Anti-Inflammatory Ergosteroid Derivatives from the Coral-Associated Fungi Penicillium oxalicum HL-44

To obtain the optimal fermentation condition for more abundant secondary metabolites, Potato Dextrose Agar (PDA) medium was chosen for the scale-up fermentation of the fungus Penicillium oxalicum HL-44 associated with the soft coral Sinularia gaweli. The EtOAc extract of the fungi HL-44 was subjected to repeated column chromatography (CC) on silica gel and Sephadex LH-20 and semipreparative RP-HPLC to afford a new ergostane-type sterol ester (1) together with fifteen derivatives (2–16). Their structures were determined with spectroscopic analyses and comparisons with reported data. The anti-inflammatory activity of the tested isolates was assessed by evaluating the expression of pro-inflammatory factors Tnfα and Ifnb1 in Raw264.7 cells stimulated with LPS or DMXAA. Compounds 2, 9, and 14 exhibited significant inhibition of Ifnb1 expression, while compounds 2, 4, and 5 showed strong inhibition of Tnfα expression in LPS-stimulated cells. In DMXAA-stimulated cells, compounds 1, 5, and 7 effectively suppressed Ifnb1 expression, whereas compounds 7, 8, and 11 demonstrated the most potent inhibition of Tnfα expression. These findings suggest that the tested compounds may exert their anti-inflammatory effects by modulating the cGAS-STING pathway. This study provides valuable insight into the chemical diversity of ergosteroid derivatives and their potential as anti-inflammatory agents.


Introduction
Marine organisms are a rich source of steroids with potent anti-inflammatory activity.They perform functions by attenuating the activity of the immune system and suppressing inflammation [1].The ergosteroids are the vast majority of these steroids and construct the main steroid of fungi [2].
Penicillium oxalicum is a frequently isolated fungus exhibiting a wide spectrum of physiological activities that are of relevance in agriculture, biotechnology, food quality assessments, and medicine [3].Previous chemical investigations of P. oxalicum led to the isolation of alkaloids [4,5], polyketides [6,7], meroterpenoids [8,9], and steroids [10,11], exhibiting bioactivities of brine shrimp lethality and anti-Rhizoctonia Solani, anti-neuroinflammatory, antipancreatic tumor, anti-HAB (harmful algal bloom), antiviral, and antibacterial properties.P. oxalicum is known for its ability in biotransformation.The fungi were found to be able to act as hyperproducers of chitin deacetylase for converting chitin to chitosan, transforming protopanaxadiol-type saponins to ginsenoside compound K, and promoting the biotransformation of ethinylestradiol 1 [12,13].
Research conducted in our group rests on the chemical and pharmacological investigation of secondary metabolites from marine invertebrates and associated fungi.Recently, we focused on the molecules having immunomodulatory and neuronal modulatory activities.A drimane meroterpenoid characterized by a thioglycerate moiety [14] and a drimane meroterpenoid with a unique borate ring system [15] were obtained from the fungi Alternaria sp.ZH-15 associated with the soft coral Lobophytum crassum collected from the Dongsha Atoll in the South China Sea.These compounds displayed potential as novel anti-epileptic agents due to their significant inhibitory activities of spontaneous synchronous Ca 2+ oscillations (SCO) and 4-aminopyridine-induced epileptic discharges in the low micromolar concentration range.Sixteen 9,10-secosteroids were isolated from the gorgonian Verrucella umbraculum collected from the Xisha islands in the South China Sea.These compounds exhibited significant suppressive effects on CD4 + T lymphocyte cell differentiation in an in vitro bioassay, representing the first report of 9,10-secosteroids to exhibit immunomodulation activity [16].As part of ongoing screening for bioactive metabolites from China marine sources, a fungus strain of P. oxalicum HL-44 was isolated from the soft coral Sinularia gaweli collected from the Xisha area of the South China Sea.Chemical investigation of this fungi led to the isolation of a new ergostane-type sterol ester (1) and fifteen known derivatives (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).Their structures were determined with extensive spectroscopic analyses and comparisons with reported data.We investigated the anti-inflammatory activities of these isolates in vitro by examining their effects on the expression of proinflammatory cytokines Tnfα and Ifnb1 in Raw264.7 cells stimulated with LPS or DMXAA.In the present study, we describe the isolation, structure elucidation, and anti-inflammatory activities of these compounds.

