Anti-inflammatory compounds from the mangrove endophytic fungus Amorosia sp. SCSIO 41026

Three new chlorinated compounds, including two propenylphenol derivatives, chlorophenol A and B (1 and 2), and one benzofuran derivative, chlorophenol C (3), together with 16 known compounds, were isolated from the mangrove endophytic fungus Amorosia sp. SCSIO 41026. 7-Chloro-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarine (4) and 2,4-dichloro-3-hydroxy-5-methoxy-toluene (5) were obtained as new natural products. Their structures were elucidated by physicochemical properties and extensive spectroscopic analysis. Compounds 1, 4, 7, 9, 13, 15, 16, and 19 possessed inhibitory effects against the excessive production of nitric oxide (NO) and pro-inflammatory cytokines in lipopolysaccharide (LPS)-challenged RAW264.7 macrophages without obvious cytotoxicity. Moreover, 5-chloro-6-hydroxymellein (13) further alleviated the pathological lung injury of LPS-administrated mice and protected RAW264.7 macrophages against LPS-induced inflammation through PI3K/AKT pathway in vivo. Our research laid the foundation for the application of compound 13 as a potential anti-inflammatory candidate.


Introduction
As an adaptive response, inflammation is triggered by harmful stimuli and conditions that prompt the host to produce pro-inflammatory cytokines including interferon, interleukin, chemokines, and tumor necrosis factor against pathogens (Al-Lamki et al., 2001). A controlled inflammatory response is generally thought to be beneficial, in providing protection against infection for example, but it can become harmful or even fatal if it is maladjusted (e.g., rheumatoid arthritis, systemic infection, and septic shock). Nitric oxide (NO) is synthesized by inducible nitric oxide synthase type 2 (NOS-2) in many cells involved in immunity and inflammation. Studies have shown that overproduction of NO is rapidly oxidized to reactive nitrogen oxides (RNOs; Coleman, 2001), which are able to modify key signal molecules such as kinases and transcription factors. In addition, RNOs restrain crucial enzymes in mitochondrial respiration, resulting in intra-cellular ATP depletion. In addition, a high concentration of NO inhibited the activity of antigen-presenting cells and T-cell proliferation (Kim et al., 2002;Hortelano et al., 2003). Therefore, targeting the reduction of these pro-inflammatory mediators can be an effective way to control and prevent chronic inflammatory diseases to some extent.
Lipopolysaccharide (LPS) is a virulence factor of the outer membrane of gram-negative bacterial cell wall, of which lipid A is the immunologically active component and can cause an inflammatory response. LPS induces the translocation of the transcription factor NF-κB into the nucleus by binding to cells expressing toll-like receptor 4 (TLR4), resulting in dephosphorylation of phosphorylated interferon regulatory factor 3 (IRF3) and stimulating the production of type I interferons, which causes subsequent inflammatory response (Yoo et al., 2014;Peerapornratana et al., 2019;Zhuo et al., 2019). Although NF-κB signaling has been historically considered a classic LPS-induced inflammatory pathway, it is of great significance to enthusiastically search for the in-depth understanding of the inflammatory response mechanism and the development of target drugs.
Halogenated organic molecules are generally rare in nature and could simply be classified as fluorinated, chlorinated, brominated, or iodinated compounds (Wang et al., 2013). Over 5,000 halogenated natural products had been reported as of 2011 with structural and functional diversity (Gribble, 2015;Zeng and Zhan, 2019). As a key part of halogenated molecules, chlorinated compounds have demonstrated a broad range of remarkable activities (Gribble, 2015), including antibacterial activity (Bracegirdle et al., 2021;Saetang et al., 2021) and anticancer activity (Luo et al., 2018). Recently, three new natural chlorinated 3-phenylpropanoic acid derivatives were found with significant and selective inhibitory activities toward Escherichia coli and Staphylococcus aureus (Shaala et al., 2020).
Naturally occurring chlorinated compounds were mainly isolated from the marine environment and microorganisms (Wang et al., 2013. Mangrove forests are complex ecosystems with the frequent tide, which contained a high concentration of chloride ions. Therefore, mangrove-derived fungi are proved to be a promising source of novel and unique chlorine-containing bioactive secondary metabolites (Xu, 2015;Chen et al., 2021a). As part of our continuous efforts to discover structurally novel and bioactive secondary metabolites from mangrove endophytic fungi (Chen et al., 2021b,d), three new chlorine-containing compounds, chlorophenol A − C (1-3), and two new natural products (4 and 5), together with 14 known compounds, were isolated from the mangrove-derived fungus Amorosia sp. SCSIO 41026. Herein, the isolation, structure elucidation, and biological effects of compounds 1-19 (Figure 1) are described.

