Chlorogenic Acid

Chlorogenic acid-enriched extract of Ilex kudingcha C.J. Tseng tea inhibits neutrophil recruitment in injured zebrafish by promoting reverse migration via the focal adhesion pathway

Abstract

Neutrophil-regulated inflammation plays crucial roles in tissue damage and repair. Dysregulation of the neutrophil response system can contribute to diseases such as cancer. Clearance of excessive neutrophils at the site of inflammation by reverse migration provides a promising strategy to mitigate the negative effects. Chlorogenic acid treatment of injured zebrafish embryos showed low-developmental toxicity. Using a transgenic zebrafish Tg (mpx: egfp) model, chlorogenic acid-enriched kuding- cha extract promoted neutrophil reverse migration via phosphorylation of ERK and AKT. Using i-TRAQ analysis, differentially expressed proteins involved in focal adhe- sion were identified, such as: Cdc42, SRC, MLC, ITGA, and Calpain. In support of this, ERK and AKT proteins are involved in the focal adhesion pathway. Real time qPCR determined that CGA downregulates genes associated with cancer metastasis, such as: HSPA5, YWHAZ, RP17, and ITGAV. Together, these results suggest that CGA- enriched Kudingcha extract may have potential as an anticancer or anti-inflammatory therapeutic agent.

Practical applications

Ilex kudingcha C.J Tseng, commonly referred to as the large-leaved kudingcha, is a tea variety naturally rich in chlorogenic acid. Chlorogenic acid, the ester of caffeic and quinic acids, has antioxidant, antibacterial, anticancer, and anti-inflammatory, activities. Kudingcha has several known biological functions, including: anticancer, anti-inflammatory, antidiabetic, and hypolipidemic effects. Treatment with kuding- cha extract reduces the recruitment of neutrophils, potentially by inhibiting focal adhesion, and activation of cancer metastasis-related genes. Importantly, kudingcha extract could be used as an alternative nutritional supplement for anticancer or anti- inflammation via its ability to suppress neutrophil recruitment.

Neutrophils are derived from hematopoietic stem cells in the bone marrow and account for 50%–70% of the total number of periph- eral blood cells. Neutrophils are a major immune cell type that play key roles in the release of inflammatory mediators, phagocytosis of invading organisms, degranulation, and even the release of DNA strands to eliminate pathogens. Neutrophilic recruitment is a highly regulated immune response to prevent tissue damage from chronic inflammation (Nathan, 2006). Dysregulation of the neutrophil re- sponse system can lead to diseases with an inflammatory patho- genesis, such as atherosclerosis, Alzheimer’s disease, and cancer (Hoodless et al., 2016). Therefore, an attractive therapeutic strategy for treating acute-inflammatory disease is clear excessive cells at the inflammatory site by restricting neutrophil recruitment via apopto- sis or reverse migration (Duffin, Leitch, Fox, Haslett, & Rossi, 2010). Unlike circulating neutrophils, neutrophils involved in the im- mune response have an extended lifespan during an inflammatory response. Non-inflammatory and immune-quiescent cells are elim- inated by the intracellular apoptotic process and engulfed by resi- dent macrophages (Lahoz-Beneytez et al., 2016). After engulfing the apoptotic cell, the macrophages change their phenotype to promote resolution of inflammation and trigger tissue repair mech- anisms. Abnormal neutrophil clearance may trigger chronic inflam- matory conditions, such as rheumatoid arthritis and cystic fibrosis. Therefore, promoting neutrophil clearance is a promising therapeutic strategy via induction of apoptosis or reverse or forward migration for mitigating the pro-inflammatory response (Elzbieta & Paul, 2013). Previously, we reported that Kudingcha tea is highly enriched with chlorogenic acid (CGA) as determined by HPLC-photo-diode array and HPLC-nuclear magnetic resonance analysis (Zhong et al., 2017). Kudingcha has several known functions, including anticancer (Zhu et al., 2014), anti-inflammatory (Song, Qian, Li, & Zhao, 2013), antidiabetic (Song, Xie, Zhou, Yu, & Fang, 2012), and hypolipidemic effects (Zheng et al., 2015). Green tea and coffee beans also contain CGA and have similar antioxidant, antibacterial, anticancer, and an- ti-inflammatory properties (Zhu et al., 2014). Zheng and colleagues reported that CGA suppresses lipopolysaccharide-induced neu- trophil recruitment in zebrafish (Zheng et al., 2015). However, the molecular mechanism for how CGA or CGA-containing herbs pre- vent inflammatory-mediated neutrophil recruitment has not been reported.

