Echinacea purpurea extract (cichoric acid) exerts an anti-inflammatory effect on yak PBMCs and regulates the TLR4 signalling pathway

Inflammation is a localised protective response elicited by injury or destruction of tissues that serves to eliminate, dilute, or sequester the injurious agent and injured tissues (10). Inflammation damages the health of livestock frequently and consequentially, thus causing huge economic losses to commercial animal husbandry. Some inflammatory processes not only lead to reduced livestock productivity, but also have a significant impact on human health. Echinacea (Echinacea purpurea) is a perennial herb of the Chrysanthemum genus which is native to North America and Canada. Studies have shown that it has antiviral, anti-inflammatory, and immunoregulatory properties (13, 17, 30). The lipopolysaccharide (LPS) is a strongly pro-inflammatory molecule outside the cell wall of Gram-negative bacteria that is comprised of conserved lipid A and polysaccharides. This molecule activates the toll-like receptor (TLR)4 signalling pathway, which leads to the release of inflammatory cytokines, while the release of LPS causes an inflammatory response (3, 14, 26, 28). The aim of this study was to evaluate the effects of echinacea extract (cichoric acid – CA) on the yak peripheral blood mononuclear cell (PBMC) inflammatory response and these cells’ TLR4 pathway to provide a theoretical basis for the rational development of utilisations of echinacea.

Animals. Six grazing yaks were provided by the Datong Breeding Farm (Qinghai, China). Cichoric acid was purchased from Sigma Xi’an Rui Bo Biological Technology Company (Shanxi, China). Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Shanghai, China), foetal bovine serum (Hangzhou Sijiqing Co., Ltd., Hangzhou, China), LPS (Solarbio, Beijing, China), a cell-counting kit (CCK)-8 (Jiangsu Haimen Biyuntian Biotechnology Research Institute, Jiangsu, China), myeloid differentiation primary response (MyD)88 antibody (Cell Signaling Technologies, Danvers, MA, USA), tumour necrosis factor (TNF) receptor associated factor (TRAF)6 antibody (Bioworld Technology, Bloomington, MN, USA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) antibody (Abcam, Cambridge, UK), and interferon regulatory factor (IRF)5 antibody (Proteintech Group, Manchester, UK) were used in experiments as described below. In order to avoid non-specific responses of immunocompetent cells, a bacterial LPS-free CA solution was prepared by dissolving 0.18 g of cichoric acid soluble powder in 1 mL of dimethyl sulphoxide (DMSO) solution, and then diluting this with 4.88 mL of medium to make a stock solution which was stored in a refrigerator at −20°C and diluted to the required concentration. In the early stage of this preparation, the research group verified that the concentration of DMSO used (< 0.1%) was not toxic to the cells (25).

Instruments and equipment. A super-clean worktable (Jiangsu Tongjing Purification Equipment Co., Jiangsu, China), MU-5810E CO₂ incubator (Nuaire, Plymouth, MN, USA), V18R horizontal centrifuge (Dynamica Scientific, Livingston, UK), GelDoc XR Biorad System (Bio-Rad, Hercules, CA, USA), power supply (Bio-Rad), T100 thermal cycler T100 (Bio-Rad), Amnis flow cytometer (Luminex, Seattle, WA, USA), and Power Wave XS2 absorbance microplate reader (Bio-Tek Instruments, Winooski, VT, USA) were used in the experiments described below.

Cell culture. Peripheral blood mononuclear cells were isolated by Ficoll (TBD, Shanghai, China) density gradient centrifugation and cultured in RPMI-1640 medium containing 10% foetal bovine serum and were then placed in a humidified incubator at 37℃, 5% CO₂ and 95% humidity. The medium was changed once every 24 h.

CCK-8 assay of cell viability. Yak PBMCs were seeded into a 96-well plate at a density of 1×10⁶ cells/well. Then, 2.5 μg/mL of ConA (100 μL) was added, and the final LPS concentration was adjusted to 0.1 μg/mL, 1 μg/mL, 5 μg/mL, and 10 μg/mL. The concentration of CA (60 μg/mL) was determined as it also had been by this research group in a previous experiment and used at the same concentration in the CA and CA + LPS groups. Each group was provided with three wells and cultured in a 5% CO₂ humidified incubator at 37°C for 48 h. The 96-well plate was removed, the instructions of the CCK-8 kit were followed, and the absorbance at 450 nm was measured with the Power Wave XS2 microplate reader.

Inflammatory cytokine determination. The treated cells were used to detect inflammatory cytokines. The cells were inoculated into 96-well plates (1×106cells/well). The concentration of CA was 60 μg/mL, LPS was 1μg/mL, and the optimal concentration was determined by CCK-8. Each experiment was repeated three times. The cells were cultured in an incubator at 37℃ under 5% CO₂ for 48 h. Interleukin 6, 8, 10 and 1β and TNF-α and INF-γ ELISA kits were used according to the protocols provided by the manufacturer (MyBioSource, San Diego, CA, USA ). The absorbance values of related factors were determined by the microplate reader.

RT-PCR. Evaluation of the mRNA expression of INF-γ, TNF-α, IL-10, IL-1β, IL-8, NF-κB, IRF5, TRAF6, and MyD88 was made with an RT-PCR. Total RNA was extracted from yak PBMCs using Trizol reagent according to the manufacturer’s instructions (Takara, Japan). An M-MLV reverse transcriptase kit was used to reverse transcribe RNA in accordance with the manufacturer’s instructions (Tiangen Biotechnology, Beijing, China) and detected according to the instructions for the real-time fluorescent RT-PCR kit (Tiangen Biotechnology, Beijing, China). GAPDH expression was included as an internal housekeeping gene control. Primer sequences as listed in Table 1 were designed and synthesised by Shanghai Shenggong (Shanghai, China). The levels of gene expression were analysed using the 2_-^△△CT method.

