Baicalin modulates NF-κB and NLRP3 inflammasome signaling in porcine aortic vascular endothelial cells Infected by Haemophilus parasuis Causing Glässer’s disease

Haemophilus parasuis (H. parasuis) can cause vascular inflammatory injury, but the molecular basis of this effect remains unclear. In this study,we investigated the effect of the anti-inflammatory, anti-microbial and anti-oxidant agent, baicalin, on the nuclear factor (NF)-κB and NLRP3 inflammasome signaling pathway in pig primary aortic vascular endothelial cells. Activation of the NF-κB and NLRP3 inflammasome signaling pathway was induced in H. parasuis-infected cells. However, baicalin reduced the production of reactive oxygen species, apoptosis, and activation of the NF-κB and NLRP3 inflammasome signaling pathway in infected cells. These results revealed that baicalin can inhibit H. parasuis-induced inflammatory responses in porcine aortic vascular endothelial cells, and may thus offer a novel strategy for controlling and treating H. parasuis infection. Furthermore, the results suggest that piglet primary aortic vascular endothelial cells may provide an experimental model for future studies of H. parasuis infection.

tissue injury 12,13 . Importantly, some downstream targets of NF-κB, such as interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α, may in turn activate the NF-κB signaling pathway 14 . IL-6 is associated with acute and chronic inflammation 15 , while IL-8 elicits the recruitment and activation of neutrophils, which subsequently release reactive oxygen species (ROS) and cause local tissue injury and inflammation 11,16 . TNF-α has an important effect on both local and systemic inflammation 17 , and IL-10 and TNF-α have been shown to be involved in the adaptive response, which might contribute to protection against H. parasuis infection 18 . Pro-inflammatory cytokines may thus affect the inflammatory response induced by H. parasuis.
The immune system involves important protection mechanisms that defend against pathogens, such as bacteria and viruses. Innate immune cells, such as macrophages, can initiate inflammation, leading to the release of inflammatory cytokines during infection 19 . Inflammasomes are molecular platforms that elicit activation of caspase-1, resulting in the maturation of proinflammatory cytokines 20 . For example, NLRP3 inflammasomes are composed of NLRP3, apoptosis-associated speck-like protein including a CARD (N-terminal caspase recruitment domains) (ASC), and pro-caspase-1 21 . Excessive release of IL-1β has been reported to be involved in some systemic inflammatory diseases 22,23 , and was shown to cause local inflammation triggered by bacterial or viral infection, representing an important mechanism for fibrogenesis production 24,25 . Previous research demonstrated that NLRP3 inflammasomes in hepatic stellate cells were activated by Schistosoma japonicum infection, leading to the initiation of the inflammatory response and resulting in liver fibrosis 26 . Activation of the NLRP inflammasome also led to the stretch-induced inflammatory response in human periodontal ligament cells 27 . Although the innate adaptive immune response may protect animals from certain diseases, inappropriate activation of the NLRP3 inflammasome may contribute to disease progression and tissue injury, including neurodegenerative diseases 28,29 , metabolic diseases 30,31 , and sepsis 32,33 . H. parasuis has been shown to activate the NLRP3 inflammasome 34 , but the mechanism of vascular inflammation induced by H. parasuis remains unclear. We therefore determined if activation of the NLRP3 inflammasome triggered vascular inflammation leading to Glässer's disease in porcine aortic vascular endothelial cells (PAVECs) infected with H. parasuis.
Baicalin (BA) is an effective plant-derived flavonoid and a traditionial Chinese medical herb isolated from Scutellaria baicalensis Georgi (Huang Qin). The chemical structure of BA has been determined 35 . BA possesses important functions, including anti-inflammatory [36][37][38] , anti-microbial 39,40 , and anti-oxidant activities 41,42 . Previous studies demonstrated that BA could reduce ROS production, suppress apoptosis, and inhibit the activation of the NF-κB and NLRP3 inflammasome signaling pathway in piglet mononuclear phagocytes treated with H. parasuis 34 . BA treatment also reduced T lymphocyte infiltration, gene expression of proinflammatory factors, and tissue damage in mice 37 . Furthermore, BA ameliorated LPS-induced inflammation and apoptosis in bovine mammary epithelial cells via inhibition of NF-κB activation and HSP72 upregulation 43 . BA also upregulated IRF4 protein expression and reversed LPS-induced macrophage subset redistribution, contributing to amelioration of inflammatory bowel diseases 44 . Moreover, BA significantly and selectively inhibited the viability of ovarian cancer cells, demonstrating its anti-cancer activity 45 . Overall, these results suggest that BA may inhibit the vascular inflammatory response induced by H. parasuis.
We previously showed that BA significantly inhibited the activation of NF-κB and the NLRP3 inflammasome during H. parasuis infection in piglet primary mononuclear phagocyte 34 . Importantly, some clinical phenotypes of Glässer's disease, such as endocarditis and meningitis, are directly related to endothelial cells. However, the role of NF-κB and inflammasomes in porcine vascular endothelial cell injury has not been well-studied.
In the present study, we further explored the molecular mechanisms responsible for mediating the H. parasuis-induced activation of NF-κB and the NLRP3 inflammasome in porcine vascular endothelial cells. Our results revealed that BA could inhibit the H. parasuis-elicited inflammatory responses in PAVECs, and may thus represent a novel strategy for controlling and treating H. parasuis infection in pigs.

