Macrolactins from Marine-Derived Bacillus subtilis B5 Bacteria as Inhibitors of Inducible Nitric Oxide and Cytokines Expression

In order to find new natural products with anti-inflammatory activity, chemical investigation of a 3000-meter deep-sea sediment derived bacteria Bacillus subtilis B5 was carried out. A new macrolactin derivative was isolated and identified as 7,13-epoxyl-macrolactin A (1). Owing to the existence of the epoxy ring, 1 exhibited a significant inhibitory effect on the expression of inducible nitric oxide and cytokines, compared with previously isolated known macrolactins (2–5). Real-time Polymerase Chain Reaction (PCR) analysis showed that the new compound significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Reverse transcription-PCR analysis demonstrated that the new compound reduced the mRNA expression level of IL-1β in a concentration-dependent manner.


Introduction
The process of inflammation is the result of immune system activation which coordinates the normal defense mechanism of our body in response to microbial infection. Uncontrolled inflammation is believed to play crucial roles in the pathogenesis of various diseases, such as cardiovascular diseases [1], inflammatory bowel disease [2], cancer [3], diabetes [4], asthma [5], and Alzheimer's disease [6]. During the inflammatory process, biochemical parameters, such as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and 5-lipoxygenase [7,8], and levels of tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1 (IL-1), and interleukin-6 [9,10] are overexpressed during inflammation. Thus, inhibition of the production of these inflammatory mediators is an important target in the treatment of inflammatory diseases [11].
Several types of drugs are used to treat inflammatory disorders, such as biological, steroidal, and nonsteroidal anti-inflammatory drugs. However, they cause adverse side effects, and biological treatment is expensive. Natural products are alternatives to these drugs and offer hope for discovering bioactive lead compounds that may be developed into drugs for treatment of inflammatory disorders [12]. Plenty of unique marine natural products and their derivatives, such as sesquiterpenoid, polysaccharide, steroid/sterol and alkaloid, are found to manifest an anti-inflammatory action [13][14][15][16][17][18].

Structural Identification of 7,13-Epoxyl-macrolactin A (1)
Compound 1 (7,13-epoxyl-macrolactin A) was isolated as a yellow amorphous powder. The molecular formula of C24H32O4, which gave 9 unsaturation degrees, was established by the HR-ESI-MS ion peak at m/z 407.2190 [M + Na] + . The IR spectrum showed the presence of OH groups (3464 cm −1 ), olefinic protons (1450 cm −1 ) and carbonyls (1664 cm −1 ). The UV maximum absorption wave length at λmax (log ε): 233 (3.88) nm indicating the presence of conjugated carbonyls. The 1 H and 13 C NMR spectra, including DEPT, clearly showed two carbonyl carbons and 12 olefinic methines belonging to 6 ethylenic bonds, in the sp 2 low field region. The sp 3 high field region showed the existence of a methyl, four oxygenated methines, and six methylenes.
Most of the 1D NMR spectral data of 1 approach to those of macrolactin A (3), a typical macrolactin isolated from culture of a deep-sea marine bacterium [23], indicating the presence of a macrolactin nucleus in 1. The molecular formula of 1, when compared with that of 3, found that an H2O unit was lost, suggesting the existence of an epoxy moiety. The 13 C NMR signals assigned to C-8 and C-12 of macrolactin A were up-field shifted from δ 137.9 to δ 130.6 and from δ 36.0 to δ 31.3 respectively in 1, indicating that the epoxy moiety was formed by the condensation between 7-OH and 13-OH. The chemical shifting effect was also observed in 1 H NMR spectra, in which the H-7 signal of macrolactin A was down-field shifted from δ 4.19 to δ 4.56 in 1, while 7-OH and 13-OH