The zebrafish is a model organism with conserved mechanisms of innate immune function that is particularly suitable for live im- aging following pharmacological and genetic manipulation in vivo. The transgenic zebrafish line Tg (mpx: egfp) labels neutrophils with green fluorescent protein (eGFP) under the control of the myeloper- oxidase promoter. Use of this reporter line enables live monitoring of neutrophil behavior in vivo. Neutrophil forward migration occur at 0 to 3 hpi; neutrophil reverse migration occur at 3 to 24 hpi (Renshaw et al., 2006).

To the best of our knowledge, this study is the first to elevate the positive effects upon neutrophil behavior in zebrafish by treat- ment with CGA or kudingcha extract. I-TRAQ analysis identified differentially expressed proteins that may play a mechanistic role in the pharmaceutical activity of CGA-enriched kudingcha.

2 | MATERIAL S AND METHODS

2.1 | Plant material

Leaves of Ilex kudingcha were collected from the Kudingcha Institute, Hainan University, Hainan province, China, on July 2017. Kudingcha leaves were identified according to their morphological characteris- tics, dried, and sealed in plastic bags, and then, stored in desiccators at room temperature (23°C).

2.2 | Preparation of kudingcha extracts

Dried and ground Kudingcha leaves were first passed through a sieve (24 mesh), and the powder (100 g) was extracted twice with 4 L of 100% MeOH (AR) at room temperature (23°C) for 2 hr under ultrasonication. A dry extract (20 g) of Kudingcha was obtained by removing the solvent with a rotary evaporation apparatus, dissolv- ing it in EtOAc, and repeating solvent removal. The resulting EtOAc fraction (3.06 g) was used for subsequent assessment of the effect of kudingcha extract on neutrophil behavior.

2.3 | Zebrafish lines and maintenance

The transgenic Tg (mpx: egfp) zebrafish line was obtained from the China Zebrafish Resource Center in Wuhan, Hubei province, China. Zebrafish were maintained by standard procedures and staged in hours postfertilization (hpf) as per standard criteria (Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995).

2.4 | Kudingcha extract treatments

De-chorionated larvae were placed in 6-well plates at a density of 10 embryos per well. Embryos at 75% of the epiboly stage (8 hpf) or more were treated with 1-phenyl-2-thiourea (0.2 mM, PTU; Sigma- Aldrich, USA). At 72 hpf postfertilization, the embryos were tran- sected from the tip of the notochord (Renshaw et al., 2006) prior to treatment with Kudingcha extract. Neutrophil counts were assessed in the adult zebrafish tail at 4 hr and 24 hr post-injury by Image pro- plus analysis (Media Cybernetics, Maryland, USA). The site of injury was determined as an area within 3 × 9 mm2 from the wounded edge with reduced blood vasculature.

2.5 | Cell death assay

Larvae at 24 hr post-injury were stained by incubation in culture medium containing 2 µg/ml of the vital dye acridine orange (AO,Sigma, St. Louis, MO, USA) for 45 min in the dark to minimize AO color fading. The larvae were then washed three times with deion- ized water to remove excess AO. Individual cell death events ap- peared as bright green dots under a fluorescence microscope (Olympus IX71, Tokyo, Japan). Tiled images were merged into a single image to determine the total number of cell death events at the injury site.

2.6 | Western blotting

Kudingcha extract-treated (0, 200, 400, and 600 μg/ml) embryos were homogenized with syringes and then, centrifuged for protein extraction. Protein extracts were loaded onto a 10% SDS–PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck, Darmstadt, Germany). Membranes were incubated at 4°C with specific primary antibodies, including: p-ERK (sc-136521), p- AKT(sc-293125), p-AMPK (sc-19129), and β-actin (sc-69879) (all purchased from Santa Cruz Biotechnology, Inc.,Texas, USA). After hybridization with appropriate secondary antibodies, protein bands were visualized using a chemiluminescence detection kit (ATTO, Tokyo, Japan) and analyzed by imageJ (Media Cybernetics, Maryland, USA).

