2 Agricultural College, Yanbian University, Yanji 133002, China;
3 Department of Urology, College of Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea
Inflammation is a basic immune response to infection, irritation, and other injuries [1-2]. However, the excessive production of inflammatory factors may lead to pulmonary fibrosis, rheumatoid arthritis and pulmonary atherosclerosis [3-5]. Macrophages release nitric oxide (NO), prostaglandin E2 (PGE2), and other cytokines, such as IL-1β, TNF-α, and IL-6 during lipopolysaccharide (LPS) stimulation . Thus, the production of these inflammatory mediators and cytokines considerably affects the treatment of inflammatory diseases.
Toll-like receptor 4 (TLR4) is a toll-like receptor subtype that can activate endogenous or exogenous immune responses after LPS stimulation . TLR4 can activate two signaling pathways. The first signaling pathway is the MyD88-dependent pathways of mitogen-activated protein kinase (MAPK) and NF-κB that induce the release of IL-1β, TNF-α, and IL-6. The second signaling pathway is the TRLF-dependent pathway that facilitates TLR4 signaling, which leads to the activation of interferon regulatory factor 3 (IRF3) .
Cardamine komarovii is a cruciferous plant of genus Cardamine, and is mainly distributed in Korea and Northeast China. C. komarovii flower (CKF) is an edible and medicinal plant. For example, CKF can maintain hemostasis, reduce blood pressure, and induce sedation [9-10]. CKF consumption increases resistance to oxidative stress. However, reports on the anti-inflammatory effect of CKF remain insufficient.Materials and Methods Chemical
Enzyme-linked immunosorbent assay (ELISA) kits were acquired from BD Bioscience (SD, USA). SyBr and PrimeScriptTM PT reagent kit were obtained from TaKaRa (Shiga, Japan). p-ERK, p-JNK, p-p38 and other antibodies were obtained from Cell Signaling Technology (MA, USA). NF-κB p65 was purchased from Santa Cruz Biotechnology (CA, USA). Alexa Fluor 594-conjugated AffiniPure Goat Anti-mouse IgG was purchased from Jackson ImmunoResearch (PA, USA).Plant authentication and extraction
CKFs were collected in Helong (GPS Coordinates: N42°31′17.26″ E128°38′47.27″; altitude: 1276 m, June 2015, China) and were authenticated (voucher specimen: YBU-0846) by Prof. LV Hui-Zi, a taxonomist from Yanbian University, China. Dried CKF (50 g) was soaked in 500 mL of ethanol for reflux extraction, and recovery percent was 10.0 %. After freeze-drying, the samples were stored at -20 ℃ before use.Animals
C57BL/6 mice (20 ± 2 g, 5-6 weeks old) were obtained from Changchun Yisi Experimental Animal Co. (Changchun, China; SPF, SCXK (J) 2017-0001). The mice were fed in an experimental animal room under a constant temperature of 23 ± 3 ℃, humidity of (50 ± 3)%, and a light of 12 h and dark of 12 h. Food and drinks were sufficient. All experiments were conducted in accordance with the guidelines of Yanbian University Experimental Animal Center.High-performance liquid chromatography (HPLC) analysis of CKF extracts
CKF extracts were dissolved in methanol and were filtered through a membrane filter (0.45 μm) prior to chromatographic detection. Flavonoids were determined, as previously described .Cell viability test
Cell viability was determined as previously described . The medium was discarded, and dimethyl sulfoxide was added to each well to solubilize formazan crystals. Optical density value was measured at 540 nm by using a spectrophotometer. The cell viability of the treatment groups was compared with the control group.Determination of NO and PGE2 production
The effect of CKF on NO concentration was determined as previously described . PGE2 was measured based on the manufacturer's instructions.ELISA
ELISA was performed as previously described . Different kits were used to measure the levels of IL-1β, TNF-α, and IL-6, in accordance with the manufacturer's instructions. Optical density was measured at 450 nm by using a spectrophotometer.Quantitative PCR
Cells (2.7 × 106) were seeded in each well plate. Different concentrations of CKF (12.5, 25, and 50 μg·mL-1) with or without LPS were added to the plates. RNA was extracted using TRIzol, reverse-transcribed into cDNA, and subjected to quantitative PCR (qPCR), as previously described . The housekeeping gene, Cyclopilin A, was used to normalize tested genes, and data quantification was calculated using the 2-ΔΔCT method.Immunoblot analysis