Structural Identification of 7,13-Epoxyl-macrolactin A (1)
Compound 1 (7,13-epoxyl-macrolactin A) was isolated as a yellow amorphous powder. The molecular formula of C 24 H 32 O 4 , which gave 9 unsaturation degrees, was established by the HR-ESI-MS ion peak at m/z 407.2190 [M + Na] + . The IR spectrum showed the presence of OH groups (3464 cm −1 ), olefinic protons (1450 cm −1 ) and carbonyls (1664 cm −1 ). The UV maximum absorption wave length at λ max (log ε): 233 (3.88) nm indicating the presence of conjugated carbonyls. The 1 H and 13 C NMR spectra, including DEPT, clearly showed two carbonyl carbons and 12 olefinic methines belonging to 6 ethylenic bonds, in the sp 2 low field region. The sp 3 high field region showed the existence of a methyl, four oxygenated methines, and six methylenes.
Most of the 1D NMR spectral data of 1 approach to those of macrolactin A (3), a typical macrolactin isolated from culture of a deep-sea marine bacterium [23], indicating the presence of a macrolactin nucleus in 1. The molecular formula of 1, when compared with that of 3, found that an H 2 O unit was lost, suggesting the existence of an epoxy moiety. The 13 C NMR signals assigned to C-8 and C-12 of macrolactin A were up-field shifted from δ 137.9 to δ 130.6 and from δ 36.0 to δ 31.3 respectively in 1, indicating that the epoxy moiety was formed by the condensation between 7-OH and 13-OH. The chemical shifting effect was also observed in 1 H NMR spectra, in which the H-7 signal of macrolactin A was down-field shifted from δ 4.19 to δ 4.56 in 1, while 7-OH and 13-OH signal at δ 5.05 and δ 4.60 of macrolactin A were missing. Comprehensive 1 H-1 H COSY and HMBC analysis allowed the complete assignment of the proton and carbon signals for 1 (Table 1 and Figure 2). As a result, the structure of 1 was elucidated as 7,13-epoxyl-macrolactin A. It was a newly isolated compound produced by deep-sea sediment of the Pacific Ocean. Many isolated macrolactins including epoxyl-macrolactin that have been isolated [19][20][21][22] and all macrolactins contain three separate diene structure elements. signal at δ 5.05 and δ 4.60 of macrolactin A were missing. Comprehensive 1 H-1 H COSY and HMBC analysis allowed the complete assignment of the proton and carbon signals for 1 (Table 1 and Figure 2). As a result, the structure of 1 was elucidated as 7,13-epoxyl-macrolactin A. It was a newly isolated compound produced by deep-sea sediment of the Pacific Ocean. Many isolated macrolactins including epoxyl-macrolactin that have been isolated [19][20][21][22] and all macrolactins contain three separate diene structure elements.

Cytotoxicity
Cytotoxicity of compounds 1-5 in RAW 264.7 cells were tested by using the MTS assay. The result showed that compounds 1-5 did not exhibit obvious cytotoxic effect at the employed

Cytotoxicity
Cytotoxicity of compounds 1-5 in RAW 264.7 cells were tested by using the MTS assay. The result showed that compounds 1-5 did not exhibit obvious cytotoxic effect at the employed concentrations (10-40 µM) (Figure 3). Interestingly, Compound 1 can promote cell proliferation under the low concentration 10 µM. This may be because, in this case, it activates the cell survival. The detailed mechanism still needs further research. concentrations (10-40 μM) (Figure 3). Interestingly, Compound 1 can promote cell proliferation under the low concentration 10 μM. This may be because, in this case, it activates the cell survival. The detailed mechanism still needs further research. As shown in Figure 4A-C, pretreatment of LPS activated cells with compounds 1 and 5 resulted in significant reduction of the mRNA expression of IL-1β, IL-6 and iNOS. Compounds 2 and 4 reduced the production of IL-1β and iNOS but had little effect on the expression level of IL-6. Compound 3 only slightly reduced the mRNA expression of IL-1β.
Based on the above results, we further investigated the effects of 1 on IL-1β mRNA expressions in LPS-stimulated RAW 264.7 macrophages by Reverse Transcription-PCR analysis. As shown in Figure 4D, compound 1 inhibited IL-1β mRNA expression in a concentration-dependent manner, while the house-keeping β-actin mRNA expression was unchanged by compound 1 under the same condition.

Inhibitory Effect of Compounds on LPS-Induced iNOS, IL-1β and IL-6 mRNA Expression
LPS can evoke innate immune response by stimulating the expression of several factors such as nitric oxide (NO) and pro-inflammatory cytokines, known to be involved in the immune response in macrophages. Compared with controls, upon LPS stimulation, macrophages strongly expressed the mRNA of iNOS, IL-1β and IL-6. Herein, compounds 1-5 were tested for in vitro anti-inflammatory activity and were found to suppress the mRNA expressions of iNOS, IL-1β and IL-6 in LPS-stimulated RAW 264.7 macrophages.
As shown in Figure 4A-C, pretreatment of LPS activated cells with compounds 1 and 5 resulted in significant reduction of the mRNA expression of IL-1β, IL-6 and iNOS. Compounds 2 and 4 reduced the production of IL-1β and iNOS but had little effect on the expression level of IL-6. Compound 3 only slightly reduced the mRNA expression of IL-1β.

General Experimental Procedures
The HR-ESI-MS analysis was performed on the Thermo Q-Exactive Orbitrap Mass spectrometer (Thermo Fisher Scientific Corporation, Waltham, MA, USA) equipped with electrospray ionization source (ESI). The preparative HPLC was performed with Varian binary  Based on the above results, we further investigated the effects of 1 on IL-1β mRNA expressions in LPS-stimulated RAW 264.7 macrophages by Reverse Transcription-PCR analysis. As shown in Figure 4D, compound 1 inhibited IL-1β mRNA expression in a concentration-dependent manner, while the house-keeping β-actin mRNA expression was unchanged by compound 1 under the same condition.

Bacteria and Fermentation
The bacterial strain B5 was isolated from deep-sea sediments which were collected at water depths of 3000 m Pacific Ocean by the Third Institute of Oceanography of China, and was identified as Bacillus subtilis B5 by complete 16S rRNA gene sequence. This bacterium was cultivated on 160 L scale using 250 mL shake flasks containing 100 mL of the seed medium (tryptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.4) cultured at 37 • C for 18 h at 200 rpm. One percent of the seed culture was inoculated into 100 L fermentors containing 80 L of fermentation medium consisting of soluble starch 0.5%, yeast extract 1%, K 2 HPO 4 1%, NaNO 3 0.5%, pH 6.8-7.0 for 2 days at the same condition mentioned above, and fermentations were conducted twice.

