Chemical Constituents of the Mushroom Dictyophora indusiata and Their Anti-Inflammatory Activities

As an edible and medicinal fungus, Dictyophora indusiata is well-known for its morphological elegance, distinctive taste, high nutritional value, and therapeutic properties. In this study, eighteen compounds (1–18) were isolated and identified from the ethanolic extract of D. indusiata; four (1–4) were previously undescribed. Their molecular structures and absolute configurations were determined via a comprehensive analysis of spectroscopic data (1D/2D NMR, HRESIMS, ECD, and XRD). Seven isolated compounds were examined for their anti-inflammatory activities using an in vitro model of lipopolysaccharide (LPS)-simulated BV-2 microglial cells. Compound 3 displayed the strongest inhibitory effect on tumor necrosis factor-α (TNF-α) expression, with an IC50 value of 11.9 μM. Compound 16 exhibited the highest inhibitory activity on interleukin-6 (IL-6) production, with an IC50 value of 13.53 μM. Compound 17 showed the most potent anti-inflammatory capacity by inhibiting the LPS-induced generation of nitric oxide (NO) (IC50: 10.86 μM) and interleukin-1β (IL-1β) (IC50: 23.9 μM) and by significantly suppressing induced nitric oxide synthase (iNOS) and phosphorylated nuclear factor-kappa B inhibitor-α (p-IκB-α) expression at concentrations of 5 μM and 20 μM, respectively (p < 0.01). The modes of interactions between the isolated compounds and the target inflammation-related proteins were investigated in a preliminary molecular docking study. These results provided insight into the chemodiversity and potential anti-inflammatory activities of metabolites with small molecular weights in the mushroom D. indusiata.


Introduction
Dictyophora indusiata, belonging to the family Phallaceae (phylum Basidiomycetes) of the Agaricomycetes class of fungi, is famous for its beautiful morphological features. Due to its delicious taste, distinctive fragrance, and medicinal properties, D. indusiata has a long history as a healthy food and as a component of folk medicine [1]. Traditional Chinese medicine has widely recognized that D. indusiata benefits some chronic diseases by alleviating inflammatory symptoms [2,3]. It is reasonable to suppose the existence of metabolites in D. indusiata that offer therapeutic potential.
Most studies on the molecular basis of the nutritional and medicinal values of D. indusiata have focused on fungal biomacromolecules, such as carbohydrates and proteins. Polysaccharides are found in the highest concentrations in the mature fruiting body of D. indusiata and, therefore, are the most intensively studied chemical constituents of D. indusiata [4].
It has been proven that D. indusiata polysaccharides (DIPs) have effective anti-tumor, anti-oxidative, anti-inflammatory, immune-enhancing, and anti-diabetes properties [5]. Compared to the polysaccharides, the bioactive compounds with small molecular weights in D. indusiata have not been sufficiently investigated [1]. From this mushroom species, three eudesmane-type sesquiterpenes and five monoterpene alcohols were reported to be identified by Kawagishi et al. [6,7]; albaflavenone and two derivatives (9,10-dihydroxyalbaflavenone and 5-hydroxy-albaflavenone) were isolated by Huang et al. and Zhang et al., respectively [8,9]; two linear sesquiterpene carboxylic acids (Phallac acids A and B) were obtained and determined by Lee et al. [10]. In addition to the terpenoid compounds, Lee et al. identified three alkaloids, Dictyoquinazols A, B, and C, from D. indusiata [11]; and Sharma et al. reported the isolation of 5-hydroxymethyl-2-furfural (HMF) [12]. The biological investigations revealed that some of these small molecules isolated from D. indusiata possess neuroprotective, anti-tyrosinase, anti-glucosidase, and some anti-inflammatory activities [6,[9][10][11][12]. However, the role of small-molecular-weight metabolites in the antiinflammatory property of the mushroom D. indusiata has yet to be fully elucidated.
Inflammation is a natural response of the immune system to damage from physical, chemical, and pathogenic factors. The activation of the nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB) pathway plays a central role in acute and chronic inflammation [13]. Abnormally high NF-κB activity is highly associated with aberrant expressions of a series of inflammatory cytokines and other mediators, and it is further associated with many diseases, such as cancer, diabetes, Alzheimer's disease, and cardiac diseases. It has been revealed that the efficient strategy for alleviating inflammatory symptoms involves either reducing the expression of inflammatory cytokines via mediation of the related signaling pathways or by inhibiting their actions using antibodies. In the clinical therapies used to cure inflammatory disease, several cytokine inhibitors, such as Etanercept, Adalimumab, and Infliximab, have been successfully applied. However, the high costs and the side effects of adverse immunological reactions limit their applications [14]. Hence, the discovery of safe and efficacious anti-inflammatory agents is still in urgent demand.
Edible and medicinal fungi are currently seen as important sources of active pharmaceutical ingredients, which can serve as lead molecules in the development of novel drugs. In this context, numerous types of bioactive natural products have been isolated from various fungi [15][16][17]. In the literature, it is abundantly clear that the anti-inflammatory effect of D. indusiata polysaccharides has long been recognized [5,[18][19][20]. However, the potential anti-inflammatory capacity and underlying mechanism of other ingredients in D. indusiata are still unclear. Herein, we report on the systematical isolation and identification of the chemical constituents in the ethanolic extract of D. indusiata. The inhibitory effects of the isolated compounds against lipopolysaccharide (LPS)-induced inflammatory responses in mouse BV-2 microglial cells were evaluated, and their anti-inflammatory mechanisms of action were investigated using molecular docking simulations. The results of this study might contribute to the knowledge of the chemistry of D. indusiata and the discovery of fungal natural products possessing potent anti-inflammatory efficiency with reliable safety profiles.
Compound 4 was purified as a yellowish oil, and its molecular formula, C 13