2.7 | iTRAQ-labelling

Protein extracts were obtained from embryos using a total protein extraction kit (Bestbio, Shanghai, China), according to the manufac- turer’s instructions. Protein concentrations were determined by the Bradford assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin as the standard. Proteins were labeled using the iTRAQ reagents multiplex kit (Applied Biosystems, Foster City, CA, USA). Sample preparation involved denaturation, alkylation, and trypsin di- gestion of proteins.

2.8 | High pH reverse-phase fractionation

After labeling, the combined and lyophilized peptide mixtures were dissolved in a high pH reverse-phase (HP-RP) solvent A (20 mM am- monium formate, pH 10) and fractionated on a Shimadzu LC-30A system with a Durashell-C18 column (4.6 × 250 mm, 5 µm 100 Å; Bonna-agela Technologies, Wilmington, DE, USA). The product was further eluted with solvent B (20% acetonitrile, 20 mM ammonium formate, pH 10), and absorbance was measured at 220 nm.

2.9 | Nano HPLC–MS/MS

To analyse each peptide fraction, a splitless ultra 2D-plus system (Eksigent, Dublin, CA, USA) was combined with a cHiPLC Nanoflex microchip system in Trap-Elute mode. The peptide sample was loaded onto the nano cHiPLC trap (200 µM × 0.5 mm ChromXP C18-CL 3 µM 120 Å; Eksigent), washed with solvents (acetonitrile/ formic acid/water, A: 2/0.1/98; B: 98/0.1/2), and then, separated on a nano cHiPLC analytical column (75 µM × 15 cm ChromXP C18-CL 3 µM 300 Å; Eksigent). MS analysis was performed with the triple- TOP 5,600 system (AB SCIEX; Concord, ON, USA) using an accu- mulation time of 250 ms per spectra (350–1500 m/z mass range) in high-resolution mode (>3,000).