Results and Discussion
To obtain the optimal fermentation condition, UPLC-MS was employed for the chemical analysis of fungal metabolites produced with strain HL-44 in five candidates of media, including Czapek Dox Agar (CZA) medium, Glucose Peptone Yeast (GPY) medium, PDA medium, Rose Bengal Medium (RBM), and Rice medium.The Total Ion Chromatography (TIC) of the PDA medium showed more abundant metabolites with ion peaks (m/z) in a mass spectrum ranging from 408 to 688.In particular, the ion peak at m/z 663.534 was fairly interesting.Thus, the PDA medium was chosen for the scale-up fermentation of the fungi HL-44.
and Wnt and promoting the expression of apoptosis-related genes such as Bcl-2, Bcl-xL, and Mcl-1.Consequently, this inhibits macrophage proliferation and promotes macrophage apoptosis.Moreover, the inhibitory effect of compound 7 on the viability of Raw264.7 cells at a concentration of 40 µM also suggests its potential to inhibit the proliferation and differentiation of tumor cells.Cell apoptosis in macrophages is intricately associated with macrophage polarization, allowing macrophages to alter their phenotype and carry out diverse functions in response to changes in the microenvironment.The two main polarization states are classically activated macrophages (M1) and alternatively activated macrophages (M2).M1 macrophages exhibit characteristics such as pro-inflammatory mediator production, promotion of cell cytotoxicity, and antimicrobial abilities.Conversely, M2 macrophages possess anti-inflammatory properties, participate in tissue repair, regulate the immune response, and demonstrate anti-inflammatory capabilities.M1 macrophages induce cell death by releasing toxic molecules such as toxins, oxidants, and proteases, thereby augmenting their antimicrobial and antitumor effects.Nonetheless, excessive activation of M1 macrophages can result in tissue damage and inflammatory responses [44].However, excessive activation of M1 macrophages can lead to tissue damage and inflammatory responses.The experimental findings revealed that compound 7 exerted a more pronounced inhibitory effect on the survival of Raw264.7 cells at a concentration of 40 µM compared to other compounds.This suggests that compound 7 may promote the polarization of M1 macrophages while inhibiting the expression of growth-related proteins such as P38, Akt, and Wnt and promoting the expression of apoptosis-related genes such as Bcl-2, Bcl-xL, and Mcl-1.Consequently, this inhibits macrophage proliferation and promotes macrophage apoptosis.Moreover, the inhibitory effect of compound 7 on the viability of Raw264.7 cells at a concentration of 40 µM also suggests its potential to inhibit the proliferation and differentiation of tumor cells.
The expression of inflammatory factors in Raw264.7 cells, such as Tnfα and Ifnb1, which were stimulated by LPS, was suppressed by the administration of compounds 1-16 (Figure 5).The above results demonstrate that the compounds exhibit good inhibitory activity against type I interferon, and the cGAS-STING pathway in the innate immune response is an important mechanism for regulating type I interferon responses [45][46][47].The expression levels of Tnfα and Ifnb1 in the STING pathway have practical importance in immune regulation, inflammatory diseases, and therapeutic interventions, serving as key indicators of immune activation, effectiveness against pathogens, and the extent of inflammation [48,49].Therefore, we further used the specific activator DMXAA of the cGAS-STING pathway to establish an activation model and evaluate the activity of these compounds.Using astin C (20 nM) as a positive control and a natural inhibitor of the STING pathway [50], the results indicate that all compounds significantly inhibit the expression levels of Tnfα and Ifnb1 (Figure 6).In the stimulation of Raw264.7 cells with LPS, compounds 2, 9, and 14 showed significant inhibition of the inflammatory cytokine Ifnb1 expression, while compounds 2, 4, and 5 exhibited strong inhibition of the inflammatory cytokine Tnfα expression.When Raw264.7 cells were stimulated with DMXAA, compounds 1, 5, and 7 effectively suppressed the inflammatory cytokine Ifnb1 expression, whereas compounds 7, 8, and 11 demonstrated the best inhibition of the inflammatory cytokine Tnfα expression.Notably, newly isolated compounds 2 and 11 demonstrated potent anti-inflammatory activity.Hence, it is probable that compounds 1-16 exert their anti-inflammatory effects through the modulation of the cGAS-STING pathway.The above results demonstrate that the compounds exhibit good inhibitory activity against type I interferon, and the cGAS-STING pathway in the innate immune response is an important mechanism for regulating type I interferon responses [45][46][47].The expression levels of Tnfα and Ifnb1 in the STING pathway have practical importance in immune regulation, inflammatory diseases, and therapeutic interventions, serving as key indicators of immune activation, effectiveness against pathogens, and the extent of inflammation [48,49].Therefore, we further used the specific activator DMXAA of the cGAS-STING pathway to establish an activation model and evaluate the activity of these compounds.Using astin C (20 nM) as a positive control and a natural inhibitor of the STING pathway [50], the results indicate that all compounds significantly inhibit the expression levels of Tnfα and Ifnb1 (Figure 6).In the stimulation of Raw264.7 cells with LPS, compounds 2, 9, and 14 showed significant inhibition of the inflammatory cytokine Ifnb1 expression, while compounds 2, 4, and 5 exhibited strong inhibition of the inflammatory cytokine Tnfα expression.When Raw264.7 cells were stimulated with DMXAA, compounds 1, 5, and 7 effectively suppressed the inflammatory cytokine Ifnb1 expression, whereas compounds 7, 8, and 11 demonstrated the best inhibition of the inflammatory cytokine Tnfα expression.Notably, newly isolated compounds 2 and 11 demonstrated potent anti-inflammatory activity.Hence, it is probable that compounds 1-16 exert their anti-inflammatory effects through the modulation of the cGAS-STING pathway.

