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Article

Anti-Inflammatory Activity of Chlorogenic Acid on Macrophages: A Simplified Simulation of Pharmacokinetics Following Ingestion Using a Windup Syringe Pump

by
Lei Cao
1,
Won Han
2,
Sang Gil Lee
3,4 and
Joong Ho Shin
1,2,*
1
Department of Biomedical Engineering, Pukyong National University, Busan 48513, Republic of Korea
2
Industry 4.0 Convergence Bionics Engineering, Pukyong National University, Busan 48513, Republic of Korea
3
Department of Food Science and Nutrition, Pukyong National University, Busan 48513, Republic of Korea
4
Department of Smart Green Technology Engineering, Pukyong National University, Busan 48513, Republic of Korea
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(1), 627; https://doi.org/10.3390/app13010627
Submission received: 16 November 2022 / Revised: 24 December 2022 / Accepted: 30 December 2022 / Published: 3 January 2023
(This article belongs to the Special Issue New Insights into Bioactive Compounds: At the Crossroads between Nutrition and Pharmacology)
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Versions Notes

Abstract

:
Cell-culture-based drug tests are usually performed in an instantaneous delivery manner. However, in vivo pharmacokinetic studies have shown a steady increase in the concentration of bioactive compounds in the plasma following oral administration, with the maximum concentration observed after several hours. Here, a novel palm-sized syringe pump powered by the manual winding of a spring was utilized for sustained delivery of chlorogenic acid (CHA) to lipopolysaccharide (LPS)-challenged RAW 264.7 macrophages over 2 h. When delivered in a sustained manner and simulating the in vivo pharmacokinetics following oral administration, CHA showed a stronger inhibitory effect on LPS-induced expression of inducible nitric oxide synthase and the transcription and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α. It also enhanced the mRNA expression of the gene encoding heme oxygenase 1. The suppression of phosphorylation of p38 but not the nuclear translocation of nuclear factor-κB was affected by the sustained delivery of CHA. High-performance liquid chromatography analysis indicated that the sustained delivery model showed a higher concentration of CHA in the conditioned medium two hours after starting the delivery. A stronger anti-inflammatory effect of CHA was observed upon sustained delivery to the cell medium, simulating an in vivo pharmacokinetic release profile following oral administration.

1. Introduction

Polyphenols are a group of naturally occurring phytochemicals characterized by multiple phenol units. They exert antioxidant and anti-inflammatory effects both in vitro and in vivo [1,2]. When polyphenols are tested in vitro, they are typically delivered as a bolus instantaneously. At the physiological level, plasma concentrations increase with time after oral administration, and the maximum level is typically observed after several hours. For example, the maximum plasma concentrations of the green tea polyphenols epigallocatechin-3-gallate and quercetin aglycone were observed two hours after oral administration in human subjects [3,4,5]. Similarly, pharmacokinetic studies have shown that after oral administration of chlorogenic acid (CHA), its concentration in plasma steadily increases and reaches a maximum approximately two hours after oral administration in humans [6]. CHA is an ester of caffeic and quinic acids and is one of the most widely consumed polyphenols because of its abundance in foods, especially coffee. The protective effect of CHA against inflammation has been reported in several studies, under both in vitro and in vivo conditions [7,8,9]. CHA inhibits the activation of mitogen-associated protein kinase (MAPK) and nuclear factor (NF)-κB signaling pathways and decreases the production of pro-inflammatory cytokines [10,11,12].
In vivo studies have shown that different administration approaches, such as injection and ingestion of nutraceuticals, exhibit very different pharmacokinetics. While the concentration of nutraceuticals, such as CHA, in plasma reaches maximum levels immediately after injection and then starts to reduce, a sustained increase in its concentration in plasma is observed over a couple of hours after ingestion [13,14]. In cell studies, an instantaneous delivery method is typically employed without considering pharmacokinetic differences. Although the release of nutraceuticals, including polyphenols, can be tailored via microencapsulation, accurate control of their release profile is difficult [15]. Microencapsulation is a technology used for packing active substances, such as polyphenols, in small-sealed capsules. Packaged materials are called core materials, and packing materials are called wall materials. For instance, CHA is encapsulated in chitosan via ionic gelation [16]. The in vitro release profile of the core material is affected by various factors, including the use of the microencapsulation method, choice of the wall material, and particle size. The in vitro anti-inflammatory activity of bioactive compounds is strongly affected by different encapsulation approaches [17,18,19]. Variable encapsulation efficiency and incomplete release of the core material from these encapsulation particles also limits the direct comparison of different delivery profiles. For instance, in vitro release kinetics have shown incomplete release of CHA from chitosan nanoparticles in various buffers [16].
A syringe pump is a motor-driven precision pump that involves the use of a syringe to deliver precise and accurate amounts of fluid into subjects [20]. The bulky size and electrical outlet requirements of a common syringe pump limit its application in cell studies. A palm-sized windup syringe pump was previously devised in our lab [21]. Its compact size and cord-free design render it suitable for cell studies. In this study, it was used to actualize the controllable sustained delivery of CHA into the culture medium. Additionally, a two hour duration was adopted since CHA showed an approximate 2 h time to maximum plasma concentration after ingestion [6]. To the best of our knowledge, this is the first study to deliver bioactive compounds to macrophages using a syringe pump. The objective of this study was to simulate the pharmacokinetics of CHA in the plasma after oral administration and to compare the anti-inflammatory activity of CHA between this sustained delivery model and a regular instantaneous delivery model.

