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

Zhao, Wai-Rong; Shi, Wen-Ting; Zhang, Jing; Zhang, Kai-Yu; Qing, Ye; Tang, Jing-Yi; Chen, Xin-Lin; Zhou, Zhong-Yan

doi:https://doi.org/10.1155/2021/6628561

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Background Materials and Methods Results Discussion Conclusion Abbreviations Data Availability Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2021 | Article ID 6628561 | https://doi.org/10.1155/2021/6628561

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

Wai-Rong Zhao,¹Wen-Ting Shi,¹Jing Zhang,¹Kai-Yu Zhang,¹Ye Qing,¹Jing-Yi Tang,¹Xin-Lin Chen,¹and Zhong-Yan Zhou^1,2

Academic Editor: Weicheng Hu

Received20 Oct 2020

Revised17 Dec 2020

Accepted05 Jan 2021

Published12 Feb 2021

Abstract

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.

1. 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]. The 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]. 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 [9]. 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) [10]. 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 [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.

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.

2.2. Chemicals

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 [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. 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.

2.7. Data Analysis

Data were presented as the mean ± S.E.M. from at least three independent experiments. The student’s paired t-test or analysis of variance (ANOVA) was used for statistical evaluation of difference between two groups. indicated a significant difference.

3. Results

3.1. 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.

(a)

(b)

(c)

Figure 1

The cytotoxicity of BJL and BJL-decreased LPS-induced NO release in RAW 264.7 cells. (a) RAW 264.7 cells were treated LPS (0.3 μg/ml) with or without various concentrations of BJL (3, 10, and 30 μg/ml) for 24 h and then followed by NO release assay. (b) RAW 264.7 cells were treated with various concentrations of BJL (3, 10, 30, 50, 80, and 100 μg/ml) for 24 h and the cell viability was measured by MTT assay. (c) RAW 264.7 cells were treated LPS (0.3 μg/ml) with or without various concentrations of BJL (3, 10, and 30 μg/ml) for 24 h and the cell viability was measured by MTT assay. Data were presented as the percentages of the control group. Results were means ± S.E.M. of more than 3 independent experiments. versus the control group. , , and versus the LPS-treated group.

3.2. BJL Ameliorated Fin Transection or LPS-Induced Migration and Aggregation of the Neutrophils in Zebrafish

The 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 anti-inflammatory 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)). These results suggested that BJL could inhibit the migration and aggregation of neutrophils during the inflammatory process.

(a)

(b)

(c)

(d)

Figure 2

BJL ameliorated fin transection or LPS-induced migration and aggregation of the neutrophils in zebrafish. (a-b) The transgenic zebrafish line Tg(MPO:GFP) which expresses green fluorescence protein in neutrophils was employed in this experiment. Three DPF zebrafish embryos were treated with various concentrations of BJL (3, 10, and 30 μg/ml) for 2 h and then zebrafish embryos were cut a part of the fin using a sharp needle and treated with BJL for another 2 h The photos were taken under a fluorescent stereoscopic microscope. The red line indicates the amputation site. The migrated cell numbers of neutrophils were calculated in each zebrafish embryo. (c-d) Three DPF zebrafish embryos were injected with LPS (0.3 μg/ml) and then treated with various concentrations (3, 10, and 30 μg/ml) of BJL for 24 h. The red circle indicates the injection site. The fluorescence intensity of the injection site was calculated by Image J data which were presented as the folds of the control group. Results were the means ± S.E.M. of more than 3 independent experiments. and versus the blank control group. and versus the LPS-treated group.

3.3. 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)). Thus, 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.

(a)

(b)

(c)

(d)

(e)

Figure 3

BJL partially inhibited LPS-induced upregulations of NF-κB, iNOS, and proinflammatory cytokines in RAW 264.7 cells. RAW 264.7 cells were treated LPS (0.3 μg/ml) with or without BJL (30 μg/ml) for 24 h. (a) The intracellular protein level of NF-κB was tested by western blotting using the specific primary antibody. (b–e) The mRNA expression levels of TNF-α, IL-6, IL-1β, and iNOS were detected by real-time PCR. Data were presented as the folds of the control group. Results were means ± S.E.M. of more than 3 independent experiments. 5, , and versus the control group. , , and versus the LPS-treated group.

