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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 64  |  Issue : 5  |  Page : 232-243

Inhibition of pro-inflammatory mediator expression in macrophages using wood vinegar from griffith's ash


1 Division of Wood Cellulose, Taiwan Forestry Research Institute, Taipei, Taiwan
2 Animal Technology Research Center, Agricultural Technology Research Institute, Hsinchu, Taiwan
3 Department of Laboratory Medicine, Linsen, Chinese Medicine and Kunming Branch, Taipei City Hospital; Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
4 Department of Biotechnology and Animal Science, National Ilan University, Yilan; Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
5 Department of Animal Science and Biotechnology, Tunghai University, Taichung, Taiwan

Date of Submission16-Jun-2021
Date of Decision24-Aug-2021
Date of Acceptance30-Sep-2021
Date of Web Publication27-Oct-2021

Correspondence Address:
Dr. Tz-Chuen Ju
Department of Animal Science and Biotechnology, Tunghai University, No. 1727, Sec. 4, Taiwan Blvd., Xitun Dist, Taichung City 40704
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjp.cjp_54_21

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  Abstract 


Macrophages are essential for host defense as they control foreign pathogens and induce acquired immune responses. Activated macrophages secrete pro-inflammatory reactive substances causing local cell and tissue inflammatory response, which helps an organism resist the invasion of foreign pathogens. Excessive or chronic inflammation can cause several diseases. Previous studies have reported that vinegar treatment decreases the levels of several inflammatory cytokines and biomarkers, including mitogen-activated protein kinases, cyclooxygenase-2, inducible nitric oxide synthase (iNOS), and nitric oxide (NO). However, the benefits of wood vinegar produced from Griffith's ash (Fraxinus formosana Hayata) in reducing inflammation have not been investigated yet. Thus, assuming that wood vinegar exerts anti-inflammatory effects in macrophages, in this study, we investigated the potential anti-inflammatory effects of the wood vinegar from Griffith's ash using a lipopolysaccharide (LPS)-induced inflammatory response model in RAW264.7 macrophages. We showed that the wood vinegar inhibited the production of iNOS, NO, and interleukin 6. In addition, we found that the wood vinegar reduced the phosphorylation levels of p38 and protein kinase C-α/δ in the LPS-stimulated RAW264.7 macrophages. Based on these results, we suggest that the produced wood vinegar can reduce inflammation in LPS-activated macrophages.

Keywords: Griffith's ash, inflammation, macrophage, wood vinegar


How to cite this article:
Ho CL, Lin CS, Li LH, Hua KF, Ju TC. Inhibition of pro-inflammatory mediator expression in macrophages using wood vinegar from griffith's ash. Chin J Physiol 2021;64:232-43

How to cite this URL:
Ho CL, Lin CS, Li LH, Hua KF, Ju TC. Inhibition of pro-inflammatory mediator expression in macrophages using wood vinegar from griffith's ash. Chin J Physiol [serial online] 2021 [cited 2021 Dec 2];64:232-43. Available from: https://www.cjphysiology.org/text.asp?2021/64/5/232/329360