2. Materials and Methods

2.1. Materials

Chlorogenic acid was purchased from the Tokyo Chemical Industry Co. (Tokyo, Japan). All products for cell culture were obtained from Thermo Fisher (Waltham, MA, USA). All chemicals used for HPLC analysis were of HPLC grade and were purchased from Samchun Chemicals (Seoul, Republic of Korea). Type 1 ultrapure water was used for all experiments.

2.2. Syringe Pump Fabrication

The manufacturing of the wind-up precision pump has been previously described in detail [21]. The 3D-printed parts were designed using the SOLIDWORKS software (Dassault Systèmes SolidWorks Co., Waltham, MA, USA). A polylactic acid (PLA) filament was used to print the body of the pump using a Sindoh 3D printer (3DWOX 1, Sindoh, Republic of Korea). The rack gear was modified to better fit syringes with a volume of 1 mL (Figure 1).

2.3. Cell Culture and Treatment

Macrophages (RAW264.7 cells) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and grown in an incubator at 37 °C in a humidified atmosphere with a 5% CO2. Cells were seeded at a density of 1.0 × 106 cells/well in a six-well plate with a complete medium. After 24 h, the culture medium was aspirated and replaced with 2 mL of FBS-free medium containing 100 ng/mL lipopolysaccharide (LPS), followed by the immediate addition of CHA. Regular instantaneous delivery was performed via pipetting. FBS-free medium (200 µL) containing 1.1 mM or 4.4 mM CHA was pipetted into the culture medium to obtain a medium with a volume of 2.2 mL containing 100 µM or 400 µM CHA. Pharmacokinetics following ingestion were simulated using a windup syringe pump. FBS-free medium (200 µL) containing 1.1 mM or 4.4 mM CHA was delivered using a 1.0 mL syringe to the culture medium at a flow rate of 0.1 mL/h by pushing the piston of the syringe via the windup pump. After 2 h, a total of 0.2 mL of medium (containing 0.22 µmol or 0.88 µmol of CHA) was delivered to the culture medium.