3.4. BJL Suppressed LPS-Induced Phosphorylation of Akt, MEK, ERK, P38, and JNK in RAW 264.7 Cells

In order to explore the action mechanisms underlying the inhibition effect of BJL on LPS-triggered expression of inflammatory factors, we examined the effect of BJL on the phosphorylation of the key proteins in Akt/MAPK signaling pathways which regulated the proinflammatory response [12]. We found that LPS dramatically stimulated the phosphorylation of Akt, MEK, ERK, P38, and JNK, while cotreatment BJL with LPS significantly suppressed their phosphorylation (Figures 3(a)–3(e)). Thus, the action mechanisms of the anti-inflammatory effect of BJL might be associated with the inhibition of Akt/MAPK signaling cascades.

4. Discussion

Inflammatory response is one of the vital cellular physiological processes which maintain normal tissue functions, and the anti-inflammatory modulation by phytomedicine is beneficial for suppression of tissue damage during inflammatory response. In the present study, we evaluated the anti-inflammatory effect of Tribulus terrestris L. extract (BJL) in both transgenic zebrafish line Tg (MPO:GFP) in vivo and mice macrophage RAW 264.7 cells in vitro, and the action 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 [33–35], 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)). Thus, 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 [20–26]. 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)). These results revealed that both fin transection and LPS injection significantly induced the migration and aggression of neutrophils, and treatment BJL reduced inflammatory response.

The 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 LPS-induced 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 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)). Thus, 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]. The inhibition of Akt/MAPKs is one of the essential mechanisms underlying the anti-inflammatory effect of natural products [42–44]. So, we proposed that the action mechanism underlying the anti-inflammatory 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. Thus, the inhibition of Akt/MAPK signaling cascades was likely involved in the anti-inflammatory activity of BJL.

(a)

(b)

(c)

(d)

(e)

The 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-trans-ρ-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.

5. 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). Thus, 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.

Abbreviations

AP1:	Activator protein-l
BCAP:	B-cell adapter for PI3K
BJL:	Tribulus terrestris L. extract
DPF:	Day post fertilization
GFP:	Green fluorescent protein
HPLC:	High performance liquid chromatography
iNOS:	Inducible nitric oxide synthase
IL-1β:	Interleukin-1 beta
IL-6:	Interleukin-6
IRF1:	Interferon regulatory factor 1
LPS:	Lipopolysaccharides
MAPKs:	Mitogen-activated protein kinases
NF-κB:	Nuclear factor kappa B
NO:	Nitric oxide
STAT1:	Signal transducer and activator of transcription 1
TNF-α:	Tumor necrosis factor-alpha
TLR:	Toll-like receptor
TT:	Tribulus terrestris L.

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 co-first authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Authors’ Contributions

Wai-Rong Zhao, Wen-Ting Shi, and Jing Zhang contributed equally to this work. Wai-Rong Zhao wrote the manuscript. Wen-Ting Shi, Jing Zhang, and Wai-Rong Zhao conducted the experiments. Kai-Yu Zhang analyzed the data. Qing Ye and Jing-Yi Tang designed the experiments. Zhong-Yan Zhou and Xin-Lin Chen designed the experiments and revised the manuscript.

Acknowledgments

This study was supported by National Nature and Science Foundation of China (82074371), Health Commission of Shanghai (2020JP019, GWIV-28, and ZY-(2018–2020)-FWTX-8001), Longhua Hospital (Y1807), Science and Technology Commission of Shanghai (16ZR1437600), and Shanghai University of Traditional Chinese Medicine (A1-N19205010302). The authors thank Prof. Reng-Yue Gu (Longhua Hospital, Shanghai University of Traditional Chinese Medicine) and Prof. Da-Yuan Zhu (Shanghai Institute of Materia Medica, Chinese Academy of Sciences) who prepared the BJL samples.

Supplementary Materials

Figure S1: chemical profile analysis of BJL by high performance liquid chromatography (HPLC). . (Supplementary Materials)