  Introduction Top


Griffith's ash (Fraxinus formosana Hayata), also commonly called Baijiyou, Shankulian, and Taiwan Bailashu, is a semi-deciduous tree endemic to Taiwan belonging to the Oleaceae family and Fraxinus genus.[1] Griffith's ash bears white flowers and imparipinnate opposite compound leaves, each consisting of 2–5 pairs of smooth-edged obovate-lanceolate leaflets (5–14 cm long and 2.5–4 cm wide). It is a drought-resistant tree that requires ample sunlight and is widely distributed throughout broad-leaved forests located at medium and low altitudes on the island of Taiwan. Wood vinegar, commonly known as wood acid, is an auburn liquid with a smoky odor. It is produced from the separation of wood tar via the condensation and clarification of flue gas generated from the dry distillation and carbonization of wood.[2],[3] It contains various organic compounds, including acids, alcohols, phenols, and neutral compounds.[4] Wood vinegar has been used for various purposes, such as for promoting the growth of crops and edible mushrooms, inhibiting plant growth,[5] accelerating fruit ripening,[6] and increasing the rate of nutrient uptake by chickens,[7] egg collagen,[4] and intestinal villi in piglets.[8] In addition, wood vinegar is widely applied in the agricultural,[5] animal husbandry,[9] and food processing industries.[10] Griffith's ash is widely cultivated as street and landscape trees owing to its pleasant appearance and fast growth. However, street and landscape trees must be trimmed, resulting in abandoned branches that contribute to carbon dioxide in the natural environment. Therefore, these branches can be used to produce charcoal and harvest wood vinegar; the produced charcoal can be used for carbon fixation, producing activated carbon,[11],[12] and deodorizing.[9],[13] The immune system generates inflammatory responses crucial for the defense of organisms. In general, the body regulates the immune response after exposure to foreign substances, such as pathogens and endotoxins, to activate the macrophages that defend the body against infection and secrete pro-inflammatory cytokines.[14],[15],[16] Although local inflammation can have a positive outcome, extended inflammatory responses can lead to few diseases.[17],[18],[19] Lipopolysaccharides (LPSs) are well-studied pathogen-associated molecular patterns generated from Gram-negative bacterial cell wall. LPSs can be recognized by toll-like receptor-4 (TLR4), one of immune cells' most important pattern recognition receptors.[20] Activation of TLR4 by LPS induces multiple intracellular signaling pathways, including reactive oxygen species (ROS), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and nuclear factor-kappa B (NF-κB), which regulate the expression of pro-inflammatory mediators, such as nitric oxide (NO), interleukin (IL-6), and tumor necrosis factor-alpha (TNF-α).[20],[21],[22],[23] In particular, activation of PKC occurs by physiological stresses, which then trigger a downstream signal transduction cascade via MAPK pathways, such as extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase1/2 (JNK1/2), and p38 MAP kinase.[24] Previous studies have reported that vinegar treatment decreases the levels of inflammatory cytokines[25],[26] and biomarkers, such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, and MAPK.[25],[26],[27] For instance, bamboo vinegar (BV) reduced iNOS expression, IL-6 secretion, and NO production in LPS-activated RAW264.7 macrophages by decreasing ROS production and PKC-α/δ activation. However, BV did not affect the TNF-α secretion, MAPK signaling, and COX-2 expression.[25] In addition, cudrania tricuspidata fruits vinegar inhibited the production of NO, iNOS, TNF-α, IL-6, and MCP-1 induced by LPSs present in RAW264.7 macrophages, 3T3-L1 adipocytes, and co-culture systems.[28] Moreover, vinegar supplementation decreased the secretion of TNF-α, IL-6, and NO in a clinical study.[29],[30] However, whether wood vinegar produced from Griffith's ash is beneficial in inflammation remains unknown. Thus, in this study, we investigated the potential anti-inflammatory effects of the wood vinegar from Griffith's ash using an LPS-induced inflammatory response model in the mouse macrophage RAW264.7 cell line.


  Materials and Methods Top


Preparation and analyses of the wood vinegar from Griffith's ash

Preparation of wood vinegar from Griffith's ash for cell viability assay

Wood vinegar is a liquid collected during the process of wood carbonization. Herein, wood vinegar was prepared during the carbonation process of Griffith's ash wood in an earth oven. The internal structure of the earth oven can be divided into the upper and bottom layers, as shown in [Figure 1]. The temperatures of the chimney outlet, upper layer, and bottom layer were approximately 150°C, 360°C, and 240°C–280°C, respectively. Hemicellulose, cellulose, and lignin in the wood could be pyrolyzed at 200°C–260°C, 260°C–310°C, and 310°C–450°C, respectively. In the early stages of carbonization, smoke, such as gas and water vapor, was collected from the chimney outlet of the earth oven in the temperature range 80°C–162°C, and condensed in a device consisting of a smoke collection flask, tube, chamber, and a condenser tube, as shown in [Figure 2]. The condensate formed on the tube wall was collected. Wood vinegar was collected from Griffith's ash at five different temperature ranges, namely 80°C–82°C, 90°C–92°C, 100°C–108°C, 120°C–125°C, and 140°C–162°C. The basic properties and composition analysis of the wood vinegar at these temperatures were provided by Dr. Zhenlong He from the Taiwan Forestry Research Institute of the Council of Agriculture. This study formulated a neutral wood vinegar from Griffith's ash for the cell viability assay. All vinegar samples were neutralized to pH 7 using NaOH for all the cell studies performed.
Figure 1: Dimensions of the earth oven and measurement points of the carbonization temperature. The temperatures of the chimney outlet, upper layer, and bottom layer were approximately 150°C, 360°C, and 240°C–280°C, respectively. Hemicellulose, cellulose, and lignin in the wood can be pyrolyzed at 200°C–260°C, 260°C–310°C, and 310°C–450°C, respectively. In the early stages of carbonization, the smoke, such as gas and water vapor, was collected from the chimney outlet of the earth oven in the temperature range 80°C–162°C.