Effect of baicalin on PAVECs viability in vitro.
We determined the optimal concentration of BA by examining the viability of PAVECs at different concentrations. A decrease in the final concentration of BA from 250 μg/mL to 12.5 μg/mL for 12 h increased the cell viability from 83.9% to 97.7% (Fig. 1). BA demonstrated no significant cytotoxicity at concentrations of 12.5 μg/mL to 100 μg/mL for 12 h (P > 0.05) (Fig. 1). We also observed a dose-time relationship among BA concentration, stimulation time, and cell viability (Fig. 1). Based on these results, we used BA at 100 μg/mL for 12 h in subsequent experiments.

Establishment of H. parasuis infection model using PAVECs.
We explored the effect of the multiplicity of infection (MOI) of H. parasuis in the PAVEC infection model. Secretion of the inflammatory cytokines IL-1β, IL-18, and TNF-α into the supernatants by PAVECs tended to increase following H. parasuis infection at a MOI of 1:10 compared with negative control cells, but the difference was not significant (P > 0.05) (Fig. 2). However, secretion of these inflammatory cytokines after 12 h was significantly increased compared with the negative control group at MOIs of 1:1, 10:1, and 100:1 (P < 0.05) (Fig. 2). An MOI of 1:1 was therefore considered to represent the best infection model of the inflammatory response triggered by the H. parasuis.

Effect of baicalin on production of proinflammatory cytokines triggered by H. parasuis in
PAVECs. We explored the release of proinflammatory cytokines from endothelial cells following H. parasuis infection. H. parasuis significantly induced the secretion of the proinflammatory cytokines IL-6, IL-8, IL-10, prostaglandin E 2 (PGE2), cyclooxygenase (COX-2), IL-1β, IL-18, and TNF-α from PAVECs compared with the negative controls (P < 0.01), as measured by enzyme-linked immunosorbent assay (ELISA) (Fig. 3A-H). We also analyzed the induction of proinflammatory cytokines by NAC alone as a positive control, and found that NAC significantly inhibited the release of the above proinflammatory cytokines, compared with PAVECs infected with H. parasuis (P < 0.01) (Fig. 3A-H). Furthermore, pretreatment with BA at a final concentration at 50 or 100 μg/

Effect of baicalin on H. parasuis-induced ROS release and cell apoptosis in PAVECs.
We measured ROS production in PAVECs following stimulation with H. parasuis by measuring the fluorescence intensity. ROS production was markedly increased following H. parasuis infection for 12 h (P < 0.01) (Fig. 5), while BA treatment (final concentration 12.5-100 μg/mL) reduced ROS production (P < 0.01) (Fig. 5). Furthermore, NAC also markedly inhibited the generation of ROS in PAVECs compared with cells infected with H. parasuis (P < 0.01) (Fig. 5). Fluorescence microscopy analysis showed that the intracellular generation of ROS was decreased in a dose-dependent manner in both BA-and NAC-treated cells (Fig. 5).
We also investigated the effect of H. parasuis on apoptosis in PAVECs, and showed that cells in the late stages of apoptosis were significantly increased following H. parasuis infection for 12 h compared with the negative control (P < 0.01) (Fig. 6). BA (final concentration 12.5-100 μg/mL) and NAC both significantly reduced the endothelial cells in the late stages of apoptosis triggered by H. parasuis (P < 0.01) (Fig. 6).