Cytotoxicity Assay
The cytotoxicity of compounds 1-5 were assessed by CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA). RAW 264.7 cells (1 × 10 4 cells/well) plated on 96-well plates were treated with different concentrations of samples (1-40 µM in DMSO) for 24 at 37 • C in 5% CO 2 . With a pipet, we put 20 µL of CellTiter 96 ® AQueous One Solution Reagent (MTS) into each well of the 96-well assay plate containing the samples in 100 µL of culture medium. Then, the plate was incubated at 37 • C for 3 h in a humidified, 5% CO 2 atmosphere and then the absorbance was recorded at 490 nm with a 96-well plate Microplate reader MK3 (Thermo Fisher Scientific Corporation, Waltham, MA, USA). The optical density of formazan formed in control cells (without treatment with samples) was taken as 100% viability.

Total RNA Isolation
RAW 264.7 cells were plated onto 6-well plates at a density of 1 × 10 6 cells/well and incubated with compounds at the indicated concentration for 1.5 h prior to LPS (100 ng/mL) stimulation. After 12 h, cells were lysed and total RNA extraction was performed by using Trizol reagent (Life Technologies, Carlsbad, MA, USA). Cells were homogenized in 200 µL of Trizol reagent, and then samples were left to rest at room temperature for 5 min. After that, 40 µL of chloroform was added and the tubes were vigorously shaken for 15 s and allowed to rest at room temperature for 5 min. Tubes were then centrifuged at 12,000× g (Eppendorf Centrifuge 5424 R, Eppendorf Instruments, Hamburg, Germany) 4 • C for 15 min. The aqueous phase was transferred to a new tube. Isopropyl alcohol (100 µL) was added to the aqueous phase: the tube was then gently mixed and incubated at room temperature for 10 min. After incubation, samples were centrifuged at 12,000× g, 4 • C for 10 min. The supernatant was poured out and the pellet was washed by 200 µL of 75% ethanol and centrifuged at 12,000× g, 4 • C for 5 min. The washing step and centrifuge were repeated. The final supernatant was removed and the pellet was dried until it was colorless. Total RNA was then dissolved in 20 µL of DEPC H 2 O, incubated at 65 • C for 5 min, and stored at −80 • C until used. 5 -CCAGTTGGTAACAATGCCATGT-3 ) was determined by real-time RT-PCR. The cDNA was synthesized from total RNA using oligo (dT) 18 primers. FastStart Universal SYBR Green Master (Rox, Roche, Basel, Switzerland) and Stepone Real-Time PCR Detection System (Applied Biosystems, Foster, CA, USA) were used for Real-time PCR analysis. Levels of all mRNAs were normalized to that of GAPDH mRNA.

Reverse Transcription-PCR Analysis of IL-1β mRNA
The inhibitory effect of compound 1 at the indicated concentrations on IL-1β mRNA expressions was determined by Reverse Transcription-PCR. Primers used were as follows: IL-1β (forward: 5 -GGGCCTCAAAGGAAAGAATC-3 , reverse: 5 -TACCAGTTGGGGAACTCTGC-3 ), β-actin (forward: 5 -CCACAGCTGAGAGGGAAATC-3 , reverse: 5 -AAGGAAGGCTGGAAAAGAGC-3 ). The total RNA was converted to cDNA and analyzed. PCR amplifications were performed on an Eppendorf traditional PCR machine (Eppendorf Instruments, Hamburg, Germany). The cDNA was amplified with 30 cycles of 95 • C for 45 s, 60 • C for 15 s, and 72 • C for 45 s. After amplification, the CR products were separated on 1.5% agarose gel for 30 min at 100 V.

Conclusions
A new macrolactins derivatives 7,13-epoxyl-macrolactin A (1), was isolated from a marine-derived bacterial strain Bacillus subtilis B5. Evaluation of its anti-inflammatory effect indicated that the new compound inhibited the expression of proinflammatory cytokines IL-1β, IL-6 and iNOS induced by LPS in RAW 264.7 macrophages, similar to the effect of 7-O-succinyl macrolactin A (5). 7-O-2 E-butenoyl macrolactin A (2) and 7-O-malonyl macrolactin A (4) reduced the production of IL-1β and iNOS but had no effect the level of IL-6. Macrolactin A (3) only slightly reduced the production of IL-1β. Additionally, 1 inhibited IL-1β mRNA expressions in in a concentration-dependent manner. Based on the anti-inflammatory effects of compounds 1-5 in LPS-stimulated RAW 264.7 macrophages, it could be suggested that different substituents at the position 7 might be key structural features for the anti-inflammatory activity. The presence of the epoxy moiety was special in the structure of the new compound and might greatly affect the anti-inflammatory activity.