2.10 | Data processing

The MS/MS spectra were processed using ProteinPilot 4.5 software (AB SCIEX; Foster City, CA, USA) and the Paragon algorithm (Shilov et al., 2007). The data were compared with those in the zebrafish UniProt database. The Proteinpilot Descriptive Statistics Template was used for statistical analysis. iTRAQ ratios were normalized to the control group to estimate upregulated and downregulated pro- teins (iTRAQ ratios: > 2 or < 0.5). 2.11 | Protein classification A method described by Zheng et al. (2015) was modified to clas- sify the identified proteins according to their gene ontology (GO) functional annotations (biological process, molecular function, and cellular component). Enrichment analysis was conducted by GO Term Mapper (http://go.princeton.edu/cgi-bin/GOTermMap- per). The KEGG Orthology-Based Annotation System (KOBAS, http://kobas.cbi.pku.edu.cn) was applied to identify which bio- logical pathways were associated with the differently expressed proteins. 2.12 | RNA isolation and RT-qPCR Tail-injured zebrafish (72 hpf stage) were treated with 60 and 130 μg/ml CGA prior to RNA extraction. Total RNA was isolated using TRIzol reagent (Invitrogen, Thermo Fisher, Guangzhou, China) and then, reverse-transcribed using the SuperScript II cDNA Synthesis Kit (Takara, Takara Bio Inc. Dalian, China). RT- qPCR (Real-time quantitative PCR) was performed with cDNA samples, primers (Table 1) and the QuantStudioTM 6 Flex SYBR® Green Reagents system (Applied Biosystems, Thermo Fisher, Guangzhou, China). The 2(−ΔΔCT) analysis method was used to de- termine relative mRNA expression. 2.13 | Statistical analysis The experimental and control group were compared by one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test. A p-value of < .05 was considered as statistically significant. 3 | RESULTS 3.1 | CGA treatment-induced low developmental toxicity during embryogenesis To evaluate CGA-associated developmental toxicity, zebrafish embryos were treated with CGA in a dose-dependent manner. Treatment with 250 µg/ml CGA caused a 50% mortality rate in 72 hpf stage embryos (Figure 1a), while 100% of 96 hpf stage embryos perished with the same treatment (Figure 1b). 3.2 | Kudingcha extract suppresses neutrophil recruitment post-injury To determine whether Kudingcha extract had an effect on neutro- phil recruitment, the total number of neutrophils was compared between the tail-wound site and the entire body. To determine neutrophil number, live-imaging analysis of Tg(mpx:eGFP) larvae treated with Kudingcha extract was performed at 4 hpi (hour post- injury) and 24 hpi. At 30 hpi, treatment with Kudingcha extract at 400 and 600 µg/ml reduced the number of neutrophils at the wound site compared with the control (Figure 2a‒d). No difference in neutrophil number was found between the whole body and the control. 3.3 | Kudingcha extract reduced cell death in the tail following injury A reduction in neutrophil number can be caused by cell death or reverse migration. To assess whether Kudingcha extract induced cell death, the tail-injured zebrafish larvae were stained with AO at 30 day post-fertilization (dpf). Treatment with Kudingcha extract caused a decrease in the number of cell death events in a dose-de- pendent manner (Figure 2e,f). This indicated that neutrophil loss was not associated with apoptotic cell death. 3.4 | Kudingcha extract induce phosphorylation of ERK, AKT Previously, the ERK, AKT, and MAPK proteins have been reported to be involved in neutrophil reverse migration (Da-Long et al., 2015). Western blotting analysis revealed increased expression of phosphorylated ERK and AKT proteins, in protein extracts from tail-injured zebrafish larvae treated with Kudingcha extract in a dose-dependent manner (Figure 3a‒c). Phosphorylated MAPK protein was absent, suggesting that ERK and AKT alone were responsible for Kudingcha extract-induced neutrophil reverse migration. FI G U R E 2 Chlorogenic acid-enriched Kudingcha extract suppresses neutrophil recruitment and the number of cell death events in tail- injured Tg(mpx:egfp) zebrafish larvae. (a) and (c) show representative fluorescence images of zebrafish embryos at 3 and 30 hr posttreatment with kudingcha extract (KDCE,200, 400 and 600 μg/ml). (b and d). No difference was observed in the overall number of neutrophils in the whole body at 3 and 30 hr post-injury with KDCE (means ± SEM, n = 20, ANOVA; 300 × 300 DPI resolution). KDCE significantly decreased neutrophil recruitment at the tail wound site 30 hr post-injury (means ± SEM, n = 20, ANOVA; 3 × 9 mm2, 300 × 300 DPI resolution). (e) and (f) show representative fluorescence images of zebrafish embryos after treatment with KDCE (200, 400 and 600 µg/ml) at 30 hr post-injury. KDCE at doses of 400 and 600 μg/ml decreased cell death at the tail wound site (means ± SEM, n = 20, ANOVA; 3 × 9 mm2, 300 × 300 DPI resolution). 3.5 | Identification and quantification of the zebrafish embryonic proteome The underlying molecular mechanisms of how CGA-enriched Kudingcha extract induces neutrophil reverse migration was unclear. To identify differentially expressed proteins (DEPs) in- volved in neutrophil reverse migration, quantitative proteomic profiling was performed following CGA treatment. Tail-injured ze- brafish larvae at 72 hpf were exposed to CGA (A: no treatment con- trol; B: 60 μg/ml CGA; C: 130 μg/ml CGA) for 30 hr. Quantitative profiling revealed 2,664 DEPs between the B versus A group; 2,673 between the C versus A group; and 2,672 between the C versus B group (p ≤ .05). Most of the identified proteins were within a 2 log2 iTRAQ ratio, and distributed close to 0 in log-normal distribution (Figure 4a). 3.6 | Functional classification of DEPs The GO enrichment analysis of DEPs categorized them into ei- ther biological process, molecular function, or cellular component (Figure 4b). The top 10 terms associated with the DEPs were cell- substrate adherence junction, cell-substrate junction, melanosome, pigment granule, extracellular space, focal adhesion, basolateral plasma membrane, adherence junction, anchoring junction, and brush border membrane. Out of the top 10 terms, focal adhesion represented 14.7% of the cluster frequency and 8% of protein fre- quency of use (p = .0002, Table 2). 3.7 | Differential expression of target protein involved in Focal adhesion To determine the upregulated or downregulated proteins associ- ated with the focal adhesion pathway, KEGG pathway analysis was performed. The upregulated genes associated with CGA-treatment were Cdc42 and MLC; while ITGA, SRC, Calpain, and MLC genes were downregulated (Figure 5). 3.8 | Chlorogenic acid suppressed cancer cell-related proteins

