Anti-inammatory Property of the Essential Oil From Cinnamomum Camphora (Linn.) Presl Leaves and the Evaluation of Its Underlying Mechanism by Using Metabolomics Analysis

Background: Cinnamomum camphora (Linn.) Presl has been widely used in traditional Chinese medicine for a variety of purposes. Our previous study indicated the antibacterial mechanism of the essential oil (EO) from C. camphora; however, the anti-inammatory activity of EO and its underlying mechanism have not been clearly demonstrated. The present study aims to evaluate the anti-inammatory principle and mechanism of EO. Methods: The anti-inammatory activity of EO was evaluated in lipopolysaccharide (LPS)-induced BV2 microglial cells. Nitric oxide (NO) production was measured by NO assay kit. The mRNA expression levels of inducible NO synthase (iNOS), interleukin-6 (IL-6), IL-18 and IL-1β were examined by real time-PCR (RT-PCR). The secretion of pro-inammatory cytokines in cell supernatants, including IL-6, IL-18 and IL-1β, were assessed by ELISA kits. Furthermore, the metabolic pro�le of BV2 microglial cells treated with or without EO was explored by GC-MS-based metabolomics analysis. Phosphofructokinase (PFK) and pyruvate kinase (PK) activities were detected by commercial kits. Results: EO signi�cantly decreased the release of NO and the mRNA expression of iNOS in LPS-induced BV2 microglial cells. EO also attenuated LPS-induced increase in the mRNA expression and secretion of in�ammatory cytokines including IL-6, IL-18 and IL-1β. 39 metabolites were identi�ed with signi�cantly different contents, including 21 upregulated and 18 downregulated ones, in the metabolomics analysis. Five pathways were enriched by shared differential metabolites. Additionally, compared with the control group, the glucose level was decreased, while the lactate level was increased, in the culture supernatant of LPS-induced BV2 microglia cells, which were reversed by EO treatment. Besides, compared to the LPS-treated group, the activities for PK and PFK in EO group were decreased by 17.59% and 18.23%, respectively. Conclusions: The EO from C. camphora acts as an anti-inammatory agent, which might be mediated through attenuating the glycolysis capacity of microglial cells.

In ammation, a complex series of interactions among cells and soluble factors, causes persistent tissue damage by leukocytes, lymphocytes or collagen 13 .Dysregulated in ammation, such as long-term chronic in ammation, has been considered to play key roles in the onset and development of metabolic disorders, neurodegeneration, and cardiovascular diseases 13,14 .It is of great signi cance to attenuate the increases of various in ammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and nitric oxide (NO) [15][16][17] .In addition, in ammatory response is usually associated with metabolic changes 18 .Metabolomics is considered to be a powerful platform to explore the potential mechanisms of drugs at the biochemical level.A recent study investigated the anti-neuroin ammatory potential of Clinacanthus nutans leaf extracts towards lipopolysaccharide (LPS)-induced BV2 microglial cells by using metabolomics approach 19 .The anti-in ammatory activity of EO and its potential mechanisms remain largely unelucidated.
In recent years, increasing evidence indicates that glycolysis plays an important role in the development of in ammation 20,21 .Glycolysis, a ubiquitous metabolic pathway in mammals, is regulated by multiple glycolytic enzymes, including phosphofructokinase (PFK) and pyruvate kinase (PK) 22,23 .Activation of the glycolytic pathway provides essential biomacromolecules, including amino acids and fatty acids, to synthesize in ammatory mediators in cells.LPS-induced cells undergo changes in glycolytic metabolism 24 .The increased level of lactate is also related to glycolytic metabolism 25,26 .Furthermore, lactate has been reported to enhance LPS-stimulated proin ammatory responses 27 .Therefore, the anti-in ammatory activity of molecules might be related to their effect on glycolytic pathway.Previous study showed that lidocaine inhibits the secretion of in ammatory cytokines in LPS-induced peritoneal macrophages by inhibiting the glycolytic pathway 28 .
In order to assess the anti-in ammatory activity of EO and explore the underlying mechanisms, this study was designed to detect NO production, cytokines secretion, and mRNA expression of inducible NO synthase (iNOS), IL-6, IL-18, and IL-1β in LPS-induced BV2 microglial cells.Furthermore, GC-MS-based metabolomics analysis and the glycolytic enzyme activity were investigated.Herein, we desire to provide better insight into the anti-in ammatory potential of EO.

