Penazaphilones J–L, Three New Hydrophilic Azaphilone Pigments from Penicillium sclerotiorum cib-411 and Their Anti-Inflammatory Activity

Penazaphilones J–L (1–3), three new hydrophilic azaphilone pigments, as well as six known compounds, were discovered from the filamentous fungus Penicillium sclerotiorum cib-411. Compounds 1–3 were structurally elucidated by the detailed interpretation of their 1D and 2D NMR spectroscopic data. Compound 1 is an unprecedented hybrid of an azaphilone and a glycerophosphate choline. Compounds 2 and 3 each contain an intact amino acid moiety. The bioassay showed that compound 3 exhibited significant anti-inflammatory activity. Concretely, compound 3 significantly suppressed the NO production, the expression levels of COX-2, IL-6, IL-1β, and iNOS mRNA in LPS-stimulated RAW264.7 cells. Moreover, treatment of compound 3 prevented the translocation of NF-κB through inhibiting the phosphorylation of PI3K, PDK1, Akt, and GSK-3β. Thus, the inhibition of compound 3 against LPS-induced inflammation should rely on its inactivation on NF-κB.

Our previous study on Penicillium sclerotiorum cib-411 discovered a series of new azaphilone alkaloids bearing an S configuration at C-7, which displayed significant antiinflammatory activity [13]. In this study, we aimed at the exploitation of minor azaphilone pigments with high hydrophilicity from this fungus and at investigation of the underlying mechanisms of its anti-inflammation. As a result, three new hydrophilic pigments, penazaphilones J-L (1-3), together with six known compounds, were obtained from the methanol extract of the fermented rice culture of P. sclerotiorum cib-411. Penazaphilone J (1) represents The 1 H NMR spectra of 1, 2, and 3 were measured at 400 MHz. The 13 C NMR spectrum of 1 was acquired at 100 MHz, and 2 and 3 were acquired at 150 MHz.
Compound 3 was also purified as a red amorphous solid and had a molecular formula C 26 H 33 ClN 2 O 6 by HR-ESI-MS analysis (m/z 505.2079 [M + H] + ) with 11 degrees of unsaturation. The UV spectrum showed similar maximal absorptions at 479, 372, and 227.5 nm to that of 2. The 1 H and 13 C NMR spectra of 3 also showed similarities to those of 2. The comparison of the NMR spectra of 3 and 2 indicated that 3 contained one less methene and that the side chain was an ornithine residue [C-2 (C-1 )/C-3 /C-4 /C-5 (NH 2 )]. Detailed analyses of the HSQC and HMBC correlations supported the above speculation. The configurations of C-7 and C-13 were biogenetically determined to be S as in 1 and 2. Therefore, the structure of 3 was elucidated and named penazaphilone L ( Figure 1A).
Penazaphilones J-L belong to sclerotioramine pigments, which contain an isoquinoline skeleton, possibly formed via sclerotiorin, respectively reacting with a glycerophosphate choline, lysine, and ornithine. Fungal secondary metabolites with a glycerophosphate choline moiety have seldom been reported [16]. Penazaphilone J is the first azaphilone pigment that contains a glycerophosphate choline moiety. This study enriches the structural diversity of this class of azaphilones.

