A novel TGR5 agonist Sauchinone ameliorates IMQ induced murine psoriasis by regulating macrophage polarization

Graphical abstract

Highlights

• •Sauchinone possesses TGR5 agonistic activity.
• •Sauchinone inhibits macrophage M1 polarization through targeting TGR5.
• •Topical use of Sauchinone cream ameliorates IMQ induced psoriasis.
• •Sauchinone cream regulates macrophage polarization in psoriatic skins.

Abstract

Introduction

G-protein-coupled bile acid receptor (TGR5) is a member of G-protein-coupled receptor (GPCR) superfamily that participates in regulating macrophage polarization and resolving inflammatory diseases. Sauchinone is Saururus chinensis derived natural product with anti-inflammatory activity. Still, whether Sauchinone could regulate macrophage polarization and its direct target remain to be explored.

Objectives

This study aims to demonstrate the direct target of Sauchinone, its influences on macrophage polarization and its pharmacological actions on imiquimod (IMQ) induced mouse psoriasis model.

Methods

We detected the TGR5 agonistic activity of Sauchinone in mouse/human TGR5/ cAMP response elements (CRE)/HEK293 stable cell lines and verified its direct effect on mouse/human macrophages by Cellular thermal shift assay (CETSA) and by examining downstream CREB phosphorylation. Afterwards, we discovered the activity of Sauchinone on regulating macrophage M1/M2 polarization in Bone marrow-derived macrophages (BMDM) by detecting M1/M2 markers through Enzyme-linked immunosorbent assay (ELISA), Real-time polymerase chain reaction (RT-qPCR), Western blot and Fluorescence-activated cell sorting (FACS). We further utilized macrophages derived from Tgr5^-/- mice or introduced TGR5 specific inhibitor, TGR5 si-RNA and PKA inhibitor to determine whether Sauchinone regulated macrophage polarization through TGR5. We then prepared Sauchinone cream formulation to disclose its pharmacological action in IMQ induced mouse psoriasis model and used FACS and immunofluorescence to verify its action on macrophage polarization in psoriatic skin. Moreover, we tested the protective actions of Sauchinone cream in IMQ treated Tgr5^-/- mice to verify that Sauchinone alleviated psoriasis in TGR5 dependent manner.

Results

Sauchinone is a novel TGR5 agonist without human/mouse species selectivity. Sauchinone rectified macrophage M1 polarization through activating TGR5. Topical use of Sauchinone cream ameliorated IMQ induced psoriasis and regulated macrophage polarization in psoriatic skins. Sauchinone cream alleviated psoriasis in TGR5 dependent manner.

Conclusion

Our work identified Sauchinone as a novel TGR5 agonist that could ameliorate IMQ induced murine psoriasis by regulating macrophage polarization.

Introduction

Psoriasis is a kind of chronic skin inflammatory disease characterized with infiltration of immune cells in lesional skin and abnormal differentiation and proliferation of keratinocytes [1]. Mounting evidences indicated that aberrant macrophage polarization plays indispensable role in psoriasis progression. Clinical studies showed that in psoriatic patients, the M1/M2 macrophage polarization ratio was elevated in peripheral CD14⁺ monocytes [4] and psoriasis lesional skin [5,6]. As for murine studies, this phenomenon was also observed in imiquimod (IMQ) induced murine psoriasis [7]. On the other side, approaches to correct the bias toward macrophage M1 polarization provide attractive therapies for psoriasis [[8], [9], [10]].

G-protein-coupled bile acid receptor (TGR5) is a member of G-protein-coupled receptor (GPCR) family that transduces the signal by increasing intracellular cAMP concentration and subsequent phosphorylation of downstream kinases. Besides its regular function in regulating bile acid metabolism and energy homeostasis, the expression of TGR5 on macrophages arises the interest of scientists to study the role of TGR5 in regulating macrophage mediated inflammation [11]. Indeed, researchers have proved that TGR5 activation participates in inhibiting macrophage M1 polarization [[12], [13], [14], [15], [16]] and promoting macrophage M2 polarization. Moreover, activation of TGR5 on macrophage by its ligand bile acid or INT-777 is able to ameliorate inflammatory diseases, such as atherosclerosis [18], Lipopolysaccharides (LPS)-induced inflammation [12] and colitis [15] in mice. Therefore, targeting TGR5 on macrophages is considered as an emerging strategy in resolving inflammation and treating inflammatory diseases [19].