Sample collection and extraction
Leaves of C. camphora were collected on March 2019 from adult trees located in the campus of Sun Yat-Sen University, and identi ed by Professor Depo Yang, School of Pharmaceutical Sciences, Sun Yat-Sen University.A voucher specimen for C. camphora (NO.A20190301) has been deposited at School of Pharmaceutical Sciences, Sun Yat-Sen University.The EO was extracted by hydrodistillation in a Clevenger-type apparatus for 2 h, and then dehydrated by ltration with anhydrous sodium sulfate.

Cell culture
The BV2 microglial cells were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China).Cells were cultured in DMEM with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin in a humidi ed incubator (Thermo Fisher, San Diego, CA, USA) containing 5% CO 2 at 37 °C.A TPVG solution (0.2% trypsin, 0.05% glucose and 0.2% EDTA in phosphate buffer saline solution) was used for cell dissociation.

Determination of cell viability
Cell viability was measured by MTT assay as described in previously reported protocol 29 .
Measurement of NO production BV2 microglial cells (5.5 × 10 4 cells/well) were incubated in a 96-well plate for 24 h and then treated with EO at different concentrations in the absence or presence of LPS (25 ng/mL).The cells treated with LPS alone were set as controls, and those treated with neither EO nor LPS were set as blank controls.After 24 h, NO production was determined by measuring the amount of nitrite in cell culture supernatants using the nitric oxide assay kit.
Measurement of IL-18, IL-1β and IL-6 production BV2 microglial cells (5 × 10 4 cells/well) were seeded in a 12-well plate and incubated for 24 h.After treated with EO at different concentrations (100, 150, 200 µg/mL) and stimulated with LPS (25 ng/mL) for 24 h, the release of IL-18, IL-1β and IL-6 in the culture supernatants was quanti ed by using the corresponding ELISA kits according to the manufacturers' protocols.
Total RNA extraction and RT-PCR BV2 microglial cells (1 × 10 6 cells/well) were incubated into 6-well plates for 24 h.The cells were treated with different concentrations of EO in the presence or absence of LPS (25 ng/mL) for 24 h.The RNA samples were isolated with Trizol reagent, and reversely transcribed into complementary DNA using HiScript II Q RT SuperMix for qPCR.Quantitative RT-PCR was performed using the Hieff™ qPCR SYBR® Green Master Mix according to the manufacturer's protocol and detected by the LightCycler 96 Real-Time PCR System.All experiments were performed in triplicate.Relative mRNA levels were calculated using 2 −ΔΔCt method.GAPDH was used as a housekeeping gene.The primer sequences were shown in Table 1.

Primer
Sequences Metabolomics analysis BV2 microglial cells were collected by centrifugation at 1,000 rpm for 3 min, washed with PBS for three times, and resuspended in methanol.The cells were homogenized by ultrasonic, followed by centrifugation at 12,000 rpm at 4 °C for 10 min.The supernatant of each sample was collected, and 10 µL ribitol (0.1 mg/mL) was added as the internal standard for analysis.Then the supernatants were concentrated in a rotary vacuum centrifuge device (LABCONCO).The dried extracts were used for GC-MS analysis, as described in previous study 12 .Each sample contained six biological and two technical replicates.
Measurement of lactate and glucose levels BV2 microglial cells were treated under the same conditions as those of RNA extraction.Lactate and glucose levels in the culture supernatants were estimated by using the lactate and glucose assay kits, respectively.

Measurement of enzyme activity
Cells were collected by centrifugation at 1,000 rpm for 3 min and then washed with PBS for three times.
The PFK and PK activities were analyzed spectrophotometrically by using commercially available kits according to manufacturer's instructions.Inhibition rate % = [(sample -control) / control] *100%.

Statistical analysis
All data were expressed as mean ± SD with the statistical method of one-way analysis of variance (ANOVA) and Student's t-test using the SPSS 19.0 software.A P value less than 0.05 was considered as a statistically signi cant difference.Metabolomics data was analyzed based on the technique described in our previous study 12 .

Cytotoxic effect of EO on BV2 microglial cells
To exclude the cytotoxicity, the BV2 microglial cells were treated with EO at concentrations ranging of 7.81-250 µg/mL for 48 h.The viability of BV2 microglial cells was measured by the MTT assay.The results showed that EO did not obviously affect cell viability even at a concentration up to 250 µg/mL Thus, the doses of 7.81-250 µg/mL of EO were selected for further experiments.

Effects of EO on the production of NO and the expression of iNOS
In order to investigate the anti-in ammatory effect of EO, NO production was evaluated in LPS-induced BV2 microglial cells.As shown in Fig. 1A, the NO production in LPS exposed cells was signi cantly increased, in compared with the control group, whereas EO treatment dose-dependently decreased NO production in BV2 microglial cells.Subsequently, RT-PCR results showed that EO suppressed the LPSinduced increase of iNOS expression in a dose-dependent manner (Fig. 1B).These results indicated that EO inhibited NO production through downregulating iNOS mRNA level in LPS-induced BV2 microglial cells.