Effects of baicalin on activation of the NF-κB signaling pathway triggered by H. parasuis.
We investigated the effect of baicalin pretreatment on the NF-κB signaling pathway in PAVECs infected with H. parasuis by measuring levels of nuclear NF-κB p65 subunit by ELISA. Expression of nuclear NF-κB p65 subunit in PAVECs was markedly increased by H. parasuis infection for 12 h (P < 0.01) (Fig. 7A), but this increase was significantly inhibited by pretreatment with BA (final concentration of 25-100 μg/mL) compared with H. parasuis infection alone (Fig. 7A). We also detected the NF-κB p65 subunit by immunofluorescence microscopy and showed that p65 was upregulated in PAVECs by H. parasuis infection (Fig. 7B), and reduced again in cells pretreated with BA (final concentration 12.5-100 μg/mL) (Fig. 7B). NAC treatment (positive control) significantly reduced the p65 levels in endothelial cells (Fig. 7B).
We also examined the expression levels of cleaved (active) caspase-1 protein in PAVECs following H. parasuis infection for 12 h by western blot. Activated caspase-1 protein expression was significantly up-regulated by H. parasuis compared with negative control cells, while NAC (positive control) significantly inhibited activated caspase-1 expression compared with H. parasuis infection (P < 0.01) (Fig. 8D). Pretreatment with BA (final concentration 12.5-100 μg/mL) significantly down-regulated activated caspase-1 protein expression compared with H. parasuis stimulation alone, in accord with the results for mRNA expression levels (P < 0.01) (Fig. 8D).