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Figure 2: Collection device for wood vinegar. The condensate formed on the tube wall was collected. The wood vinegar was collected from Griffith's ash at five different temperatures: 80°C–82°C, 90°C–92°C, 100°C–108°C, 120°C–125°C, and 140°C–162°C.

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Basic properties of the produced wood vinegar

The basic properties of the wood vinegar collected from Griffith's ash at five different temperatures are tabulated in [Table 1]. The specific gravity and pH of the vinegar were in the range 1.009–1.012 and 2.64–3.25, respectively, and both factors decreased inversely with the increasing temperature of the chimney outlet. Conversely, the acidity was in the range of 5.71%–13.01% and increased with an increase in the temperature of the chimney outlet. The contents of tar, ash, and ignition residue varied from 0.0550% to 0.2179%, 0.0730% to 0.3417%, and 0.0599% to 0.2780%, respectively.
Table 1: The general property test results of Formosan ash wood vinegar collected at five different temperatures

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Composition analysis of the produced wood vinegar

Water constituted approximately 75%–85% of the total composition of the wood vinegar produced at all temperatures, and the analyses of the other components are tabulated in [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]. The composition analysis showed that the wood vinegar consisted of 36 organic compounds that could be further classified into acids, alcohols, phenols, and neutral compounds, with acids being the main constituent (48.2%–62.6%). Further, the acid composition analysis revealed that the wood vinegar contained four acids: acetic (the major acid present), propionic, butanoic, and crotonic acids. The acid content of wood vinegar increased with the increasing temperature of the chimney outlet [Table 3]. The composition analysis of phenols showed that the wood vinegar contained 18 phenolic compounds, predominantly syringol, acetol, guaiacol, phenol, m-methylphenol, and 4-methylsyringol. Phenols were the second most abundant constituent of wood vinegar (21.3%–24.0%). The phenol content reduced with increasing chimney outlet temperature, except for the one collected at 120°C–125°C [Table 4]. The composition analysis of alcohols revealed that the wood vinegar contained 10 alcohols (the main one being methanol). The alcohol content was reduced with an increase in the chimney outlet temperature [Table 5]. The composition analysis of neutral compounds showed that the wood vinegar contained four types of neutral compounds, butyrolactones were the main among them. Neutral compounds were the least abundant constituent of the wood vinegar (4.8%–7.1%), as listed in [Table 6].
Table 2: Composition analysis of wood vinegar from Griffith's ash

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Table 3: The acid compounds of Formosan ash wood vinegar

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Table 4: The phenolic compounds of Formosan ash wood vinegar

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Table 5: The alcohols compounds of Formosan ash wood vinegar

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Table 6: The total concentration of neutral compounds of Formosan ash wood vinegar

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Cell cultures

The murine macrophage cell lines RAW264.7 were propagated in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Biological Industries Ltd., Kibbutz Beit Haemek, Israel), and 2-mM L-glutamine (Life Technologies, Carlsbad, CA, USA) at 37°C in a 5% CO2 incubator. The macrophage cell lines RAW Blue were propagated in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum and 200-μg/mL Zeocin at 37°C in a 5% CO2 incubator.[31]

Cell survival

Cell survival was quantified using the alamarBlue assay (InvivoGen, San Diego, CA, USA).[32] In brief, RAW 264.7 macrophages (1 × 105 in 0.1 mL of medium) were seeded in 96-well plates. Cells were incubated in 10% volume of the culture medium of alamarBlue at 37°C in a 5% CO2 incubator for 2–3 h. The fluorescence was detected using a fluorescence plate reader (excitation/emission wavelengths: 570 nm/600 nm).

