Inflammation response is a regulated cellular process and excessive inflammation has been recognized in numerous diseases, such as cardiovascular disease, neurodegenerative disease, inﬂammatory bowel disease, and cancer. Tribulus terrestris L. (TT), also known as Bai Jili in Chinese, has been applied in traditional Chinese medicine for thousands of years while its anti-inflammatory activity and underlying mechanism are not fully elucidated. Here, we hypothesize Tribulus terrestris L. extract (BJL) which presents anti-inflammatory effect, and the action mechanism was also investigated. We employed the transgenic zebrafish line Tg(MPO:GFP), which expresses green fluorescence protein (GFP) in neutrophils, and mice macrophage RAW 264.7 cells as the in vivo and in vitro model to evaluate the anti-inflammatory effect of BJL, respectively. The production of nitric oxide (NO) was measured by Griess reagent. The mRNA expression levels of inflammatory cytokines and inducible nitric oxide synthase (iNOS) were measured by real-time PCR, and the intracellular total or phosphorylated protein levels of NF-κB, Akt, and MAPKs including MEK, ERK, p38, and JNK were detected by western blot. We found that BJL significantly inhibited fin transection or lipopolysaccharide- (LPS-) induced neutrophil migration and aggregation in zebrafish in vivo. In mice macrophage RAW 264.7 cells, BJL ameliorated LPS-triggered excessive release of NO and transcription of inflammatory cytokine genes including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). BJL also reduced the LPS-induced elevations of intracellular iNOS and nuclear factor kappa B (NF-κB) which mediate the cellular NO and inflammatory cytokine productions, respectively. Moreover, LPS dramatically increased the phosphorylation of Akt and MAPKs including MEK, ERK, p38, and JNK in RAW 264.7 cells, while cotreatment BJL with LPS suppressed their phosphorylation. Taken together, our data suggested that BJL presented potent anti-inflammatory effect and the underlying mechanism was closely related to the inhibition of Akt/MAPKs and NF-κB/iNOS-NO signaling pathways.
Inflammatory response defenses the foreign attacks including tissue injury, pathogens, infections, and irritants and restores normal tissue function . It plays a pivotal role in the physiological process of immunomodulation , and excessive inflammation response is also the main pathological feature in numerous chronic diseases, including cardiovascular disease, neurodegenerative disease, inﬂammatory bowel disease, rheumatoid arthritis, and cancer . Many types of inflammatory cells, such as neutrophils, eosinophils, macrophages and mononuclear phagocytes, are involved in the inflammatory response . The blood resident neutrophil is commonly the primary cell that migrates to the inflammatory position and initiates inflammatory action . It is well-known that macrophages interact with neutrophils and clean the damage tissue and the other inflammatory cells by phagocytosis and consequently suppress the inflammation action [5, 6]. However, macrophages produce proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, which mediate by the transcript factor NF-κB during the tissue repairing process [7, 8]. The production of NO is an important feature of inflammatory response mediated by macrophages, which causes cell oxidative damage and is dramatically regulated by iNOS . The transcription of iNOS gene is regulated by various transcript factors including NF-κB, activator protein-l (AP-l), interferon regulatory factor 1 (IRF1), and signal transducer and activator of transcription 1 (STAT1) . Moreover, the activation of Akt/MAPKs signaling cascade was considered as one of the important physiological procedures on LPS-induced inflammation both in macrophage and zebrafish [11, 12]. Thus, pharmacological inhibition of Akt/MAPKs and NF-κB/iNOS-NO signaling pathways might be effective for the suppression of tissue injury during inflammatory response.
Tribulus terrestris L. (TT, Zygophyllaceae family), which contains active components including alkaloids, steroidal saponins, ﬂavonoids, tannins, amino acids, quinines, and phenolic compounds, is a commonly used traditional Chinese herbal medicine, also known as Bai Jili in Chinese, for treating various diseases including hypertension, edema, eye problems, sexual dysfunction, and rheumatoid arthritis in clinics for thousands of years [13, 14]. The prosexual, cardiac-protective, muscle protective, neuroprotective, and osteoprotective effects of TT were most studied in the recent years [14–19], and the anti-inflammatory effect of TT might contribute to these broad range of biological effects . In our previous studies, we have established multiple drug screening models using zebrafish, such as proangiogenesis [20, 21], antiangiogenesis , cerebral hemorrhage [23, 24], and neuroprotection [25, 26]. Zebrafish also has been supposed as one of the wildly used anti-inflammatory drug screening in vivo models, with several advantages including low cost, easy observation, and short test period [4, 27, 28]. In the present study, we evaluated the anti-inflammatory effect of BJL in zebrafish in vivo and mice macrophage RAW 264.7 cells in vitro, and the action mechanisms were also partially elucidated.
2. Materials and Methods
2.1. Ethic Statement
Zebrafish were kindly provided by Prof. Simon Lee from University of Macau and maintained by the Laboratory Animal Service Center, Longhua Hospital, Shanghai University of Traditional Chinese Medicine. All experiments were in accordance with the Longhua Hospital-Animal Experimentation Ethics Committee of Shanghai University of Traditional Chinese Medicine.
