The oxygenated products of cryptotanshinone by biotransformation with Cunninghamella elegans exerting anti-neuroinflammatory effects by inhibiting TLR 4-mediated MAPK signaling pathway CURRENT STATUS: POSTED

Background: Neuroinflammatory processes are critical in the development and progression of Alzheimer's disease (AD). The potent anti-neuroinflammatory inhibitors are expected as the candidates to treat AD. Cryptotanshinone ( 1 ), a major bioactive constituent in the traditional Chinese medicinal herb Dan-Shen Salvia miltiorrhiza Bunge, has been reported to possess remarkable pharmacological activities, especially anti-oxidation and anti-inflammation. Methods: Cryptotanshinone ( 1 ) was biotransformed with the fungus Cunninghamella elegans AS3.2028 to improve its bioactivities and physicochemical properties. The structures of transformed products were elucidated by comprehensive spectroscopic analysis including HRESIMS, NMR and ECD data. Their anti-neuroinflammatory activities were assessed by ELISA, transcriptome analysis, western blot, and immunofluorescence methods. Results: Three oxygenated products ( 2 – 4 ) at C-3 of cryptotanshinone ( 1 ) were obtained, among them 2 was a new compound. All of the biotransformed products ( 2 – 4 ) were found to inhibit significantly lipopolysaccharide-induced nitric oxide production in BV2 microglia cells with the IC 50 values of 0.16‒1.16 μ M, approximately 2‒20 folds stronger than the substrate ( 1 ). These biotransformed products also displayed remarkably improved inhibitory effects on the production of inflammatory cytokines (IL-1β, IL-6, TNF-α, COX-2 and iNOS) in BV-2 cells via targeting TLR4 compared to substrate ( 1 ). The underlying mechanism of 2 was elucidated by comparative transcriptome analysis, which suggested that it reduced neuroinflammatory mainly through mitogen-activated protein kinase (MAPK) signaling pathway. Western blotting results revealed that 2 downregulated LPS-induced phosphorylation of JNK, ERK, and p38 in MAPK signaling pathway. Conclusion: The biotransformed products of cryptotanshinone exhibit potent anti-neuroinflammatory activities. These findings provide a basal material for the discovery of candidates in treating AD.


Introduction
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder and the most common form of dementia among the elderly 1 . The multiple mechanisms about its occurrence and development have been revealed 2 . Many therapeutic approaches and clinic trials to AD have been reported 3 . Until 2018, only five small molecular chemicals are used clinically as drugs for the treatment of AD 3 . In 2019, one sodium oligomannate derived from a traditional Chinese medicine seaweed, GV-971, was approved as a drug in China to relieve AD by inhibiting β-amyloid protein aggregation, remodeling gut microbiota and suppressing gut bacterial amino acids-shaped neuroinflammation 4 . It should be noted that neuroinflammatory processes are critical in the development and progression of AD [5][6][7][8] . Therefore, the potent anti-neuroinflammatory inhibitors derived from natural products are expected as the candidates to treat AD 9 .
Tanshinones are a class of lipophilic abietane diterpenes mainly isolated from Dan-Shen Salvia miltiorrhiza Bunge which is a well-known traditional Chinese medicine 10 . Modern pharmacological studies have demonstrated that tanshinones possess a variety of bioactivities, such as antiinflammatory, anti-oxidative, acetylcholinesterase inhibitory, β-amyloid protein aggregation inhibitory, and neuroprotective activities 11,12 . These tanshinones from S. miltiorrhiza have shown an exciting prospect for the treatment of AD 13,14 . Among the tanshinones, sodium tanshinone IIA sulfonate has been developed successfully as a clinically effective drug to treat angina pectoris and coronary artery disease 12 . It has attracted extensive attentions that cryptotanshinone, another representative bioactive component in tanshinones, has been proved to be a promising candidate to treat AD 15,16 . However, the poor water solubility of cryptotanshinone limits its bioavailability and possible clinical applications 16,17 . Under the above considerations, it is urgent to discover cryptotanshinone derivatives featuring better bioavailabilities with high activities.
A literature survey revealed that cryptotanshinone has been remoulded to obtain modified derivatives by chemical and biological approaches 14,18−21 . Compared to chemical approaches, biotransformation of cryptotanshinone has proven to be an attractive alternative to improve the physicochemical and pharmacological properties. For example, five anhydrides of cryptotanshinone with anti-influenza A virus activities were obtained via biotransformation by fungus Mucor rouxii 20 . Three oxygenated products were harvested under the action of fungus Cunninghamella elegans in a gentle manner without changing the skeleton of ortho-naphthoquinone of cryptotanshinone 21 . It should be noted that the fungal genus Cunninghamella could be used as a microbial model to mimic mammalian drug biotransformation and as an in vitro model for drug metabolism studies 22 . It has been reported that C. elegans could be used as an in vitro model for the achievement of the in vivo minor metabolites of cryptotanshinone 21,23 .
In the present work, cryptotanshinone (1) was biotransformed using the fungus C. elegans AS3.2028 to obtain its new derivatives with improved physicochemical properties and bioactivities. As a result, multiple transformed products of cryptotanshinone have been emerged in the fermentation broth in 72 h. Three metabolites (2 − 4) ( Fig. 1) have been isolated and characterized from the biotransformed products. The metabolite 2, (15R)-3-keto-cryptotanshinone, was found to exhibit potential antineuroinflammatory activity, representing a promising candidate for the discovery of AD drugs. Herein, we report the biotransformation of cryptotanshinone (1), and the isolation, structure elucidation, biological evaluation and preliminary mechanism interpretation of the biotransformed products.