Nitric oxide production assay

RAW264.7 macrophages (1 × 105 in 0.5 mL of medium) were seeded in 24-well plates and then incubated with or without LPSs (1 μg/mL) in the absence or presence of wood vinegar for 24 h. The NO production of wood vinegar was measured indirectly by analyzing nitrite levels using the Griess reaction.[17] The absorbance was recorded at a wavelength of 570 nm in a microplate reader (OPTImax tunable plate reader, Molecular Device, Sunnyvale, CA, USA).[32]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting

After treatment, the cells were collected and lysed at 4°C in lysis buffer (25-mM Tris-HCl, pH 7.5; 100-mM NaCl; 2.5-mM EDTA; 2.5-mM EGTA; 20-mM NaF; 1-mM Na3VO4; 20-mM sodium β-glycerophosphate; 10-mM sodium pyrophosphate, and 0.5% Triton X-100) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Then, the whole cell lysate was separated by SDS-PAGE and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane.[15],[16] Briefly, proteins were electrophoretically fractionated through an 8% or 10% SDS-polyacrylamide gel and electrotransferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated for 1 h at room temperature in a blocking solution (5% nonfat milk in PBS with 0.1% Tween-20), and then the membranes were incubated for 2 h at room temperature with a specific primary antibody in blocking solution. The following primary antibodies were applied to the blots at the indicated dilutions: anti-p-ERK, anti-p-JNK1/2, anti-p38, anti-PKC, and anti-COX2 (each of them was in ratio 1:1000 and was acquired from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-iNOS (1:1000; Millipore, Bedford, MA, USA). The immunoreactive signals were detected using enhanced chemiluminescence reagents (PerkinElmer Life and Analytical Sciences, Boston, MA, USA).

Enzyme-linked immunosorbent assay

RAW 264.7 macrophages (1 × 105 in 0.5 mL of medium) were seeded in 24-well plates and treated as indicated. Secretion of TNF-α and IL-6 was measured by ELISA (DuoSet ELISA Development System, R&D Systems, McKinley Place, MN, USA) following the manufacturer's protocol.[32] Briefly, 96-well microplates were coated overnight with the TNF-α or IL-6 antibodies, blocked with 1% BSA in PBS, and washed extensively. Samples or standards (100 μL) were added to the microplates, incubated at room temperature for 2 h, and washed extensively, followed by a 2-h incubation with the biotin-conjugated detection antibody and a 30-min incubation with 100-μL streptavidin–horseradish peroxidase plus substrate for signal development. Following the addition of 100-μL stop solution into each well, the optical density of each well was detected using a microplate reader set to a wavelength of 450 nm using an ELISA Reader (OPTImax tunable plate reader, Molecular Device, Sunnyvale, CA, USA).

Nuclear factor-kappa B activity assay

RAW-Blue cells, an NF-κB reporter cell line generated from RAW264.7 cells, were purchased from Invivogen (CA, USA). These cells were incubated with wood vinegar for 30 min, followed by 1-μg/mL LPS stimulation for 24 h. The transcriptional activity of NF-κB was analyzed using detection medium QUANTI-Blue purchased from Invivogen following the instructions on the enclosed product data sheet.[32]

Statistical analysis

Results were expressed as mean ± standard deviation (SD) of triplicate samples. Each experiment was repeated at least three times to confirm the reproducibility of findings. Multiple groups were analyzed by one-way analysis of variance followed by a post hoc Student–Newman–Keuls test. Differences were considered statistically significant if P < 0.05.


  Results Top


General properties of the produced wood vinegar

The general property test results of Formosan ash wood vinegar collected at five different temperatures are listed in [Table 1]. Formosan ash wood vinegar has specific gravity and pH in the ranges 1.009–1.012 and 2.64–3.25, respectively, both values decrease with an increasing chimney temperature. In addition, its acidity ranges from 5.71% to 13.01% and increases with increasing chimney temperature. It also contains 0.0550%–0.2179% tar, 0.0730%–0.3417% ash, and 0.0599%–0.2780 ignition residue.