References

G. R. Kim, J. Y. Yang, K. S. Hwang et al., “Anti-inflammatory effect of a novel synthetic compound 1-((4-fluorophenyl)thio)isoquinoline in RAW264.7 macrophages and a zebrafish model,” Fish Shellfish Immunology, vol. 87, pp. 395–400, 2019.
View at: Publisher Site | Google Scholar
R. Jakhar, C. Sharma, S. Paul, and S. C. Kang, “Immunosuppressive potential of astemizole against LPS activated T cell proliferation and cytokine secretion in RAW macrophages, zebrafish larvae and mouse splenocytes by modulating MAPK signaling pathway,” International Immunopharmacology., vol. 65, pp. 268–278, 2018.
View at: Publisher Site | Google Scholar
J. W. Jeong, H. J. Cha, M. H. Han et al., “Spermidine protects against oxidative stress in inflammation models using macrophages and zebrafish,” Biomolecules & Therapeutics, vol. 26, no. 2, pp. 146–156, 2018.
View at: Publisher Site | Google Scholar
S. A. Renshaw, C. A. Loynes, D. M. I. Trushell, S. Elworthy, P. W. Ingham, and M. K. Whyte, “A transgenic zebrafish model of neutrophilic inflammation,” Blood, vol. 108, no. 13, pp. 3976–3978, 2006.
View at: Publisher Site | Google Scholar
B. Chen, S. Huang, Y. Su et al., “Macrophage Smad3 protects the infarcted heart, stimulating phagocytosis and regulating inflammation,” Circulation Research., vol. 125, no. 1, pp. 55–70, 2019.
View at: Publisher Site | Google Scholar
M. R. Elliott, K. M. Koster, and P. S. Murphy, “Efferocytosis signaling in the regulation of macrophage inflammatory responses,” The Journal of Immunology, vol. 198, no. 4, pp. 1387–1394, 2017.
View at: Publisher Site | Google Scholar
W. B. Zhang, F. Yang, Y. Wang et al., “Inhibition of HDAC6 attenuates LPS-induced inflammation in macrophages by regulating oxidative stress and suppressing the TLR4-MAPK/NF-κB pathways,” Biomedicine & Pharmacotherapy, vol. 117, Article ID 109166, 2019.
View at: Publisher Site | Google Scholar
Z. Zhong, A. Umemura, E. Sanchez-Lopez et al., “NF-κB restricts inflammasome activation via elimination of damaged mitochondria,” Cell, vol. 164, no. 5, pp. 896–910, 2016.
View at: Publisher Site | Google Scholar
J. Wang, M. Y. Wu, H. Su et al., “iNOS interacts with autophagy receptor p62 and is degraded by autophagy in macrophages,” Cells, vol. 8, no. 10, p. 1255, 2019.
View at: Publisher Site | Google Scholar
R. E. Simmonds and B. M. Foxwell, “Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation,” Rheumatology (Oxford), vol. 47, no. 5, pp. 584–590, 2008.
View at: Publisher Site | Google Scholar
J. H. Hwang, K. J. Kim, S. J. Ryu, and B. Y. Lee, “Caffeine prevents LPS-induced inflammatory responses in RAW264.7 cells and zebrafish,” Chemico-Biological Interactions, vol. 248, pp. 1–7, 2016.
View at: Publisher Site | Google Scholar
J. L. Yeh, J. H. Hsu, Y. S. Hong et al., “Eugenolol and glyceryl-isoeugenol suppress LPS-induced iNOS expression by down-regulating NF-kappaB AND AP-1 through inhibition of MAPKS and AKT/IkappaBalpha signaling pathways in macrophages,” International Journal of Immunopathology and Pharmacology, vol. 24, no. 2, pp. 345–356, 2011.
View at: Publisher Site | Google Scholar
H. H. Lee, E. K. Ahn, S. S. Hong, and J. S. Oh, “Anti-inflammatory effect of tribulusamide D isolated from Tribulus terrestris in lipopolysaccharide-stimulated RAW264. 7 macrophages,” Molecular Medicine Reports, vol. 16, no. 4, pp. 4421–4428, 2017.
View at: Publisher Site | Google Scholar
Y. Ma, Z. Guo, and X. Wang, “Tribulus terrestris extracts alleviate muscle damage and promote anaerobic performance of trained male boxers and its mechanisms: roles of androgen, IGF-1, and IGF binding protein-3,” J Sport Health Sci, vol. 6, no. 4, pp. 474–481, 2017.
View at: Publisher Site | Google Scholar
V. Neychev and V. Mitev, “Pro-sexual and androgen enhancing effects of Tribulus terrestris L.: fact or Fiction,” Journal of Ethnopharmacology, vol. 179, pp. 345–355, 2016.
View at: Publisher Site | Google Scholar