A link between TGR5 and psoriasis has also been established. Oral administration of bile acids, the natural ligand of TGR5, has been reported to improve IL-23 induced psoriasiform [20]. However, the inhibitive effects of bile acids on IL-17A production and IL-17A–induced CCL20 production in keratinocytes were independent of TGR5 [20]. Nevertheless, the role of TGR5 in psoriasis cannot be neglected because TGR5 mainly localized to dermal macrophages in skin [21]. Understanding the action of agonizing TGR5 on macrophage in skin might offer attractive prospect for the potential application of novel TGR5 agonist for psoriasis treatment.

Sauchinone (C₂₀H₂₀O₆) (Fig. 1A) is a pharmacologically active natural product that isolated from the root of Saururus chinensis. Sauchinone shows beneficial effects in different inflammatory disease models, such as ulcerative colitis (UC) [[22], [23], [24]], osteoarthritis (OA) [25,26], acute lung injury (ALI) [27] and allergic asthma [28]. Also, it has been reported that Sauchinone suppresses LPS induced inflammatory response by antagonizing NF-κB pathway and c-raf/MEK1/2/ERK1/2 pathway in macrophages [[29], [30], [31]]. However, whether Sauchinone could modulate macrophage polarization and the potential target remains to be illustrated. Meanwhile, the application of Sauchinone externally applied formulation in psoriasis, whose progression is closely associated with aberrant macrophage polarization, has not been reported.

Fig. 1.

Sauchinone possesses TGR5 agonizing activity. (A) The structure of Sauchinone. (B) Mouse or human TGR5/CRE/HEK293 stable cell lines were incubated with different concentrations of Sauchinone or INT-777. The TGR5 agonist activity was calculated by determining the luciferase activity in cell lysate. (C) CRE/HEK293 stable cell lines were incubated with different concentrations of Sauchinone or Forskolin. The CRE agonist activity was calculated by determining the luciferase activity in cell lysate. (D) Human FXR/FXRE/Huh7 cells were incubated with different concentrations of Sauchinone or OCA. The FXR agonist activity was calculated by determining the luciferase activity in cell lysate. (E) The thermal stability of TGR5 protein in BMDM and THP-1 cells treated with or without Sauchinone (30 μM) was measured by CETSA. (F) BMDM or THP-1 cells were incubated with different concentrations of Sauchinone for 1 h. The protein level of phosphorylated CREB in cell lysates was detected by western blot. Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05 compared as indicated.

In this research, we first determined the TGR5 agonistic activity of Sauchinone in mouse/human TGR5/ cAMP response elements (CRE)/HEK293 stable cell lines and confirmed its direct action on TGR5 in macrophages. Afterwards, we studied the influence of Sauchinone on macrophage M1/M2 polarization and investigated whether Sauchinone modulates macrophage polarization through targeting TGR5 by utilizing Tgr5^-/- mice, TGR5 specific inhibitor SBI-115 and TGR5 si-RNA. Furthermore, we evaluated therapeutic effects of Sauchinone cream in mouse model of psoriasis where abnormal macrophage polarization happens in lesional skins. In the end, we constructed psoriasis model on Tgr5^-/- mice and tested the protective effects of Sauchinone cream to verify that Sauchinone alleviated psoriasis through activating TGR5.

Materials and methods

Reagents

Sauchinone was kindly supplied by professor Zhao Qinshi at Kunming Institute of Botany. Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT, USA). IMDM, DMEM and Opti-MEM medium were obtained from Gibco (Grand Island, NY, USA). INT-777, Forskolin, Obeticholic acid (OCA), SBI-115 and H89 were obtained from MCE (Monmouth Junction, NJ, USA). Recombinant Murine M−CSF, Recombinant Murine IFN-γ, Recombinant Murine IL-13, Recombinant Murine IL-4, Recombinant Human IFN-γ and Recombinant Human TNF-α were obtained from Peprotech (Rocky Hill, NJ, USA). LPS and collagenase Ⅳ were obtained from Sigma Aldrich (St. Louis, MO, USA). IMQ cream (0.05 %, w/w) was obtained from Sichuan Mingxin Pharmaceutical (Sichuan, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Mouse IL-1β ELISA kit and kit for BCA protein assay were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Mouse IL-6, TNF-α, IL-12, IL-10, IFN-γ and IL-17 ELISA kits were obtained from ebioscience (San Diego, CA, USA). SDS lysis buffer was obtained from Beyotime (Shanghai, China). Lipofectamine™ RNAiMAX Transfection Reagent was obtained from Invitrogen (Waltham, MA, USA). TGR5 si-RNA was obtained from Ribobio (Guangzhou, China). RNA extraction kit was obtained from Tiangen (Beijing, China). Kits for cDNA synthesis and RT-qRCR were obtained from Yeasen (Shanghai, China). HRP-conjugated anti-GAPDH (KC-5G5) was obtained from Kangcheng (Shanghai, China). HRP-conjugated secondary antibody (1706515) was obtained from Bio-Rad (Richmond, CA, USA). Anti-mouse STAT-1 (9712L), anti-mouse p-STAT-1 (7649L), anti-mouse Inducible nitric oxide synthase (INOS) (13120S), Anti-mouse STAT-6 (9362S), anti-mouse p-STAT-6 (56554S), anti-mouse PKA (5842S), anti-mouse p-CREB (9197S), anti-mouse CREB (9198L),anti-mouse p-IκB (9246L), anti-mouse IκB (4812S), anti-mouse p-p65 (3033L), anti-mouse p65 (4764S), anti-mouse ASC (67824S) and anti-mouse NLRP3 (15101S) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-GPCR TGR5 (ab72608) and anti-mouse Caspase-1 (ab179515) were obtained from Abcam (Cambridge, MA, USA). FITC-anti-Gr-1 (553127), FITC-anti-CD86 (561962), Percp-cy5.5-anti-CD11b (550993), APC-anti-CD11c (550261), APC-anti-CD86 (558703), BUV395-anti-CD45 (565967), and Fixable Viability Stain 780 (565388) were obtained from BD Biosciences. AF488-anti-INOS (53-5920-82), PE-anti-F4/80 (12-4801-82), Percp-cy5.5-anti-F4/80 (45-4801-82), APC-anti-CD206 (17-2061-82) and anti-CD16/CD32 (2.4G2) (14-0161-86) were obtained from ebioscience. PE-anti-CD301 (145704) was obtained from Biolegend (San Diego, CA, USA).