Biological Activity
Numerous fungal metabolites have been reported to have anti-inflammatory activities [34,35]. According to the structural information of these reported compounds and based on our research interests, compounds 1, 3, 4, 11, 15, 16, and 17 were selected for further biological evaluations.

Effects of the Compounds on Cell Viability
The cytotoxicity of compounds 1, 3, 4, 11, 15, 16, and 17 on BV-2 cells was evaluated using a thiazolyl blue tetrazolium bromide (MTT) assay [36]. As shown in Figure 5, after 24 h of exposure, the tested compounds at the examined concentrations did not induce significant cytotoxicity in the BV-2 cells (p > 0.05); therefore, subsequent in vitro anti-inflammatory experiments were performed with these concentrations.

Effects of the Compounds on NO Production in LPS-Stimulated BV-2 Cells
During inflammation, the induced nitric oxide synthase (iNOS) would be activated and produce large amounts of nitric oxide (NO) [37]. The inhibitory effects of the selected compounds on NO production in LPS-induced microglia BV-2 cells were evaluated by measuring the nitrite content accumulated in the culture medium based on the Griess reaction [38]. Among the tested compounds (Table 2), compound 17 exhibited the most potent inhibitory effect against LPS-induced NO production in the BV-2 cells, with an IC 50  ties [34,35]. According to the structural information of these reported compounds and based on our research interests, compounds 1, 3, 4, 11, 15, 16, and 17 were selected for further biological evaluations.

Effects of the Compounds on Cell Viability
The cytotoxicity of compounds 1, 3, 4, 11, 15, 16, and 17 on BV-2 cells was evaluated using a thiazolyl blue tetrazolium bromide (MTT) assay [36]. As shown in Figure 5, after 24 h of exposure, the tested compounds at the examined concentrations did not induce significant cytotoxicity in the BV-2 cells (p > 0.05); therefore, subsequent in vitro anti-inflammatory experiments were performed with these concentrations.