2.4. Protein Extraction and Western Blot

Proteins were extracted at the indicated time points. The medium was aspirated, and the cells were washed with cold PBS. CETi lysis buffer with protease and phosphatase inhibitors (TransLab, Daejeon, Republic of Korea) was used for whole-protein extraction. Nuclear and cytoplasmic extractions were performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher). Protein extracts were mixed with 5× Laemmli loading buffer and heated at 90 °C for 5 min. Electrophoresis was performed on 8 or 10% SDS-PAGE gels for 30 min at 60 V followed by subjecting the samples to 120 V for one hour. Semi-transfer was conducted using Pierce G2 Turbo Blot (Thermo Fisher) into a nitrocellulose membrane. After blocking, membranes were incubated with primary antibodies at 4 °C overnight. On the second day, the membranes were incubated with secondary antibodies at room temperature for one hour and then developed with Pierce ECL Western Blotting substrate. The antibodies used in this study are listed in Table S1 (Supplementary Materials). Images were captured with ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA) and analyzed using Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA).

2.5. RNA Extraction, cDNA Synthesis, and Real-Time Polymerase Chain Reaction (PCR)

RNA was extracted using TRIzol Reagent (Thermo Fisher) following the manufacturer’s instructions. RNA concentrations were measured using the Cytation 5 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA, USA). cDNA synthesis was performed using a SmartGene Compact cDNA synthesis kit (SamJung Bioscience, Daejeon, Republic of Korea). Real-time PCR was performed using a SmartGene Real-Time PCR master mix (SamJung Bioscience). The real-time PCR conditions comprised polymerase activation and initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 5 s and annealing/extension at 60 °C for 30 s. The primers used in this study are listed in Table S2 (Supplementary Materials).

2.6. Enzyme-Linked Immunosorbent Assay for Tumor-Necrosis Factor (TNF)-α and Interleukin (IL)-1β

After RAW 264.7 macrophages were treated with LPS and CHA for 24 h, the conditioned medium was collected to measure TNF-α and IL-1β levels using QuantikineTM ELISA kits obtained from R&D Systems (Minneapolis, MN, USA) following the manufacturer’s protocols.

2.7. High-Performance Liquid Chromatography (HPLC) Analysis

An HPLC system (Agilent Model 1200, Palo Alto, CA, USA) equipped with a binary pump, a UV-Vis spectrophotometer, and a C18 reverse-phase symmetry analytical column (5 µm × 250 mm × 4.6 mm, YMC, Koyoto, Japan) was used. All samples were filtered using nitrocellulose filter paper (pore size 0.22 µm) before injection. Samples were separated using a gradient elution program at a flow rate of 1.0 mL/min. The mobile phase comprised a mixture of water containing 50 mM phosphoric acid (phase A) and acetonitrile containing 50 mM phosphoric acid (phase B). The gradient elution program is described as follows: 92–89% A (0–4 min), 89–76% A (4–10 min), 76–30% A (10–16 min), 30–92% A (16–20 min), and 92% A (20–25 min). The UV-Vis spectra were recorded at 330 nm for CHA quantification and peak area calculation. The injection volume for each sample was 20 µL.

2.8. Statistical Analysis

Results from triplicate experiments are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc analysis or Student’s t-test as indicated in the figure legends was performed using Prism (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Effect on the Expression of Inducible Nitric Oxide Synthase (iNOS) and Cyclooxygenase (COX)-2

The CHA dose was determined based on the regulation of LPS-induced iNOS protein expression. Serial concentrations ranging from 5 to 400 µM were used to treat LPS-stimulated macrophages via instantaneous delivery. Inhibitory effects on the induction of iNOS protein expression were observed with CHA concentrations of 100, 200, and 400 µM. Therefore, 100 µM was selected as the low dose, and 400 µM as the high dose for further analysis (Figure 2).
Low doses of CHA showed higher inhibition of LPS-induced expression of both iNOS mRNA and protein when administered via sustained delivery than that via instantaneous delivery. However, this difference was not observed at a high dose.
In contrast to iNOS expression, CHA treatment did not affect COX-2 protein expression. Additionally, its mRNA level was elevated by low-dose CHA in the sustained delivery model and by high-dose CHA in both instantaneous and sustained delivery models.