Cell cultures and treatment

For the differentiation of BMDM, wild type (WT) and Tgr5^-/- C57BL/6 mice were euthanatized and bone marrow was achieved from the tibia and femur bones, followed by stimulating with 10 ng/ml Recombinant Murine M−CSF in IMDM for 7 d. On d 4, fresh IMDM medium containing 10 ng/ml Recombinant Murine M−CSF was added. After 7 d, BMDM were digested with 0.05 % trypsin and adjusted to 4 × 10⁵/ml for following experiments. The purity of BMDM (CD11b⁺, F4/80⁺) was detected with FACS and was consistently > 98 %. For the activation of macrophage M1/M2 polarization, BMDM were stimulated with 1 μg/ml LPS plus 50 ng/ml murine IFN-γ or 20 ng/ml murine IL-4 plus 20 ng/ml murine IL-13 for 24 h respectively. The culture supernatants were harvested for ELISA or NO assay. Cells were collected for RT-qPCR, western blot, and FACS assay.

THP-1 cells (ATCC, Manassas, VA, USA) were cultured in sterile RPMI 1640 medium supplemented with 10 % FBS, 1 % penicillin/streptomycin at 37 °C incubator containing 5 % CO₂ and passaged twice weekly. THP-1 monocytes were stimulated with 200 ng/ml PMA for 2 d for the differentiation of macrophage.

HaCaT cells (ATCC, Manassas, VA, USA) were cultured in sterile DMEM medium supplemented with 10 % FBS, 1 % penicillin/streptomycin at 37 °C incubator containing 5 % CO₂ and passaged twice weekly. For the activation of keratinocytes, HaCaT cells were stimulated with 10 ng/ml TNF-α and 10 ng/ml human IFN-γ for 24 h. Cells were collected for RT-qPCR assay.

In vitro TGR5 agonistic activity screening assay

TGR5 agonistic activity was tested in mouse/human TGR5/CRE/HEK293 stable cell lines and CRE/HEK293 cells (without TGR5) [32]. Briefly, cells in 96-well plates were incubated with different concentrations of Sauchinone or INT-777or Forskolin in fresh DMEM medium containing 10 % FBS and for 5.5 h. Then the luciferase activity in cell lysate was determined using the Steady-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The efficacy of INT-777 (20 μM) in TGR5/CRE luciferase assay and the efficacy of Forskolin (20 μM) in CRE luciferase assay were set as 100 %.

FXR activity assay

Human FXR (hFXR) eukaryotic expression vector and reporter plasmid containing receptor response sequence were transferred into Huh7 cells to detect the agonistic activity of Sauchinone on FXR [33]. Briefly, cells in 96-well plates were incubated with different concentrations of Sauchinone or OCA in fresh DMEM medium containing 10 % FBS and for 16 h. Then the luciferase activity in cell lysate was determined using the Steady-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The efficacy of OCA (10 μM) was set as 100 %.