Effects of the Compounds on NO Production in LPS-Stimulated BV-2 Cells
During inflammation, the induced nitric oxide synthase (iNOS) would be activated and produce large amounts of nitric oxide (NO) [37]. The inhibitory effects of the selected compounds on NO production in LPS-induced microglia BV-2 cells were evaluated by measuring the nitrite content accumulated in the culture medium based on the Griess reaction [38]. Among the tested compounds (Table 2), compound 17 exhibited the most potent inhibitory effect against LPS-induced NO production in the BV-2 cells, with an IC50 of 10.86 μM, followed by another sterol compound, compound 15 (IC50: 42.41 μM). Compound 16 was the epimer of compound 15; however, its IC50 value was 66.12 μM, indicating a comparatively weak inhibitory capacity. The IC50 values of three tested previously undescribed compounds, compounds 1, 3, and 4, were 46.30 μM, 89.12 μM, and 62.15 μM, respectively. Moreover, compound 11 possessed no NO production inhibitory activity at the tested concentrations.  To examine the inhibitory effects of the selected compounds on the LPS-induced production of pro-inflammatory cytokines, we investigated the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in LPS-treated BV-2 cells using ELISA. As shown in Table 2, the treatments with compounds 1, 3, 11, and 15 exhibited inhibitions of TNF-α production. Among them, compound 3 had the strongest inhibitory activity (IC 50 : 11.9 µM). The expression of LPS-induced IL-1β could be only inhibited by compound 17, with an IC 50 value of 23.9 µM. Regarding the LPS-induced generation of IL-6, the inhibitory effects of compounds 1, 3, 11, 15, and 16 were observed, and compound 16 displayed the lowest IC 50 value of 13.53 µM.

Effects of the Compounds on Expressions of iNOS and p-IκB-α in LPS-Stimulated BV-2 Cells
A Western blot analysis was employed to further determine the inhibitory effects of the selected compounds on two key enzymes involved in the inflammatory response: iNOS and phosphorylated nuclear factor-kappa B inhibitor-α (p-IκB-α) [39]. Figure 6A,B show that compounds 15 and 17 significantly reduced iNOS expression at concentrations of 20 µM (p < 0.05) and 5 µM (p < 0.01), respectively. This finding is consistent with the results of the previous assay, where compounds 15 and 17 possessed the highest NO production inhibition activity. As shown in Figure 6C, compound 17 significantly inhibited p-IκB-α expression (p < 0.01). The treatments with the other tested compounds exhibited no or weak effects on the levels of iNOS and p-IκB-α, with no statistical differences ( Figure S2).
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Molecular Docking Simulation
A molecular docking study was conducted based on the results obtained from the previous in vitro experiments to further explore the anti-inflammatory mechanisms of the selected compounds.
As compounds 15 and 17 exhibited good inhibitory activity on the LPS-induced gen-

