Tribulus terrestris L. Extract Protects against Lipopolysaccharide-Induced Inflammation in RAW 264.7 Macrophage and Zebrafish via Inhibition of Akt/MAPKs and NF-κB/iNOS-NO Signaling Pathways

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.


Background
Inflammatory response defenses the foreign attacks including tissue injury, pathogens, infections, and irritants and restores normal tissue function [1]. It plays a pivotal role in the physiological process of immunomodulation [2], 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 [3].
Many types of inflammatory cells, such as neutrophils, eosinophils, macrophages and mononuclear phagocytes, are involved in the inflammatory response [1]. e blood resident neutrophil is commonly the primary cell that migrates to the inflammatory position and initiates inflammatory action [4]. 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]. e production of NO is an important feature of inflammatory response mediated by macrophages, which causes cell oxidative damage and is dramatically regulated by iNOS [9]. e 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) [10]. Moreover, the activation of Akt/MAPKs signaling cascade was considered as one of the important physiological procedures on LPSinduced inflammation both in macrophage and zebrafish [11,12]. us, 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]. e prosexual, cardiac-protective, muscle protective, neuroprotective, and osteoprotective effects of TT were most studied in the recent years [14][15][16][17][18][19], and the anti-inflammatory effect of TT might contribute to these broad range of biological effects [13]. In our previous studies, we have established multiple drug screening models using zebrafish, such as proangiogenesis [20,21], antiangiogenesis [22], 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.  ). 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. e TT powder was continually boiled with 800 ml distilled water for 1 h and the supernatant was collected.

Materials and Methods
ese two supernatants were combined and stored at 5-10°C for 12 h. en, the crude TTextract 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. e yield of TT extract (BJL) was 4.92%. e 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.

Zebrafish Maintenance and Morphological Observation.
e transgenic zebrafish line Tg (MPO:GFP), which expresses green fluorescence protein (GFP) in neutrophils [29], 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. ree 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. en, 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 LPSinjection 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. e fluorescence intensity of injection site in each embryo was calculated by Image J. e cell viability was measured by MTT assay. e supernatant medium was used for NO release assay by total NO detection kit (Griess reagent, Beyotime Biotechnology). e results were presented as the percentage or folds of the control group.

Western Blot
Analysis. RAW 264.7 cells were seeded in 60 mm × 15 mm Petri dish with a density of 5 × 10 5 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.
e cell samples were collected and centrifuged (12000 g) for 15 min at 4°C. e supernatant of each sample was transferred to a new ice cool tube and its protein concentration was determined by BCA kit ( ermo 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). en, the separated protein was transferred to a PVDF membrane (0.22 μm) by the semidry transfer system (Bio-Rad). e 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. e membrane was washed with PBST 3 times and incubated with HRPlinked 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). e intensity of each band was calculated by Image J.

BJL Reduced LPS-Induced Excessive NO Release in RAW
264.7 Cells. Nitric oxide (NO) production by macrophage is an essential process of inflammatory action. We found that LPS significantly promoted the NO production in mice macrophage RAW 264.7 cells, and BJL (3, 10, and 30 μg/ml) concentration dependently inhibited the LPS-induced NO release (Figure 1(a)). As a result from the cell viability assay, BJL did not cause any cytotoxicity in Raw 264.7 cells at 50 μg/ml (Figure 1(b)) and cotreatment BJL with LPS also did not cause any cytotoxicity at the indicated concentrations (Figure 1(c)). So, we could conclude that BJL reduced LPS-induced excessive NO release which was not associated with the cytotoxicity of BJL in RAW 264.7 cells.