Fungal Material
P. oxalicum HL-44 was isolated from the soft coral S. gaweli that was collected from the Xisha area of the South China Sea at a depth of 15 m in Aug 2018 and identified as P. oxalicum using 18sRNA sequence (GenBank accession number MG585101.1).A voucher strain of this fungus (internal strain No. HL-44) was deposited at Tongji University, Shanghai, China.

Fungal Material
P. oxalicum HL-44 was isolated from the soft coral S. gaweli that was collected from the Xisha area of the South China Sea at a depth of 15 m in Aug 2018 and identified as P. oxalicum using 18sRNA sequence (GenBank accession number MG585101.1).A voucher strain of this fungus (internal strain No. HL-44) was deposited at Tongji University, Shanghai, China.

Screening Culture Medium and Cultivation
Five media, including Czapek Dox Agar (CZA) medium, Glucose Peptone Yeast (GPY) medium, Potato Dextrose Agar (PDA) medium, Rose Bengal Medium (RBM), and Rice medium, were used in the fermentation to compare the chemical diversity of fungal HL-44.UPLC-MS analysis of five extracts indicated that the PDA medium allowed the strain to produce metabolites with large molecular weights and more chemical diversity (m/z range 1094, 1033, 970, 801 cm −1 ; 1 H and 13 C NMR data see Table 1; HRESIMS m/z 663.53448 [M + H] + (calcd for C 44 H 71 O 4 , 663.53469).

Cell Viability Detection
The viability of Raw264.7 cells was evaluated using the CCK-8 assay.Initially, the cells were seeded at a density of 3 × 10 4 cells/well in 96-well plates and allowed to incubate overnight.The cells were treated with the tested compounds at concentrations ranging from 1.25 to 40 µM for 24 h.After removing the culture medium, a CCK-8 solution diluted in Dulbecco's modified Eagle medium was added to each well.Following a 1-h incubation, the absorbance was measured at 450 nm using a multifunctional microplate spectrophotometer.Cell viability was then calculated as a percentage relative to the blank group [51].
3.6.Quantitative Real-Time PCR (qPCR) Raw264.7 cells were pretreated with tested compounds at a concentration of 20 µM, along with astin C [50] at a concentration of 20 nM, for 2 h.Subsequently, the cells were stimulated with LPS at a concentration of 100 ng/mL and DMXAA at a concentration of 25 µg/mL for 6 h [52].Total RNA was extracted from Raw264.7 cells using a triazole reagent supplied by Thermo Fisher Scientific.cDNA synthesis was performed using SuperScript III reverse transcriptase obtained from Invitrogen.Real-time PCR analysis was conducted using a PrimeScript RT reagent kit from Takara.The relative expression levels of the target genes were quantitatively normalized to the expression level of Gapdh using the ∆∆ct method.Primer sequences are Ifnb1, forward 5 -GCACTGGGTGGAATGAGACT-3 and reverse 5 -AGTGGAGAGCAGTTGAGGACA-3 ; Tnfα, forward 5 -GTCCCCAAAGGGATGAGAAGTT-3 and reverse 5 -GTTTGCTACGA-CGTGGGCTACA-3 [52].