Immunoblot analysis was performed as previously described . CKF extracts with different concentrations were added to plates containing peritoneal macrophages (PMs, 2.5 × 106 cells/well). PMs were stimulated with LPS or were untreated. The cells were treated with RIPA, and the supernatant was removed. The protein concentration of each sample was measured, and the same amount of protein was added to 12% SDS-PAGE protein sample buffer to separate the samples and was transferred to PVDF membranes. The membranes were incubated overnight with primary antibodies at 4 ℃ after overnight blocking with 0.1% Tris-buffered saline, polysorbate 20 (TBS-T), and 5% skim milk. The membranes were incubated with secondary antibodies for 1 h and visualized by using an ECL detection kit (ChemiDocTM XRS+ System, Bio-Rad, USA).Immunocytochemistry
Immunocytochemistry analysis was performed as previously described . CKF extracts (50 μg·mL-1) and DEX (0.5 mmol·L-1) was added to plates containing peritoneal macrophages (PMs, 2.5 × 106 cells/well). PMs were stimulated with LPS or were untreated for determining the nuclear translocation of NF-κB.BALF cytokine and cell population measurement
An LPS-induced ALI model was established as previously described . LPSs were instilled in the intratracheal of mice after administration of CKF extracts (5 and 10 mg·kg-1). Mice were sacrificed to collect BALF by washing with ice PBS. After centrifugation, part of cell-free supernatants was used to detect the levels of inflammatory factors, such as IL-1β, TNF-α, and IL-6 in BALF by using ELISA kits based on the instructions. Cellularity was determined by using a flow cytometer after the suspension of cell precipitation in PBS solution. FITC-labeled anti-Gr1 (neutrophils) were purified from BALF cells by using BD FACSVerseTM. Data were analyzed in FlowJo 10.0.Lung histopathology
The right lung was soaked in formalin and was embedded in paraffin. The paraffin tissue was cut into 5 μm sections and was stained with H & E. The lung histopathology scores as previously described .Statistical analysis
All data were expressed as the mean ± standard error of at least three separate experiments conducted in triplicate. Statistical analysis was performed through variance analysis combined with rank-sum test. P < 0.05 was considered statistically significant.Results Extract standardization through HPLC
Fig. 1 presents the HPLC profiling analysis results of methanol-extract material of CKF. The total flavonoid, myricetin, and quercetin contents of the extracts are 41.13, 2.88, and 0.36 mg·g-1, respectively.
The anti-inflammatory effect of CKF was investigated, and MTT assay was conducted to determine the cell viability of CKF. CKF extracts with concentrations of up to 50 μg·mL-1 did not exert any toxic effect on PMs. Therefore, we detected the in vitro anti-inflammatory activities of 12.5, 25, and 50 μg·mL-1 CKF extracts (Fig. 2). We evaluated the NO, PGE2, and their synthesis-related enzymes iNOS and COX-2 accumulation in PMs to investigate the effect of CKF extracts on LPS-stimulated NO and PGE2 production. PMs were stimulated with or without LPS after treatment with different concentrations of CKF extracts. NO (Fig. 3A) and PGE2 (Fig. 3B) levels remarkably decreased with the treatment of CKF extract in a dosage-dependent manner (P < 0.05). iNOS and COX-2 are precursors of NO and PGE2. Therefore, we measured NO and PGE2 levels by conducting an immunoblot assay. As shown in Fig. 3C, iNOS and COX-2 expression levels notably decreased in the CKF group, compared with model group (P < 0.05).
Pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, play important roles in various inflammatory diseases [15-16]. CKF treatment can remarkably reduce IL-1β (Fig. 4A), TNF-α (Fig. 4B), and IL-6 (Fig. 4C) secretion in a dosage-dependent manner. In addition, qPCR analysis showed that CKF treatment decreases IL-1β (Fig. 4D), TNF-α (Fig. 4E), and IL-6 (Fig. 4F) mRNA expression levels.
We evaluated the effect of CKF extracts on the phosphorylation of MAPKs, such as p38, ERK, and JNK, in LPS-induced PMs to investigate whether they can regulate the MyD88-dependent pathway. As shown in Fig. 5, the phosphorylation levels of ERK, and JNK has no significantly decreased, but the phosphorylation levels of p38, decreased under CKF treatment in a dosage-dependent manner compared with the model group (P < 0.05). Immunocytochemistry staining (Fig. 6) showed that CKF extracts attenuate the LPS-induced translocation of NF-kB p65 subunit into the nucleus from the cytoplasm. Therefore, our findings suggested that CKF plays an anti-inflammatory role by regulating the NF-κB signaling pathway. The attenuation of CKF on the production of pro-inflammatory mediators through the TRIF-dependent signaling pathway was investigated. As shown in Fig. 7, CKF remarkably inhibits p-IRF3, p-STAT1, and p-STAT3 production compared to the model group (P < 0.05). These findings suggested that CKF has an anti-inflammatory effect through the regulation of the TRIF-dependent signaling pathway.