Composition analysis of the produced wood vinegar

The water content of Formosan ash wood vinegar collected at five different temperatures is around 75%–85%. Other components are listed in [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]. The analysis results show that Formosan ash wood vinegar consists of 36 organic compounds, including acids, alcohols, phenols, and neutral compounds, with acids being the principal component constituting 48.2%–62.6% [Table 2]. Acid analysis reveals four acid compounds in wood vinegar: acetic acid (present in the highest concentration), propionic acid, butanoic acid, and crotonic acid. The acid compounds of Formosan ash wood vinegar increase with rising chimney temperature [Table 3]. The phenolic content analysis results show that the vinegar contains 18 phenols, more diverse than the acids. The major phenolic compounds are syringol, acetol, guaiacol, phenol, m-methylphenol, and 4-methylsyringol. Phenolic compounds are the second most abundant with a total concentration ranging between 21.3% and 24.0%. Except at 120°C–125°C, Formosan ash wood vinegar's phenolic content decreases with the increasing chimney temperature [Table 4]. Alcoholic content analysis indicates that the vinegar contains 10 alcohols, mainly methanol, and their amount decreases with the increasing chimney temperature [Table 5]. The neutral compound analysis shows four neutral compounds, of which butyrolactone has the highest concentration. The total concentration of neutral compounds is 4.8%–7.1%, which is the lowest among the components of Formosan ash wood vinegar [Table 6].

Effects of the produced wood vinegar on cell viability and nitric oxide production

The cell viability of RAW264.7 macrophages was evaluated by adding alamarBlue after treating with 250–4000 μg/mL of the wood vinegar collected from Griffith's ash at different temperatures. At all four temperatures and concentrations tested, the viability of RAW264.7 macrophages was unaffected in RAW264.7 macrophages [Figure 3]a. Since NO plays a crucial role in LPS-induced inflammatory reactions in macrophages, we evaluated the effect of wood vinegar on NO production in LPS-activated macrophages. RAW264.7 macrophages were incubated with or without 250, 500, 1000, 2000, and 4000 μg/mL wood vinegar for 30 min followed by LPS stimulation for 24 h (each group containing 0.1% DMSO in the culture medium). We found that wood vinegar reduced NO production in a dose-dependent manner [Figure 3]b. Based on the NO and cell viability assays results, the wood vinegar collected at 90°C–92°C with different concentrations was selected for subsequent experiments.
Figure 3: Wood vinegar from Griffith's ash inhibited NO generation in RAW264.7 macrophages. (a) RAW264.7 macrophages were incubated for 24 h with or without wood vinegar. Cell viability was measured by the alamarBlue assay. Data are expressed as mean ± SD of three independent experiments. *P < 0.05 compared to LPS alone (n = 3). (b) RAW264.7 macrophages were treated for 30 min with 0–4000-μg/mL wood vinegar, followed by incubation with or without 1-μg/mL LPS for 24 h. NO production in the culture medium was measured by the Griess reaction. The data are presented as the mean ± SD from three independent experiments. *P < 0.05 compared to LPS alone. LPS: Lipopolysaccharide, NO: Nitric oxide, SD: Standard deviation.

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Effects of produced wood vinegar on pro-inflammatory mediator expression in  Escherichia More Details coli-lipopolysaccharide-activated macrophages

The wood vinegar from Griffith's ash was tested to determine its effect on the protein expression of iNOS. RAW264.7 macrophages were treated with 250–1000-μg/mL wood vinegar for 30 min. Subsequently, the cells were incubated with 1-μg/mL LPS for 24 h and then harvested for western blot assays to determine the protein expression of iNOS and COX-2. The results showed that the expression of iNOS increased after LPS addition, whereas the treatment with wood vinegar tended to reduce the level of iNOS at 1000 μg/mL. These results indicated that wood vinegar inhibited LPS-induced iNOS expression in RAW264.7 macrophages [Figure 4]a and [Figure 4]b. However, the expression of COX-2 in RAW264.7 macrophages was increased by LPS addition but was reduced by an increase in the concentration of wood vinegar. Therefore, we concluded that the wood vinegar did not affect LPS-induced COX-2 expression in RAW264.7 macrophages [Figure 4]a and [Figure 4]c. In addition to activating the expression of iNOS, COX-2, and NO, LPS-stimulated RAW 264.7 macrophages produced pro-inflammatory antibodies, such as TNF-α and IL-6. The cells were treated with 250–1000 μg/mL of wood vinegar for 30 min and incubated with 1 μg/mL of LPS for 24 h. The supernatant was then harvested for determining the TNF-α and IL-6 levels using ELISA. The secretions of TNF-α and IL-6 increased with LPS addition. After treatment with wood vinegar, the secretion of IL-6 in RAW264.7 macrophages decreased, but this trend was not observed for TNF-α. The results suggested that wood vinegar was an effective inhibitor of the LPS-induced secretion of IL-6 [Figure 4]d but did not affect the secretion of TNF-α [Figure 4]e.
Figure 4: Wood vinegar from Griffith's ash inhibited pro-inflammatory mediators in RAW264.7 macrophages. RAW264.7 macrophages were treated for 30 min with 250, 500, or 1000 μg/mL of wood vinegar, followed by incubation with or without 1 μg/mL of LPS for 24 h. The lysates were assessed by western blotting analyses. The expression levels of COX-2 and iNOS in the cell lysates were measured by western blotting analyses (a-c). The levels of IL-6 (d) and TNF-α (e) in the culture media were determined by ELISA. Western blotting results are representative of three different experiments. ELISA data are presented as mean ± SD of three independent experiments. *P < 0.05 compared to LPS alone (n = 3). iNOS: Inducible nitric oxide synthase, IL-6: Interleukin-6, TNF-α: Tumor necrosis factor-alpha, LPS: Lipopolysaccharide, SD: Standard deviation.

