Bioactive Aspergteroids G–J from Soft-Coral-Associated Symbiotic and Epiphytic Fungus Aspergillus terreus EGF7-0-1

Two new disubstituted maleimides, aspergteroids G–H (1–2), and two trisubstituted butenolides aspergteroids I–J (3–4), along with four known analogs, were isolated and structurally identified from the fermentation extract of soft-coral-associated symbiotic and epiphytic fungus Aspergillus terreus EGF7-0-1. The structures of the new compounds were established mainly via spectroscopic data analyses, and their absolute configurations were determined via X-ray diffraction analysis and comparison of the calculated and experimental electronic circular dichroism. Myocardial protection assays showed that compounds 1, 2, 5, and 6 possess protective effects against tert-butyl hydroperoxide (TBHP)-induced H9c2 (rat myocardial cells) apoptosis at low concentrations. Based on the analyses of the protein–protein interaction (PPI) network and Western blotting, compound 1 may inhibit the apoptosis and inflammatory response of cardiomyocytes after TBHP induction and improve the antioxidant capacity of cardiomyocytes. We speculate that the anti-inflammatory response of compound 1 is suppressed by the glycogen synthase kinase-3 beta (GSK-3β), downregulated by the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation, and suppressed by the expression of cysteinyl aspartate specific proteinase-3 (caspase-3) and B-cell lymphoma-2 associated X protein (Bax).


General Experimental Procedures
Details of the instrumentations and materials used in this work are included in the Supplementary Materials.

Fungal Materials, Extraction, and Fermentation
Strain EGF7-0-1 was isolated from soft coral in the South China Sea and identified as Aspergillus terreus based on the sequencing of its ITS region (GenBank no. KY425727.1) with 100% similarity. The soft coral was taxonomically identified as Sinularia scabra Tixier-Durivault. by associate research fellow Xin-ming Liu (Institute of Marine Drugs, Guangxi Key Laboratory of Marine Drugs, Guangxi University of Chinese Medicine). The strain and soft coral voucher specimen were stored in the Laboratory of Marine Natural Medicine, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine (Strain No. EGF7-0-1).
The rice culture medium included 100 g of rice, 3.3% sea salt, and 110 mL of pure water and was pH-neutral. A 10 mL seed solution was inoculated into the rice culture medium (100 g/1 L/flask) at 28 °C for 30 days, and a total of 200 L was cultured. The fermented media were exhaustively extracted with EtOAc (500 mL) and evaporated under reduced pressure to afford the EtOAc extracts (500 g).

General Experimental Procedures
Details of the instrumentations and materials used in this work are included in the Supplementary Materials.

Fungal Materials, Extraction, and Fermentation
Strain EGF7-0-1 was isolated from soft coral in the South China Sea and identified as Aspergillus terreus based on the sequencing of its ITS region (GenBank no. KY425727.1) with 100% similarity. The soft coral was taxonomically identified as Sinularia scabra Tixier-Durivault. by associate research fellow Xin-ming Liu (Institute of Marine Drugs, Guangxi Key Laboratory of Marine Drugs, Guangxi University of Chinese Medicine). The strain and soft coral voucher specimen were stored in the Laboratory of Marine Natural Medicine, School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine (Strain No. EGF7-0-1).
The rice culture medium included 100 g of rice, 3.3% sea salt, and 110 mL of pure water and was pH-neutral. A 10 mL seed solution was inoculated into the rice culture medium (100 g/1 L/flask) at 28 • C for 30 days, and a total of 200 L was cultured. The fermented media were exhaustively extracted with EtOAc (500 mL) and evaporated under reduced pressure to afford the EtOAc extracts (500 g).

Structural Characterizations of Compounds
Crystal data for 1.  Table S1.

Quantum Chemical Calculations
Conformational searches were carried out on compounds 3 and 4 using the MMFF94 molecular force field via the SYBYL X 2.1.1 program with an energy cutoff of 10 kcal/mol. The optimized structures were obtained at the B3LYP/6-31+G(d) gas-phase level by using Gaussian09 software (Semichem Inc, Kansas, MO, USA). Furthermore, the electron circular dichroism (ECD) of methanol was calculated at the B3LYP/6-31+G(d) level. The resulting ECD spectra of compounds 3 and 4 were then weighted by Boltzmann distribution and compared with experimental spectra, which were simulated using SpecDis 1.70.1 software (Torsten Bruhn, Berlin, Germany).

Cell Viability Assays
The cell viability of compounds 1-8, a Cell Counting Kit-8 (CCK-8) was used in accordance with the manufacturer's instructions. H9c2 cells were plated in a 96-well plate at a density of 5000 cells per well and incubated with the test samples for 24 h. Following this 24 h incubation, 10 µL of the CCK-8 solution was added to each well, and the plate was further incubated for 2 h. The optical density values were then measured at 450 nm using a microplate reader, and cell viability was calculated using the following equation:

Targets Prediction
The prediction of potential targets of compound 1 and the construction of PPI networks were modeled in accordance with the methods we previously reported [20].

Western Blot Assays
H9c2 cells were cultured in a 6-well plate at a density of 2 × 10 5 cells/per well and incubated at 37 • C under a 5% CO 2 atmosphere. After 24 h, the cells were stimulated at concentrations of 1 µM, 5 µM, and 10 µM for an additional 24 h. Following a 1 h incubation with DMEM medium (including 10% FBS and 1% penicillin)containing 200 µM TBHP, the cells were washed with PBS before protein was extracted using RIPA lysis buffer containing protease or phosphatase inhibitors. Protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit. Next, equal amounts of protein extract were separated on a 12% dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a 0.45 µm polyvinylidene fluoride (PVDF) membrane. The membranes were then blocked with 5% nonfat milk before being incubated overnight at 4 • C with specific antibodies targeting B-cell lymphoma-2 (Bcl-2), Bax, caspase-3, NLRP3, GSK-3β, and βactin. After incubation with the appropriate HRP-conjugated secondary antibodies, protein bands were detected using an enhanced chemiluminescence kit and imaged, and band intensities were quantified using Image J 1.46 r (National Institutes of Health, Bethesda MD, USA).
Similarly, the absolute configuration of 3 was established as 5R according to biological pathways [25] and the quantum chemical ECD calculation.
Similarly, the absolute configuration of 4 was determined to be 5R through both the biological pathways [25] and the quantum chemical ECD calculations.