Effects of Compound 3 on the Viability of RAW264.7 Cells
Macrophages are an efficient immune weapon for preventing invading pathogens through phagocytosis or secreting multiple pro-inflammatory cytokines [22,23]. Upon stimulation with LPS, excessive levels of the inflammatory mediator NO can motivate the release of proinflammatory cytokines such as PGE 2 , TNF-α, and IL-6 [24,25]. Therefore, inhibiting the overproduction of these pro-inflammatory cytokines could be a highly effective way to explore new anti-inflammatory agents in vitro.
Our previous studies showed that penazaphilones can significantly inhibit the release of NO in LPS-induced RAW264.7 macrophages [17], but the underlying molecular mechanism is unclear. In this study, we first examined the anti-inflammatory activity of compounds 1-3. As shown in Table S2, 3 exhibited the most significant activity among them. Therefore, the anti-inflammatory activity of 3 was deeply investigated. To evaluate whether 3 has a cytotoxic activity to RAW264.7 cells, cells were co-incubated with different concentrations of 3 (6.25, 12.5, 25, 50, 100 µM) for 24 h. The results showed that 3 did not exhibit a significant cytotoxic effect under the concentration of 50 µM (Figure 2A). Therefore, the concentration range of 6.25-50 µM was considered valuable in subsequent investigations.
NO has been identified as a pro-inflammatory molecule and is associated with chronic inflammatory diseases and cancers [26,27]. NO production is generally regarded as a marker of macrophage activation, which is closely related to the pathogenesis of inflammatory diseases [28]. In the following study, we first tested whether 3 can affect the NO production of RAW264.7 cells. As shown in Figure 2B NO has been identified as a pro-inflammatory molecule and is associated with chronic inflammatory diseases and cancers [26,27]. NO production is generally regarded as a marker of macrophage activation, which is closely related to the pathogenesis of inflammatory diseases [28]. In the following study, we first tested whether 3 can affect the NO production of RAW264.7 cells. As shown in Figure

Compound 3 Reduced LPS-Induced over Expression of COX-2, IL-6, IL-1β, and iNOS
As a main inflammatory mediator, NO is synthesized in response to inflammatory stimuli [29,30]. It is reported that a high level of NO not only promotes the inflammatory response but also increases oxidative stress [31,32]. The COX enzymes transform arachidonic acid into prostaglandins, promoting oxidative stress and chronic inflammatory conditions [33]. Thus, down-regulation of these inflammatory intermediaries provides a potential therapeutic strategy for modulating inflammation-related disorders. To further reveal the anti-inflammatory activity of 3, RAW264.7 cells were pretreated with 3 for 30 min and then stimulated with LPS for 6 h. The transcriptional expression levels of these cytokines and inflammatory enzymes were measured by RT-PCR, and the results demonstrated that the stimulation of RAW 264.7 macrophages with LPS notably elevated the mRNA levels of COX-2 ( Figure 3A), IL-6 ( Figure 3B), IL-1β ( Figure 3C), and iNOS ( Figure  3D). Compound 3 significantly attenuated the mRNA levels of pro-inflammatory (iNOS and COX-2) and cytokines (IL-6 and IL-1β). Each value is presented as means ± SD (n = 3). * indicates a significant difference between the LPS group and the drug groups (p < 0.05). # indicates a difference between the LPS group and the control group (p < 0.05).

Compound 3 Reduced LPS-Induced over Expression of COX-2, IL-6, IL-1β, and iNOS
As a main inflammatory mediator, NO is synthesized in response to inflammatory stimuli [29,30]. It is reported that a high level of NO not only promotes the inflammatory response but also increases oxidative stress [31,32]. The COX enzymes transform arachidonic acid into prostaglandins, promoting oxidative stress and chronic inflammatory conditions [33]. Thus, down-regulation of these inflammatory intermediaries provides a potential therapeutic strategy for modulating inflammation-related disorders. To further reveal the anti-inflammatory activity of 3, RAW264.7 cells were pretreated with 3 for 30 min and then stimulated with LPS for 6 h. The transcriptional expression levels of these cytokines and inflammatory enzymes were measured by RT-PCR, and the results demonstrated that the stimulation of RAW 264.7 macrophages with LPS notably elevated the mRNA levels of COX-2 ( Figure 3A), IL-6 ( Figure 3B), IL-1β ( Figure 3C), and iNOS ( Figure 3D). Compound 3 significantly attenuated the mRNA levels of pro-inflammatory (iNOS and COX-2) and cytokines (IL-6 and IL-1β).