3.2. Effects on the Secretion of Pro-Inflammatory Cytokines

Activated macrophages initiate inflammation by producing pro-inflammatory cytokines, such as TNF-α and IL-1β. Secretion of TNF-α into the conditioned medium was remarkably induced by LPS and suppressed by high doses of CHA at sustained delivery (Figure 3). Neither low-dose CHA nor high-dose CHA administered via instantaneous delivery showed a significant alteration in TNF-α levels.
The secretion of IL-1β into the conditioned medium was decreased by low doses of CHA in the sustained delivery model and high doses of CHA in both delivery models. Different delivery models did not show any differences in IL-1β secretion.

3.3. Effect on mRNA Expression of Pro-Inflammatory Cytokines and Antioxidant Genes

Consistent with its secretion level, Tnf-α mRNA expression was significantly reduced by high doses of CHA in the sustained delivery model (Figure 4). Neither a low dose nor a high dose of CHA administered via instant delivery showed significant alterations in mRNA expression.
Although sustained delivery of a high dose of CHA suppressed the elevation in Il-1b mRNA, unlike the secretion level of IL-1β, sustained delivery of a low dose and instantaneous delivery of a high dose of CHA did not exert an inhibitory effect on Il-1β mRNA expression. This suggests that CHA modulates the secretion of IL-1β through both transcriptional and post-transcriptional regulation. In addition, mRNA levels of the other two cytokines, Il-10 and monocyte chemoattractant protein-1 (Mcp-1), were not altered by either LPS or CHA.
LPS can produce intracellular reactive oxygen species and induce oxidative stress in RAW 264.7 cells [22]. LPS suppressed the mRNA expression of the antioxidant genes glutathione peroxidase 1 (Gpx1) and catalase (Cat). Sustained delivery of low doses of CHA showed a significant elevation in Gpx1 mRNA levels, and instantaneous delivery of high doses of CHA showed a significant elevation in Cat mRNA expression. However, no differences were observed between different CHA treatments. Heme oxygenase 1 (Hmox1) mRNA expression was elevated by LPS exposure and further augmented by CHA treatment. While high doses of CHA showed a higher ability to induce the expression of Hmox1 than low doses, sustained delivery enhanced Hmox1 1 expression at low doses but not at high doses.

3.4. Regulation of the MAPK and NF-κB Pathways

MAPK signal transduction pathways comprise a family of protein kinases that mediate biological processes and cellular responses to external stress signals. Increased activity of MAPK, particularly p38 MAPK, is involved in the regulation of transcription and translation of inflammatory mediators (21). In the present study, inhibition of LPS-induced phosphorylation of p38 by CHA showed a pattern very similar to that of the suppression of iNOS expression. The inhibition of phosphorylation was enhanced by both higher doses and sustained drug delivery (Figure 5). Such regulation by CHA was not observed for extracellular signal-regulated kinase (ERK), another MAPK pathway component.
NF-κB is also activated by a variety of stimuli and regulates the expression of a panel of pro-inflammatory genes (22). Although LPS-induced nuclear translocation of NF-κB was inhibited by CHA treatment, neither dose-dependent nor delivery model-dependent differences were observed (Figure 6). This suggests that NF-κB may not be involved in the enhanced anti-inflammatory activity of higher doses of CHA and its sustained delivery.

3.5. Changes in CHA Levels in the Conditioned Medium

The CHA concentration in the conditioned medium was determined two hours after starting the treatment by HPLC analysis. When the cells were treated with low doses of CHA, no significant differences were observed in CHA concentrations (Figure 7). When a high dose of CHA was applied, a significantly higher amount of CHA was detected in the conditioned medium when added using the sustained delivery model. This may explain the stronger anti-inflammatory activity observed with the sustained delivery model.

