Essential Oils from Zingiber striolatum Diels Attenuate Inflammatory Response and Oxidative Stress through Regulation of MAPK and NF-κB Signaling Pathways

Zingiber striolatum Diels (Z. striolatum), a widely popular vegetable in China, is famous for its medicinal and nutritional values. However, the anti-inflammatory effects of essential oil from Z. striolatum (EOZS) remain unclear. In this study, EOZS from seven regions in China were extracted and analyzed by GC–MS. LPS-induced RAW264.7 cells and 12-O-Tetradecanoylphorbol 13-acetate (TPA)-stimulated mice were used to evaluate the anti-inflammatory effects of EOZS. Results show that 116 compounds were identified in EOZS from seven locations. Samples 2, 4 and 5 showed the best capability on DPPH radical scavenging and NO inhibition. They also significantly reduced the production of ROS, pro-inflammatory cytokines, macrophage morphological changes, migration and phagocytic capability. Transcriptomics revealed MAPK and NF-κB signaling pathways may be involved in the anti-inflammatory mechanism, and the predictions were proven by Western blotting. In TPA-induced mice, EOZS reduced the degree of ear swelling and local immune cell infiltration by blocking the activation of MAPK and NF-κB signaling pathways, which was consistent with the in vitro experimental results. Our research unveils the antioxidant capability and potential molecular mechanism of EOZS in regulating inflammatory response, and suggests the application of EOZS as a natural antioxidant and anti-inflammatory agent in the pharmaceutical and functional food industries.


Analysis of Chemical Compounds of EOZS
The chemical compounds of EOZS were analyzed by a Shimadzu instrument (GCMS-QP2010 Ultra), equipped with a Rxi-5 ms silica capillary column (30 m × 0.25 mm, 0.25 µm film thickness). The operating method was performed according to a previous study [21] with few modifications. The temperature of the injector was set to 250 • C. The oven's initial temperature was set to 60 • C, and raised to 195 • C at a rate of 3 • C/min. Then, the temperature was increased to 300 • C at 15 • C/min and held at this temperature for 10 min. Helium was used as a carrier gas and its linear velocity was adjusted to 36.5 cm/s (1.0 mL/min). Injected volumes of the sample were 1 µL in split mode (oil 50 µL: hexane 500 µL). The ionization energy was set to 70 eV. The transfer line temperatures and ionization source were kept at 250 • C and 200 • C, respectively. Mass spectra were obtained in the range of 35-400 m/z, with a scanning speed of 0.3 s/scan. The retention index of each compound was calculated by using n-alkanes standards (C 8 -C 40 ). The compounds were identified by comparing the mass spectra of each chromatographic peak in the NIST05 library and comparing their calculated retention indexes with the retention indexes in the literature [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37].

Antioxidant Assay
The DPPH radical scavenging assay was used to evaluate the antioxidant capacity of EOZS. In short, 0.1 mM DPPH (dissolved in methanol) was added to a 96-well plate in a volume of 150 µL per well. Subsequently, gradient concentrations of EOZS (80, 40, 20, 10 µg/mL) were added. The 96-well plate was protected from light and incubated for 30 min at room temperature. The absorbance values were measured at 517 nm using a microplate reader. DPPH scavenging capacity was calculated using the following formula, and the results were presented as percentage: DPPH scavenging capacity (%) = [1 − (OD sample /OD control )] × 100% 2.5. Cell Culture and Detected of Cytotoxicity RAW 264.7 macrophage cells were purchased from the Shanghai Zhong Qiao Xin Zhou Biotechnology Co.; Ltd (Shanghai, China). The cells were maintained in DMEM/highglucose containing 10% FBS, 100 U/mL penicillin and 100 mg/L streptomycin. Cells were grown in a humidified atmosphere supplemented with 5% CO 2 at 37 • C. The cytotoxicity of EOZS on RAW 264.7 cells were determined by MTT. In brief, RAW 264.7 cells (5 × 10 4 cells/mL) were plated in a 96-well plate and maintained for 24 h. Then, the cells were treated with EOZS at various concentrations (0, 5, 10, 20, 40, 80 µg/mL) and incubated for another 24 h. After removing the spent medium, 100 µL MTT solutions (dissolved in serum-free DMEM, 500 µg/mL) were added to each well and cultured for 4 h. Then, the supernatant was discarded and formazan crystals were dissolved in DMSO (150 µL/well). Finally, the absorbance values were detected at 570 nm. Data were presented in the form of percentages.