General experimental procedures
The UV and IR spectra were recorded on a Shimadzu UV-2600 PC spectrometer (Shimadzu) and an IR Affinity-1 spectrometer (Shimadzu). Optical rotations were determined with an Anton Paar MPC 500 polarimeter. HRESIMS spectra were recorded with a Bruker maXis Q-TOF mass spectrometer. The NMR spectra were recorded on a Bruker Avance-500 or 700 spectrometers (Bruker BioSpin International AG, Switzerland) using tetramethylsilane as internal standard, and chemical shifts were recorded as δ-values. Semipreparative high-performance liquid chromatography (HPLC) was performed on the Hitachi Primaide with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 mm × 250 mm, 5 μm). Thin-layer chromatography analysis (TLC) and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10-40 μm) and over silica gel (200-300 mesh; Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), respectively. Spots were detected on TLC (Qingdao Marine Chemical Factory) under 254 nm UV light. All solvents employed were of analytical grade (Tianjin Fuyu Chemical and Industry Factory).

Fungal material
The endophytic fungal strain SCSIO 41026 was isolated from the leaf of Avicennia marina (Forsk.) Vierh. collected from the mangrove wetland in Zhanjiang, Guangdong province, China (21.235° N,110.451° E). The strain was stored on MB agar (malt extract 15 g, sea salt 10 g, agar 16 g, H 2 O 1 l, and pH 7.4-7.8) slants at 4°C and deposited at the Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences. The ITS1-5.8S-ITS4 sequence region (GenBank accession No. OL826791) of strain SCSIO 41026 was amplified by PCR. The DNA sequencing showed it shared significant homology to the sequence of Amorosia littoralis (accession No. AM292047.1), so it was designated as Amorosia sp. SCSIO 41026.

X-ray crystallographic analysis
The clear light colorless crystals of 4 and 12 were obtained in MeOH by slow evaporation. Crystallographic data for the structures have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, United Kingdom [fax: +44(0)-1,223-336,033 or e-mail: deposit@ ccdc.cam.ac.uk].
For LPS induction, RAW264.7 cells were cultured in a 24-well plate at a density of 1 × 10 5 cells/well (1 ml medium/well) overnight. After 24 h of exposure to 0.1 μg/ml LPS (#L2630, Merck, Shanghai, China) and being separately co-treated with the compounds (final concentration was maintained at 10 μM), the culture supernatant was collected to measure the production of NO, IL-6, TNF-α, and MCP-1, and cells were digested and harvested for RNA extraction.

Cell viability assay
3 × 10 4 cells/well (100 μl medium/well) were seeded in a 96-well plate overnight; then, they were exposed to DMEM supplemented with the compounds (final concentration was maintained at 10 μM) for 24 h, respectively. At the same time, the wells without cells were set as blank group and the drug-free cells as control group. Whereafter, 10 μl CCK-8 solution (#C0037, Beyotime Institute of Biotechnology, Shanghai, China) was added to each well and incubated at 37°C for 1 h. The absorbance was read at 450 nm with a microplate reader (Thermo, Waltham, MA, United States). Cell viability was calculated by the formula (experimental group absorbance value/control group absorbance value) × 100%.

Nitric oxide production assay
Nitric oxide production was assessed by a Nitric Oxide Assay Kit (#S0021, Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions; in the meanwhile, L-NMMA was used as a positive control of compound treatment since it is the total NOS inhibitor.

Cytokine detection
After 24 h of LPS induction, the cell culture supernatant was collected to measure the release of tumor necrosis factor α (TNFα), interleukin 6 (IL-6), and monocyte chemoattractant protein 1 (MCP-1). Cytokine levels were quantified in the samples using an automatic ELISA (ELLA, protein simple).

Quantitative real-time PCR analysis
Total RNA was isolated from cells using TRIzol ® reagent (Ambion, Austin, Texas, United States) and was reversetranscribed into cDNA using EasyScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Gene transcript levels of IL-6, TNF-α, MCP-1, IL-10, IL-4, and Arg-1 were quantified using the TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer's protocol. β-actin was used as housekeeping control. Primers used for quantitative PCR are listed in Supplementary Table 1.

RNA sequencing
After 24 h of co-treatment of LPS and compound 13, RAW264.7 macrophages were collected for RNA extraction and transcriptome sequencing. RNA sequencing was performed on the Annoroad Illumina-HiSeq platform. Differentially expressed genes were defined as values of p < 0.01. Pathway enrichment analysis using Gene Set Enrichment Analysis (GSEA).