To determine whether CGA affects cancer cell metastasis via the focal adhesion pathway, the DEGs identified by i-TRAQ analysis were screened against known metastasis-related genes (Figure 6a) and further validated by RT-qPCR analysis (Figure 6b‒g). Downregulated proteins associated with cancer metastasis included: heat shock protein 5, ywhai, ribosomal protein L7, laminin alpha 5, cadherin 17, and ITGAV. At the mRNA level, only laminin alpha 5 and cadherin 17 were shown to be upregulated, which may be a compensatory response to increased protein production (Ma et al., 2019).

4 | DISCUSSION

Resolution of inflammation via reductions in pro-inflammatory mediators and neutrophil recruitment, or promotion of neutrophil reverse migration or apoptosis provides the most promising thera- peutic strategy for treating acute inflammatory diseases.Previously, our work suggested that the pharmaceutical action of Kudingcha is most likely due to its CGA content (Zhong et al., 2017). According to HPLC analysis (data not shown) the CGA content of Kudingcha methanol extract is roughly 16%. In this study, CGA treatment to zebrafish embryos-induced low-development toxicity. Based on this, treatment with Kudingcha extract at doses lower than 600 µg/ml was considered safe. Despite the well-known anti-inflam- matory activity of CGA (Gong, Su, Zhan, & Zhao xxxx), the molecular mechanism is still undetermined. Using a tail injured-Tg (mpx: eGFP) zebrafish model, the effect of CGA and Kudingcha extract treatment was examined on neutrophil behavior. Treatment of tail-injured lar- vae with Kudingcha-extract resulted in a reduction of neutrophil re- cruitment at the site of injury.

Protein Ratio Distribution

ITGAV gene, undergoes posttranslational cleavage to yield a disul- fide-linked heavy and light chain. These two chains combine with multiple integrin beta chains to form different integrins (Ballestrem, Hinz, Imhof, & Wehrle-Haller, 2001). The cell division control pro- tein 42, encoded by the CDC42 gene, is involved in cell cycle regu- lation (Flier & Sonnenberg, 2001). The calpain protein, encoded by the CAPN3 gene, is a muscle-specific member of the calpain large subunit family that specifically binds to titin (Franco & Anna, 2005). The myosin light chain (MLC), the light chain of myosin protein, is a member of the Ca + binding protein family and contains two Ca + binding EF-hand motifs. The SRC protein, a non-receptor tyrosine kinase encoded by the SRC gene, phosphorylates a specific tyrosine residue in other proteins (Lazar & Garcia, 1999).

The ERK and AKT proteins also have known roles in focal ad- hesion. Together, we speculated that Kudingcha extract-induced neutrophil reverse migration through the focal adhesion pathway. The association between endothelial cells and the extracellular ma- trix plays an integral role in neutrophil transmigration by providing structural support for endothelial conformational changes, or by re- cruiting intracellular molecules that constitute the hyperpermeabil- ity signaling cascades (Yuan, Qiang, Rigor, & Wu, 2012).

Neutrophils are known to kill harmful microorganisms by the formation of NETs. Under severe chronic inflammatory conditions, NETs promote the development and progression of cancer cell metastases (Cools-Lartigue et al., 2013; Park et al., 2016; Tohme et al., 2016). Albrengues and colleagues proposed that during chronic inflammation, NETs remodeled the formation of laminins by modifying the integrin α3β1-activating epitope. This leads to activa- tion of FAK/ERK/MLCK/YAP proteins involved in the focal signaling pathway that contributes to cancer cell initiation and metastasis.

FI G U R E 4 Proteome profiling and comparative analysis of the protein expression level in zebrafish embryos. (a) show the distribution of iTRAQ ratio; (b) show gene ontology categories (biological process, molecular function, cellular component). Only categories with five or more associated proteins are shown.