Determination of NO Production
RAW 264.7 cells (5 × 10 5 cells/mL) were seeded in a 24-well plate and incubated overnight. After discarding the supernatant, the cells were pre-treated with EOZS at different concentrations for 2 h, and then incubated with LPS (1 µg/mL) for 24 h [38]. NO production was measured using a NO assay kit according to the specification. In short, 50 µL supernatant of each well was transferred to another 96-well plate, followed by mixing with an equal volume of Griess reagents for 10 min. The absorbance values at 540 nm were determined using a microplate reader. The concentrations of NO were calculated according to the standard curve of NaNO 2 .

Determination of Intracellular ROS
Intracellular ROS produced by LPS-induced RAW 264.7 cells were detected using the DCFH-DA fluorescent probe, which can be oxidized by ROS in cells to produce green fluorescent DCF. Thus, the level of intracellular ROS is proportional to the fluorescence intensity of DCF. Briefly, RAW 264.7 cells were seeded in 6-well plate at a density of 1 × 10 6 cells per well and maintained for 24 h. The cells were treated with 40 µg/mL of S2, S4 and S5 for 2 h, and were co-treated with LPS (1 µg/mL) for an additional 24 h. Then, the cells were incubated with DCFH-DA for 30 min and subsequently washed with PBS for three times. After blowing down the cells, the cells were resuspended with PBS and 10 µL was taken in a counting plate and placed in Countess™ II FL Automated Cell Counter (ThermoFisher, Waltham, MA, USA) for counting and photographing.

ELISA Assay
The level of pro-inflammatory cytokines (TNF-α, IL-6 and PGE 2 ) was determined using ELISA kits according to the manufacturer's instructions. In brief, 50 µL of standards, samples or blank were added to the wells pre-coated with antibodies, and incubated at 37 • C for 30 min followed by washing each well five times with 200 µL of wash solution. Then, 50 µL of HRP-conjugate reagent with antibodies was added to each well and incubated at 37 • C for 30 min. After a washing step with 200 µL of wash solution for five times, 100 µL of TMB substrate solution was added and incubated at 37 • C for 10 min. Then, 50 µL of stop solution was added to each well to stop the reaction. The absorbance values were obtained at 450 nm. The concentrations of cytokines were calculated according to the relevant standard curve.

Analysis of Cell Morphology and Size
RAW 264.7 cells (5 × 10 5 cells/mL) were plated in a 6-well plate and incubated overnight. After pre-treatment with 40 µg/mL of S2, S4 and S5 for 2 h, the cells were co-treated with LPS (1 µg/mL) for another 12 h and monitored with a HoloMonitor M4 instrument (PHI, Lund, Sweden). Time-lapse images of each well were captured from three random positions. Data on the optical volume, area and average optical thickness of RAW 264.7 cells were obtained from HoloStudio M4 software.

Cell Migration Assay
An in vitro scratch assay was used to assess the ability of cell migration. Briefly, RAW 264.7 cells were seeded in a 12-well plate and incubated overnight to form over a 90% confluent monolayer. Then, cells were pre-treated with indicated EOZS in 1% FBS medium for 2 h, followed by scratching using a pipette tip and co-treatment with LPS (1 µg/mL) for 24 h. Images were captured at 0 h and 24 h, and cell migration distance was measured by using image J (NIH Image J system, Bethesda, MD, USA).

Determination of Phagocytosis
Phagocytic uptake of RAW 264.7 cells was detected by using FITC-dextran. RAW 264.7 cells (1 × 10 6 cells per well) were seeded in a 6-well plate and incubated for 24 h. Cells were then treated with S2, S4 and S5 for 2 h, followed by co-treatment with LPS (1 µg/mL) for 24 h. After discarding the supernatant, cells were incubated with fresh medium containing FITC-dextran (1 mg/mL) at 37 • C for 1 h. The phagocytosis of cells on FITC-dextran were analyzed by Flowsight flow cytometry (Merck, Schwalbach, Germany).