Western blot analysis. After being treated for 48 h, cells were lysed with a protein extraction solution, harvested, and evaluated in terms of protein concentrations by using BCA (the binding reaction of caffeic acid with bovine serum albumin). Gel was prepared for SDS-PAGE electrophoresis, and samples were added to the wells for electrophoresis. Resolution through 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transfer onto PVDF membranes took place. The membrane was sealed with skim milk at room temperature for 1h. The primary antibody was diluted and incubated on the PVDF membrane overnight at 4℃, then decolorised and washed three times on the shaker with tris-buffered saline with Tween (TBST) for 5 min at room temperature. The secondary antibody was diluted 3000-fold with TBST, incubated on the PVDF membrane at room temperature for 30 min, then decolorised with TBST and washed three times on the shaker for 5 min each. The electrochemiluminescent A and B reagents were mixed in a medium-volume centrifuge tube with the protein on the PVDF membrane facing the mixture. After 5 min, the residual liquid was removed, wrapped, and put into a cassette for exposure. The Bio-Rad gel imaging system was used for visualisation.

Statistical analysis. Data analysis was performed using SPSS 22.0 (IBM, Armonk, NY, USA). All data were expressed as the mean ± SD. Statistical significance was determined by Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests, and P < 0.05 was considered a statistically significant difference.

LPS is an important component of the outer membrane of Escherichia coli and is considered to be the main pathogenic factor in Gram-negative bacteria (9). The inflammatory response is considered to be a potential biological protective mechanism for epithelial cell damage, which may be caused by chemical factors (such as toxic organic compounds) and foreign bodies (including various metals, wood chips, and sawdust and dust entering the body) (16). Echinacea extract enhances immunity, exhibits anti-inflammation, anti-oxidation, anti-virus, and anti-tumour activities, and regulates cell apoptosis. Echinacea is one of the few medicinal plants currently known to have immunity-enhancing and anti-inflammatory effects. It has a wide range of uses in treatment, and stable and discriminating curative effects (2, 4). Studies have shown that macrophages, peripheral blood monocytes and ATCII synthesise and secrete many cytokines and inflammatory mediators, including TNF-α, IL-6, and IL-8 (6, 24). Tumour necrosis factor-α leads to the loss of epithelial barrier function and enhances the permeability of monocytes, and also acts as a pro-inflammatory cytokine to promote the secretion of IL-1β, IL-6, and IL-8 (27). Interleukin-1β activates macrophages and neutrophils (7). Interleukin-6 activates the proliferation of neutrophils and B lymphocytes (21), and IL-8 is a chemokine factor that activates neutrophils and changes their morphology, thus enabling neutrophils to migrate to the reaction site (19). In the current study, yak PBMCs treated with LPS had raised levels of IL-6, IL-8, IL-1β, INF-γ and TNF-α, and the expression of each gene in the CA+LPS treatment group was significantly lower than in the LPS treatment groups. The inhibitory effect of CA on LPS-induced inflammatory factors was verified.

Toll-like receptor 4 is the most important member of the TLR family. It resists the invasion of pathogenic microorganisms via the innate immune response by mediating the expression of NF-κB (12), which is a group of important transcription factors with a wide range of biological functions. The cellular biological effects include stress-induced, immune, and inflammatory responses, and the development of haematopoietic cells, lymphatic organs, and keratinocytes, as well as cell proliferation and apoptosis, tumorigenesis, and neuronal synaptic formation (23). Interferon regulatory factor 5 plays an important role in regulating the transcription of interferon, immune response to pathogens, signal transduction of cytokines, and immune regulation (1, 15, 18). Myeloid differentiation primary response 88, as the first identified toll-like/IL-1 (TIR) domain containing adaptor protein, is recruited by all TLRs except TLR3 in mammals (11). The principal functional domains of MyD88 consist of the C-terminal TIR domain and the N-terminal death domain (8). The TIR domain interacts with the TIR domain of activated TLRs, and the death domain is involved in recruiting the downstream molecule IL-1 receptor-associated protein kinase (IRAK) (22). Subsequently, the MyD88-IRAK complex induces the auto-ubiquitination of TRAF6, which is the only member of the TRAF family that participates in the MyD88-dependent pathway (5, 20). Then, the MyD88-IRAK complex activates NF-κB and activator protein 1, and inflammatory cytokines are produced and eventually eliminate pathogens (29). In addition, the TRAF6 gene is a key node gene in the TLR pathway and can directly affect the cell cycle, apoptosis, and growth. These structures indicate that CA may directly or indirectly affect the immune regulatory mechanism through the TLR pathway. The results showed that in yak PBMCs treated with LPS, the levels of MyD88, NF-κB and TRAF6 were augmented, and their levels in the CA+LPS treatment group were significantly diminished from those in the groups treated only with LPS. The results also showed that CA can regulate the TLR4 signalling pathway in yak PBMCs and that it enhances immune regulation and anti-inflammatory mechanisms. Immune regulation was mainly manifested in inhibition of the immune response, thereby enhancing the immune function of grazing yak.

In summary, CA inhibited the LPS-induced inflammatory response of yak PBMCs and gene and protein expression of key nodes of the TLR4 signalling pathway in these cells. This study suggests that CA has anti-inflammatory and immunomodulatory effects on yak PBMCs through the TLR4 pathway.

* These authors contributed equally to this study and should be considered co-first authors.