2 cells were stimulated by LPS (1µg/mL) with or without Pec for 24 h, the cell supernatant was collected and centrifuged at 1000 r/min for 5 min. Then, the supernatant was diluted with the sample dilution buffer at appropriate ratio. The levels of TNF-α and IL-6 were examined by using ELISA Kit (Neobioscience Technology Co., Ltd., China) according to the manufacturer's instruction.

Real-time quantitative PCR (RT-PCR)
Total RNA of the cells was extracted by using Trizol (Life Technologies, Shanghai, China) according to its protocol. Total RNA was reverse-transcribed using an All-In-One RT Master Mix (Applied Biological Materials Inc., Nanjing, China). Real-time quantitative PCR was performed by using AceQ Universal SYBR® qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) and an ABI 7500 sequence system.
The primer sequences were shown in Table 1. and transferred to a polyvinylidene uoride (PVDF) membrane. The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with the indicated antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST three times and incubated with the secondary antibody for 1 h at room temperature. The protein bands were visualized using High sensitivity ECL kit (Wanlei bio, Shanghai, China) by LuminesCent image analyzer (Amersham Imager 600, GE Healthcare).
Grayscale of each band was performed using Image J software (NIH, USA).

Statistical analysis
The experiment data were presented as Mean ± SD. The statistical signi cance was analyzed by one-way analysis of variance by GraphPad Prism 6.0. Differences were considered signi cant at the 95% con dence level (p 0.05). All experiments were performed at least three times.

Effects of Pec on cell survival
Before determining the effects of Pec on anti-in ammation, we rst examined the cytotoxicity of Pec ( In ammatory cytokines such as TNF-α and IL-6 are involved in in ammatory process in LPS-induced BV 2 cells. We investigated whether Pec inhibited the secretion of TNF-α and IL-6. Pretreatment with or without Pec (50 µM and 100 µM) for 1 h and then treat with LPS (1 µg/mL) for 24 h, TNF-α and IL-6 expression was measured by RT-PCR. As shown in Fig. 2C and D, the mRNA expression of TNF-α and IL-6 was signi cantly inhibited by pretreatment with Pec. On the other hand, the culture medium was collected to detect the protein level of TNF-α and IL-6 by ELISA. The results showed that Pec suppressed LPS-induced production of TNF-α and IL-6 at the protein level in BV 2 cells ( Fig. 2A and B).

Pec inhibited the expression of NO and COX-2 induced by LPS in BV 2 cells
In addition to pro-in ammatory cytokines release, many in ammatory mediators were also involved in in ammatory process in BV 2 cells, such as NO, and in ammatory enzymes iNOS, COX-2. To examine the effects of Pec on NO production of LPS-stimulated BV 2 cells, the supernatant was tested by Griess regents. As shown in Fig. 3C, LPS signi cantly augmented NO production, while Pec treatment decreased the expression of NO in BV 2 cells. Then, we investigated the expression of iNOS and COX-2, Pec signi cantly reduced the LPS-stimulated increase of iNOS ( Fig. 3A and B) and COX-2 ( Fig. 4) compared with LPS treatment. These data indicated that Pec inhibited the accumulation of NO by regulating the iNOS and COX-2 expression, and it might be a potential inhibitor of microglial activation.

Pec inhibited LPS-stimulated in ammatory response via NF-κB pathway
NF-κB plays a crucial role in the development of in ammation and regulates the expression of in ammatory cytokines and mediators. Thus, the effects of Pec on NF-κB pathway in LPS-induced BV 2 cells were investigated. As shown in Fig. 5A-C, pretreatment with Pec inhibited the degradation of IκBa and the phosphorylation of IKKα/β compared with the LPS-induced group. In addition, the level of p65 was measured by Western blot. For total protein of p65, LPS stimulation increased phosphorylation of p65. Pretreatment with Pec dramatically decreased the level of phosphorylated p65 ( Fig. 6A and C). For the nuclear translocation of p65, the level of p65 in the nucleus was signi cantly elevated with the treatment of LPS, whereas pretreatment with Pec obviously reduced the p65 nuclear translocation ( Fig.   6A and B). These results suggested that Pec inhibited in ammatory response in LPS-stimulated BV 2 cells via NF-κB signaling pathway.