RNA Sequencing
Three groups (three samples per group) were used for the analysis of RNA-seq, including control, LPS, LPS + S4 (briefly indicated as S4). Total RNA from three groups were obtained using Trizol reagent (Invitrogen, Carlsbad, CA, USA). After the RNA purity, concentration and integrity of each sample were tested for eligibility, cDNA libraries were constructed and sequenced using the Illumina Novaseq platform by PE150 strategy.

Western Blotting
RIPA lysis buffer containing Protease and Phosphatase Inhibitor Cocktail (Beyotime Technology, Shanghai, China) was added to RAW 264.7 cells and then incubated in ice for 10 min, followed by centrifugation at 12,000 rpm for 10 min at 4 • C. After collecting the supernatant, the total protein concentration of each sample were determined using a BCA Protein Assay Kit (Beyotime Technology, Shanghai, China). Specifically, the protein of cell nucleus and cytoplasm were separated, respectively, using a NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA, USA). The protein samples were mixed with loading buffer and boiled at 100 • C for 10 min to denature. Subsequently, the proteins were separated by 8% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Burlington, MA, USA) and blocked with 5% BSA (Aladdin, Shanghai, China). The membranes were incubated with the primary antibodies (1:1000) diluted in Primary Antibody Dilution Buffer (Beyotime Technology, Shanghai, China) at 4 • C overnight, and then incubated with secondary antibodies (1:2000) diluted in TBST for 2 h. At last, the proteins were determined using Immobilon Western Chemilum HRP Substrate (Millipore, USA).

Immunofluorescence Staining
RAW 264.7 cells were seeded in 35 mm confocal dishes at a density of 5 × 10 5 cells/ dish and maintained for 24 h. After treating with S2, S4 and S5 (40 µg/mL) for 2 h, the cells were co-incubated with LPS (1 µg/mL) for another 24 h. After fixing with 4% paraformaldehyde solution for 10 min, the cells were permeabilized by 0.5% Triton X-100 solution treatment for 15 min and incubated with primary antibody (p65, 1:400) at 4 • C overnight. Then, the cells were incubated with secondary antibody conjugated with Alexa Fluor 555 for 2 h at room temperature, followed by treatment with DAPI solution (10 µg/mL) for 5 min. Image acquisitions were performed using Zeiss LSM-800 scanning confocal microscope (Jena, Germany).

TPA-Induced Mouse Ear Edema Model
A total of twenty-nine male Kunming (6-week-old) mice were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Mice were acclimatized for seven days under temperature (25 • C) and relative humidity (50%) controlled conditions and a 12 h light/dark cycle before the experiments. Subsequently, all the mice were randomly divided into six groups, including control (n = 4), TPA (n = 5), dexamethasone (DXM, n = 4), S2 (n = 6), S4 (n = 5) and S5 (n = 5). DXM (5 mg/kg), S2 (50 mg/kg), S4 (50 mg/kg) and S5 (50 mg/kg) were, respectively, administrated to the right ear of mice for two days. On the third day, one hour after administration with DXM, S2, S4 and S5, 20 µL of TPA (50 µg/mL) was applied to the right ear of mice, except for the control group. After six hours, mice were euthanized and the right ear tissues (6 mm diameter) obtained from Antioxidants 2021, 10, 2019 6 of 21 each mouse were weighed. All procedures in the experiment were performed according to the national legislation and local guidelines of Wuyi University (Approval number: CN2021001).

Hematoxylin-Eosin Staining
Mice ear tissues were fixed with 4% paraformaldehyde, embedded with paraffin, and made into sections. The tissue sections were stained using hematoxylin-eosin (HE) observed and photographed using Olympus IX71 microscope (Tokyo, Japan).

Immunohistochemistry Staining
After dewaxing and rehydrating with a gradient of ethanol and distilled water, the sections were doused with 3% H 2 O 2 for 30 min. Then, the sections were blocked with 10% goat serum for 30 min, incubated with primary antibodies against p-p65, p-p38, p-ERK1/2, p-JNK1/2 at 4 • C overnight and then incubated with secondary antibody. Finally, the sections were stained with 3,3 -diaminobenzidine and photographed using Olympus IX71 microscope.