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Effects of the produced wood vinegar on protein kinase C-α/δ/ε phosphorylation

It has been demonstrated that LPS increases the expression levels of inflammatory mediators via the MAPK and PKC signaling pathways in macrophages.[23] We examined the effects of wood vinegar on the phosphorylation levels of PKC [Figure 5] and MAPK [Figure 6] in LPS-activated macrophages. RAW264.7 macrophages were treated with 1000 μg/mL of wood vinegar for 30 min and then, with 1 μg/mL of LPS, after which the expression of phosphorylated PKCα, PKCδ, and PKCε proteins was determined at different time points by western blotting analysis. The results showed that the phosphorylation of PKCα, PKCδ, and PKCε was strong after 60 min of LPS stimulation. The addition of wood vinegar did not affect the phosphorylation of PKCα, PKCδ, and PKCε initially (0 min). However, it reduced the LPS-induced p-PKCα [Figure 5]a and p-PKCδ [Figure 5]b protein expression levels subsequently. Thus, the LPS-induced p-PKCα and p-PKCδ expression could be inhibited by wood vinegar [Figure 5].
Figure 5: Wood vinegar from Griffith's ash reduced the phosphorylation levels of PKCα and PKCδ in RAW264.7 macrophages. RAW264.7 macrophages were treated with 1000 μg/mL of wood vinegar, followed by incubation with or without 1 μg/mL of LPS for 0–60 min. The phosphorylation levels of (a) PKCα, (b) PKCδ, and (c) PKCε in the cell lysates were assayed by western blotting. The data are presented as the mean ± SD from three independent experiments. * and *** indicate a significant difference at the level of P < 0.05 and P < 0.001, respectively, compared to LPS alone. PKC: Protein kinase C, SD: Standard deviation, LPS: Lipopolysaccharide.

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Figure 6: Wood vinegar from Griffith's ash reduced the phosphorylation levels of p38 in RAW264.7 macrophages. RAW264.7 macrophages were treated with 1000 μg/mL of wood vinegar, followed by incubation with or without 1 μg/mL of LPS for 0–60 min. The phosphorylation levels of (a) ERK1/2, (b) JNK1/2, and (c) p38 in the cell lysates were assayed by western blotting. The data are presented as the mean ± SD from three independent experiments. *P < 0.05 compared to LPS alone. SD: Standard deviation, LPS: Lipopolysaccharide.

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Effects of the produced wood vinegar on the mitogen-activated protein kinase signal transduction pathway

RAW264.7 macrophages were treated with 1000-μg/mL wood vinegar for 30 min, and then, 1-μg/mL LPS was added before the expression of phosphorylated ERK1/2, JNK1/2, and p38 (labeled as p-ERK1/2, p-JNK1/2, and p-p38, respectively) was determined at various time points via western blotting assay. The results showed that p-ERK1/2 and p-JNK1/2 were initially expressed after 10 min of LPS stimulation, and the expressions became strong after 20 min of stimulation [Figure 6]a and [Figure 6]b. However, the addition of wood vinegar did not affect the LPS-induced expression of p-ERK1/2 and p-JNK1/2 [Figure 6]a and [Figure 6]b. In contrast, p38 was activated at the beginning (0 min) of LPS stimulation and strongly expressed after 30 min. The addition of wood vinegar did not affect the phosphorylated p38 protein level at the beginning (0 min); however, subsequently, it reduced the LPS-induced p-p38 expression [Figure 6]c. The results showed that the treatment with wood vinegar inhibited the LPS-induced expression of p-p38 [Figure 6]c.