BJL Ameliorated Fin Transection or LPS-Induced Migration and Aggregation of the Neutrophils in Zebrafish.
e migration and aggregation of neutrophils initiated the inflammatory action. In this study, we employed the transgenic zebrafish line Tg(MPO:GFP) which expressed green fluorescent protein in neutrophils to evaluate the antiinflammatory effect of BJL. We found that fin transection significantly increased the cell number of the neutrophils which migrated and aggregated to the wound, and BJL (3, 10, and 30 μg/ml) concentration dependently decreased these cell numbers in the amputation site (Figures 2(a) and 2(b)). Consistently, injection of LPS (0.3 μg/ml) dramatically increased the fluorescent intensity on the injection site in zebrafish, which indicated cell number of the neutrophils increased on the injection site, and treatment BJL (3, 10, and 30 μg/ml) with LPS (0.3 μg/ml) reduced the fluorescent intensity (Figures 2(c) and 2(d)). ese results suggested that BJL could inhibit the migration and aggregation of neutrophils during the inflammatory process.

BJL Partially Inhibited LPS-Induced Upregulation of NF-κB, iNOS, and Inflammatory Cytokines including TNF-α, IL-6,
and IL-β in RAW 264.7 Cells. NF-κB plays an important role in the inflammatory action which initiates the transcription of inflammatory cytokines such as TNF-α, IL-6, and IL-1β. We found that LPS significantly increased the intracellular amount of NF-κB, and cotreatment BJL with LPS decreased the intracellular NF-κB (Figure 3(a)). Considering the results from Figures 3(b)-3(d), we found that LPS upregulated mRNA expressions of TNF-α, IL-6, and IL-1β genes, and BJL significantly suppressed LPS-induced mRNA expressions of these proinflammatory cytokine genes. In addition, LPS also upregulated the mRNA expression level of iNOS which mainly mediated the secretion of NO induced by LPS in RAW 264.7 cells (Figure 3(e)). us, we could conclude that BJL significantly reduced the elevations of inflammatory cytokines stimulated by LPS, which might be related to the suppression of the intracellular protein level of NF-κB.   -TTC TCA TTC CTG CTT GTG G-3′  5′-ACT TGG TGG TTT GCT ACG- us, the action mechanisms of the anti-inflammatory effect of BJL might be associated with the inhibition of Akt/MAPK signaling cascades.