Statistical Analysis
The statistical analyses were conducted using GraphPad Prism 8.0.A one-way analysis of variance (ANOVA) was applied to the data, followed by Tukey's test for comparisons against the blank and control groups.A significance level of p < 0.05 was considered statistically significant.

Conclusions
With chemical analysis of the PDA medium extract of the fungus P. oxalicum HL-44 associated with the soft coral S. gaweli, a new ergostane-type sterol ester (1) was isolated together with fifteen derivatives (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).Their structures were determined using spectroscopic analyses and comparisons with reported data.This is the first report of compounds 2 and 11 from the family of Eurotiaceae.The isolation of ergosteroid derivatives with varying degrees of oxidation revealed a remarkable range of chemical diversity, expanding the ergosteroid family associated with this fungus.In an in vitro biotest, these compounds demonstrated potent anti-inflammatory activities at a concentration of 20 µM, effectively suppressing the expression of inflammatory factors such as Tnfα and Ifnb1 in Raw264.7 cells induced with LPS or DMXAA.Among DMXAA-induced Raw264.7 cells, compounds 7 and 8 exhibited the highest level of inhibition in terms of Tnfα and Ifnb1 expression.Conversely, in LPS-induced Raw264.7 cells, compounds 2 and 4 displayed the most pronounced inhibitory effects on Tnfα and Ifnb1 expression.This study provides valuable insight into the chemical diversity of ergosteroid derivatives and their potential as antiinflammatory agents.

Figure 1 .
Figure 1.Structures of compounds 1-16 isolated from the fungi P. oxalicum HL-44, having a c sponding β-methyl in X or α-methyl in Y.

Figure 1 .
Figure 1.Structures of compounds 1-16 isolated from the fungi P. oxalicum HL-44, having a corresponding β-methyl in X or α-methyl in Y.

Molecules 2023 , 13 Figure 5 .
Figure 5. Raw264.7 cells were pretreated with compounds 1-16 at a concentration of 20 µM for 2 h.Subsequently, the cells were stimulated with LPS (100 ng/mL) for 6 h.Total RNA was then extracted from the cells and subjected to a quantitative real-time polymerase chain reaction (qRT-PCR) to analyze the expression of a panel of genes associated with the innate immune response.** p < 0.01 vs.Control group; ## p < 0.01 vs. Blank group, n = 3.

Figure 5 .
Figure 5. Raw264.7 cells were pretreated with compounds 1-16 at a concentration of 20 µM for 2 h.Subsequently, the cells were stimulated with LPS (100 ng/mL) for 6 h.Total RNA was then extracted from the cells and subjected to a quantitative real-time polymerase chain reaction (qRT-PCR) to analyze the expression of a panel of genes associated with the innate immune response.** p < 0.01 vs.Control group; ## p < 0.01 vs. Blank group, n = 3.

Figure 6 .
Figure 6.Raw264.7 cells were preincubated with compounds 1-16 (20 µM) and Astin C (20 nM) for a duration of 2 h, followed by stimulation with DMXAA (25 µg/mL) for 6 h.Total RNA was extracted from the cells and subjected to qRT-PCR analysis to evaluate the expression of a panel of genes associated with innate immune-responsive genes.** p < 0.01 vs.Control group; ## p < 0.01 vs. Blank group, n = 3.

Figure 6 .
Figure 6.Raw264.7 cells were preincubated with compounds 1-16 (20 µM) and Astin C (20 nM) for a duration of 2 h, followed by stimulation with DMXAA (25 µg/mL) for 6 h.Total RNA was extracted from the cells and subjected to qRT-PCR analysis to evaluate the expression of a panel of genes associated with innate immune-responsive genes.** p < 0.01 vs.Control group; ## p < 0.01 vs. Blank group, n = 3.