Okanin, effective constituent of the flower tea Coreopsis tinctoria, attenuates LPS-induced microglial activation through inhibition of the TLR4/NF-κB signaling pathways

The EtOAc extract of Coreopsis tinctoria Nutt. significantly inhibited LPS-induced nitric oxide (NO) production, as judged by the Griess reaction, and attenuated the LPS-induced elevation in iNOS, COX-2, IL-1β, IL-6 and TNF-α mRNA levels, as determined by quantitative real-time PCR, when incubated with BV-2 microglial cells. Immunohistochemical results showed that the EtOAc extract significantly decreased the number of Iba-1-positive cells in the hippocampal region of LPS-treated mouse brains. The major effective constituent of the EtOAc extract, okanin, was further investigated. Okanin significantly suppressed LPS-induced iNOS expression and also inhibited IL-6 and TNF-α production and mRNA expression in LPS-stimulated BV-2 cells. Western blot analysis indicated that okanin suppressed LPS-induced activation of the NF-κB signaling pathway by inhibiting the phosphorylation of IκBα and decreasing the level of nuclear NF-κB p65 after LPS treatment. Immunofluorescence staining results showed that okanin inhibited the translocation of the NF-κB p65 subunit from the cytosol to the nucleus. Moreover, okanin significantly inhibited LPS-induced TLR4 expression in BV-2 cells. In summary, okanin attenuates LPS-induced activation of microglia. This effect may be associated with its capacity to inhibit the TLR4/NF-κB signaling pathways. These results suggest that okanin may have potential as a nutritional preventive strategy for neurodegenerative disorders.

. Anti-inflammatory activity of Coreopsis tinctoria Nutt. in BV-2 microglial cells. BV-2 microglial cells were treated with extracts of Coreopsis tinctoria Nutt. in the presence of LPS (100 ng/mL) for 24 h at the indicated concentrations. Aliquots of the culture supernatants were removed and analyzed for nitrite production. Cell viability was determined by MTT assay. Data are expressed as means ± SEM (n = 3). ### P < 0.001 compared with the untreated control group, *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the cells treated with LPS only. (EtOH: ethanol, EtOAc: ethyl acetate, n-BuOH: n-butanol, NO: nitric oxide, LPS: lipopolysaccharide).

Effects of the EtOAc extract of C. tinctoria Nutt on LPS-induced microglia activation in vivo.
We next determined the anti-neuroinflammatory effect of the EtOAc extract in vivo. Mice were pre-treated orally with the extract, then microglial activation was induced by LPS administered intracerebroventricularly. The microglial response was detected immunohistochemically using an antibody against Iba-1, a protein that is strongly up-regulated in activated microglia. Shapiro-Wilk test showed that the data were not normally distributed. Kruskal-Wallis test showed significant difference among different groups [χ (2) 2 = 26.825, P < 0.001, Fig. 4]. Mann-Whitney test further indicated that mice treated with LPS had significantly more Iba-1-positive cells compared to the control group (P < 0.001). Mice treated with the EtOAc extract (200 mg/kg) and LPS had significantly fewer Iba-1 positive cells than mice treated with LPS alone (control group: 36.4 ± 3.8, LPS group: 73.0 ± 2.5, EtOAc extract group: 43.8 ± 2.9).
The content of okanin in EtOH, EtOAc and n-BuOH extracts of C. tinctoria Nutt. The okanin used in our experiment was prepared from the EtOAc extract of the flower tea C. tinctoria Nutt. by means of a chromatography method. Its purity was identified as 99.1% by HPLC. The content of okanin in EtOH, EtOAc and n-BuOH extracts was determined as 1.61%, 4.94% and 0.33%, respectively (Fig. 5A,B and C) by means of UPLC chromatography (Waters Acquity UPLC H-Class system equipped with a Sample Manager-FTN, Quaternary Solvent Manager, PDA Detector and Empower software). Therefore, okanin was concentrated most effectively in the EtOAc extract.

Effects of okanin on LPS-induced iNOS expression.
At the beginning, MTT assay was carried out.
The results showed that okanin did not reduce the cell viability (Fig. 6A), excluding a possible effect of reduced viability on mRNA or protein expression in the following experiments.