Effect of 3 on NF-κB Translocation
The nuclear translocation of NF-κB transcriptional subunit p65 is a crucial procedure for the activation of most pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α [34,35]. LPS can induce inflammation by triggering transcriptional activation of inflammation-inducing genes through the migration of NF-κB to the nucleus [36]. To evaluate if NF-κB signaling could be regulated by 3, the nuclear protein levels of p65 were assayed in RAW264.7 cells treated with different concentrations of 3. As shown in Figure 4A, LPS up-regulated p65 levels in the RAW264.7 cell nucleus, which were inhibited, especially in the nucleus, by 3. Furthermore, immunofluorescent staining demonstrated that NF-κB p65 is mainly distributed in the cytoplasm. After LPS treatment, the majority of intracellular p65 translocated to the nucleus, as indicated by the strong nuclear staining for NF-κB p65 ( Figure 4B). However, pre-treatment of 3 significantly reduced the p65 nuclear accumulation ( Figure 4B). Therefore, treatment of 3 prevented the translocation of NF-κB in RAW264.7 cells induced by LPS.

Effect of 3 on NF-κB Translocation
The nuclear translocation of NF-κB transcriptional subunit p65 is a crucial procedure for the activation of most pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α [34,35]. LPS can induce inflammation by triggering transcriptional activation of inflammation-inducing genes through the migration of NF-κB to the nucleus [36]. To evaluate if NF-κB signaling could be regulated by 3, the nuclear protein levels of p65 were assayed in RAW264.7 cells treated with different concentrations of 3. As shown in Figure 4A, LPS up-regulated p65 levels in the RAW264.7 cell nucleus, which were inhibited, especially in the nucleus, by 3. Furthermore, immunofluorescent staining demonstrated that NF-κB p65 is mainly distributed in the cytoplasm. After LPS treatment, the majority of intracellular p65 translocated to the nucleus, as indicated by the strong nuclear staining for NF-κB p65 ( Figure 4B). However, pre-treatment of 3 significantly reduced the p65 nuclear accumulation ( Figure 4B). Therefore, treatment of 3 prevented the translocation of NF-κB in RAW264.7 cells induced by LPS.

Compound 3 Suppressed NF-κB Activity Induced by LPS
The PI3K/Akt pathway has been documented to positively regulate NF-κB and Tolllike receptor-mediated proinflammatory responses in LPS-stimulated RAW 264.7 cells [37,38]. We, therefore, examined the potency of 3 on the PI3K/Akt signaling pathway, and the results showed that the phosphorylation of Akt, PI3K, and PDK1 induced by LPS were significantly suppressed by 3 in RAW264.7 cells ( Figure 5A). The rapid phosphorylation of GSK-3β is another important step for the accumulation of p65 in the nucleus, so the

Compound 3 Suppressed NF-κB Activity Induced by LPS
The PI3K/Akt pathway has been documented to positively regulate NF-κB and Toll-like receptor-mediated proinflammatory responses in LPS-stimulated RAW 264.7 cells [37,38]. We, therefore, examined the potency of 3 on the PI3K/Akt signaling pathway, and the results showed that the phosphorylation of Akt, PI3K, and PDK1 induced by LPS were significantly suppressed by 3 in RAW264.7 cells ( Figure 5A). The rapid phosphorylation of GSK-3β is another important step for the accumulation of p65 in the nucleus, so the phosphorylation level of GSK-3β was also determined. The result showed that 3 significantly suppressed the phosphorylation level of GSK-3β ( Figure 5B), thus inhibiting the activation of NF-κB induced by LPS. This result suggested that the protection of 3 against LPS-induced inflammation should rely on its inhibition of NF-κB activation.