Lipopolysaccharide (LPS) was bought from Sigma Aldrich (St. Louis, USA). Nitric oxide (NO) detection kit was supplied by Beyotime Biotechnology (Shanghai, China). Primary and secondary antibodies were bought from Cell Signaling Technology (MA, USA). The whole plant of Tribulus terrestris L. (TT) was identified by the Pharmacy Department, Longhua Hospital. Tribulus terrestris L. extract (BJL) was prepared by Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China). Briefly, TT was dried and smashed. 100 g TT powder was boiled within 1200 ml distilled water for 1 h and the supernatant was collected. The TT powder was continually boiled with 800 ml distilled water for 1 h and the supernatant was collected. These two supernatants were combined and stored at 5–10°C for 12 h. Then, the crude TT extract was filtered and absorbed by macroporous resin (1400, 2 : 1). Finally, the macroporous resin was washed by 60% ethanol, and the eluate was concentrated and sprayed to avoid dehydration. The yield of TT extract (BJL) was 4.92%. The chemical profile of BJL was analyzed by high performance liquid chromatography (HPLC) and is presented in supplementary materials (Figure S1). BJL was dissolved in dimethyl sulfoxide (DMSO) while other drugs were prepared in MiliQ water.
2.3. Zebrafish Maintenance and Morphological Observation
The transgenic zebrafish line Tg (MPO:GFP), which expresses green fluorescence protein (GFP) in neutrophils , was employed for anti-inflammatory activity evaluation. Zebrafish were maintained under a stander condition according to the zebrafish book (A guide for the laboratory use of zebrafish Danio (Brachydanio) rerio, by Monte Westerfield, Institute of Neuroscience, University of Oregon). Zebrafish embryos were generated by natural breeding and culture in embryo media at 28.5°C in an incubator. Three DPF (day post fertilization) zebrafish embryos were distributed in a 12-well plastic plate with 10 embryos in each group. On the fin transection model, zebrafish embryos were treated with various concentrations (3, 10, and 30 µg/ml) of BJL for 2 h, and then zebrafish embryos were cut fins using a sharp needle. Then, zebrafish embryos were incubated with BJL for another 2 h, and the number of fluorescent cells migrated to wound was observed under a fluorescent stereoscopic microscope (Nikon SMZ18, Japan). On the LPS-injection model, zebrafish embryos were injected with LPS (0.3 µg/ml), and then zebrafish embryos were treated with various concentrations (3, 10, and 30 µg/ml) of BJL for 24 h. The fluorescence intensity of injection site in each embryo was calculated by Image J.
2.4. Cell Viability and Nitric Oxide (NO) Release Assay in RAW 264.7 Macrophage
The mice macrophage RAW 264.7 cell line was purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2 in air. RAW 264.7 cells were seeded in a 96-well plate with a density of 2 × 10⁴ cells/well and further cultured for 24 h. RAW 264.7 cells were treated LPS (0.3 µg/ml) with various concentrations (3, 10, and 30 μg/ml) of BJL for 24 h. The cell viability was measured by MTT assay. The supernatant medium was used for NO release assay by total NO detection kit (Griess reagent, Beyotime Biotechnology). The results were presented as the percentage or folds of the control group.
2.5. Western Blot Analysis
RAW 264.7 cells were seeded in 60 mm × 15 mm Petri dish with a density of 5 × 10⁵ cells/dish and incubated for 24 h and then treated BJL (30 μg/ml) with or without LPS (0.3 μg/ml) for 24 h. After drug treatment, the RAW 264.7 cells were washed with ice cool PBS 3 times and lysed by RIPA buffer (Beyotime Biotechnology) on ice. The cell samples were collected and centrifuged (12000 g) for 15 min at 4°C. The supernatant of each sample was transferred to a new ice cool tube and its protein concentration was determined by BCA kit (Thermo Fisher). Each sample was denatured for 5 min at 95°C after adding the 5 × loading buffer (Beyotime Biotechnology). 30 μg protein of each sample was applied for the protein separation by SDS-PAGE electrophoresis (Bio-Rad). Then, the separated protein was transferred to a PVDF membrane (0.22 μm) by the semidry transfer system (Bio-Rad). The membrane was blocked by 5% skim milk in PBST for 2 h and then followed by primary antibodies including NF-κB, Akt, Phospho-Akt, ERK1/2, Phospho-ERK1/2, p38, Phospho-p38, JNK, Phospho-JNK, and GAPDH (1 : 1000, Cell Signaling Technology) incubation over night with gentle shacking at 4°C. The membrane was washed with PBST 3 times and incubated with HRP-linked secondary antibody for 2 h at room temperature. Finally, the membrane was washed with PBST 3 times and imaged by the AM600 image system (GE Healthcare) after adding the ECL chemiluminescence substrate (Bio-Rad). The intensity of each band was calculated by Image J.
2.6. Real-Time PCR Analysis
RAW 264.7 cells were seeded in 60 mm Petri dish with a density of 5 × 10⁵ cells/well and incubated for 24 h at 37°C in a humidified atmosphere of 5% CO2 in air. RAW 264.7 cells were treated BJL (30 μg/ml) with or without LPS (0.3 μg/ml) for 24 h. The total RNA of each group was extracted by the TriPure Isolation Reagent (Roche, Manneheim, Germany) and their RNA concentrations were detected and calculated by absorbance at 260 nm using Microplate Reader (Tecan M200, NanoQuant). The first stranded cDNA was synthesized by cDNA synthesis kit (Roche, Manneheim, Germany). Finally, the mRNA expression level of interested gene was detected by real-time PCR with specific primers (Table 1) under the SYBR green-based real-time PCR system (Roche, LC96). The mRNA expression level of each gene was calculated by 2−∆∆Ct relative quantification method with 3 independent replicates.
5′-TTC TCA TTC CTG CTT GTG G-3′
5′-ACT TGG TGG TTT GCT ACG-3′
5′-GAG GAT ACC ACT CCC AAC AGA CC -3′
5′-AAG TGC ATC ATC GTT GTT CAT ACA-3′
5′-AGA GCA TCC AGC TTC AAA T-3′
5′-CAT CTC GGA GCC TGT AGT G-3′
5′-CCT TCC GTG TTC CTA CCC-3′
5′-CAA CCT GGT CCT CAG TGT AG-3′