Statistical Analysis
PAST (Version 3.0) software was used to perform matrix plot, principle component analysis (PCA) and hierarchical cluster analysis (HCA) on 116 chemical components in EOZS from seven regions. Grayscale values of bands in Western blotting were analyzed using Image J (Rockville, MD, USA). The integral optical density (IOD) of immunohistochemical staining was analyzed using Image Pro Plus (Rockville, MD, USA). All the data in this study were presented as the mean ± standard error of mean (SEM). Statistical significance was assessed by one-way analysis of variance (ANOVA) using GraphPad Prism 7.0 software. A value of p < 0.05 was considered to represent a significant difference.

DPPH Scavenging Activity
As shown in Figure 2A, EOZS from seven regions all exhibited concentration-dependent antioxidant capacity ranging from 10 μg/mL to 80 μg/mL. At a concentration of 80 μg/mL, the DPPH scavenging rate of EOZS followed the order of S4 (37.26%) > S2 (32.79%) > S5 (26.99%) > S1 (26.26%) > S6 (17.23%) > S3 (16.86%) > S7 (15.61%).  Figure 2B). The toxicity of S1 on RAW 264.7 cells showed a concentration dependence, with significant effects occurring at concentrations greater than or equal to 20 µg/mL. However, S3, S6 and S7 exhibited no inhibition on the cell proliferation of RAW 264.7 cells at a concentration of 80 µg/mL. The safe concentration of S2, S4 and S5 on RAW 264.7 cells were thought to be 40 µg/mL. Therefore, 10 µg/mL (S1), 40 µg/mL (S2 to S7) and 80 µg/mL (S3, S6 and S7) of different EOZS were selected to clarify their effects on NO release in LPS-induced RAW 264.7 cells. As shown in Figure 2C, NO content in the LPS-treated group was elevated approximately 7.6-fold compared to the control group. After treatment with 10 µg/mL of S1, 40 µg/mL of S2 to S7 and 80 µg/mL of S3, S6, and S7, the mean NO release level of RAW 264.7 cells was decreased by 1.5~3.9-fold. In general, the EOZS from seven regions all significantly inhibited the NO produced by LPS-induced RAW 264.7 cells. In subsequent experiments, EOZS from three different regions with the best effect on NO inhibition (40 µg/mL of S2, S4, and S5) were selected to further investigate their anti-inflammatory effects and underlying molecular mechanism.
ROS, as a second messenger in the inflammatory response, is upregulated during inflammation, thereby inducing oxidative stress. As shown in Figure 2D, the expression of ROS in RAW 264.7 cells was extremely low in the control group. After treatment with LPS, the cellular ROS level was significantly increased, whereas the level of ROS expression in RAW 264.7 cells decreased with the treatment of S2, S4 and S5 at a concentration of 40 µg/mL.

EOZS Attenuated Production of Pro-Inflammatory Mediators
To further investigate the inhibitory effect of EOZS on inflammatory factors, we collected the supernatants of LPS-induced RAW 264.7 cells for detection. Compared with the control group, the expression levels of TNF-α, IL-6 and PGE 2 in LPS-induced RAW 264.7 cells were significantly increased (p < 0.0001), up-regulated by 5.40-, 6.85-and 5.24fold, respectively. Treatment with 40 µg/mL of S2, S4 and S5 caused a significant decrease in TNF-α, IL-6 and PGE 2 production ( Figure 2E-G).

EOZS Reduced Cellular Morphological Changes, Migration and Phagocytic Capability in LPS-Induced RAW 264.7 Cells
During the inflammatory process, activated macrophages alter their morphology and phenotype to rapidly respond to external stimuli. Several studies have shown that cell morphology, migration and phagocytic capability would be changed in activated RAW 264.7 cells [39][40][41]. It could be distinctly observed that 12 h after LPS stimulation, RAW 264.7 cells underwent deformation and produced elongated pseudopods, accompanied by an increase in cell volume and area as well as a decrease in cell thickness, while these conditions were improved in the S2, S4 and S5 treatment group ( Figure 3A-D). Furthermore, S2, S4 and S5 significantly inhibited the migration ability in LPS-stimulated RAW 264.7 cells ( Figure 3E,F). We also tested the effect of S2, S4 and S5 on the phagocytic activities of LPSinduced RAW 264.7 cells by using FITC-labeled dextran. Flow cytometry data showed that all three reduced the phagocytic ability in LPS-induced RAW 264.7 cells ( Figure 3G).