Discussion
To our best of our knowledge, the current study provides the first evidence for the anti-inflammatory functions of baicalin in H. parasuis-infected PAVECs via inhibiting activation of the NLRP3 inflammasome and NF-κB signaling pathway, and thereby protecting the cells from H. parasuis-evoked inflammation.
Endothelial cells have previously been shown to play an active role during the inflammatory immune response to bacteria and their products 46 . Other studies also demonstrated a central role for endothelial cells in regulating inflammation 47,48 . In addition, endothelial cell activation is necessary for leukocyte recruitment to reduce the sepsis-associated increase in vascular permeability 49 . We therefore developed a suitable cellular model for evaluating endothelial cell activation during bacterial infection, and for improving our understanding of and developing ways of modulating inflammation. H. parasuis was previously shown to participate in serum resistance, adhesion, and invasion in studies using porcine kidney epithelial cells (PK-15) 50 , porcine aortic endothelial cells (AOC-45) 51 , porcine umbilical vein endothelial cells 52 , and newborn pig tracheal cells 53 . H. parasuis infection was shown to contribute to activation of the p38/JNK/mitogen-activated protein kinase pathway predominantly linked to inflammation in PK-15 cells 54 . In the current study, we used primary endothelial cells isolated from porcine aortas. To the best of our knowledge, this is the first report of the use of primary aortic vascular endothelial  PAVECs were pre-treated with baicalin and co-cultured with H. parasuis. The release of proinflammatory cytokines in the cell culture supernatants was measured by ELISA assays. ## P < 0.01 vs. control. *Indicates significance at P < 0.05 and **indicates significance at P < 0.01.
In the present study, the secretion of IL-6, IL-8, and TNF-α by endothelial cells was significantly increased by H. parasuis, consistent with the previous finding that H. parasuis stimulated the release of IL-6 and IL-8 by newborn pig tracheal cells 53 . TNF-α has the characteristics of a multifunctional pro-inflammatory cytokine, with an important role in the pathogenesis of inflammatory diseases 9 . IκB kinase, which is activated by TNF-α, has been reported to phosphorylate IκB and induce its degradation, leading to the liberation of NF-κB, and evoking expression of a variety of genes that participate in inflammatory responses, such as IL-6 and IL-8 55 . Notably, overexpression of IL-8 has been related to some lung diseases 56 . In addition, IL-10 induced humoral immunity to kill extracellular microbial pathogens and produce protective antibodies 57,58 . H. parasuis was shown to activate the inflammatory transcription factor NF-κB in a time-and dose-dependent manner in PK-15 cells, through IκB degradation and release of IL-8 and CCL4 11,54 . Overexpression of porcine Coro1A down-regulated NF-κB and thereby inhibited the transcription of the NF-κB-mediated downstream genes IL-6, IL-8, and COX-2 59 . Our results indicated that H. parasuis could activate the NF-κB signaling pathway in aorta endothelial cells. Furthermore, this activation was significantly inhibited by baicalin, suggesting that baicalin exerted important effects resulting in reduced H. parasuis-evoked inflammation.
Inflammasomes are intracellular protein complexes that play an important role in innate immune sensing 60 . Previous research showed that Streptococcus pneumoniae could induce NLRP3-dependent IL-1β production, related to a pro-inflammatory cytokine cascade 61 . In addition, the degree of caspase-1 activation via the NLRP3 inflammasome was associated with clinical disease severity in patients with S. pneumoniae infection 62 . Staphylococcus aureus α-hemolysin was linked to an IL-1β response, which evoked NLRP3-dependent activation of caspase-1 in mouse and human monocytic cells 63,64 . To determine if activation of the NLRP3 inflammasome occurred in aorta endothelial cells stimulated with H. parasuis, and to explore the molecular mechanisms mediating inflammasome activation, we established an H. parasuis cell-infection model. We demonstrated that NLRP3, ASC, and caspase-1 aggregation, and IL-1β expression, were significantly increased in endothelial cells infected with H. parasuis. However, activation of the NLRP3 inflammasome was markedly attenuated in cells pretreated with baicalin. We therefore inferred that activation of the inflammasome was accompanied by the development of vascular inflammation, ultimately leading to Glässer's disease as a result of overexpression of proinflammatory cytokines or chemokines during H. parasuis infection. This suggests that the development of vascular inflammation might be related to activation of caspase-1 via inflammasomes, though further studies are needed to clarify the molecular mechanisms responsible for the development of local inflammation.
Previous research demonstrated that ROS released by NADPH oxidase may act as defense and signaling molecules linked to innate immunity and various kinds of cellular responses 65,66 . An imbalance between ROS and antioxidant enzymes could lead to cytotoxicity, thus contributing to the pathogenesis of chronic diseases 67 . In this study, we used NAC as a positive control, and showed that both NAC and baicalin could significantly inhibit the production of ROS. NAC has been considered as a ROS-specific inhibitor. SEA-induced NLRP3 inflammasome formation and activation in hepatic stellate cells were significantly attenuated or abolished by NAC 26 . Inhibitors of ROS release and ROS scavengers have been reported to inhibit activation of the NLRP3 inflammasome 68 ; however, ROS inhibition could not affect activation of the NLRP3 inflammasome directly, but could negatively regulate the priming step of inflammasome activation 69 . We therefore speculated that excessive ROS production may activate the relevant inflammatory signaling pathway leading to the vascular inflammatory response, which could be inhibited by baicalin. Further studies are needed to determine the specific mechanisms involved.
Overall, the results of the current study demonstrate that H. parasuis activates the NLRP3 inflammasome and NF-κB signaling pathway in PAVECs, while these effects can be significantly inhibited by baicalin. We aim to conduct further studies to validate the anti-inflammatory effect of baicalin in a pig model of H. parasuis infection in vivo.