4. Discussion

In the present study, a novel palm-sized windup syringe pump was utilized to actualize the sustained delivery of CHA to LPS-stimulated macrophages over two hours. The sustained delivery model is a simplified simulation of the in vivo pharmacokinetic profile of CHA after oral administration. Compared to traditional instantaneous delivery through a pipette, sustained delivery model showed a stronger inhibition on several LPS-induced inflammatory markers, including iNOS, TNF-α, and IL-1β. Additionally, p38 MAPK signaling pathway may be involved in this enhanced anti-inflammatory efficacy of sustained delivery.
CHA significantly inhibited LPS-induced elevation of iNOS mRNA and protein expression. As a driver of inflammation, iNOS catalyzes the overproduction of nitric oxide, which plays a pivotal role in the pathogenesis of inflammation [23]. The expression of iNOS is a direct consequence of an inflammatory process, and the activation of iNOS leads to organ destruction in some inflammatory and autoimmune diseases. Therefore, iNOS has been targeted to improve the efficacy of immunotherapies [24,25]. A low dose of CHA in the sustained delivery model showed stronger inhibition of iNOS mRNA and protein expression than that observed with the regular instantaneous model. This difference was not observed when a high dose of CHA was used. This finding suggests that the inhibitory effect of CHA on iNOS would be saturated at a certain dose. This is supported by similar iNOS mRNA expression levels observed between samples not subjected to the LPS challenge and those treated with 400 µM CHA in an instantaneous manner.
COX mediates inflammation by catalyzing arachidonic acid metabolism and prostaglandin synthesis. Among the three isoforms of COX, the expression of COX-2 is selectively induced by proinflammatory stimuli, such as LPS [26]. COX-2 inhibitors are potential therapeutics for inflammatory diseases. Although both the inhibitory and non-inhibitory effects of CHA on LPS-induced COX-2 protein expression have been reported previously [7,8,27], our results showed that CHA exhibited no effect on LPS-induced COX-2 protein expression at concentrations up to 400 µM. Noticeably, Cox-2 mRNA levels were elevated by certain CHA treatments. This may be related to the ability of CHA to activate protein kinase A (PKA) and the cAMP-responsive element-binding protein (CREB) [28]. Cox-2 mRNA expression is regulated at various stages in a complex manner. Apart from p38 MAPK and NF-κB, activation of the protein kinase A (PKA) pathway targets CREB, another major transcription factor for the Cox-2 gene [29]. Activation of PKA and CREB by CHA has been reported under in vitro and in vivo conditions [28,30,31]. Although CHA inhibits the activation of p38 and NF-κB, it may increase the transcription of Cox-2 via the PKA/CREB signaling pathway.
Cytokines are small proteins produced by almost all cells that regulate and influence the immune response. Pro-inflammatory cytokines, such as TNF-α and IL-1β, are used as biomarkers to indicate inflammation progress [32]. TNF-α is a major cytokine involved in the initiation of the inflammatory response. Its functions include the induction of the expression of other cytokines, such as IL-1 and IL-6, upregulation of the expression of adhesion molecules, and regulation of arachidonic acid metabolism. CHA suppresses both the mRNA expression and release of TNF-α and IL-1β in LPS-stimulated primary microglia and macrophages [8,33]. A significant inhibitory effect on the mRNA expression of both Tnf-α and Il-1b was observed only when high doses of CHA were delivered in a sustained manner. A notable difference between the present study and previous studies is the timing of the CHA treatment. In previous studies, pretreatment with CHA one or two hours before LPS exposure was predominantly adopted [8,27,33]. However, in the present study, to illustrate the effect of CHA administered using different delivery models, CHA was delivered to the culture medium immediately after LPS exposure. This may have influenced the regulatory effects of CHA on the inflammatory response. In terms of the release of TNF-α, an inhibitory effect was observed when a combination of a high dose of CHA and a sustained delivery model was employed. However, IL-1β secretion showed a different pattern. Low doses of CHA administered via the sustained delivery model and high doses of CHA administered using both models inhibited the release of IL-1β into the conditioned medium, and no significant differences were observed among different treatments. This suggests that CHA modulates the secretion of IL-1β via both transcriptional and post-transcriptional regulation.