Discussion
Tea is a popular beverage worldwide. The different types of tea, including green tea, red tea and black tea, originate from plants of the Camellia genus. Recently non-Camellia teas, as popular health supplements, have attracted more attention from both scientists and consumers 12 . C. tinctoria Nutt., known as "snow chrysanthemum" or "snow tea" is traditionally used as a non-Camellia tea by the Uyghur folk. However, its biological activities and the mechanisms underlying its effects have not been reported in detail.
Microglia play an essential role in defending the brain against injury or disease 13 . When exposed to stimuli such as infectious agents, they become activated and begin to release pro-inflammatory molecules including NO, TNF-α , IL-1β and IL-6. Overexpression of these pro-inflammatory mediators can cause neuronal death, pathological changes and neurodegenerative diseases [14][15][16] . Inhibition of microglial activation is therefore considered to be a valid approach to prevent and treat neuroinflammation-mediated diseases 16 . Here we report that the EtOAc extract of Coreopsis tinctoria Nutt attenuated LPS-induced microglial activation both in vitro and in vivo. Its effective constituent, okanin, was further investigated. Okanin was able to suppress LPS-induced microglial activation. This effect may be associated with its ability to inhibit the TLR4/NF-κ B signaling pathways.
To assess the anti-neuroinflammatory properties of the C. tinctoria extracts, we assayed NO production in BV-2 microglial cells that were stimulated with LPS. The total EtOH extract of Coreopsis tinctoria Nutt significantly inhibited production of NO. Further study revealed that the EtOAc extracts significantly inhibited  LPS-induced NO production, while the n-BuOH extract did not. These results suggest that the EtOAc extract contained the active agent that suppresses NO production. NO is synthesized in a variety of cells and tissues by the enzyme NO synthase (NOS) 17 . Microglia express an inducible isoform of NOS (iNOS), which produces NO continuously when the cells are activated 18 . The effect of the EtOAc extract on LPS-induced iNOS mRNA expression in BV-2 microglial cells was investigated by qRT-PCR. The EtOAc extract significantly inhibited LPS-induced iNOS mRNA expression. The results may explain why the extract has the capacity to reduce NO production.
COX-2, an important enzyme associated with inflammation, has been reported to be elevated in AD brains 19,20 . It was previously reported that COX-2 inhibitors have a neuroprotective effect by blocking microglial activation and downstream events 11,21,22 . Here, we investigated the effect of the EtOAc extract on LPS-induced COX-2 expression in BV-2 cells. The EtOAc extract significantly inhibited LPS-induced COX-2 mRNA expression.
Following the onset of inflammation, activated microglia produce three main pro-inflammatory cytokines, namely IL-1β , IL-6 and TNF-α 23 . We used qRT-PCR to examine the effect of the EtOAc extract on LPS-induced mRNA expression of these three genes in BV-2 microglial cells. The EtOAc extract significantly inhibited LPS-induced mRNA expression of IL-1β , IL-6 and TNF-α .
We next determined the anti-neuroinflammatory effect of the EtOAc extract in vivo. Mice were pre-treated orally with the extract, then microglial activation was induced by intracerebroventricular administration of LPS. The microglial response was assessed immunohistochemically using an antibody against Iba-1, a protein that is strongly up-regulated in activated microglia. The EtOAc extract significantly inhibited LPS-induced microglial activation in vivo evidenced by the fewer Iba-1 positive cells in mice after the treatment of the EtOAc extract.
Since the EtOAc extract exhibited significant anti-neuroinflammatory effects both in vitro and in vivo, it was considered to be responsible for the potential of the traditional flower tea on overactivation of microglia. In our previous research, okanin, as the major component of EtOAc extract shown in Fig. 5, exhibited significant inhibition effect in LPS-induced microglial cells. Our previous data showed that okanin was able to block the LPS-induced production of NO by BV-2 microglia. The present study revealed that okanin was also able to inhibit the LPS-induced increase in iNOS expression. The mRNA and protein levels of iNOS were both reduced by okanin, and the level of inhibition was directly related to the concentration of okanin. Furthermore, we found that okanin lowered the mRNA and protein levels of the LPS-induced pro-inflammatory cytokines IL-6 and TNF-α .   Supplementary Figures 2 and 3. The translocation of NF-κ B p65 was determined by immunocytochemistry (C). Data are expressed as means ± SEM (n = 3). ### P < 0.001 compared with the control group (untreated cells); ***P < 0.001 compared with the cells treated with LPS only. Taken together, our data showed that okanin potently inhibits a number of pro-inflammatory responses in microglial cells.
The NF-κ B complex is a well-known transcriptional activator which modulates gene expression in response to a wide variety of stressors, toxins and microbial antigens, including LPS. The NF-κ B complex is a homodimer or heterodimer of proteins in the NF-κ B and Rel families 24 . Under normal conditions, NF-κ B associates with members of the cytoplasmic Iκ B family of inhibitor proteins. Exposure of the cell to stimuli such as LPS triggers receptor-mediated phosphorylation of Iκ B, which then undergoes ubiquitination followed by proteasomal degradation 25 . Meanwhile, the NF-κ B dimer shuttles into the nucleus, where it binds to specific response elements upstream of its target genes, thus enhancing transcription 24 . As mentioned above, the NF-κ B complex is a key mediator of the LPS-induced activation of microglia, and activates the transcription of genes encoding cytokines 11,26 . Therefore, we used western blotting and immunofluorescence assays to test whether okanin affects the NF-κ B subunit p65 (RelA) and the NF-κ B inhibitor Iκ B in BV-2 cells treated with LPS. The data showed that okanin significantly inhibited the LPS-induced phosphorylation of Iκ Bα in BV-2 cells. Furthermore, in LPS-treated BV-2 cells, okanin significantly suppressed the shuttling of p65 into the nucleus.
Toll-like receptors (TLRs) recognize molecules, such as cell membrane products and nucleic acids, which are found on different kinds of microbial pathogen 27,28 . Most TLR-initiated signaling causes NF-κ B to shuttle into the nucleus, as described above, where it activates a variety of genes that encode pro-inflammatory factors 24 . In the CNS, TLRs 1-9 are expressed on microglia 29 . TLR4 is an innate immune receptor, which recognizes LPS in the outer membrane of gram-negative bacteria 30,31 . It has been shown that preventing TLR4-mediated signaling suppresses the activation of microglia and reduces the release of pro-inflammatory cytokines 31 . Our work demonstrates that the LPS-induced activation of BV-2 microglial cells was associated with up-regulated expression of TLR4, which could be decreased by okanin. This suggests that down-regulated expression of TLR4 might contribute to the inhibitory effect of okanin on the activation of BV-2 microglia.
In conclusion, this study shows for the first time that the EtOAc extract of Coreopsis tinctoria Nutt attenuates LPS-induced microglial activation both in vitro and in vivo. Its effective constituent, okanin, suppresses LPS-induced microglial activation. This effect may be associated with its ability to inhibit the TLR4/NF-κ B signaling pathways. Considering that neuroinflammation mediated by activated micoglia plays an important role in neurodegenerative diseases, okanin is a potential treatment for neurodegenerative diseases.