Molecular Docking Simulation
A molecular docking study was conducted based on the results obtained from the previous in vitro experiments to further explore the anti-inflammatory mechanisms of the selected compounds.
As compounds 15 and 17 exhibited good inhibitory activity on the LPS-induced generation of NO and the expression of iNOS in the BV-2 cells, a molecular docking study was performed to clarify their modes of interactions with iNOS. As shown in Figure 7A,B, the binding of compounds 15 and 17 with iNOS was achieved by stacking the compounds over the active region of haem and forming hydrogen bonds with the iNOS protein backbone [40]; both compounds fitted well within this pocket, with compound 15 forming three hydrogen bonds with Gln257, Pro334, and Gly365 (binding energy: −8.21 kcal/mol) and compound 17 establishing three hydrogen bonds with Glu257, Gly365, and Tyr367 (binding energy: −7.29 kcal/mol). The compounds shown to have strong bio-activities in the ELISA tests were subjected to molecular docking studies with the corresponding pro-inflammatory cytokines. TNFα has three identical receptor binding sites, as the protein possesses a unique threefold symmetry structure [41]. Compound 3 docked flexibly into the hydrophobic pocket located in the trimer core with a binding energy of −8.53 kcal/mol ( Figure 7C). The three main binding interactions were the hydrogen bond established between the hydroxyl groups at C-14 with Tyr118 residues on chain A, the hydrogen bond established between the hydroxyl groups at C-12 with Tyr118 residues on chain B, and Pi-Pi stacking from the The compounds shown to have strong bio-activities in the ELISA tests were subjected to molecular docking studies with the corresponding pro-inflammatory cytokines. TNFα has three identical receptor binding sites, as the protein possesses a unique threefold symmetry structure [41]. Compound 3 docked flexibly into the hydrophobic pocket located in the trimer core with a binding energy of −8.53 kcal/mol ( Figure 7C). The three main binding interactions were the hydrogen bond established between the hydroxyl groups at C-14 with Tyr118 residues on chain A, the hydrogen bond established between the hydroxyl groups at C-12 with Tyr118 residues on chain B, and Pi-Pi stacking from the 4-pyridone ring with Tyr59 residues on chain C. Visible conformational changes in TNF-α occurred after the docking of compound 3, the helices on chains A and B disappeared, and a large number of loop structures newly formed ( Figure 7C ). Therefore, it was speculated that compound 3 inhibited TNF-α function by changing the protein structure and disrupting the receptor binding sites. Compound 16 bounded to two loop structures of IL-6 ( Figure 7D) by forming three hydrogen bonds with Arg74, Leu89, and Gln85 (binding energy: −8.39 kcal/mol). Compound 17 matched well in the protein-binding pocket with IL-1β ( Figure 7E); it formed four hydrogen bonds with Glu50, Pro51, Lys97, and Asn102 of IL-1β with a binding energy of −4.40 kcal/mol.
The IκB-α/NF-κB complex used for this molecular docking study is composed of three subunits [42]: NF-κB-P65 (chain A), NF-κB-P50 (chain C), and IκB-α (chain D) ( Figure 7F). Compound 17 could be bound to the lower part of the central cavity of the complex and interacted with NF-κB-P65 (chain A) and IκB-α (chain D) (binding energy: −4.49 kcal/mol). The primary binding interactions were the hydrogen bonds formed between the hydroxyl group with Arg253 residues on chain A and Glu213 on chain D, the hydrogen bond established between the epoxy group and Arg201 on chain A, and the hydrogen bond formed between the carbonyl oxygen and Asn182 on chain D.