Materials
Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a HITACHI UH 5300 UV spectrophotometer. ECD data were acquired on a J-815-150S Circular Dichroism spectrometer. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets. 1 H and 13 C NMR spectra were acquired by a JEOL Eclips-500 spectrometer at 500 MHz for 1 H and 125 MHz for 13 C in CDCl 3 , using TMS as internal standard. HREIMS were measured on a Thermo MAT95XP high resolution mass spectrometer, and EIMS spectra on a Thermo DSQ EImass spectrometer. Semi-preparative HPLC was performed on a Hitachi L-2000 HPLC system coupled with a Hitachi L-2455 photodiode array detector and using a semi-preparative C 18 column (Kromasil 250 × 10 mm, 5 µm). The column temperature was set at 30 °C, and the flow rate was 2 mL/min. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 300 − 400 mesh) was used for column chromatography (CC).

Substrate
Cryptotanshinone (1) was purchased from Macklin Biochemical Co., Ltd in Shanghai, China and authenticated by comparing its physical and spectroscopic data with the reported values. Its purity was determined to be 98% by HPLC analysis.

Fungal Strain
The fungus C. elegans AS3.2028 was purchased from China General Microbiological Culture Collection Center, Beijing, China.

Biotransformation Medium
The biotransformation experiment was carried out in modified Czapek-Dox medium, which consisted of the following ingredients: glucose (

Biotransformation Procedure
A spore suspension of the fungus grown on PDA was transferred to 500 mL Erlenmeyer flasks containing 100 mL biotransformation medium, and incubated at 28 °C, 180 rpm on a rotary shaker for 24 h. Subsequently, the substrate cryptotanshinone (1) dissolved in methanol was added to each flask to achieve a final concentration of 0.1 mg/mL in a sterile condition. The cultures were incubated for another 72 h.

Extraction and Isolation
The large-scale cultures (10 L) were filtered and the filtrate was extracted with an equivalent volume of EtOAc for three times. The organic layer was collected and concentrated under vacuum at 40 °C.

Bioassay for NO Production Inhibitory Activities
The bioassay for NO production inhibitory activities was performed as described by Xia et al. 24 . The BV-2 microglia cells were seeded in 96-well plates. In each well, LPS (1 µg/mL) was added after treating with or without compounds at various concentrations for 24 h. The NO production in the supernatant was detected by the Griess reaction. The absorbance at 540 nm was measured in a microplate reader. The NO concentration and the inhibitory rate were calculated through a calibration curve. Quercetin was used as the positive control. Experiments were operated in triplicate, and the data were described as mean ± SD of three independent experiments.

ELISA Measurement
The BV-2 microglial cells were cultured in 96-well plates, grown overnight, incubated with compounds for 1 h, and then stimulated with LPS (1 µg/mL) for 24 h. The supernatant (50 µL) from the culture was collected to determine the concentrations of IL-6, IL-1β and TNF-α with ELISA kits according to the protocols.