Apoptosis is programed cell death. Pharmacological upregulation of granulocyte apoptosis can resolve inflammation (Poon, Lucas, Rossi, & Ravichandran, 2014; William et al., 2002), whereas inhib- iting apoptosis results in severe tissue damage. In this study, treat- ment with Kudingcha extract significantly suppressed cell death. It was determined that the anti-inflammatory effects of Kudingcha ex- tract was independent of apoptosis; therefore, its anti-inflammatory effects are likely mediated by neutrophil reverse migration. Western blot analysis revealed increased phosphorylation of ERK and AKT proteins in response to Kudingcha extract treatment. The ERK (Yang et al., 2014), AKT (Tell, Kimura, & Palić, 2012), and MAPK (Taylor et al., 2013) proteins play known roles in regulating neutrophil mi- gration. Therefore, these results suggest that the anti-inflammatory mechanism of Kudingcha extract involved neutrophil reverse migra- tion via the ERK and AKT proteins.

Proteomic profiling analysis by I-TRAQ identified proteins associated with the focal adhesion pathway, such as ITGA, Calpain, MLC, Cdc42, and SRC. Integrin alpha-V protein, encoded by the (Albrengues et al., 2018). Similarly, the results from this study indi- cated that, in addition to integrin alpha-V, proteins associated with metastasis such as heat shock protein 5, ywhai, ribosomal protein L7, and cadherin 17, were downregulated in response to CGA. The ywhai protein, encoded by the gene YWHAZ, is highly expressed in stomach lung, and liver cancer and plays a key role in cancer me- tastasis (Hong, Jianguo, Jian, Yongbin, & Wenxi, 2009; Nishimura et al., 2013; Yang, Wen, Chen, Lozano, & Lee, 2003; Zang, Li, & Zhang, 2010). The ribosomal protein L7, encoded by the RPL7 gene, is abnormally increased in some steroid-sensitive cancers. However, a few studies have reported a relationship between RPL7 and cancer metastasis (Graham et al., 2000; Hide et al., 2003). The heat shock protein 5, encoded by the HSPA5 gene, is highly expressed in stom- ach and liver cancer, and promotes cancer metastasis and drug re- sistance (Dezheng et al., 2011; Fernandez et al., 2000; Llana et al., 2006; Ni & Lee, 2007; Shuda et al., 2003; Wang et al., 2005; Zhang et al., 2006). Knockout of HSPA5 in mice inhibits the metastasis of tumor cells in vitro and in vivo (Zhang et al., 2006). The integrin α V subunit protein, involved in cellular adhesion to the extracellular ma- trix, regulates cancer cell migration and invasion (Jaques et al., 2014; Sarmishtha et al., 2003). The cadherin 17 protein, encoded by the CDH17 gene, accelerates the proliferation and migration of cancer cells (Takamura et al., 2010). The laminin alpha 5 protein, encoded by the LAMA5 gene, regulates cell adhesion and migration including the migration and invasion of cancer cells (Cheng et al., 2015; Nicole et al., 2011; Yamato et al., 2013).

FI G U R E 6 Hierarchical clustering and mRNA expression profiles of proteins associated with cancer metastasis. Proteins (a) and mRNA (b–g) were extracted from injured zebrafish embryos following treatment with CGA at 72 hr postfertilization. (a), control group; (b), 60 CGA µg/mL group; (c), 130 CGA µg/ml.

It is widely known Kudingcha has anticancer properties includ- ing reduction of cancer metastasis, however, the complex bio-active ingredient was undetermined (Song et al., 2013). Based on similar anticancer properties, we determined that the bio-active ingredient of Kudingcha extract was CGA, which stimulated neutrophil reverse migration via regulation of the focal adhesion pathway.

In conclusion, Kudingcha extract induces neutrophil reverse mi- gration and cancer metastasis by regulating focal adhesion. Further investigation is warranted to determine the connection between CGA-induced neutrophil reverse migration and focal adhesion molecule in tail-injured Tg (mpx: eGFP) zebrafish by loss or gain of function studies that target ITGA, Calpain, ERK, or AKT.