Diversity of Metabolites with Small Molecular Weights in D. indusiata
There are four main stages in the morphological development of the fruit body of D. indusiata: the primordia stage, the ball-shaped stage, the peach-shaped stage, and the mature stage. An integrated quantitative proteomic and metabolomic analysis has revealed that [43], compared to the three early stages, more metabolites with <15 carbon atoms in the mature fruiting body of D. indusiata are upregulated, and for metabolites with ≥15 carbon atoms, more metabolites are downregulated. This phenomenon suggests that, in the final growth stage, large amounts of natural products with comparatively small molecular weights are preferentially biosynthesized and accumulated and that these chemical constituents may be crucial contributors to the taste, fragrance, and nutritional and medicinal properties of the mature fruiting body of D. indusiata. Our result is consistent with that reported above. In this research, eighteen small-molecule compounds were isolated from the ethanolic extract of D. indusiata, of which the number of core skeleton carbon atoms in 11 isolated compounds were less than 15.
Among the four compounds newly described in this manuscript, three were terpenoids. Terpenoids are one of the largest groups of secondary metabolites found in nature. Edible fungi are considered prolific producers of structurally diverse terpenoid compounds, as these fungi possess an extensive repertoire of natural product biosynthesis pathways [44,45]. Compound 1 was a tricyclic sesquiterpenoid with a skeleton of antsorane [46,47], compound 2 was a rearranged cuparane-type sesquiterpenoid [48], and compound 4 was an acyclic norsesquiterpenoid [49,50]. The antsorane skeleton contained in compound 1 was quite unusual. As a natural product, antsorenone (synonym antsorane) was first discovered by Chazan in 1969 from the plant Elionurus tristis [46]. Several analogs were successively identified from Eremophila fraseri [51], Vetiveria nigritana [52], and Chrysopogon zizanioides [53]. The structure of antsorane was fully characterized by Gabriel P. Garcia et al. in 2019 [47]. To the best of our knowledge, compound 1 is the second natural compound ever reported with this specific sesquiterpenic ketone skeleton and the first one isolated from fungi. In Garcia et al.'s article, the high stability of the antsorane molecule was found, as it was subjected to several Brönsted and Lewis acid treatments and all the attempts to transform it into zizaen-3-one following a reverse Wagner-Meerwein rearrangement under acidic conditions failed. Accordingly, Garcia et al. hypothesized that the antsorane skeleton was the final step of the biosynthesis pathway that started with acorane. In terms of the molecular structure of our compound 1, a hydroxyl group was present on the bridged ring of the antsorane skeleton; its formation pathway and its biological role in the ecology and physiology of D. indusiata deserve further attention and investigation.
The structural diversity of fungal terpenoids contributes to functional diversity. Terpenoids are widely involved in every aspect of fungi growth, development, and stress responses [45]. Fungal terpenoids play biological signaling and defensive roles, as they often possess a volatile profile and a unique aromatic property [54]; they can mediate many ecological interactions between fungi and other organisms. In this research, we found that compound 1 had a considerable sour and smelly odor, and the newly described compound 3 had an aroma of wood mixed with caramel. Together with the known odorous compounds, such as nicotinic acid (6) and oxindole (10), these small-molecular-weight compounds are vital contributors to the distinctive fragrance of D. indusiata.
According to the classes of the enzyme involved in their biosynthesis, the major groups of fungal secondary metabolites can be classified as polyketides, non-ribosomal peptides, terpenes, and alkaloids [55]; they can serve as metabolic precursors for a wide range of other metabolites. Only a relatively small number of higher fungal species have been chemically investigated; fungal natural products represent a vast and largely untapped source of potentially potent new pharmaceuticals [56,57]. Among fungal species, the basidiomycetes are one of the most important producers of diverse bioactive secondary metabolites [58,59]. Ganoderma lucidum and Poria cocos are two well-known medicinal basidiomycetes. Based on the phytochemical reports, more than 270 secondary metabolites have been reported to be isolated from Ganoderma lucidum [60][61][62]; more than 140 terpenoids and more than 20 steroids have been identified in Poria cocos [63,64]. As reviewed by Habtemariam [1] and described in the Section 1, the small-molecule compounds identified from D. indusiata mainly include a few terpenoids and alkaloids. The chemodiversity of this edible and medicinal mushroom species has not been adequately characterized. In this study, various terpenoids, steroids, lactones, fatty acids, and heterocyclic compounds were identified. The present findings reveal that abundant small-molecular-weight metabolites with highly complicated and diverse structures exist in the mushroom D. indusiata. These small molecules may form the basis for the further exploitation of D. indusiata as a source of functional ingredients for the food, cosmetic, and pharmaceutical industries.