Bioinformatics Analysis of RNA Sequencing Data
To investigate the mechanism of EOZS-inhibiting LPS-induced RAW 264.7 cells, control, LPS and S4 groups were selected for RNA sequencing. Gene expression correlation analysis suggested Pearson correlation coefficient between different samples in the same group were between 0.973 and 0.998, indicating the excellent biological replicates among the samples ( Figure S1A). Compared with the control group, 4677 differentially expressed genes (DEGs) were identified in LPS-induced RAW264.7 cells, of which 2078 were upregulated and 2599 were down-regulated ( Figure 4A). A total of 103 DEGs were found in S4 vs. LPS group, of which 51 were up-regulated and 52 were down-regulated ( Figure 4B). Then, we calculated the intersections between 2078 up-regulated DEGs in the LPS vs. control groups and 52 down-regulated DEGs in the S4 vs. LPS group. As shown in the Venn diagram and the heat map, 45 co-regulated DEGs were obtained ( Figure 4C,D). String (https://www.string-db.org/, accessed on 21 September 2021) was performed to analyze protein-protein interaction network of 45 co-regulated DEGs (confidence score = 0.7), and disconnected nodes in the network were hided. Three hub genes with the highest degree were visualized using Cytoscape software ( Figure 4E), namely IL-1b (12), CXCL10 (11) and CSF2 (7), and their FPKM value in the three groups were presented in Figure 4F.  diagram and the heat map, 45 co-regulated DEGs were obtained ( Figure 4C,D). String (https://www.string-db.org/, accessed on 21 September 2021) was performed to analyze protein-protein interaction network of 45 co-regulated DEGs (confidence score = 0.7), and disconnected nodes in the network were hided. Three hub genes with the highest degree were visualized using Cytoscape software ( Figure 4E), namely IL-1b (12), CXCL10 (11) and CSF2 (7), and their FPKM value in the three groups were presented in Figure 4F. To gain a better understanding of the potential relationship among the 45 co-regulated DEGs, we conducted a functional enrichment analysis. GO analysis revealed that the biological process of these DEGs included the regulation of nitric-oxide synthase activity, inactivation of MAPK activity, etc. Cellular components were mainly extracellular space and extracellular region, while molecular function involved MAP kinase tyrosine/serine/threonine phosphatase activity, interleukin-1 receptor binding, etc. ( Figure S1B). In accordance with the results of KEGG enrichment, S4 treatment regulated IL-17 signaling pathway, TNF signaling pathway and MAPK signaling pathway ( Figure 4G). We further found through the KEGG map that MAPK signaling pathway and NF-kB signaling pathway were involved in the IL-17 signaling pathway and TNF signaling pathway ( Figure S1D). Based on the evidence above, Western blotting was subsequently performed to verify the inhibitory effect of S4 on the MAPK signaling pathway and NF-kB signaling pathway.