M. A. A. Marques, B. Lourenço, M. P. Reis et al., “Osteoprotective effects of tribulus terrestris L.: relationship between dehydroepiandrosterone levels and Ca(2+)-sparing effect,” Journal of Medicinal Food., vol. 22, no. 3, pp. 241–247, 2019.
View at: Publisher Site | Google Scholar
H. O. Santos, S. Howell, and F. J. Teixeira, “Beyond tribulus (Tribulus terrestris L.): the effects of phytotherapics on testosterone, sperm and prostate parameters,” Journal of Ethnopharmacology, vol. 235, pp. 392–405, 2019.
View at: Publisher Site | Google Scholar
Z. Chauhdary, U. Saleem, B. Ahmad, S. Shah, and M. A. Shah, “Neuroprotective evaluation of Tribulus terrestris L. in aluminum chloride induced Alzheimer’s disease,” Pakistan Journal of Pharmaceutical Sciences, vol. 32, no. 2 (Supplementary), pp. 805–816, 2019.
View at: Google Scholar
P. L. Reshma, P. Binu, N. Anupama et al., “Pretreatment of Tribulus terrestris L. causes anti-ischemic cardioprotection through MAPK mediated anti-apoptotic pathway in rat,” Biomedicine & Pharmacotherapy, vol. 111, pp. 1342–1352, 2019.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, L. Y. Huan, W. R. Zhao, N. Tang, Y. Jin, and J. Y. Tang, “Spatholobi Caulis extracts promote angiogenesis in HUVECs in vitro and in zebrafish embryos in vivo via up-regulation of VEGFRs,” Journal of Ethnopharmacology, vol. 200, pp. 74–83, 2017.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, Y. Xiao, W. R. Zhao et al., “Pro-angiogenesis effect and transcriptome profile of Shuxinyin formula in zebrafish,” Phytomedicine, vol. 65, Article ID 153083, 2019.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, W. R. Zhao, Y. Xiao et al., “Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo,” Acta Pharmacologica Sinica, vol. 41, no. 2, pp. 260–269, 2019.
View at: Publisher Site | Google Scholar
B. Huang, Z. Y. Zhou, S. Li et al., “Tanshinone I prevents atorvastatin-induced cerebral hemorrhage in zebrafish and stabilizes endothelial cell-cell adhesion by inhibiting VE-cadherin internalization and actin-myosin contractility,” Pharmacological Research, vol. 128, pp. 389–398, 2018.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, B. Huang, S. Li et al., “Sodium tanshinone IIA sulfonate promotes endothelial integrity via regulating VE-cadherin dynamics and RhoA/ROCK-mediated cellular contractility and prevents atorvastatin-induced intracerebral hemorrhage in zebrafish,” Toxicology and Applied Pharmacology, vol. 350, pp. 32–42, 2018.
View at: Publisher Site | Google Scholar
C. M. Chong, Z. Y. Zhou, V. Razmovski-Naumovski et al., “Danshensu protects against 6-hydroxydopamine-induced damage of PC12 cells in vitro and dopaminergic neurons in zebrafish,” Neuroscience Letters, vol. 543, pp. 121–125, 2013.
View at: Publisher Site | Google Scholar
C. M. Chong, M. Shen, Z. Y. Zhou et al., “Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP⁺-induced oxidative stress and cell death in SH-SY5Y cells,” Free Radical Biology and Medicine, vol. 74, pp. 283–293, 2014.
View at: Publisher Site | Google Scholar
S. Brugman, “The zebrafish as a model to study intestinal inflammation,” Developmental & Comparative Immunology, vol. 64, pp. 82–92, 2016.
View at: Publisher Site | Google Scholar
B. Novoa and A. Figueras, “Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases,” Advances in Experimental Medicine and Biology, Current Topics in Innate Immunity II, vol. 946, pp. 253–275, 2012.
View at: Publisher Site | Google Scholar
L. von Gersdorff Jørgensen, “The dynamics of neutrophils in zebrafish (Danio rerio) during infection with the parasite Ichthyophthirius multifiliis,” Fish and Shellfish Immunology, vol. 55, pp. 159–164, 2016.
View at: Publisher Site | Google Scholar
R. Korhonen, A. Lahti, H. Kankaanranta, and E. Moilanen, “Nitric oxide production and signaling in inflammation,” Current Drug Targets-Inflammation and Allergy, vol. 4, no. 4, pp. 471–479, 2005.
View at: Publisher Site | Google Scholar
J. Lei, Y. Vodovotz, E. Tzeng, and T. R. Billiar, “Nitric oxide, a protective molecule in the cardiovascular system,” Nitric Oxide, vol. 35, pp. 175–185, 2013.