Western blot
BV-2 cells were seeded in 6-well plates, incubated with or without compounds for 1 h, then treated with or without LPS (1 µg/mL) for 16 h. The cells were lysed with RIPA lysis buffer. The concentrations of protein were determined by Pierce Rapid Gold BCA Protein Assay Kit. The quantified protein of each sample was electrophoresed in 10% SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) western membrane. The membranes were blocked using 5% (W/V) skim milk in TBST (Trisbuffered saline with 0.1% Tween 20) for 2 h. Then the membranes were incubated with primary antibodies at 4 °C overnight. After being washed by TBST three times, the membranes were incubated with corresponding secondary antibody at room temperature for 2 h. Finally, immunoreactive signals were detected using a chemiluminescence imager (GE Amersham Imager 600, USA). Intensities of band signals were quantified using the densitometric ImageJ software (National Institutes of Health, MD).

Transcriptome sequencing
The BV-2 cells were treated with 10 µM of compound 2 (sample group) or DMSO (model group) for 1 h followed by stimulated with LPS (1 µg/mL) for 24 h. The control group was without LPS stimulation.
Total RNAs were extracted with Trizol (Invitrogen, USA) from the treated BV-2 cells. Three biological replicates were sequenced using the Illumina Hiseq × 10 platform for each group. The differentially expressed genes (DEGs) were screened out with a q value (p-adjust value) ≤ 0.01 and fold change ≥ 2 and further analyzed with KEGG.

Cell Viability Assay
The mouse hippocampal neuron cell line (HT22) was used in glutamate toxicity model to evaluate the neuroprotective activities of compounds 25 . The HT22 cells were cultured in 96-well plates and pretreated with tested compounds (1 µM and 0.1 µM) for 1 h. To each well, 5 mM glutamate was added. After treatment for 12 h, the culture medium was replaced with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (0.5 mg/ml) and incubated in the dark at 37 °C for 4 h. The MTT solution was carefully removed. Dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance was detected at 540 nm using a microplate reader. Experiments were operated in triplicate. The cell survival rates to vehicle were calculated with Graphpad prism 7.
For BV-2 cells, after being seeded into a plate, the cells were incubated with various concentrations of the compounds for 24 h. The MTT assay was used to evaluate their cytotoxic activities, consistent with the above operations.
The immunofluorescence assay was performed as described by Xia et al. 24 . Briefly, BV-2 cells were pre-treated with DMSO, LPS, and 10 µM compound 2 before LPS, respectively. The cell-seeded glass coverslips were subjected to treatment with cold 4% paraformaldehyde and 0.2% Triton X-100 (in PBS). Then, the coverslips were blocked with 5% BSA (in PBS) for 1 h and incubated with a primary antibody NF-κB p65 at 4 °C overnight, followed by addition of a secondary antibody labeled with Alexa Fluor 594 for 1 h. After being stained with DAPI, the coverslips were washed and sealed. Images were obtained by fluorescence microscope (Leica, Solms, Germany).