EOZS Inhibited Expression Level of iNOS, COX2 and Activation of MAPK and NF-κB Pathways in LPS-Induced RAW 264.7 Cells
NO and PGE 2 secreted by the cells can be catalyzed by inducible nitric oxide synthase (iNOS) and Cyclooxygenase 2 (COX2), respectively. Therefore, the above results prompted us to investigate the changes in the protein expression of iNOS and COX2. As expected, the dramatic up-regulation of iNOS and COX2 proteins was observed in the LPS treatment group, while 40 µg/mL of S2, S4 and S5 treatment reduced the expressions of both proteins ( Figure 5A). After LPS treatment for 30 min, the MAPK signaling pathway in LPS-induced RAW 264.7 cells was activated, as evidenced by significantly increased protein phosphorylation levels of p38, ERK1/2 and JNK. Interestingly, 40 μg/mL of S2, S4 and S5 treatment all reduced the expression levels of p-p38, p-ERK1/2 and p-JNK to various extents in LPS-induced RAW 264.7 cells, but did not affect the expression of p38, ERK1/2 and JNK ( Figure  5B).
NF-κB p65 protein in the LPS group was significantly increased in the nucleus and decreased in the cytoplasm, indicating the activation of NF-κB signaling pathway, while S2, S4 and S5 treatment reduced the nuclear translocation of NF-κB p65 ( Figure 5C). A After LPS treatment for 30 min, the MAPK signaling pathway in LPS-induced RAW 264.7 cells was activated, as evidenced by significantly increased protein phosphorylation levels of p38, ERK1/2 and JNK. Interestingly, 40 µg/mL of S2, S4 and S5 treatment all reduced the expression levels of p-p38, p-ERK1/2 and p-JNK to various extents in LPS-induced RAW 264.7 cells, but did not affect the expression of p38, ERK1/2 and JNK ( Figure 5B).
NF-κB p65 protein in the LPS group was significantly increased in the nucleus and decreased in the cytoplasm, indicating the activation of NF-κB signaling pathway, while S2, S4 and S5 treatment reduced the nuclear translocation of NF-κB p65 ( Figure 5C). A similar result was detected in the immunofluorescence assay. As shown in Figure 5D, NF-κB p65 marked with red fluorescence in the LPS group was mainly located in the nucleus compared to the control group. However, this condition was reduced in the S2, S4 and S5 treatment groups, suggesting that the nuclear translocation of p65 was obviously blocked.

EOZS Ameliorated Ear Edema and Infiltration of Immune Cells in TPA-Induced Mouse Model
A TPA-induced mouse ear edema inflammatory model was used to evaluate the in vivo anti-inflammatory effects of S2, S4 and S5 in the present study. This animal model is a reliable in vivo model system that mimics inflammatory skin diseases such as psoriasis and can therefore be used to assess the acute and chronic effects of drugs in skin inflammation [42]. Six hours after TPA application, the mean ear weight of mice increased from 7.6 mg to 23.4 mg, while the increase in mean ear weight in the dexamethasone, S2, S4 or S5 treatment groups was smaller than that in the TPA group. Compared with the TPA group, the mean ear weight of the S2, S4, or S5 treatment groups was 16.4 mg, 18.0 mg and 17.2 mg, respectively, which all showed significant inhibition on TPA-induced ear edema ( Figure 6A). In addition, obvious histological lesions, including dermal edema and extensive inflammatory cell infiltration, was observed in the TPA-treated group. Treatment with S2, S4 and S5 effectively improved these pathological indicators ( Figure 6B). CD45, also known as a leukocyte common antigen (LCA), is a marker of immune cells [43]. Visibly, the number of CD45 positive immune cells in a mouse ear was significantly increased by approximately 10-fold after TPA application, whereas dexamethasone, S2, S4, and S5 treatments resulted in a significant reduction in CD45 positive immune cells infiltration at the site of inflammation ( Figure 6C,D).

EOZS Ameliorated Ear Edema and Infiltration of Immune Cells in TPA-Induced Mouse Model
A TPA-induced mouse ear edema inflammatory model was used to evaluate the in vivo anti-inflammatory effects of S2, S4 and S5 in the present study. This animal model is a reliable in vivo model system that mimics inflammatory skin diseases such as psoriasis and can therefore be used to assess the acute and chronic effects of drugs in skin inflammation [42]. Six hours after TPA application, the mean ear weight of mice increased from 7.6 mg to 23.4 mg, while the increase in mean ear weight in the dexamethasone, S2, S4 or S5 treatment groups was smaller than that in the TPA group. Compared with the TPA group, the mean ear weight of the S2, S4, or S5 treatment groups was 16.4 mg, 18.0 mg and 17.2 mg, respectively, which all showed significant inhibition on TPA-induced ear edema ( Figure 6A). In addition, obvious histological lesions, including dermal edema and extensive inflammatory cell infiltration, was observed in the TPA-treated group. Treatment with S2, S4 and S5 effectively improved these pathological indicators ( Figure 6B). CD45, also known as a leukocyte common antigen (LCA), is a marker of immune cells [43]. Visibly, the number of CD45 positive immune cells in a mouse ear was significantly increased by approximately 10-fold after TPA application, whereas dexamethasone, S2, S4, and S5 treatments resulted in a significant reduction in CD45 positive immune cells infiltration at the site of inflammation ( Figure 6C,D).