HMOX1 catalyzes the degradation of heme groups in several proteins, such as hemoglobin, myoglobin, and cytochrome p450. Its expression is induced by oxidative stress, cytokines, and other mediators produced during the inflammatory processes. [34]. The induction of the expression of HMOX1 by both LPS and CHA has been reported previously [35,36,37,38]. A clear variance of CHA treatments on the mRNA expression of Hmox1 but not other antioxidant genes measured in this study may be attributed to the mediation of Hmox1 by anti-inflammatory pathways [39,40]. Other experiments, such as the intracellular oxidative status measurement, are needed to better understand the effect of different delivery motions on antioxidant efficacy.
The regulatory effect of CHA on MAPK and NF-kB pathways in LPS-treated macrophages has been reported in several studies [10,11,12,27]. In the present study, the inhibition of LPS-induced phosphorylation of p38 by CHA was enhanced more by sustained delivery than by higher doses of CHA. However, neither a high dose of CHA nor sustained delivery altered the nuclear translocation of NF-κB. These findings suggested that the phosphorylation of p38 was more sensitive to CHA treatment than to the nuclear translation of NF-κB.
The does-dependent anti-inflammatory effect of CHA has been reported [7,41,42]. This may be attributed to a higher uptake of CHA by macrophages at higher does. Although sustained delivery and instantaneous delivery of low dose CHA showed similar CHA concentrations two hours after starting delivery, slow delivery motion may enhance the overall bioavailability of CHA. This is corroborated the discovery that a slow release of encapsulated apigen and curcumin showed a stronger anti-inflammatory effect than instantly released free apigen and curcumin [43]. The mechanism of how sustainable release enhances anti-inflammatory effect of bioactive compounds needs further investigation. Notably, sustained delivery of a high dose of CHA led to a higher CHA concentration in the conditioned medium two hours after starting delivery compared to those observed with the instantaneous delivery model, which may contribute to its stronger anti-inflammatory activity. The lower concentration of CHA observed with instantaneous delivery may be attributed to a more rapid degradation of CHA in the culture medium or a higher uptake into cells within the first two hours; however, a sustained increase in CHA concentration in the culture medium over two hours exhibited an overall stronger anti-inflammatory effect. In the small intestine, quinic acid and caffeic acid are cleaved from CHA and their metabolites are absorbed and enter the blood circulation [44]. However, the metabolism of CHA by macrophages remains unclear and is needed for future research.
Although the maximum plasma concentration of CHA was observed around 2 h after oral administration in human, a much earlier time to maximum plasma concentration is usually observed in rodents [45,46]. To simulate the in vivo pharmacokinetics in rodents, a different time course needs to be employed.
This approach also has its limitations. One is the concentrations used in this study were much higher than plasma concentration in humans after consumption of CHA rich food [47]. Another is the simplification of the in vivo pharmacokinetics. This study simulated the absorption in a linear way whereas plasma concentration of CHA does not increase in an exactly linear way after oral administration. In addition, the concentration of bioavailable CHA is not proportionate with the oral dosage [47]. The difference of high dose and low dose may not be observed in vivo.