Western blot
The concentration was detected with BCA kit before total protein was extracted from RAW264.7 cells. The samples mixed with the loading buffer were separated on an 8% SDS-PAGE gel. After transferring to polyvinylidene fluoride membranes by electro-transfer, the membranes were blocked with 5% BSA at room temperature for 1 h. Next, the PVDF membranes were incubated with primary antibody overnight at 4°C and then incubated with peroxidase-conjugated secondary antibodies at room temperature for 1 h. The protein brands were captured in a gel imaging system. ImageJ was used to analyze the value of the brands.

Statistical analysis
Statistical analyses were performed with GraphPad Prism (version 8.4.3). Independent Student's t-tests were used to compare the means of numerical variables. Data were presented as the mean ± SD. Statistical significance was defined as p < 0.05.

Results and discussion
Structural characterization of these isolated compounds Chlorophenol A (1) was obtained as a colorless oil and had the molecular formula C 11 H 13 ClO 3 as determined by HRESIMS spectrum, which showed a cluster of deprotonated ion peaks at m/z 227.0476/229.0422 ([M − H] − ) with a ratio of 3:1, indicative of a monochlorinated compound (Luo et al., 2018). The 1D NMR (Table 1) and HSQC spectrum of 1 showed signals of five non-protonated sp 2 carbons (δ C 150.0, 150.0, 143.1, 129.8, and 113.0), one aromatic methine (δ H/C 6.83/106.9), two olefinic methines (δ H/C 6.53/124.3 and 6.19/127.3), two oxygenated methyls (δ H/C 3.80/60.5 and 3.68/61.1), one methyl (δ H/C 1.88/18.7), and one phenolic hydroxyl group (δ H 9.97). The aforementioned data combined with five degrees of unsaturation (DOU) suggested that compound 2 presented a benzene ring skeleton, and the 1 H-1 H COSY correlations (Figure 2) between H-7/H-8 and H-8/H 3 -9 showed the existence of a propenyl group. Besides, the HMBC correlations ( Figure 2) from H-7 to C-1, C-2, and C-6, and from H 3 -11 to C-6, revealed that the propenyl group Frontiers in Microbiology 06 frontiersin.org and one methoxy group were located at C-1 and C-6, respectively. The HMBC correlations from 3-OH to C-2, C-3, and C-4 indicated the location of the phenolic hydroxyl group (C-3). The chemical shift of C-4 (δ C 113.0) revealed that the chlorine substituent instead of the oxygenated methyl was attached at C-4. The other methoxy group was deduced to link with C-5 by the HMBC signal of H 3 -10/C-5 and the chemical shift of C-5 (δ C 150.0). The aforementioned NMR characteristics showed great similarity to those of the co-isolated 2,4-dichloro-3-hydroxy-5-methoxy-toluene (5), which was reported as a synthetic chlorinated phenolic compound (Calam and Oxford, 1939). The main differences were the appearances of a propenyl group at C-1, a methoxy group at C-6, an aromatic hydrogen at C-2 in 1 instead of a methyl at C-1, an aromatic hydrogen at C-6, and a chlorine atom at C-2 in 5, respectively. This deduction was further supported by the above 2D NMR data. Therefore, the structure of 1 was determined, as shown in Figure 1. Chlorophenol B (2) was obtained as a colorless oil, and its molecular formula was deduced to be C 11 H 12 Cl 2 O 3 by the HRESIMS peak at m/z 261.0092 [M − H] − (calcd. For C 11 H 11 Cl 2 O 3 − 261.0091), suggesting five DOU. The 9:6:1 (m/z 261.0092/263.0065/265.0033) isotopic distribution pattern suggested the presence of two chlorine atoms (El-Kashef et al., 2020). The 1 H, 13 C (Table 1) NMR, and HSQC spectra of 2 showed signals of six non-protonated sp 2 carbons (δ C 146.1, 144.5, 143.3, 122.8, 122.4, and 120.0), two olefinic methines (δ H/C 6.42/123.1 and 6.58/133.5), two methoxy groups (δ H/C 3.73/60.4 and 3.72/60.7), one methyl (δ H/C 1.89/19.6), and one phenolic hydroxyl group (δ H 9.63). The above NMR data were highly similar to those of compound 1. The main differences were the appearances of a chlorine atom at C-2, a methoxy group at C-3, and a hydroxyl group at C-6 in 2 instead of an aromatic hydrogen at C-2, a hydroxyl group at C-3, and a methoxy group at C-6 in 1, respectively. This deduction was further supported by the 2D NMR data. The HMBC spectrum of 2 showed correlations from H-7 to C-1, C-2, and C-6, and from 6-OH to C-1 and C-6, which implied that the propenyl and the hydroxyl groups were located at C-1 and C-6, respectively. Besides, the HMBC correlations from 6-OH and H 3 -10 (δ H 3.73) to C-5 (δ C 143.3) suggested that the methoxy group (δ H/C 3.73/60.4) was located at C-5. The chemical shift of C-2 (δ C 122.4) indicated one of the chlorine atoms was attached to C-2. Besides, the HMBC correlation from H 3 -11 (δ H 3.72) to C-3 (δ C 144.5) verified that the remained one methoxy group (δ H/C 3.72/60.4) was located at C-3. The above-detailed analysis of the NMR data allowed the assignment of all carbon and proton resonances of 2. Consequently, the structure of 2 was elucidated, as shown in Figure 1.
Frontiers in Microbiology 09 frontiersin.org Compound 13 protected RAW264.7 macrophages against LPS-induced inflammation through PI3K/AKT pathway To further explore the mechanism of compound 13 in ameliorating LPS-induced inflammation, high-throughput sequencing of RNAs from RAW264.7 macrophages was conducted. The results of the GO enrichment analysis indicated that chemokine activity and nitric oxide synthase regulatory activity may play key roles in inflammation induced by LPS ( Figure 6A and Supplementary Figure 27). Furthermore, combining the results of the KEGG pathway analysis uncovered that the PI3K/AKT signaling pathway was significantly activated, indicating that it might be a potential key pathway in mediating inflammatory responses in the current cell model ( Figure 6B). Consistent with this hypothesis, our subsequent research displayed that compound 13 could effectively restrain the expression levels of PI3K and AKT upregulated by LPS ( Figures 6C,D), proving that the PI3K/AKT signaling pathway was involved in this procedure regulated by compound 13, which is in accordance with another research (Zhong et al., 2016). In this study, we found that compounds 4, 9, 13, and 15 could effectively inhibit the synthesis of NO and overproduction of pro-inflammatory cytokines induced by LPS. Especially, compound 13 also exhibited an outstanding anti-inflammatory activity in vivo, suggesting the most active compound.
Mitochondria are key organelles involved in metabolic regulation, and their dysfunction is closely related to metabolic inflammatory diseases. Although the gene expression heat map showed that genes related to mitochondrial function and transcription factors were largely enriched (Supplementary Figure 27), whether they participated in LPS-induced inflammatory response of compound 13 still needs further investigation.