EOZS Suppressed MAPK and NF-κB Pathways in TPA-Induced Mouse Model
We then attempted to investigate the effects of S2, S4 and S5 on MAPK and NF-κB signaling pathways in vivo. Immunohistochemical staining was performed to detect the expression levels of phosphor-p38, phosphor-ERK1/2, phosphor-JNK and phosphor-p65.

EOZS Suppressed MAPK and NF-κB Pathways in TPA-Induced Mouse Model
We then attempted to investigate the effects of S2, S4 and S5 on MAPK and NF-κB signaling pathways in vivo. Immunohistochemical staining was performed to detect the expression levels of phosphor-p38, phosphor-ERK1/2, phosphor-JNK and phosphor-p65. The results indicated that TPA-induced mice ears showed a significant increase in the phosphorylation levels of p38, ERK1/2, JNK and p65 relative to unexposed ears. However, the S2, S4 and S5 treatment groups all reduced the expressions of these phosphorylated proteins to different degrees ( Figure 7A-E). The above results demonstrated that S2, S4 and S5 could reduce inflammatory response in mouse ear by inhibiting MAPK and NF-κB signaling pathways, which is similar to the results in vitro.

Discussion
Natural products are used as traditional medicines for the treatment or prevention of a wide variety of human diseases due to their extensive pharmacological properties. As a kind of relatively safe natural product, plant-derived essential oils are more easily accepted by consumers [44]. To reduce substance abuse, the use of essential oils to relieve or treat diseases has also been developed as an alternative therapy [45]. Z. striolatum has traditional medicinal value, but the anti-inflammatory effect of its essential oil has not yet been reported. In the present work, we mainly analyzed the composition of EOZS from seven regions and evaluated their suppressive effect on inflammatory response and oxidant stress. Sixteen common chemical compounds were identified from the EOZS of seven regions, suggesting that these characteristic peaks may be used as the index components for quality control of Z. striolatum. From the phytochemical classification, Z. striolatum from seven locations could be divided into three clusters. The factors causing these differences may be related to the geographical location, climatic conditions and soil environment. Although the main chemical components and relative contents in S1-S5 were similar, the cytotoxicity of S1 was stronger than other samples. This indicated that it may be caused by minor or trace components in the S1.
Our research showed that EOZS from seven locations exhibited a certain degree of