Discussion
In recent years, several monomers have been indicated for their potential neuroprotective effects in various neurodegenerative diseases [26][27][28][29]. Pec is widely distributed in medicinal plants. It is reported to have effects of antioxidant, anti-tumor, anti-cancer and antiviral. Moreover, Pec showed anti-in ammatory effects in animal models, which resulted in potent inhibiting like-wise carrageenan-induced mouse paw edema, arachidonic acid-induced mouse ear edema and passive cutaneous anaphylaxis [25]. However, the potential mechanism is not clear. In this study, we investigated the anti-in ammatory effects of Pec against LPS-stimulated neuro-in ammation via NF-κB signaling pathway in microglial BV 2 cells.
Accumulating evidence suggests that over-activated microglial cells were the symbol of neuroin ammation [30]. It is reported that microglial cells can be over activated by LPS and release a variety of in ammatory cytokines [31]. Therefore, targeting the pro-in ammatory cytokines secreted by microglial activation might be a promising therapeutic strategy to prevent or relieve neuro-in ammation. In general, over-activated microglial cells produce in ammatory cytokines such as TNF-α, IL-6 and IL-1β [32]. In this study, the results showed that the expression of TNF-α and IL-6 in LPS-stimulated microglial cells could be suppressed with the pretreatment of Pec. Activation of microglial cells also induce in ammatory mediators such as COX-2 and iNOS. The iNOS is a major source of NO generation, which has neurotoxicity against complex I and II in the respiratory chain and generates various deleterious reactive molecules [33]. NO generation is reduced with the decreasing of iNOS expression. Our results showed that Pec inhibited the expression of COX-2 and iNOS in LPS-stimulated microglial cells. These ndings suggest that Pec could inhibit the expressions of in ammatory cytokines and mediators in LPSstimulated microglial cells.
The NF-κB family of transcription factors is specially considered to play an important role in regulating the production of pro-in ammatory cytokines [38]. It is reported that NF-κB signaling pathway could regulate the production of TNF-α, IL-6 and IL-1β in LPS-or TNF-α-induced microglial cells. Non-activated NF-κB bounds to the inhibitor of IκBα family protein and is stayed in cytosol. With the LPS stimulation, NF-κB signaling could be activated with the IκBα kinase (IKK) activation, and the activated IKK would phosphorylate IκBα. Then, the IκBα dissociated, and the enhanced phosphorylation or degradation resulted in the downstream target p65 phosphorylation and translocation into the nucleus, which is associated with the secretion of in ammatory cytokines, such as TNF-α, IL-6 and IL-1β [39,40]. In the present study, we found that LPS could enhance IKK and p65 phosphorylation and IκBα degradation. However, with the pretreatment of Pec, these effects could be blocked, indicating that Pec inhibits the in ammatory responses in LPS-stimulated microglial cells via NF-κB signaling pathway.

Conclusions
In conclusion, the present study demonstrated the neuro-protective effects of Pec on inhibiting the expression of pro-in ammatory cytokines and in ammatory mediators in LPS-stimulated microglial cells via NF-κB signaling pathway. As a natural avonoid, Pec might provide a potential therapy for preventing and relieving the progression of neuro-in ammatory diseases. presented as means ± SD of three times.   Effects of Pec on the production of COX-2 in LPS-induced BV2 cells. Cells were pretreated with different concentrations of Pec for 1h, then treated with 1μg/mL LPS for 24h. (A, B) The COX-2 expression was determined by Western blot. β-actin was used as an internal control. All data were presented as the mean ± SD of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs. LPS-treated group.

Figure 5
Effects of Pec on NF-κB signaling pathway in LPS-induced BV2 cells. Cells were pretreated with different concentrations of Pec for 1h, then treated with 1μg/mL LPS for 24h. (A-C) IKKβ, IKKα/β phosphorylation, IκBα and β-actin expression were determined by Western blot. The non-phosphorylated form of each targeted protein was used as loading control, β-actin was used as an internal control. All data were presented as the mean ± SD of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs. LPS-treated group.