Effects of EO on the expression and secretion of IL-6, IL-18 and IL-1β
To con rm the anti-in ammatory effect of EO, the levels of in ammatory mediators, including IL-6, IL-18 and IL-1β, were evaluated on LPS-stimulated BV2 microglial cells.Compared with the control group, LPS increased IL-6, IL-18 and IL-1β transcription, which were decreased after treated with EO in dosedependent manners (Fig. 1C-E).The ELISA results indicated that the secretion of IL-6, IL-18 and IL-1β in cell supernatants were remarkably increased in LPS group compared with the control; and EO treatment alleviated the elevated levels of IL-6, IL-18 and IL-1β (Fig. 1F-H).

Metabolomics analysis
Six biological and two technical replicates yielded 36 data in this study.There were 60 metabolites for subsequent analysis after data processing.KEGG was used to retrieve the biological categories of the identi ed metabolites.The abundance of 39 metabolites in the EO group was different in comparison of the model group (Fig. 2A).The Z-value was calculated for a comparative study based on the model group, showing the variations of these metabolites.The Z-score plot displayed that it spanned from − 7.74 to 7.37 in the EO group (Fig. 2B).Speci cally, there were 18 metabolites decreased and 21 metabolites increased in the EO group, when compared with the model group.
To investigate the metabolic differences among the control group, model group and EO group, orthogonal partial least-squares discriminant analysis (OPLS-DA) was applied for the recognition of the sample patterns, followed by ranking the altered metabolites in loading.The three groups were separated obviously.(Fig. 3A).Discriminating variables were present with S-plot (Fig. 3B) while cut-off values were set as greater or equal to the 0.05 and 0.5 for the absolute value of covariance p and correlation p (corr), respectively.Alanine, hexadecanoic acid, aspartic acid, valine, and myo-inositol showed the largest correlations and covariances in the predictive component between the model group and the EO group (Fig. 3C).
Five impacted pathways were enriched by shared differential metabolites, including alanine, aspartate and glutamate metabolism, citrate cycle (TCA cycle), galactose metabolism, fatty acid biosynthesis, and pantothenate and CoA biosynthesis, between the model and EO groups (Fig. 4A).The rst four pathways were dramatically impacted (P < 0.05), where amino acid metabolism was the most signi cant one, followed by glucose metabolism.As shown in Fig. 4B, most of metabolites enriched in the ve pathways were decreased.The alanine aspartate and glutamate metabolism pathway was mostly impacted among the altered metabolic pathways under EO treatment (P < 0.05).These results indicated that antiin ammatory activity of EO might be through interfering with amino acid and glucose metabolism.

Analysis of lactate and glucose levels
To evaluate whether glycolytic activity was suppressed by EO treatment, lactate production and glucose uptake were detected in BV2 microglial cells.Compared with the control group, the glucose level was decreased, while the lactate level was increased, in the culture supernatant of LPS-induced BV2 microglia cells, indicating that LPS treatment augmented the glycolytic capacity; however, the glucose level was increased and the lactate level was decreased after EO treatment, which indicated that EO suppressed glycolytic capacity induced by LPS (Fig. 5).

Enzyme activity analysis
To further demonstrate the effect of EO on glycolysis, the activities of PK and PFK, key enzymes associated with glycolysis, were measured.As shown in Fig. 6, compared to the control group, the activities for PK and PFK increased by 3.96%, 95.74% and 24.88% after treatment with LPS, respectively, whereas, the activities for PK and PFK decreased by 17.59% and 18.23% after EO treatment.It suggested that EO could disturb the LPS-induced activation of glycolysis.