This study conducted the anti-inflammation experiment of CKF in vivo by using the ALI model. BALF and lung tissues were collected from the sacrificed mice, the number of cell populations, total protein in BALF, and secretion of pro-inflammatory mediators (IL-1β, TNF-α, and IL-6) were measured, and the pathological conditions of lung tissues were observed through H & E staining. The results showed that CKF treatment can decrease the protein of BALF (Fig. 8A). The number of neutrophils was analyzed to determine the cellular infiltration components in BALF. The results showed that the neutrophils (Fig. 8B) in the LPS group remarkably increase, whereas the neutrophils in the CKF and DEX groups remarkably decrease (P < 0.05). The production of IL-1β (Fig. 8C) was remarkably inhibited under 5 and 10 mg·kg-1 of CKF extracts (P < 0.05). However, CKF showed no obvious depressant action on TNF-α or IL-6 (data not shown). The results showed that the lungs have remarkable decrease of alveolar space, interstitial and alveolar cell infiltration after the intratracheal instillation of LPS. CKF treatment remarkably improves the LPS-induced lung histopathological injury (P < 0.05; Fig. 8D).
Medicinal plants play a vital role in the primary healthcare system. However, few studies have been reported on the anti-inflammatory effect of CKF. The results of this study confirmed that CKF suppresses the inflammatory responses in PMs through MyD88/TRIF pathways. In this study, we found that the total flavonoid content of CKF is 41.13 mg·g-1, and two flavonoid compounds (myricetin and quercetin) are determined through HPLC. These results confirmed that the anti-inflammatory activity of CKF is closely related to high flavonoid content .
LPS stimulation can induce macrophages to produce inflammatory cytokines. NO levels are affected by iNOS expression, which induce the continuous production of NO . The aberrant expression of COX-2 causes the massive release of PGE2, which leads to rheumatoid arthritis and inflammatory liver injury [19-20]. We found that CKF can inhibit NO and PGE2 expression levels by down-regulating iNOS and COX-2 levels in LPS-induced PMs. In addition, the overproduction of pro-inflammatory cytokines may accelerate the pathological changes in inflamed tissues. These changes result in shock, tissue necrosis, and endothelial dysfunction . In this study, we detected the mRNA expression of IL-1β, TNF-α and IL-6, and we found that CKF inhibits the expression levels of IL-1β, TNF-α and IL-6 in a dosage-dependent manner.
TLR4 triggers the activation of the MyD88-and/or TRIF-dependent signaling pathway. Ligation of LPS to TLR4 triggers its association with MyD88 signaling pathways, such as the MAPK and NF-κB pathways. The phosphorylation level of ERK, JNK and p38 in MAPK can regulate the production of NO and pro-inflammatory cytokines [22-23]. IκB degradation can lead to the transcription of NF-κB into the nucleus that can regulate COX-2 expression. Our results indicate that CKF inhibits the phosphorylation levels of p38 and attenuate NF-kB into the nucleus from the cytoplasm that inhibits the production of inflammatory cytokines and mediators.
The TRIF-dependent pathway lead to IFN-β expression and IRF3 phosphorylation activates the transcription and protein expression of IFN-β . The extracellular IFN-β protein binds to the IFN receptor and induces the phosphorylation of STAT1 and STAT3 . Subsequently, the down-regulation of STAT1 phosphorylation decreases iNOS expression and NO production. In addition, the induction of IFN-β expression by IRF3 phosphorylation promotes STAT3 phosphorylation, which leads to TNF-α and IL-6 production. Our data indicate that the anti-inflammatory effect of CKF is closely related to the down-regulation of the phosphorylation of vital factors (IRF3 and STAT1/3) that constitutes the TRIF-dependent signaling pathway.
We investigated the protection of CKF on mice from LPS-induced ALI to demonstrate the in vivo anti-inflammatory properties of CKF. Previous studies have shown that LPS-induced ALI is characterized by alveolar inflammatory cell infiltration, which is mainly manifested as neutrophil infiltration, and extensive release of inflammatory medium in lung tissues was observed . BALF concentration is a characteristic of alveolar capillary permeability. BALF concentration in the LPS group is higher than that in other groups. Meanwhile, CKF can decrease the BALF concentration and present certain dose dependence. Our study found that CKF can remarkably reduce the excessive production of IL-1β from the BALF in ALI. In the present study, extensive inflammatory cells, including neutrophils, entered the lung tissues after the LPS stimulation on mice. However, CKF treatment can decrease the number of inflammatory cells in the lungs. The result revealed that CKF has a good anti-inflammatory effect. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In vivo experiments demonstrated the anti-inflammatory effect of CKF.
In conclusion, CKF can inhibit the expression of inflammatory factors in LPS-induced PMs by regulating the MyD88 and TRIF signaling pathways. CKF can protect mice from LPS-induced ALI. In vitro and in vivo studies demonstrated the anti-inflammatory effect of CKF and provided a theoretical basis for subsequent studies on CKF.
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