5. Conclusions

In this study, CHA, a phenolic compound widely distributed in fruits and vegetables, was delivered to LPS-treated macrophages in a sustained manner over two hours. Compared to a regular instantaneous bolus delivery model, slow but sustainable delivery of CHA could exert a stronger anti-inflammatory effect. A higher dose and sustained delivery altered the phosphorylation of p38 but not the nuclear translocation of NF-κB. Whether other bioactive compounds could exhibit similar effects requires further investigation, this study could help us understand the pharmacology of bioactive compounds and develop effective administration strategies for anti-inflammatory compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app13010627/s1, Table S1: List of the antibodies used in the present study; Table S2: List of the primers used in the present study.

Author Contributions

Conceptualization, L.C. and J.H.S.; methodology, L.C. and W.H.; software, W.H. and J.H.S.; validation, J.H.S. and S.G.L.; formal analysis, L.C.; resources, J.H.S. and S.G.L.; data curation, L.C. and W.H.; writing—original draft preparation, L.C. and W.H.; writing—review and editing, J.H.S. and S.G.L.; visualization, L.C. and W.H.; supervision, J.H.S.; project administration, J.H.S.; funding acquisition, J.H.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (Nos. 2020R1C1C1003567 and 2022R1A5A8023404).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Illustration of the experimental setup. (a) CAD model of the color-coded assembled wind-up precision syringe pump. (b) CAD model of color-coded and 3D-printed wind-up syringe pump components. (c) A photo of two syringe pumps with 1 mL syringes used for cell treatment.
Figure 1. Illustration of the experimental setup. (a) CAD model of the color-coded assembled wind-up precision syringe pump. (b) CAD model of color-coded and 3D-printed wind-up syringe pump components. (c) A photo of two syringe pumps with 1 mL syringes used for cell treatment.
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Figure 2. Effect of different delivery models on iNOS and COX-2 expression. (a) Cells were treated with a serial concentration of CHA with regular instantaneous delivery. Protein expression of iNOS was determined. (b) Low doses and high doses of CHA were delivered to a culture medium using a pipette or syringe pump. Representative images of iNOS and COX-2 protein expression are shown. (c) Protein expression levels were quantified. (d) mRNA levels of iNos and Cox-2 were measured. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; iNOS: inducible nitic oxide synthase; COX: cyclooxygenase; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
Figure 2. Effect of different delivery models on iNOS and COX-2 expression. (a) Cells were treated with a serial concentration of CHA with regular instantaneous delivery. Protein expression of iNOS was determined. (b) Low doses and high doses of CHA were delivered to a culture medium using a pipette or syringe pump. Representative images of iNOS and COX-2 protein expression are shown. (c) Protein expression levels were quantified. (d) mRNA levels of iNos and Cox-2 were measured. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; iNOS: inducible nitic oxide synthase; COX: cyclooxygenase; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
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Figure 3. Effect of different delivery models on the secretion of TNF-α and IL-1β. Cells were treated with LPS (100 ng/mL) and CHA for 24 h. (a) TNF-α concentration in the conditioned medium and (b) IL-1β concentration in the conditioned medium were determined. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; TNF: tumor necrosis factor; IL-interleukin; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
Figure 3. Effect of different delivery models on the secretion of TNF-α and IL-1β. Cells were treated with LPS (100 ng/mL) and CHA for 24 h. (a) TNF-α concentration in the conditioned medium and (b) IL-1β concentration in the conditioned medium were determined. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; TNF: tumor necrosis factor; IL-interleukin; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
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Figure 4. Effect of different delivery models on mRNA expression of pro-inflammatory cytokines and antioxidant genes. Cells were treated with LPS (100 ng/mL) and CHA for 6 h. (a) mRNA levels of Tnf-α, Il-1β, Il-10, and Mcp-1 were determined 6 h after treatment. (b) mRNA levels of Hmox-1, Sod, Cat, and Gpx-1 were determined 6 h after treatment. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; TNF: tumor necrosis factor; IL: interleukin; Hmox: Heme oxygenase; Sod: superoxide dismutase; Mcp: monocyte chemoattractant protein; Cat: catalase; Gpx: glutathione peroxidase; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: fmapk.