Discussion
Natural products are used as traditional medicines for the treatment or prevention of a wide variety of human diseases due to their extensive pharmacological properties. As a kind of relatively safe natural product, plant-derived essential oils are more easily accepted by consumers [44]. To reduce substance abuse, the use of essential oils to relieve or treat diseases has also been developed as an alternative therapy [45]. Z. striolatum has traditional medicinal value, but the anti-inflammatory effect of its essential oil has not yet been reported. In the present work, we mainly analyzed the composition of EOZS from seven regions and evaluated their suppressive effect on inflammatory response and oxidant stress. Sixteen common chemical compounds were identified from the EOZS of seven regions, suggesting that these characteristic peaks may be used as the index components for quality control of Z. striolatum. From the phytochemical classification, Z. striolatum from seven locations could be divided into three clusters. The factors causing these differences may be related to the geographical location, climatic conditions and soil environment.
Although the main chemical components and relative contents in S1-S5 were similar, the cytotoxicity of S1 was stronger than other samples. This indicated that it may be caused by minor or trace components in the S1.
Our research showed that EOZS from seven locations exhibited a certain degree of DPPH scavenging activity. Furthermore, they also significantly inhibited the production of NO and ROS in LPS-induced RAW 264.7 cells. Interestingly, EOZS from Huaihua City (S2), Zhangjiajie City (S4) and Enshi City (S5) showed a stronger inhibitory effect on NO production in LPS-induced RAW 264.7 cells. From the results of PCA and HCA, it can be seen that the main difference between cluster III (S1-S5) and other clusters included βphellandrene, β-pinene, α-pinene and α-humulene. Lee et al. identified β-phellandrene as the main component of Zanthoxylum schinifolium essential oil, and found that Zanthoxylum schinifolium essential oil significantly inhibited the mRNA transcription level of inducible nitric oxide synthase [46]. There was direct evidence that α-pinene and α-humulene suppressed NO produced by activated macrophages or monocytes [47,48]. In addition, as the main component of the essential oil of Xylopia parviflora, β-pinene has been shown to reduce the NO production in LPS-induced RAW 264.7 cells [49]. The above evidence may partly explain the anti-inflammatory activity of S2, S4 and S5. S1 was not compared with other samples in cluster III at the same concentration because of its stronger cytotoxicity. However, the main components of S3 and S2, S4 and S5 were similar, whilst the effect of S3 was weaker, indicating that there may be an antagonistic effect between different components in S3.
Transcriptomics is a method to study the expression of RNA and the regulation of transcription in cells [50]. In recent years, transcriptomics has been widely used to reveal differences in the expression of genes in biological functions and to discover the mechanisms or targets of drugs. By using transcriptomics analysis, Li et al. found that Rebaudioside A could protect Caenorhabditis elegans from oxidative stress via regulating TOR and PI3K/Akt pathways [51]. RNA sequencing results of this study indicated that the MAPK and NF-κB pathways may be involved in the potential anti-inflammatory mechanism of EOZS. The MAPK signaling pathway plays an important role in extracellular signaling to the inner nucleus, and it can be activated by external environmental stress or stimulation by inflammatory mediators [52]. It has been shown that the conditional knockdown of the p38α MAPK gene results in a significant reduction in TNF-α production by LPS-induced microglia in mice, thus preventing the neuronal damage produced by LPS induction [53]. NF-κB, as a core regulator of inflammatory response, has been a popular research target in the field of inflammation and immunity. When canonical NF-κB signaling pathway is activated, IκB is phosphorylated via the IκB kinase (IKK) complex and subsequently polyubiquitinated and degraded by the 26S proteasome. NF-κB is then phosphorylated and enters the nucleus, leading to the initiation and protein expression of pro-inflammatory genes such as TNF-α and IL-6 [54]. All of this evidence suggests that the activation status of MAPK and NF-κB signaling pathways has a critical impact on the production of downstream pro-inflammatory mediators. Therefore, the inhibition of the excessive activation of MAPK and NF-κB signaling pathways is a strategy for the treatment of inflammation-related diseases.
During inflammation, cells damaged by irritation recruit immune cells to migrate towards the site of inflammation by releasing cytokines and chemokines, while immune cells produce more inflammatory mediators through activated MAPK and NF-κB signaling pathways [55], creating a positive feedback loop that may further exacerbate the progression of inflammation. In this study, we demonstrated that EOZS inhibited the activation of macrophage and production of pro-inflammatory mediators through the regulation of MAPK and NF-κB signaling pathways (Figure 8). In vivo experiments also showed that EOZS could reduce the infiltration of immune cells to the site of inflammation to alleviate inflammation. Indeed, ample evidence suggests that plant volatile oils possess anti-inflammatory pharmacological activity in vivo and in vitro [56][57][58].

Conclusions
In conclusion, this was the first study to clarify the anti-inflammatory molecular mechanism of EOZS, and these findings indicate that EOZS may have the potential to prevent or treat inflammation-related diseases. However, there are differences in the antiinflammatory effects of EOZS from different locations. Therefore, it is necessary to establish relevant standards to control the quality of EOZS in order to promote its industrialization.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Geographic information of Z. striolatum collected from seven different habitats. Figure S1

Conclusions
In conclusion, this was the first study to clarify the anti-inflammatory molecular mechanism of EOZS, and these findings indicate that EOZS may have the potential to prevent or treat inflammation-related diseases. However, there are differences in the antiinflammatory effects of EOZS from different locations. Therefore, it is necessary to establish relevant standards to control the quality of EOZS in order to promote its industrialization.

Conflicts of Interest:
The authors declare no conflict of interest.