Bioassay for AChE Inhibitory Activities
The AChE inhibitory activities of compounds were evaluated in 96 well microplates using a spectrophotometric method 26,27 . The inhibition rates were calculated by comparing the rates of enzyme reaction of samples relative to that of blank (DMSO). Huperzine A was used as a positive control. All experiments were performed in triplicates independently. Metabolite 2 was obtained as a red powder. Its molecular formula was assigned as C 19 H 18 O 4 based on the [M + H] + ion peak in HRESIMS with 11 degrees of unsaturation, suggesting the introduction of one oxygen but less two hydrogens than the substrate cryptotanshinone (1). In the 1 H NMR spectrum, two aromatic protons (δ H 7.62 and δ H 7.65), one methine, three methylenes (one oxygenated), and three methyl groups were observed ( Table 1). The 13 C NMR spectrum indicated the presence of 19 carbons, including three ketone carbonyls, eight aromatic carbons (two protonated and six quaternary), one quaternary carbon, one methine, three methylenes (one oxygenated) and three methyl groups (Table 1). These spectroscopic features of 2 were very similar to those of 1. The significant difference was that a ketone carbonyl signal at δ C 212.5 was presented in 2, by concomitant disappearance of a methylene signal compared to 1 (δ H 1.66 (2H, m) and δ C 37.8). The HMBC correlations of 2 from H-1/H-18/H-19 to C-3 indicated that the ketone carbonyl was anchored at C-3. The absolute configuration of C-15 was determined as R by comparison of the experimental and calculated electronic circular dichroism (ECD) curves generated by time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d,p) level (Fig. 2). Thus, the structure of 2 was characterized as (15R)-3keto-cryptotanshinone.  inflammation. Inhibitors of NO production represent the potential therapeutic agents for inflammatory diseases 29 . Cryptotanshinone (1) has been reported to possess potent anti-inflammatory activities 30-neuroinflammatory activities. All of the compounds showed potent inhibitory activities against lipopolysaccharide (LPS)-induced NO production in BV2 microglia cells ( Table 2). The substrate (1) showed better activity with the IC 50 value of 3.30 µM, approximately 2.5 folds than that of the positive control quercetin (8.50 µM). Compared to 1, metabolites 2 − 4 exhibited improved inhibitory activities against NO production in BV2 cells with the IC 50 values of 0.49, 1.16, and 0.16 µM, about 6.7, 2.8 and 20.6 folds more potent than 1, respectively. It is also interesting to observe that metabolites 3 and 4, featuring respective β-and α-orientation hydroxyl groups at C-3, exhibited significantly discriminating activities, the latter displaying 7 folds higher activity than the former. It indicated that the oxygenated group at C-3, hydroxyl or carbonyl, might be beneficial to improve the anti-neuroinflammatory activity of cryptotanshinone. NO levels in culture supernatants were measured using the Griess reagent, and IL-1β, IL-6, and TNF-α in culture supernatants were determined with an ELISA kit. Values were given as the mean ± SD (µM) from three independent experiments. The overproduction of pro-inflammatory cytokines in nerve cells usually reflects the activated stress state of cell34. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the culture supernatant of LPS-induced BV-2 cells were evaluated by ELISA method. All of the tested compounds (1 − 4) showed remarkable inhibitory effects on the release of IL-1β, IL-6 and TNF-α (Table 2). In comparison to substrate 1, the biotransformed products 2 − 4 showed significantly improved inhibitory effects especially for IL-1β with 4.3, 1.6 and 2.0 folds, respectively. In addition, to exclude the influence of cytotoxicity of compounds to their inhibitory activities, a cytotoxicity assay was conducted against BV-2 cells. As a result, all compounds showed no cytotoxicity at the concentration of 10 µM. Based on the above results, it was confirmed that cryptotanshinone (1) and its metabolites (2 − 4) exhibited antineuroinflammatory activities in BV2 cells.

The Inhibitory Effects of Expressions of iNOS, COX-2, and TLR4 in LPS-Induced BV-2 Cells
In order to further interpret the anti-neuroinflammatory effects of the compounds, the production of two inflammatory mediators in LPS-induced BV-2 cells, iNOS and COX-2, was assayed by western blot.
Compared to 1, compounds 2 − 4 exhibited improved inhibitory effects on the level of expressed proteins (Fig. 3). Consequently, the initial inflammatory target TLR4 was detected, which plays an important role in the immune and inflammation system to recognize LPS 35 . BV-2 cells were pretreated with compounds for 1 h and then exposed to LPS for 16 h. As a result, the expression of TLR4 was stimulated by LPS, while cryptotanshinone (1) and its metabolites (2 − 4) reduced its expression level (Fig. 3). Compared to 1, compounds 2 and 4 showed significantly improved effects on inhibiting the expression of TLR4.

The Targeted Signal Pathway of Compound 2 in LPS-Induced BV-2 Cells
RNA-seq was used to investigate the gene expression profiles of BV-2 cells. It was found that LPS upregulated 1212 genes in the transcript profiles of BV-2 cells, while compound 2 downregulated 306 genes of them (FC ≥ 2-fold change, q value ≤ 0.01) (Fig. 4a and Table S1). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these differential expressed genes were significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway (Fig. 4b).
Subsequently, the phosphorylation levels of c-Jun NH 2 -terminal protein kinase (JNK), extracellular regulated protein kinases (ERK), and p38 in MAPK signaling pathway were determined with western blot. Compound 2 was founded to inhibit significantly the phosphorylation of JNK, ERK, and p38 with a dose-depend manner (Fig. 5). These findings suggested its potential use to attenuate neuroinflammatory through MAPK signaling pathway. The phosphorylation of JNK, ERK, and p38 usually lead to the activation of NF-κB in nucleus. As expected, compound 2 was also found to reduce the effect of p65 in LPS-stimulated BV-2 cells (Fig. 6), which activated the production of proinflammatory cytokines.