View at: Publisher Site | Google Scholar
D. Tousoulis, A. M. Kampoli, C. Tentolouris, N. Papageorgiou, and C. Stefanadis, “The role of nitric oxide on endothelial function,” Current Vascular Pharmacology, vol. 10, no. 1, pp. 4–18, 2012.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, J. Q. Xu, W. R. Zhao et al., “Ferulic acid relaxed rat aortic, small mesenteric and coronary arteries by blocking voltage-gated calcium channel and calcium desensitization via dephosphorylation of ERK1/2 and MYPT1,” European Journal of Pharmacology, vol. 815, pp. 26–32, 2017.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, W. R. Zhao, W. T. Shi et al., “Endothelial-dependent and independent vascular relaxation effect of tetrahydropalmatine on rat aorta,” Frontiers in Pharmacology, vol. 10, p. 336, 2019.
View at: Publisher Site | Google Scholar
Z. Y. Zhou, W. R. Zhao, J. Zhang, X. L. Chen, and J. Y. Tang, “Sodium tanshinone IIA sulfonate: a review of pharmacological activity and pharmacokinetics,” Biomedicine and Pharmacotherapy, vol. 118, Article ID 109362, 2019.
View at: Publisher Site | Google Scholar
T. J. Guzik, R. Korbut, and T. Adamek-Guzik, “Nitric oxide and superoxide in inflammation and immune regulation,” ndian Journal of Physiology and Pharmacology, vol. 54, no. 4, pp. 469–487, 2003.
View at: Google Scholar
M. Ghasemi, “Nitric oxide: antidepressant mechanisms and inflammation,” Advances in Pharmacology, Neuropsychotherapeutics, vol. 86, pp. 121–152, 2019.
View at: Publisher Site | Google Scholar
M. Gliozzi, M. Scicchitano, F. Bosco et al., “Modulation of nitric oxide synthases by oxidized LDLs: role in vascular inflammation and atherosclerosis development,” International Journal of Molecular Sciences, vol. 20, no. 13, 2019.
View at: Publisher Site | Google Scholar
J. D. Bailey, M. Diotallevi, T. Nicol et al., “Nitric oxide modulates metabolic remodeling in inflammatory macrophages through TCA Cycle regulation and itaconate accumulation,” Cell Reports, vol. 28, no. 1, pp. 218–230.e217, 2019.
View at: Publisher Site | Google Scholar
L. Shang, T. Wang, D. Tong, W. Kang, Q. Liang, and S. Ge, “Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways,” Cell Proliferation, vol. 51, no. 6, Article ID e12516, 2018.
View at: Publisher Site | Google Scholar
T. D. Troutman, W. Hu, S. Fulenchek et al., “Role for B-cell adapter for PI3K (BCAP) as a signaling adapter linking Toll-like receptors (TLRs) to serine/threonine kinases PI3K/Akt,” Proceedings of the National Academy of Sciences, vol. 109, no. 1, pp. 273–278, 2012.
View at: Publisher Site | Google Scholar
M. Tanaka, Y. Kishimoto, M. Sasaki et al., “Terminalia bellirica (gaertn.) roxb. Extract and gallic acid attenuate LPS-induced inflammation and oxidative stress via MAPK/NF-κB and Akt/AMPK/Nrf2 pathways,” Oxidative Medicine and Cellular Longevity, vol. 2018, Article ID 9364364, 15 pages, 2018.
View at: Publisher Site | Google Scholar
Y. Nie, Z. Wang, G. Chai et al., “Dehydrocostus lactone suppresses LPS-induced acute lung injury and macrophage activation through NF-κB signaling pathway mediated by p38 MAPK and Akt,” Molecules, vol. 24, no. 8, p. 1510, 2019.
View at: Publisher Site | Google Scholar
H. Harikrishnan, I. Jantan, M. A. Haque, and E. Kumolosasi, “Anti-inflammatory effects of Phyllanthus amarus Schum. & Thonn. through inhibition of NF-κB, MAPK, and PI3K-Akt signaling pathways in LPS-induced human macrophages,” BMC Complementary and Alternative Medicine, vol. 18, no. 1, p. 224, 2018.
View at: Publisher Site | Google Scholar
H. J. Ko, E. K. Ahn, and J. S. Oh, “N-trans-ρ-caffeoyl tyramine isolated from Tribulus terrestris exerts anti-inflammatory effects in lipopolysaccharide-stimulated RAW 264.7 cells,” International Journal of Molecular Medicine, vol. 36, no. 4, pp. 1042–1048, 2015.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2021 Wai-Rong Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

1213

Downloads

910

Citations