Materials and Methods
General. UPLC-PDA analysis was performed on a Waters Acquity UPLC H-Class system fitted with an

Preparation of Okanin. Okanin (30 mg) was prepared from the EtOAc extract of flower tea C. tinctoria
Nutt. according to the method described in our previous work 5 . Its structure was identified on the basis of NMR spectra analysis.

Quantitative real-time PCR (qRT-PCR). For detection of mRNA expression of pro-inflammatory factors
by qRT-PCR, BV-2 microglial cells were pretreated with extract of C. tinctoria Nutt or okanin for 2 h, then exposed to LPS for 4 h. Total RNA was extracted from the cells with Trizol reagent. After cDNA synthesis, qRT-PCR assays were carried out using a GoTaq one-step real-time PCR kit with SYBR green on a CFX Connect TM real-time PCR system (Bio-Rad, Hercules, CA, USA). Expression levels of iNOS, COX-2, IL-1β , IL-6 and TNF-α were normalized to those of GAPDH (see Table 1 for primer sequences).
Enzyme-linked immunosorbent assay (ELISA). The protein levels of IL-6 and TNF-α were determined using ELISA. BV-2 cells were pretreated with okanin for 2 h and then stimulated with 100 ng/ml LPS for 24 h. The levels of IL-6 and TNF-α in the culture medium were measured by ELISA kits as previously reported 33 . Briefly, standards or samples were added to the wells of the microplate pre-coated with a specific monoclonal antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody was pipetted into the wells. The substrate solution was added to the wells after a wash to remove any unbound antibody-enzyme reagent. The color development was finally stopped and the absorbance was read at 450 nm using a plate reader (Bio-Tek, Winooski, VT, USA).

Western blot analysis.
Western blotting was carried out as previously reported 11,34,35 . To detect NF-κ B p65 and phosphorylation of Iκ Bα , BV-2 cells were pretreated with okanin for 2 h and then exposed to LPS for 30 min. To detect TLR4, BV-2 cells were pretreated with okanin for 2 h and then exposed to LPS for 24 h. Cells were lysed in lysis buffer after washing in ice-cold PBS 35 . The protein content of the lysates was measured using the BCA assay, then the samples were separated by 12% SDS-PAGE. The proteins were transferred to a membrane, which was incubated sequentially with blocking buffer, primary antibodies, and horseradish peroxidase-conjugated secondary antibodies.
in the LPS groups were treated orally with vehicle and injected with LPS. Mice in the EtOAc group were treated orally with the EtOAC extract of C. tinctoria Nutt. and injected with LPS. All groups received daily oral administration of vehicle or extract of C. tinctoria Nutt at a dose of 200 mg/kg/day. The animals were injected intracerebroventricularly with LPS (40 μ g/mouse) or PBS on day 7 and were sacrificed 3 days later. Seven mice were used in each group.
Immunohistochemical analysis of mouse brain sections. To prepare brain tissues for immunohistochemistry, mice were first anesthetized with chloral hydrate (400 mg/kg, intraperitoneal injection). The animals were then perfused transcardially with heparin (10 U/ml in saline solution) followed by paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4). The brains were removed and incubated in paraformaldehyde for 8 h, then in 30% sucrose solution for 24 h. The samples were stored at − 20 °C until required. Coronal sections (20 μ m) from the hippocampal region were cut on a freezing microtome (LEICA CM1850), then washed in 10 mM PBS (pH 7.4). Slides and sections were then treated as reported previously 36,37 . Cells positive for Iba-1 staining were counted under a light microscope (LEICA DMI 3000 B). The observation of immunohistochemistry was obtained from two sections for each animal. The researcher was blinded to the treatment status of the slides.