The Neuroprotective Activities of Compounds against HT22 Neuronal Cells
Glutamate is an endogenous excitatory neurotransmitter. Glutamate toxicity has been used as a common model to investigate oxidative stress-induced neuronal cell death 36,37 . Extracellular glutamate with a high concentration could deplete glutathione (GSH) and accumulate reactive oxygen species (ROS) in neurons, which may lead to cell death 38 . In our study, the substrate cryptotanshinone and its converted products were evaluated for their neuroprotective activities against glutamate-induced oxidative toxicity in HT22 neuronal cells. As shown in Fig. 7,

Physicochemical Properties of Compounds Predicted in silico
The physicochemical properties of tanshinones are critical to their bioavailabilities in vivo 11 . The physicochemical properties of the metabolites of cryptotanshinone (1) were predicted by using the ACD/Labs Percepta platform-PhysChem Module software. The substrate cryptotanshinone (1) and its metabolites all complied with the Lipinski rule of five, predicted in silico ( Table 3). The calculated octanol/water partition coefficient (Log P) and water solubility (Log S) of metabolites 2 − 4 were improved compared to those of 1, especially metabolite 2. These results suggested that the water solubility of the metabolites have been improved relative to the liposoluble substrate.

Discussion
It has been well documented that tanshinones from Chinese traditional medicine Salvia miltiorrhiza Bunge (Danshen) exhibit potential anti-inflammatory activities, like tanshinone IIA, tanshinone I, dihydrotanshinone, and cryptotanshinone [40][41][42][43] . They all possess a common active core structure of furanophenanthraquinone. It is hard to make them a direct clinical application due to their high hydrophobicity and poor absorbed property when given orally or intraperitoneally 11 . Sodium tanshinone IIA sulfonate is one of water-soluble derivatives of tanshinone IIA without change of basic skeleton 11 . Biotransformation has been considered as a mild and effective approach to improve the water solubility and activities for natural products 44,45 . In our study, cryptotanshinone was biotransformed by only oxidation at C-3 of its cyclohexane moiety without destruction of the active core. The obtained three oxygenated products thereby possess improved physicochemical properties.
Our next work is to dig the effective tool enzymes from Cunninghamella elegans AS3.2028 for further amplified preparation, especially for 2.
Our bioassay results indicated that the three oxygenated products of cryptotanshinone could significantly suppress the LPS-induced production of NO, IL-1β, IL-6 and TNF-α. Excessive NO and proinflammatory cytokines may lead severe diseases such as septic shock, inflammatory diseases and neurotoxicity 31 (Table S1), which mostly enriched in MAPK pathway. MAPK signaling pathway was regarded as an attractive target due to its capability of reducing the synthesis of proinflammatory cytokines and their signaling 48 50 . In turn, TAK1 phosphorylates MAPK, eventually resulting in the activation of NF-κB, which plays an important role in the LPS-induced production of pro-inflammatory cytokines ( Fig. 8) 51 . In the present study, as shown in Fig. 5, LPS intensely increased the phosphorylation of JNK, ERK and p38 in BV-2 cells. While when cells were pre-treated with compound 2, their phosphorylation levels were significantly decreased. In addition, the immunofluorescence results suggested that compound 2 could also attenuate the action of p65 in the nucleus of LPS-stimulated BV-2 cells (Fig. 6). Thus, compound 2 could relieve the stimulation of LPS to BV-2 cells and downregulate the expression level of TLR4 to influence the downstream signaling and inhibit the production of proinflammatory factors to exhibit anti-neuroinflammatory effect (Fig. 8).
Intriguingly, compound 2 also displayed the neuroprotective activities against glutamate-induced oxidative toxicity in HT22 neuronal cells and modest AChE inhibitory activity. These results suggested that 2 could be considered as a promising candidate for development of AD drug. The absorption, bioavailability and metabolism in vivo of 2 need to be further investigated.

Consent for publication
All the authors have approved the manuscript.

Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Competing interests
The authors declare that they have no competing interests. Structures of cryptotanshinone (1) and its biotransformed products (2−4).

Figure 2
Experimental and calculated ECD spectra of 2.    The effects of compound 2 on the nuclear translocation of NF-κB p65 in LPS-stimulated BV2 cells. Immunofluorescence staining detected the distribution of NF-κB p65 by the colocalization of receptors (p65, red) with nuclei (DAPI, blue).

Figure 7
Neuroprotective activities of compounds 1−4 against glutamate-induced HT22 neuronal cell death. The cell viability after treating with 5 mM glutamate in the presence or absence of compounds. Data are the mean ± standard error of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the Glu-treated group.

Figure 8
The proposed signaling mechanism for the inhibitory effects of compound 2 on LPS stimulated neuroinflammation in BV2 cells.