Evaluation of Cell Viability
The cytotoxicities of compounds 1-8 against H9c2 cell lines were tested using a CCK-8 assay kit. The results showed that compounds 1-8 did not exhibit obvious cytotoxic effects in concentrations below 10 µM ( Figure 5).

Evaluation of Cardioprotective Effects
Since oxidative stress contributes to many cardiovascular diseases [26], the antioxidant effects of butenolides were the focus of our attention. All compounds in this study were evaluated for their protective effects against TBHP-induced H9c2 apoptosis. H9c2 cells were treated with TBHP-induced cell injury. The cell injury was estimated in terms of cell viability. Treatment with TBHP significantly decreased cell viabilities (p < 0.05, Figure 6). As shown in Figure 6, compounds 1, 2, 5, and 6 showed protective effects while compounds 3, 4, 7, and 8 could not improve the cell viability at these three concentrations, suggesting that the cardioprotective effects of disubstituted maleimides and trisubstituted butenolides could be related to their phenyl-and benzyl-disubstituted types and nitrogenous element.
were evaluated for their protective effects against TBHP-induced H9c2 apoptosis. H9c2 cells were treated with TBHP-induced cell injury. The cell injury was estimated in terms of cell viability. Treatment with TBHP significantly decreased cell viabilities (p < 0.05, Figure 6). As shown in Figure 6, compounds 1, 2, 5, and 6 showed protective effects while compounds 3, 4, 7, and 8 could not improve the cell viability at these three concentrations, suggesting that the cardioprotective effects of disubstituted maleimides and trisubstituted butenolides could be related to their phenyl-and benzyl-disubstituted types and nitrogenous element. To investigate the mechanism underlying the cardioprotective effects of compound 1, the top 100 genes associated with myocardial protection for compounds 1 and 2 were predicted [27]. A Venn diagram approach identified 24 overlapping genes. Subsequently, a protein-protein interaction (PPI) network was constructed using the STRING 11.5 database [28] and Cytoscape 3.9.0 software [29]. The PPI analysis revealed that glycogen synthase kinase-3β (GSK-3β) had the highest degree score, suggesting it may play a crucial role in mediating the cardioprotective effects of compound 1 (Figure 7). To investigate the mechanism underlying the cardioprotective effects of compound 1, the top 100 genes associated with myocardial protection for compounds 1 and 2 were predicted [27]. A Venn diagram approach identified 24 overlapping genes.
To investigate the mechanism underlying the cardioprotective effects of compound 1, the top 100 genes associated with myocardial protection for compounds 1 and 2 were predicted [27]. A Venn diagram approach identified 24 overlapping genes. Subsequently, a protein-protein interaction (PPI) network was constructed using the STRING 11.5 database [28] and Cytoscape 3.9.0 software [29]. The PPI analysis revealed that glycogen synthase kinase-3β (GSK-3β) had the highest degree score, suggesting it may play a crucial role in mediating the cardioprotective effects of compound 1 (Figure 7). Many studies have indicated that GSK-3β is a promising target for the treatment of cardiovascular diseases [30]. Additionally, the Bcl-2/Bax and caspase-3 proteins are important regulators of cell survival and apoptosis. We found that after TBHP induced oxidative stress injury of cardiomyocytes, 1 could inhibit GSK-3β activity to downregulate the activation of NLRP3 inflammatorome and reduce the inflammatory response. The expression changes in Bcl-2/Bax and caspase-3 indicated that 1 could effectively inhibit cell apoptosis and protect myocardial cells, as shown in Figure 8. Many studies have indicated that GSK-3β is a promising target for the treatment of cardiovascular diseases [30]. Additionally, the Bcl-2/Bax and caspase-3 proteins are important regulators of cell survival and apoptosis. We found that after TBHP induced oxidative stress injury of cardiomyocytes, 1 could inhibit GSK-3β activity to downregulate the activation of NLRP3 inflammatorome and reduce the inflammatory response. The expression changes in Bcl-2/Bax and caspase-3 indicated that 1 could effectively inhibit cell apoptosis and protect myocardial cells, as shown in Figure 8. Figure 8. The effects of 1 on Bcl-2, Bax, caspase-3, NLRP3, and GSK-3β proteins in H9c2 cells were determined by Western blot analysis. All data are expressed as means ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. treated group.

Conclusions
Four new and four known compounds were isolated from the fermentation extract of the soft-coral-associated symbiotic and epiphytic fungus Aspergillus terreus EGF7-0-1. The new natural disubstituted maleimides identified in this research expand the knowledge of the chemical space and biological diversity of microorganisms of marine origin. Because of structural specificities, compound 1 showed significant protective effects against the TBHPinduced H9c2 apoptosis at low concentrations. We further attempted to explore the possible mechanism of oxidative stress injury in cardiomyocytes through network pharmacology and the TBHP-induced injury model. The results suggested that compound 1 may reduce the inflammatory response and protect cardiomyocytes through GSK-3β/NLRP3 pathway. At the same time, the expression of caspase-3 and Bax was regulated, the antiapoptotic gene Bcl-2 was upregulated, the antioxidant capacity of cells was improved, and apoptosis was inhibited.