Methods
Bacterial strain, growth conditions and drug. H. parasuis SH0165 strain, isolated from the lung of a commercial pig with arthritis, fibrinous polyserositis, hemorrhagic pneumonia, and meningitis, is a highly virulent strain of serovar 5 70,71 . The SH0165 strain was grown in tryptic soy broth (Difco Laboratories, USA) or tryptic soy agar (Difco Laboratories) supplemented with 10 μg/ml of NAD (Sigma, USA) and 10% newborn calf serum (Gibco, USA) under 37 °C. PAVECs were isolated and identified as described previously 72 . Briefly, the uptake of Ac-LDL was determined by incubating PAVECs with 10 μg/ml of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)-labelled Ac-LDL (Invitrogen, USA) in cell medium for 12 h at 37 °C. Then the cells were washed three times with PBS, detached by trypsinization and analyzed by fluorescence microscopy (Fig. S1). The PAVECs were cultured as described previously, with some minor modifications 73 . Briefly, endothelial cells were obtained in small sheets after treatment of the aorta lumen (20 min, 37 °C) with 0.1% type I collagenase (Sigma, USA) in M-199 medium (Gibco, USA) containing penicillin-streptomycin solution (Gibco, USA). The suspension was centrifuged at 100 × g for 10 min, and the cells from one aorta were resuspended in 5 mL of M-199 containing 20% fetal bovine serum (FBS) (Gibco, USA), and then plated in a T-25 tissue-culture plate (Costar, USA). Endothelial cells were counted and their viability was determined by Trypan blue exclusion.

Effect of dosing schedule on PAVECs viability in vitro. The viability of PAVECs was measured by Cell
Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Japan) 74 . Briefly, PAVECs were seeded into 96-well plates (Costar, USA) at 1 × 10 5 cells/well and then treated with baicalin at a final concentration of 0, 12.5, 25, 50, 100, 250, 500, or 1000 μg/mL for 6, 12, 24, or 48 h at 37 °C under 5% CO 2 . CCK-8 solution (10 μL) was added to each well and incubated for 90 min at 37 °C, and the optical density was then measured at 450 nm. Cell viability was calculated according to the following formula: cell viability (%) = (experimental well − blank well/ control well − blank well) × 100%. The data were expressed as mean ± standard deviation of triplicate samples from at least three independent experiments.