Ergostane-Type Steroids Isolated from D. indusiata and Their Anti-Inflammatory Activities
The scientific literature on the biological activities of small-molecule metabolites in D. indusiata remains very scarce. Only two derivatives of albaflavenone isolated from D. indusiata were evaluated for their anti-inflammatory activities; these two compounds showed moderate inhibitory effects on LPS-induced NO production and TNF-α secretion in BV-2 cells [9]. In this research, three tested ergostane-type steroids (15, 16, and 17) isolated from D. indusiata exhibited good anti-inflammatory activities. Ergosterol is the primary sterol in fungal membranes and presumably contributes to membrane fluidity and function [65]. Ergosterol and its related derivatives are metabolites essential for fungal growth, development, and adaptation to various stresses [66]. Ergosterol biosynthesis can be divided into three modules: mevalonate, farnesyl pyrophosphate (farnesyl-PP), and ergosterol biosynthesis [67]. Ergosterol and some of its biosynthetic intermediates are critical natural products of great economic value. In the pharmaceutical industry, ergosterol is a direct precursor of vitamin D2 and steroid drugs [68].
Pharmaceutical steroids, non-steroidal anti-inflammatory drugs (NSAIDs), and antihistamines have been widely applied in anti-inflammatory therapies [69]. In some cases, the marketed anti-inflammatory drugs may not deliver a radical cure and may cause side effects [70,71]. Therefore, the search for novel and safe anti-inflammatory agents or lead compounds in nature has become the focus of biologists and chemists. The extensive efforts devoted to searching for promising ergostane-type steroid compounds with high anti-inflammatory activities in fungi and to elucidating their mechanisms of action have recently been well-reviewed [34,35,66]. In the present study, the best anti-inflammatory activities (reflected by the smallest IC 50 values) were exerted by compound 15 against TNFα production (IC 50 = 65.5 µM), compound 16 against IL-6 production (IC 50 = 13.53 µM), and compound 17 against NO and IL-1β production (IC 50 = 10.86 µM and 23.9 µM, respectively). Moreover, the evident suppression of iNOS and p-IκB-α expressions by compound 17 was observed. Since the experimental models vary among the related research, it is not easy to appropriately compare our results to the data reported in the literature.
Nevertheless, comparing the data obtained within our in vitro anti-inflammatory assays and in silico analysis may provide new insights into the structure-activity relationship of isolated sterols. Compounds 15 and 16 were two ergosterol derivatives with a rearranged tetracyclic skeleton [72]; they were epimers, and the stereo configuration of their methoxy at C-11 was on the same face and on the opposite face to the hydroxyl group on their molecules, respectively ( Figure 1). Compound 15 showed higher inhibitory activity on LPS-induced NO production than compound 16. From the visualized results of molecular docking, the hydroxyl group of compound 15 formed a hydrogen bond with Gly365 of iNOS. In addition, the methoxy group, which was on the same face as the hydroxyl group, formed another hydrogen bond with Gln257. Notably, compound 17, the most potent NO production inhibitor found in this research, also established hydrogen bonds with the amino acid residues of Gly365 and Gln257 of iNOS. It can be deduced that the methoxy and the hydroxyl groups located on the same face of the molecule conferred compound 15 the capacity to interact with Gly365 and Gln257 simultaneously, which was crucial for its inhibitory effect against iNOS. In contrast, compound 16 showed the strongest inhibitory activity against IL-6 production among the tested compounds. The structure of IL-6 contained four relatively long α-helices (A-D) and two short helices (S1 and S2) linked via loop structures [73]. The docking result demonstrated that the binding site of compound 16 was situated in the loop region; the hydroxyl group of compound 16 interacted with Arg74 on the loop structure connected to the short helix S1; and the methoxy group located on the opposite face of the molecule could establish a hydrogen bond with Leu89 on the loop structure attached to the long helix B, with this fulfilling the firm binding and inhibition of IL-6.
Compound 17 is an epoxy-7-sitosterol, and the epoxy and carbonyl groups appeared to be the key functional elements. In the molecular docking analysis, hydrogen bonds formed by the epoxy group and carbonyl group of compound 17 with the amino acid residues of IL-1β were observed. At the same time, the ergosterol derivatives without these two substituents (compounds 15 and 16) exhibited no activity against IL-1β; these two substituents of compound 17 also played essential roles in combination with the IκBα/NF-κB complex, as they could establish hydrogen bonds with the NF-κB-P65 (chain A) and IκB-α (chain D), respectively, to form an IκB-α/NF-κB-P65/inhibitor (compound 17) complex. This increased the binding affinity of IκB-α and NF-κB-P65, suggesting that the external factor-stimulated phosphorylation and degradation of IκB-α could subsequently be inhibited to certain degrees. The release of NF-κB-P65 from IκB-α; and the following relocation of NF-κB-P65 from the cytoplasm to the nucleus were suppressed, implying that the activation of NF-κB would be prevented [39]. This inference can be supported by the present data from Western blots. Thus, we can conclude that compound 17 exerts an antiinflammatory effect by affecting the NF-κB signaling pathway. This research was mainly carried out by performing experiments on in vitro cell models and computer simulations. The results cannot fully elucidate the tested compounds' actual effects and mechanisms of action. In vivo studies and clinical trials are needed to confirm these findings.