Compound 3 Suppressed NF-κB Activity Induced by LPS
The PI3K/Akt pathway has been documented to positively regulate NF-κB and Tolllike receptor-mediated proinflammatory responses in LPS-stimulated RAW 264.7 cells [37,38]. We, therefore, examined the potency of 3 on the PI3K/Akt signaling pathway, and the results showed that the phosphorylation of Akt, PI3K, and PDK1 induced by LPS were significantly suppressed by 3 in RAW264.7 cells ( Figure 5A). The rapid phosphorylation of GSK-3β is another important step for the accumulation of p65 in the nucleus, so the phosphorylation level of GSK-3β was also determined. The result showed that 3 significantly suppressed the phosphorylation level of GSK-3β ( Figure 5B), thus inhibiting the activation of NF-κB induced by LPS. This result suggested that the protection of 3 against LPS-induced inflammation should rely on its inhibition of NF-κB activation. Penazaphilones J-L belong to the sclerotiorin type of azaphilone. A previous study showed that sclerotiorin derivatives exhibit various bioactivities. Isochromophilones I and II were novel gp120-CD4 binding inhibitors and potential lead compounds for HIV therapy [39]. Sclerotiorin and isochromophilone IV showed antitumor activity by inhibiting the interaction of Grb2−Shc and bacteriostatic activity against all Gram-positive and Gramnegative bacteria [40,41]. Sclerotiorin is also an inhibitor of lipoxygenase [42]. Isochromophilones III−V and isochromophilone VI were potent inhibitors of acylCoA:cholesterol acyltransferase, promising inhibition sites for the treatment of atherosclerosis and hypercholesterolemia [43]. In this study, we found that penazaphilone L exhibited the most significant anti-inflammatory activity without obvious cytotoxicity. Therefore, we disclosed the underlying mechanism of its anti-inflammation that penazaphilone L suppressed the mRNA expression levels of COX-2, IL-6, IL-1β, and iNOS, inhibited the phosphorylation of PI3K, PDK1, Akt, and GSK-3β, thus inhibiting translocation of NF-κB and resulting in the prevention of LPS-induced inflammation.

Chemicals and Reagents
See Supplementary Materials.

Strain and Fermentation Conditions
P. sclerotiorum cib-411 was preserved in Chengdu Institute of Biology, CAS. The medium and fermentation procedures were the same as those reported [17].

Cell Line and Cell Culture
The RAW264.7 macrophage cells were provided by the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), and incubated at 37 • C in a humidified incubator with 5% CO 2 (SANYO, Tokyo, Japan).

Cell Viability Assay
The cytotoxicity against RAW264.7 cells was evaluated by MTT method [44]. Concisely, 96-well plates (Corning, Suzhou, China) containing cells at a density of 1 × 10 5 cells/well were incubated at 37 • C overnight, then treated with different compounds (0, 6.25, 12.5, 25, 50, 100 µM) for 24 h. The medium was decanted, and the MTT (0.5 mg/mL) working solution was dripped into each well and stained for an extra 4 h at 37 • C in the dark. Subsequently, the formazan crystals were dissolved by adding a stopping buffer. After thorough dissolution, the absorbance at 550 nm was measured by an Infinite M200 PRO microplate reader (Tecan, Männedorf, Switzerland) to determine the product amount.

Measurement of Nitric Oxide (NO)
NO content of the supernatant was checked by Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylenediamine dihydrochloride), similar to our previous publication [44]. In brief, RAW264.7 cells in a 96-well plate (1 × 10 5 cells/well) were incubated overnight. Cells were treated with different concentrations of samples (30 min) first, then incubated with LPS (1 µg/mL) for 6 h. After incubation, the cell supernatant (100 µL) was moved to another 96-well plate and commingled with Griess reagent (v/v, 1:1) for 10 min incubation at indoor temperature. The absorbance was determined at 570 nm.