Discussion
Inflammatory response is one of the vital cellular physiological processes which maintain normal tissue functions, Evidence-Based Complementary and Alternative Medicine 7 mechanisms underlying the anti-inflammatory activity of BJL were also studied. Nitric oxide (NO) is an essential modulator during inflammatory response and presents cytotoxicity to pathogenic microbes while also damages host tissue [30]. NO usually plays a protective role in cardiovascular disease and vascular endothelial function [31,32]. Promotion of the generation and bioavailability of NO were important mechanisms underlying the protective effect of traditional Chinese medicine in cardiovascular disease with vascular endothelial dysfunction, for example, hypertension, hyperglycemia, hyperlipidemia, and atherosclerosis [33][34][35], whereas excessive NO generation commonly plays a harmful role in the inflammatory response in macrophages [30,36]. In the present study, we found that LPS promoted the overproduction of NO in mice macrophage RAW 264.7 cells (Figure 1(a)) which mimicked the harmful role of NO during inflammation process. Modulation of the production and bioavailability of NO is one of the potent strategies for treatment of diseases involved in inflammation [37,38]. We found that the abnormal elevated NO level induced by LPS was significantly decreased in the BJL-treated group (Figure 1(a)). As we all know that the cell number and cell viability affect the NO production, LPS and BJL did not present any cytotoxicity in the indicated concentrations (Figures 1(b) and 1(c)).
us, BJL significantly inhibited LPS-induced excessive NO production regardless of its cytotoxicity in RAW 264.7 cells.
Zebrafish has been considered as one of the emerged promising animal models for drug discovery. In our previous studies, we established zebrafish disease models including angiogenesis, neuroprotection, and cerebral hemorrhage and found several active compounds isolated from traditional Chinese medicine [20][21][22][23][24][25][26]. In the present study, we employed transgenic zebrafish line Tg(MPO:GFP) which expresses specific green fluorescence protein in neutrophils as the in vivo model to evaluate the anti-inflammatory effect of BJL. We found that fin transection significantly induced the migration and aggregation of neutrophils on amputation site while the BJL-treated group dramatically decreased the numbers of neutrophils in a concentration-dependent manner (Figures 2(a) and 2(b)). Consistently, zebrafish embryos were injected with LPS and the intensity of green fluorescence on the injection site was significantly elevated (Figures 2(c) and 2(d)). However, treatment BJL with LPS injection reduced the fluorescence intensity on the injection site (Figures 2(c) and 2(d)). ese results revealed that both fin transection and LPS injection significantly induced the migration and aggression of neutrophils, and treatment BJL reduced inflammatory response.
e secretion of NO induced by LPS was mainly mediated by iNOS in macrophages [9], and iNOS gene expression is also regulated by NF-κB, which is a vital nuclear transcript factor in inflammatory response and initiates the transcription of inflammatory factors including TNF-α, IL-6, and IL-1β [12]. We found that LPS stimulated the elevation of intracellular NF-κB in RAW 264.7 cells, and treatment BJL suppressed the LPS-induced NF-κB increase (Figure 3(a)). Moreover, the mRNA expression levels of inflammatory cytokine genes including TNF-α, IL-6, and IL-1β were dramatically elevated by LPS stimulation, while treatment BJL ameliorated LPSinduced gene expression of inflammatory cytokines in RAW 264.7 cells (Figures 3(b)-3(d)). In macrophage, the upregulation of iNOS is a key character of  Evidence-Based Complementary and Alternative Medicine proinflammation [39]. We found that the mRNA expression level of iNOS was significantly increased in the LPS-treated group, while that was significantly decreased by BJL treatment (Figure 3(e)). us, we concluded that BJL presented anti-inflammatory effect in macrophage RAW 264.7 cells, which was associated with the downregulation of NF-κB and inhibition of mRNA expression of inflammatory factors including iNOS, TNF-α, IL-6, and IL-1β. LPS binds to the toll-like receptor (TLR) and stimulates the down-stream PI3K/Akt, MAPKs, and NF-κB signaling cascades by the TIR domain-containing TLR signaling adapter BCAP (B-cell adapter for PI3K), which initiated inflammatory response [40,41]. e inhibition of Akt/ MAPKs is one of the essential mechanisms underlying the anti-inflammatory effect of natural products [42][43][44]. So, we proposed that the action mechanism underlying the antiinflammatory effect of BJL was involved in Akt/MAPK signaling cascades. Considering the western blot results shown in Figure 4, LPS significantly increased the phosphorylation of Akt, MEK, ERK, P38, and JNK. Cotreatment BJL with LPS suppressed their phosphorylation in RAW 264.7 cells.
us, the inhibition of Akt/MAPK signaling cascades was likely involved in the anti-inflammatory activity of BJL. e anti-inflammation effect of Tribulus terrestris L. contributes to its effectiveness in multiple diseases while the anti-inflammatory component of Tribulus terrestris L. is not well-known. Lee et al. found that tribulusamide D, a compound isolated from Tribulus terrestris L., suppressed LPS-induced inflammatory response in RAW 264.7 cells via inhibition of P38 and NF-κB signaling cascade [13]. N-transρ-caffeoyl tyramine, which is also one of the components of Tribulus terrestris L., presented anti-inflammatory activity in LPS-stimulated RAW 264.7 cells by suppression of the protein levels of COX-2 and p-JNK [45]. So, these two compounds might be the active constituents of BJL in the present study.

Conclusion
BJL presented anti-inflammatory effect both in zebrafish in vivo and macrophage in vitro, and the underlying mechanisms were associated with the inhibition of Akt/MAPKs and NF-κB/iNOS-NO signaling cascades and the reduction of inflammatory factors including TNF-α, IL-6, and IL-1β ( Figure 5). us, we could conclude that BJL was potential for developing as treatment agent for the treatment of diseases involved in inflammation, such as atherosclerosis, inflammatory bowel disease, and rheumatoid arthritis. In addition, the active component of BJL and the related animal study should be done in the future study.

Data Availability
All the data used to support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure
Wai-Rong Zhao, Wen-Ting Shi, and Jing Zhang are the cofirst authors.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Wai