Fungi Material
The mature fruiting bodies of D. indusiata were collected from the Yibin edible fungi cultivation base (28 •

Chemicals and Reagents
The chromatographically pure reagents were purchased from Fisher-Scientific (Pittsburgh, PA, USA), ultrapure water was obtained using a PALL Lab Water Purification System

Cell Culture
The mouse microglial cell line BV-2 was obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 • C in a humidified incubator with an atmosphere of 5% CO 2 .

General Experimental Procedures
The isolation of the compounds was performed by using repeated open-column chromatography (CC), preparative thin-layer chromatography (TLC), and preparative high-performance liquid chromatography (HPLC). The chemical structure of the isolated compounds was determined by the spectra of analytical HPLC, UV, 1 H and 13 C-NMR, HRESIMS, CD, and XRD. Instrumental details were described in Supplementary Materials (Table S1).
Crystal data: C 15 Table 1. CD spectrum (MeOH) is shown in Figure 3.  Figure 3. Assay   Compounds 1, 3, 4, 11, 15, 16, and 17 were selected for in vitro investigations of antiinflammatory activities using an LPS-stimulated BV-2 microglial cell model. Three groups were designed for the experiments: the normal cell group, where the cells were cultured under normal growth conditions; the LPS group, where the cells were treated with LPS but without the tested compounds; and the sample group, where the cells were treated with LPS plus the tested compounds.

MTT Assay for the Measurement of Cell Viability
The cytotoxic effects of the selected compounds on the BV-2 cells were evaluated using an MTT assay [38]. Briefly, BV-2 cells were seeded into 96-well plates at a density of 8 × 10 3 cells/well and cultured overnight in an incubator at 37 • C with 5% CO 2 . Then, the cells were treated with different concentrations of the selected compounds diluted with DMEM. After 2 h, LPS (1 µg/mL) was added. After 24 h of incubation, 20 µM MTT was added to each well and the cells were incubated at 37 • C for 4 h. Afterward, the supernatant was discarded, and 150 µL of DMSO was added to each well to dissolve the formazan. The absorbance (optical density, OD) of each well was measured at 490 nm using a multilabel plate reader (Victor 2030, PerkinElmer, Waltham, MA, USA). The cell viability results were calculated as percentages.

Measurement of Nitric Oxide and Cytokine Production
The tested compounds were diluted in a gradient with DMEM. The BV-2 cells were seeded into 96-well plates at a density of 8 × 10 3 cells/well, cultured overnight in an incubator at 37 • C with 5% CO 2 , and then cultured with LPS (1 µg/mL) in the absence or presence of the tested compounds for 24 h. The culture supernatants were collected for the measurements. The total nitric oxide level was measured using the NO assay kit. The TNF-α, IL-1β, and IL-6 concentrations were quantified using mouse ELISA kits. All operations were conducted according to the manufacturer's instructions.

Western Blot Analysis
The protein expressions of iNOS and p-IκB-α were measured using a Western blot analysis [74]. The tested-compound-treated BV-2 cells in the logarithmic growth phase were digested in a single-cell suspension with trypsin and seeded into 6-well plates at a density of 3 × 10 5 cells/well. Then, the cells were washed twice with 4 • C phosphate-buffered saline (PBS) and harvested. Next, 200 µL of radioimmunoprecipitation assay (RIPA) buffer was added to each well, and the lysate was centrifuged at 12,000× g for 20 min after incubation at 4 • C for 30 min. The protein concentration was determined using a bicinchoninic acid (BCA) assay. The protein was collected for Western blot analyses of iNOS and p-IκB-α. After being resolved using SDS/PAGE loading buffer, the protein was electrophoretically separated at 120 V for 90 min and transferred to polyvinylidence difluoride (PVD) membranes at 300 mA for 85 min. Then, the PVD membranes were blocked with 5% skim milk in phosphatebuffered saline containing 0.05% Tween-20 (PBST) for 120 min at room temperature. The cells were incubated overnight at 4 • C in diluted primary antibodies (anti-iNOS and anti-p-IκB-α). The membrane was washed with PBST three times and incubated in a (horseradish peroxidase) HRP-conjugated secondary antibody solution for 120 min at room temperature. Finally, an appropriate amount of enhanced chemiluminescence reagent was added to detect the protein. The data for iNOS and p-IκB-α were normalized on the basis of GAPDH levels.