Quantitative RT-PCR
Cells were pre-treated with different concentrations of compound 3 for 30 min and then incubated with LPS (1 µg/mL) for 6 h. After being washed by PBS, total RNA was extracted with Trizol. Next, cDNA was synthesized by the RevertAid First Strand cDNA Synthesis Kit. The primers were used for the amplification of GAPDH, COX-2, iNOS, IL-6, and IL-1β. The semi-quantitative PCR amplified products of GAPDH were separated by 1% agarose gel electrophoresis, stained with GoldView (Solarbio, Beijing, China), and photographed using a Gel Doc XR+ with Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA). The expression levels of COX-2 mRNA, IL-6 mRNA, IL-1β mRNA, and iNOS mRNA were checked by qRT-PCR analysis with SYBR Green Premix Ex Taq (Roche, Mannheim, Germany) and performed by the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The mRNA expression levels were analyzed using the 2 −∆∆CT method. The results were normalized to GAPDH expression and quantified relative to the corresponding mRNA expression in the control group, which was normalized to 1 [44].

Western Blot
Cells were pre-treated with 3 at different concentrations for 30 min and then incubated with LPS (1.0 µg/mL) for different time points. The cells were washed with cold PBS, and then scraped out. The total protein and nuclear protein were respectively obtained using RIPA lysis buffer and Nuclear and Cytoplasmic Extraction Kit (CWBio, Beijing, China). The protein concentrations were determined by using the BCA protein assay kit (Absin, Shanghai, China). The protein samples were put into a gel loading buffer and boiled at 95 • C for 5 min. After separating on a 10% SDS-polyacrylamide gel for 2 h, the protein was transferred to PVDF membranes and blocked with 5% BSA for 2 h at room temperature, incubated overnight with primary antibodies at 4 • C, and washed three times with the 0.1% TTBS for 10 min. After that process, the secondary antibodies with horseradish peroxidase (HRP)-conjugated IgG were further incubated for 2 h at room temperature (1:2000 dilution). With an enhanced chemiluminescence detection system (Tanon 5200 Multi, Beijing, China), the blots were visualized using an eECL kit. Subsequently, the protein bands were quantified using image J [44].
3.5.6. In Vitro Cell Uptake RAW264.7 cells in 24-well plates (1 × 10 6 cells/well) were incubated at 37 • C with 5% CO 2 overnight, then pre-treated with different concentrations of 3 for 30 min, and later incubated with LPS (1 µg/mL) for 6 h. After washing with cold PBS, the cells were fixed with 4% paraformaldehyde for 15 min, blocked with 3% BSA for 1 h, and finally incubated with the primary antibody NF-κB p65 (1:400) for 1 h. After being rinsed with PBS three times, cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 for an extra 2 h. Coverslips were then mounted with DAPI containing mounting medium. Immunofluorescent images were captured using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystem, Bensheim, Germany) [44].

Data Analysis
SPSS software (SPSS Inc., Chicago, IL, USA) was employed for statistical analysis. The results are expressed as the mean ± SD. The statistical significance of the difference was analyzed by one-way ANOVA followed by Duncan's multiple range test, and p < 5% were considered statistically.

Conclusions
In a summary, this study disclosed three new hydrophilic azaphilone alkaloids (1-3) from the fungus P. sclerotiorum. Compound 1 is a structurally intriguing natural pigment and represents a novel azaphilone alkaloid containing a glycerophosphate choline moiety. Compounds 2 and 3, respectively, contain lysine and an ornithine moiety, which seldom exist in azaphilone metabolites. Compounds 1-3 possessed anti-inflammatory activity. Compound 3 exhibited the most significant bioactivity, suggesting the side chain on N-2 greatly influenced the anti-inflammatory activity. The investigation of the underlying mode of action confirmed that compound 3 might exert anti-inflammatory activity by suppressing the production of nitric oxide (NO); down-regulating the mRNA levels of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), interleukin-1β (IL-1β), and NO synthase (iNOS); and inhibiting the phosphorylation of PI3K, PDK1, Akt, and GSK-3β, as well as the nuclear translocation of nuclear factor κB (NF-κB) p65, a series of genes and proteins in the NF-κB signaling pathway. The results in this study suggested that penazaphilone L (3) is a potential functional colorant and may have a promising prospect in food industry.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1-3 are available from the authors.