PAVEC model of H. parasuis infection.
To confirm the MOI of H. parasuis in the endothelial cells, 1 × 10 5 cells were seeded into 96-well plates followed by the addition of H. parasuis at 10 4 , 10 5 , 10 6 , or 10 7 CFU/mL and co-culture at 37 °C under 5% CO 2 for 3, 6, 12, and 20 h, respectively. Release of the inflammatory cytokines TNF-α, IL-1β, and IL-18 from the cell culture supernatant was determined to explore the MOI and optimal interaction time.
Measurement of ROS and cell apoptosis. Intracellular ROS were detected by DCFH-DA staining 75 .
Briefly, 2 × 10 5 PAVECs were seeded into 24-well plates (Costar) and treated with baicalin at final concentrations of 12.5, 25, 50, and 100 μg/mL, respectively, for 1 h. H. parasuis 2 × 10 5 CFU/mL was added into the plates and co-cultured for 12 h. After 12 h, the incubations were washed three times with sterile phosphate-buffered saline and stained with 10 μM DCFH-DA and 5 μM DHE (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 37 °C under 5% CO 2 for 30 min. The fluorescence intensities were then determined using a fluorescence microplate reader (Olympus, Japan). Apoptosis was detected using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA) according to standard procedures, and examined by flow cytometry 76 . Briefly, 2 × 10 5 PAVECs were seeded into 24-well plates (Costar) and then treated with baicalin at a final concentration of 12.5, 25, 50, or 100 μg/mL, respectively, for 1 h. H. parasuis at 2 × 10 5 CFU/mL was then added into the plates and co-cultured for 12 h. The incubations were then washed three times with sterile phosphate-buffered saline and stained with FITC Annexin V and Propidium Iodide (PI). Cell apoptosis was measured by flow cytometry (Beckman Coulter, FC500, USA). Total RNA extraction and qRT-PCR determination. We also measured the gene expression levels of IL-1β, IL-18, TNF-α, IL-6, IL-8, and IL-10, as well as the NLRP1, NLRP3 (NLRP3, ASC, and caspase-1), NLRC4, and AIM2 inflammasomes in PAVECs infected with H. parasuis. PAVECs at 1 × 10 7 were seeded into 24-well plates and incubated with baicalin at a final concentration of 12.5, 25, 50, or 100 μg/mL for 1 h, followed by the addition of 1 × 10 7 CFU/mL H. parasuis for 12 h. After co-culture, the PAVECs were collected and total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA). The RNA was reverse-transcribed to cDNA using reverse transcriptase (TaKaRa, Dalian, China) and cDNA was amplified and measured using a SYBR Green PCR Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Individual transcripts in each sample were repeated at least three times and β-actin was used as the internal control. The nucleotide sequences of the primers used for qPCR are listed in Table 1.
Detection of NF-κB p65 nuclear translocation by ELISA and immunofluorescence. We determined the effects of baicalin pretreatment on NF-κB signaling in PAVECs infected with H. parasuis by measuring p65 levels in monocytes. PAVECs at 1 × 10 7 were seeded into 6-well tissue culture plates and pretreated with NAC (1 mM) or baicalin (12.5, 25, 50, 100 μg/mL), respectively, for 1 h. H. parasuis at 1.0 × 10 7 CFU/mL was then added and co-cultured for 12 h at 37 °C under 5% CO 2 . The cells were then collected and cytoplasmic and nuclear proteins were extracted using a cytosolic-nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China). Protein concentrations were measured using bicinchoninic acid protein assay reagents (Beyotime Biotechnology), according to the manufacturer's instructions. NF-κB p65 protein levels in the cytoplasm and nucleus were determined using an NF-κB ELISA kit (Blue Gene Biotechnology, Shanghai, China), and nuclear translocation of NF-κB p65 was expressed as the ratio of nuclear/cytoplasmic expression of NF-κB p65 protein. We also investigated NF-κB p65 nuclear translocation by immunofluorescence 77 . Briefly, 1 × 10 6 PAVECs were seeded into 6-well plates with cover slips. The cells were pretreated with NAC (1 mM) or baicalin (12.5, 25, 50, 100 μg/mL), respectively, for 1 h and then co-cultured with 1 × 10 6 CFU/mL H. parasuis for 12 h, followed by fixing with 4% paraformaldehyde for 1 h and permeabilization with 0.5% Triton X-100 for 30 min. The cells were blocked with goat serum for 1 h, and incubated with anti-NF-κB p65 antibody (NF-κB Activation, Nuclear Translocation Assay Kit, Beyotime) at 4 °C overnight. After washing three times, the slices were further incubated with anti-rabbit Cy3 antibody (Beyotime) for 1 h at 37 °C and counterstained with 4' ,6-diamidino-2-phenylindole. The slices were then visualized and images were captured with a fluorescence microscope. Western blotting. PAVECs at 1 × 10 7 CFU/mL were pretreated with NAC (1 mM) or baicalin (final concentration 12.5, 25, 50, or 100 μg/mL) for 1 h, respectively, and H. parasuis at 1.0 × 10 7 CFU/mL was then added into the plate wells and co-incubated for 12 h. The cells were collected and total cell protein was extracted using a total protein extraction kit (Beyotime Biotechnology). The protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma). Total cell proteins were isolated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane and blocked with 5% skim milk for 1 h at 25 °C. After washing five times in TBST, the membrane was was cultured with cleaved caspase-1 antibody or β-actin antibody (Cell Signaling Technology, USA) for 12 h at 4 °C, followed by five washes in TBST, incubation with horseradish peroxidase-linked goat anti-rabbit antibody (Proteintech, USA) at 25 °C for 1 h, and then visualized using ECL solution (Thermo Pierce ECL, USA). The expression levels of cleaved caspase-1 and β-actin were measured using a FluorChem FC2 AIC system (Alpha Innotech, USA).

Statistical analysis.
The experimental data were expressed as mean ± SD. The difference between two groups was analyzed using the two-tailed Student t test. P values of <0.05 were considered significant. *p < 0.05; **p < 0.01 and ***p < 0.001.