Molecular Docking
The protein structures of TNF-α (PDB ID: 7KP8), IL-1β (PDB ID: 8I1B), iNOS (PDB ID: 4UX6), and IL-6 (PDB ID: 2L3Y) were downloaded from the Protein Data Bank (http: //www.rcsb.org/, accessed on 1 February 2023). The structure of the IκB-α/NF-κB complex (PDB ID: 1IKN) was modeled using SwissModel (http://swissmodel.expasy.org/, accessed on 1 February 2023) to remove the repeats of subunits from the heterodimer [42]. Each structure was prepared and refined using the Protein Preparation Wizard of Maestro 11.9 (Schrodinger LLC, New York, NY, USA). Hydrogens were added, water molecules were removed, energy was optimized, and the force-field parameters were adjusted to make them a low-energy conformation satisfying the ligand structure [75]. The amino acids were modified with a flexible setting, and the polar hydrogen atoms of the specific amino acids in the binding pocket were allowed to rotate during the optimization of the docking poses. The 3D structures of compounds 3, 15, 16, and 17 were created and optimized to lower-energy conformers with the Maestro Build Panel, and they were used as the ligands for the molecular docking simulations. The glide ligand docking and induced-fit docking modules in Maestro 11.9 software were employed for the docking study and for the analysis of the results [76,77]. The docking results were visualized using USCF ChimeraX (https://www.rbvi.ucsf.edu/chimerax, accessed on 1 February 2023).

Statistical Analysis
Each experiment was repeated three times, with three replicates within each experiment. The data are expressed as mean ± standard deviations (SD). IBM SPSS software (version 23.0, SPSS Inc. Chicago, IL, USA) was used to generate graphs and perform statistical analyses. Data were analyzed using a Student's t-test and one-way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.

Conclusions
A chemical investigation of the ethyl acetate fraction of D. indusiata ethanolic extract resulted in the isolation and identification of 18 compounds (1-18), including three previously undescribed sesquiterpenoids (1, 2, and 4) and one previously undescribed quinolone derivate (3). Their structures and absolute configurations were elucidated using spectroscopic methods and chemical analyses. Among them, compound 1 was the first natural product identified from fungi with an unusual molecular skeleton of antsorane. Seven isolated compounds were evaluated for anti-inflammatory activities in an LPS-stimulated BV-2 microglial cell model. Compound 3 had the best inhibitory effect on TNF-α secretion. Compound 16, an ergosterol derivative with a rearranged tetracyclic skeleton, showed the most potent activity on the suppression of IL-6 generation. The 5α,6α-epoxy-7-sitosterol compound 17 exhibited an anti-inflammatory effect by inhibiting the production of NO and IL-1β and the expressions of iNOS and p-IκB-α. The molecular docking study conducted in this research indicated that the tested compounds could exert their anti-inflammatory activities by binding to the hydrophobic pocket of the corresponding protein. These findings suggest that, as D. indusiata is an edible and medicinal mushroom, its anti-inflammatory property would be at least partially contributed by the presence of a series of compounds with small molecular weights as active constituents. The results of this study demonstrate the chemodiversity and biological potential of the small-molecule metabolites in D. indusiata. They provide a foundation for future investigations into the molecular basis of the pharmacological effects of the mushroom Dictyophora indusiata.