Effects of the produced wood vinegar on the nuclear factor-kappa B activation

NF-κB signaling plays a central role in the pro-inflammatory responses of macrophages upon TLR signaling activation. We investigated whether wood vinegar reduced pro-inflammatory responses through the inhibition of NF-κB activation. An NF-κB reporter cell line generated from RAW264.7 cells (RAW-Blue cells) was incubated with wood vinegar for 30 min, followed by 1-μg/mL LPS stimulation for 24 h. The transcriptional activity of NF-κB was analyzed by the reporter assay. We found that LPS treatment significantly increased the transcriptional activity of NF-κB; however, wood vinegar did not inhibit NF-κB activity. Wood vinegar even slightly increased the NF-κB activity in the LPS-activated cells at high concentrations [Figure 7]. This result indicated that the anti-inflammatory activities of wood vinegar were not due to its NF-κB inhibitory activity.
Figure 7: Effect of wood vinegar on LPS-mediated NF-κB activation in RAW264.7 macrophages. RAW264.7 macrophages were treated with 1000-μg/mL wood vinegar, followed by incubation with or without 1-μg/mL LPS for 24 h. The transcriptional activity of NF-κB was analyzed using detection medium QUANTI-Blue. The data are presented as the mean ± SD from three independent experiments. *P < 0.05 compared to untreated cells. SD: Standard deviation, LPS: Lipopolysaccharide, NF-κB: Nuclear factor-kappa B.

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  Discussion Top


In this study, mouse RAW264.7 macrophages were stimulated with LPSs, which transmit signals into the cells by binding to the TLR4,[33] thereby inducing the inflammatory responses. This study aimed to understand the effects of wood vinegar from Griffith's ash on NO, iNOS, COX-2, IL-6, and TNF-α, and further investigate the activation mechanisms of various signal transduction pathways, including the MAPK, PKC, and NF-κB pathways.

NO is a biologically active gaseous molecule that serves as an essential intercellular signaling regulator. LPS-stimulated mouse RAW264.7 macrophages generate an extensive amount of NO, a critical inflammation indicator.[34] Excessive NO generation is closely associated with inflammatory symptoms and plays a crucial role in detecting inflammatory responses.[35],[36] Although a low NO concentration is sufficient to defend the body against foreign pathogens, an excessive NO concentration results in cytotoxicity.[37],[38] This study determined the total amount of NO produced in LPS-induced RAW264.7 macrophages after treating cells with the wood vinegar collected from Griffith's ash at different temperatures. The LPS-induced NO generation decreased within the wood vinegar in a dose-dependent manner, indicating the effective reduction of NO generation by the wood vinegar.

The effects of wood vinegar on the cell viability of RAW 264.7 macrophages were determined using alamarBlue dye. The untreated group with 100% viability was considered to be the control group. RAW264.7 macrophages treated with 250–4000 μg/mL of wood vinegar remained over 80% viable. Hence, the concentration range of this study did not cause any cytotoxic effects on the cells and was suitable for investigating the vinegar's anti-inflammatory activity. From the aforementioned results, it was apparent that the reduction of LPS-induced NO production in RAW264.7 macrophages by wood vinegar was not due to the inhibitory effect caused by cell death.

iNOS is the most important NO source for inducing inflammatory responses and eliminating pathogens and can only be found in activated cells. It is currently believed that causative factors of iNOS expression include LPSs and cytokines. LPSs induce iNOS, generating an extensive amount of NO that causes adverse effects in organisms, such as inflammatory responses, cellular senescence, and apoptosis. This study found that protein expression of iNOS was effectively reduced by wood vinegar. Therefore, the wood vinegar from Griffith's ash plays a crucial role in protecting cells against LPS stimulation by reducing NO and iNOS levels effectively. Exposure of cells and tissues to stimuli, such as cytokines, endotoxins, oncogenes, and TNFs, leads to the expression of COX-2, the elevation of prostaglandin production, and subsequent inflammatory responses.[39],[40] This study found that wood vinegar did not reduce COX-2 expression.