Figure 4. Effect of different delivery models on mRNA expression of pro-inflammatory cytokines and antioxidant genes. Cells were treated with LPS (100 ng/mL) and CHA for 6 h. (a) mRNA levels of Tnf-α, Il-1β, Il-10, and Mcp-1 were determined 6 h after treatment. (b) mRNA levels of Hmox-1, Sod, Cat, and Gpx-1 were determined 6 h after treatment. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; TNF: tumor necrosis factor; IL: interleukin; Hmox: Heme oxygenase; Sod: superoxide dismutase; Mcp: monocyte chemoattractant protein; Cat: catalase; Gpx: glutathione peroxidase; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: fmapk.
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Figure 5. Effect of different delivery models on the phosphorylation levels of MAPK. Cells were treated with LPS (100 ng/mL) and CHA for 2 h. (a) Representative images of phosphorylated p38, total p38, phosphorylated ERK, and total ERK. (b) Quantification of the proportion of phosphorylated p38 and total p38 and phosphorylated ERK and total ERK. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; ERK: extracellular signal-regulated kinase; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
Figure 5. Effect of different delivery models on the phosphorylation levels of MAPK. Cells were treated with LPS (100 ng/mL) and CHA for 2 h. (a) Representative images of phosphorylated p38, total p38, phosphorylated ERK, and total ERK. (b) Quantification of the proportion of phosphorylated p38 and total p38 and phosphorylated ERK and total ERK. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; ERK: extracellular signal-regulated kinase; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
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Figure 6. Effect of different delivery models on nuclear translocation of NF-κB. Cells were treated with LPS (100 ng/mL) and CHA for 2 h. (a) Representative images of NF-κB in the nuclear fraction. Lamin B1 was used as a loading control. (b) The protein expression level was quantified. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; NF: nuclear factor; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
Figure 6. Effect of different delivery models on nuclear translocation of NF-κB. Cells were treated with LPS (100 ng/mL) and CHA for 2 h. (a) Representative images of NF-κB in the nuclear fraction. Lamin B1 was used as a loading control. (b) The protein expression level was quantified. Bars without a common letter indicate significant differences (p < 0.05). CHA: chlorogenic acid; LPS: lipopolysaccharide; NF: nuclear factor; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
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Figure 7. Effect of different delivery models on CHA concentration in the conditioned medium. (a) Upper panel: Representative HPLC chromatogram of standard CHA. Middle panel: Representative HPLC chromatogram of the conditioned medium with CHA observed 2 h after starting delivery. Lower panel: Representative HPLC chromatogram of the conditioned medium without CHA. (b) The concentration of CHA in the conditioned medium observed 2 h after starting delivery. Statistical analysis was performed using Student’s t-test (L vs. LS; H vs. HS). (c) A qualitative model of the pharmacokinetics of CHA in the conditioned medium with two different delivery models. CHA: chlorogenic acid; LPS: lipopolysaccharide; NF: nuclear factor; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
Figure 7. Effect of different delivery models on CHA concentration in the conditioned medium. (a) Upper panel: Representative HPLC chromatogram of standard CHA. Middle panel: Representative HPLC chromatogram of the conditioned medium with CHA observed 2 h after starting delivery. Lower panel: Representative HPLC chromatogram of the conditioned medium without CHA. (b) The concentration of CHA in the conditioned medium observed 2 h after starting delivery. Statistical analysis was performed using Student’s t-test (L vs. LS; H vs. HS). (c) A qualitative model of the pharmacokinetics of CHA in the conditioned medium with two different delivery models. CHA: chlorogenic acid; LPS: lipopolysaccharide; NF: nuclear factor; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump.
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Cao, L.; Han, W.; Lee, S.G.; Shin, J.H. Anti-Inflammatory Activity of Chlorogenic Acid on Macrophages: A Simplified Simulation of Pharmacokinetics Following Ingestion Using a Windup Syringe Pump. Appl. Sci. 2023, 13, 627. https://doi.org/10.3390/app13010627

AMA Style

Cao L, Han W, Lee SG, Shin JH. Anti-Inflammatory Activity of Chlorogenic Acid on Macrophages: A Simplified Simulation of Pharmacokinetics Following Ingestion Using a Windup Syringe Pump. Applied Sciences. 2023; 13(1):627. https://doi.org/10.3390/app13010627

Chicago/Turabian Style

Cao, Lei, Won Han, Sang Gil Lee, and Joong Ho Shin. 2023. "Anti-Inflammatory Activity of Chlorogenic Acid on Macrophages: A Simplified Simulation of Pharmacokinetics Following Ingestion Using a Windup Syringe Pump" Applied Sciences 13, no. 1: 627. https://doi.org/10.3390/app13010627

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