Discussion
In ammation, an important protective response of organism, aims to eliminate or scavenge harmful stimuli, including damaged cells and pathogens, so as to restore normal tissue structure and function.A wild in ammation causes harmful effects to tissues and microcirculatory systems, and leads to severe complications 13 .Therefore, alleviation of in ammatory responses is the key for tissues to recover from different stimulating factors 30 .NO plays an important role in the immune system and the maintenance of body quiescence, but excessive NO causes a variety of in ammatory related diseases.In addition, the upregulation of in ammatory factors persisting at an in ammatory site is important, such as IL-1β, IL-18 and IL-6.Overproduction of these mediators recruits downstream effector cells (such as neutrophils), and directs the natural evolution of the in ammatory response 31 .Therefore, it is of great signi cance to explore the mechanisms of in ammation by detecting and observing the changes of these in ammatory factors 32 .The model of LPS-induced NO production in BV2 microglial cells is widely employed in screening anti-in ammatory agents for neuroin ammation and related neurodegenerative diseases 33 .
Kim et al. found that ginsenosides exert anti-in ammatory activities by suppressing the in ammatory enzymes, such as iNOS and COX-2, as well as the production of proin ammatory cytokinases such as TNF-α, IL-1β, and IL-6, in LPS-stimulated macrophages and microglial cells 30 .Currently, EO was found to inhibit LPS-induced NO production via suppression of iNOS expression at the mRNA level.In addition, the production of pro-in ammatory cytokines (IL-16, IL-18, and IL-1β) was signi cantly reduced by EO in BV2 microglial cells.These results showed that EO notably inhibits the expression of pro-in ammatory mediators, suggesting that it could inhibit the occurrence of in ammatory responses or reduce the aggravation of in ammatory mediators.
Recent ndings suggested that in ammation is closely related to metabolic disorders, thus the metabolites of biological organisms can be used as important indicators to re ect the inhibition or activation of related metabolic pathways under in ammation 34 .The signi cant disparation of metabolites can be witnessed by metabolomics pro ling analysis 21 .Previous investigations have shown that LPS activates BV2 microglial cells resulting in altered metabolism.Therefore, this study applied metabolomics to elucidate the anti-in ammatory mechanism of EO in LPS-induced BV2 microglial cells.
A previous study showed the changes in the metabolite levels were related to the anti-in ammatory effects of volatile oils from Angelica sinensis (VOAS), after the intervention of VOAS in acute in ammation displayed a trend of restoring the level of biomarkers to normal 35 .Similarly, the relative concentrations of metabolites between the model group and the EO group further demonstrated that physiological metabolism of LPS-induced BV2 microglial cells was restored after EO treatment.
According to the metabolomics analysis, most were amino acids among the 39 signi cantly different metabolites, which play essential roles in an organism.Further enrichment analysis and pathway analysis pointed mainly to the involvement of amino acid, glucose metabolism and fatty acid metabolism.Based on metabolic pathway analysis, TCA cycle plays the most important role in the amino acid metabolism, and the major metabolic part of galactose metabolism is glycolysis 35 .
Recent studies have shown that pro-in ammatory responses of microglia are driven by glycolysis, which is associated with high level of glucose uptake, revealing the crucial function of glycolysis in in ammation responses 16 .Lactate is an important product of glycolysis, and high lactate level is associated with a number of cellular events that lead to in ammation 27 .Therefore, the contents of glucose and lactate in the cell supernatant can re ect the glycolytic capacity of BV2 microglial cells.The experimental results showed that EO inhibits LPS-induced increase of glucose consumption and lactate production, suggesting that EO might inhibit the glycolytic ability in LPS-induced BV2 microglia cells.
Growing evidence indicates that glycolysis is closely linked with several key enzymes, which are crucial for the regulation of glycolysis, including PFK and PK 18 .Wang

Conclusions
In summary, a metabolomics approach was adopted to investigate the anti-in ammatory mechanisms of EO.Our studies showed that EO exerts anti-in ammatory effects by inhibiting iNOS gene expression and NO production, as well as the expression and secretion of in ammatory cytokines (IL-6, IL-18 and IL-1β) in LPS-treated BV2 microglial cells.We demonstrated that LPS promotes signi cant metabolic changes, resulting in increasing glycolysis.Whereas, EO suppresses the increase of glucose consumption and lactate production in LPS-stimulated BV2 microglial cells, which was related to the decreasing activities of key enzymes in glycolysis.Therefore, these results elucidated that EO acts as an anti-in ammatory agent via attenuating the LPS-induced glycolysis capacity.Furthermore, these data elucidated a molecular pathway that directly links glycolysis to the anti-in ammatory program of BV2 microglial cells, suggesting a potential role for metabolic therapies in treating in ammation.

Abbreviations
Effects

Figure 6
Figure 6 et al. demonstrated that the deacetylase Sirtuin5 plays a role in regulating in ammatory response by regulating the PK activity of PKM2; besides, withaferin A, a bioactive compound derived from Withania somnifera, considerably improved hepatic in ammation by regulating glycolysis-related enzyme genes in the liver of obese mice 36 .Similarly, our results veri ed that cells treated with EO suffered a decrease in the activities of PK and PFK and disrupted glycolysis.Glycolysis converts glucose into pyruvate, which is subsequently converted into lactate, and secreted, or gets into TCA cycle, which pointed out the importance of glycolysis.Therefore, further study about the factors affecting glycolysis can be the direction of our research.