TNF-α has an important role in the inflammatory response and exhibits a wide range of biological activities, including cell growth, cell death, immune responses, and tumorigenesis.[41] Moreover, it stimulates the release of other cytokines, such as IL-4 and IL-6. However, the experimental results showed that wood vinegar did not affect TNF-α expression but inhibited the NO, IL-6, and iNOS expression. Therefore, this study further explored its effects on the MAPK, and PKC signal transduction pathways and observed that the reduction of iNOS protein expression might be attributed to inhibition via the MAPK or PKC pathways.

The LPS stimulation of macrophages activates ERK1/2 (p44/p42), p38, and JNK1/2, activating the downstream transcription factors via phosphorylation to regulate cell growth and differentiation.[42],[43] The treatment of LPS-stimulated RAW264.7 macrophages with wood vinegar did not inhibit p-ERK1/2 and p-JNK1/2 but effectively reduced the expression of p-p38. PKC can activate MAPK to induce the production of pro-inflammatory cytokines (IL-6 and TNF-α) in cells.[23],[44] The treatment of LPS-stimulated RAW264.7 macrophages with wood vinegar almost completely inhibited the phosphorylation of PKC. The pro-inflammatory mediators iNOS, NO, COX-2, IL-6, and TNF-α were regulated by multiple signaling pathways: ROS, PKC, MAPKs, and NF-κB. We found that wood vinegar inhibited PKCα, PKCδ, and p38 in LPS-activated macrophages, suggesting that wood vinegar could theoretically inhibit all the pro-inflammatory mediators. However, wood vinegar inhibited iNOS, NO, and IL-6, but not COX-2 and TNF-α expression, suggesting that although PKCα, PKCδ, and p38 were inhibited by wood vinegar, COX-2 and TNF-α can be produced by other complementary pathways in LPS-activated macrophages. In addition, not all the pro-inflammatory mediators were inhibited by wood vinegar, suggesting that wood vinegar did not cause overall immune suppression in the host during an infection.

It has been reported that basal diet supplemented with 0.1% wood vinegar significantly improved the body weight gain of the grower-finisher pig due to the enhanced total-track digestibility of nutrients.[8] In addition, drinking water supplemented with 1.0% (v/v) wood vinegar improved intestinal morphology in broiler chicks, and no significant side effects were observed.[7] Furthermore, hens fed with charcoal powder containing wood vinegar (9.9 g/kg) increased the collagen content of the egg yolk.[4] Notably, wood vinegar prolonged the lifespan of Caenorhabditis elegans by reducing oxidative stress.[45] In the current study, we demonstrated the anti-inflammatory potential of wood vinegar. Considering previous and present studies provide a potential nutritional exploration of wood vinegar as functional foods, food additives, or feed additives for human or animals consumption. However, the potential toxicity of wood vinegar should be evaluated carefully before use.


  Conclusion Top


In summary, the wood vinegar from Griffith's ash did not cause harmful effects to RAW264.7 cells; however, it effectively inhibited the LPS-stimulated activation of the MAPK and PKC signaling pathways in macrophages, which further reduced the protein expression of iNOS in cells, the extracellular level of inflammatory substances (NO), and the release of pro-inflammatory cytokines (IL-6). Collectively, the experiments confirmed the anti-inflammatory activity of the wood vinegar from Griffith's ash [Figure 8]. Based on these results, we suggest that wood vinegar is able to reduce inflammation in LPS-activated macrophages, and it could be developed as a possible anti-inflammatory agent in the future.
Figure 8: Overview of the putative mechanisms by which wood vinegar from Griffith's ash attenuated the inflammatory response in macrophage. Wood vinegar from Griffith's ash inhibited LPS-induced inflammatory response in RAW264.7 macrophages by reducing the phosphorylation levels of p38 and PKC signaling pathways. LPS: Lipopolysaccharide, PKC: Protein kinase C.

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Financial support and sponsorship

This work was supported by grants from the Ministry of Science and Technology of Taiwan (No. MOST 106-2311-B-029-005 and No. MOST 107-2311-B-029-002), Taiwan.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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