Conclusion
In summary, the chemical investigation of the mangrove endophytic fungus Amorosia sp. SCSIO 41026 resulted in the isolation and identification of 19 secondary metabolites, including three new chlorine-containing fungal metabolites and two new natural products. Our results demonstrated that  compounds 1, 4, 7, 9, 13, 15, 16, and 19 inhibited LPS-induced overproductions of NO and pro-inflammatory cytokines including IL-6, TNF-α, and MCP-1 both in mRNA and protein levels with a premise that none of these compounds showed significant cytotoxicity. Among these metabolites, compound 13 was identified as the most active compound with an outstanding ability of anti-LPS-induced inflammation in RAW264.7 macrophages and in ALI mice, probably by inhibiting the PI3K/AKT signaling pathway. Thus, this study demonstrates the potential value of mangrove endophytic fungus as a promising source of bioactive compounds. Compound 13 weakens the pathological lung injury of LPS-challenged mice. BALB/c mice were intragastric injected with compound 13 for 24 h before the LPS administration. Mice were killed after 6 h of LPS injection (5 mg/kg), and lung histopathology was performed with H&E staining (A). Inflammation score was measured independently by three pathologists blinded to the experiment (B, n = 6). The levels of anti-inflammation cytokines in BALF were assayed (C). ****p < 0.001 vs. control group, n = 6. p value was assessed by two-tailed Student's t-test.
Frontiers in Microbiology 10 frontiersin.org PI3K/AKT signaling pathway was involved in the regulation of LPS-induced inflammation by compound 13 in RAW264.7 macrophages.

Ethics statement
The animal study was reviewed and approved by the Medical Ethics Committee of Peking Union Medical College. Written informed consent was obtained from the owners for the participation of their animals in this study.

Author contributions
XR and CC performed the experiments and wrote the paper. YY, ZX, and XL helped with the analysis of the data. PG, XL, and YL designed and supervised the experiments. All authors contributed to the article and approved the submitted version.