Cellular thermal shift assay (CETSA)

CETSA was performed under the guidance of a previously reported protocol [34]. Briefly, BMDM or THP-1 cells were pre-incubated with Sauchinone (30 μM) for 2 h. Cells were harvested with cell scraper before resuspending in PBS containing protease inhibitor. Then the cell suspensions were divided into 5 aliquots and heated at various temperature. After freezing and thawing twice in liquid nitrogen, the suspensions were centrifuged at 10,000 g for 20 min at 4 °C to separate the soluble fraction from precipitates. The supernatants were collected for SDS-PAGE to detect TGR5 expression.

Cell counting Kit-8 (CCK-8) assay

BMDM in 96-well were treated with or without different concentrations of Sauchinone. After 24 h, the supernatants were changed with IMDM medium containing 10 % CCK-8 regents. After 2 h, OD450 (OD570 for calibration) was detected by microplate reader (Molecular Devices, Sunnyvale, CA, USA) to indicate the cell viability.

Western blot

Total proteins from mice dorsal skins and cell samples were first isolated with SDS lysis buffer. Then, the concentration was measured by BCA protein assay kit and adjusted to equal amount. After that, proteins were separated in SDS-PAGE system and subsequently transferred to nitrocellulose membranes. After blocking with milk powder at room temperature for 1 h. the membranes were precut and incubated at 4 °C for 12 h with primary antibodies. The membranes were incubated with HRP-conjugated anti-GAPDH or HRP-conjugated secondary antibody for 1 h. Protein signals were finally visualized in Chemidoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA) with ECL substrate (Thermo Fisher Scientific).

Enzyme-linked immunosorbent assay (ELISA)

Mice dorsal skins were homogenized with PBS and then centrifuged to achieve clear supernatants for measurement. BCA protein assay kit was first utilized to determine the protein levels in skin homogenates. Then ELISA kit was utilized to detected cytokines in serum, skin homogenates and cell culture supernatants. Briefly, samples were added to the 96 well plate that pre-coated with capture antibody. After 2 h incubation, the detection antibody and HRP were added successively before TMB coloration. The OD450 (OD650 for calibration) was detected by microplate reader (Molecular Devices). The concentrations of cytokines in skin homogenates were shown as pg/mg protein and the concentrations of cytokines in serum and cell culture supernatants were shown as pg/ml.

Real-time polymerase chain reaction (RT-qPCR)

First, total RNA in mice dorsal skins or cell samples was extracted by RNA extraction kit. Then cDNA was acquired with cDNA synthesis kits and RT-qPCR was subsequently conducted on 7500 Fast RT-qPCR System (Applied Biosystems, Foster city, CA, USA) with specific primers and RT-qPCR SYBR Green kits. Primer sequences information was shown in Table S1.

Si-RNA transfection

TGR5 si-RNA or NC si-RNA were transfected into BMDM transiently with Lipofectamine™ RNAiMAX Transfection Reagent according to the manufacturer’s instructions. Briefly, si-RNA was diluted in Opti-MEM medium and then mixed with Opti-MEM medium containing Lipofectamine™ RNAiMAX Transfection Reagent. After incubating at room temperature for 5 mins, the mixture was added to BMDM. 72 h later, the medium was removed and BMDM were stimulated for M1/M2 polarization in the presence or absence of Sauchinone.

Preparation of Sauchinone cream

The oil phase and aqueous phase were prepared first. The aqueous phase includes distilled water 3.8 ml, glycerol 400 mg, triethanolamine 20 mg, methyl paraben 20 mg, the oil phase includes octadecanol 100 mg, stearic acid 560 mg, liquid paraffin 100 mg, Sauchinone 0 mg (0 %) or 100 mg (2 %) or 200 mg (4 %). Then the oil phase (heated to 85 ℃) was slowly added to the aqueous phase (heated to 75 ℃) with continuously stirring. The cream was subsequently obtained by cooling the mixture to room temperature with continuously stirring.

Animals

C57BL/6 and BALB/c mice (8–10 weeks, 20 g) were purchased from Shanghai Lingchang Biotechnology Co. Ltd. Tgr5^-/- mice with C57BL/6 background were gifted by Professor Xie Xin at Shanghai Institute of Materia Medica (SIMM) kindly. Tgr5^-/- mice were constructed by deleting 23 bp of gene encoding area. For genetic identification, genomic DNA was extracted from WT or Tgr5^-/- mice tails and amplified by PCR and analyzed by agarose gel electrophoresis. The mRNA level of Tgr5 in skin tissues were also measured by RT-qPCR (Fig. S1). All animals were kept in the specific pathogen-free animal facilities at SIMM. All experiments conducted in this study were under the supervision of IACUC at SIMM.

IMQ induced murine psoriasis-like skin lesion model

To evaluate the pharmacological effects of Sauchinone on psoriasis, female BALB/c mice were divided into 5 groups (Normal, Model, 4 % Sauchinone, 2 % Sauchinone and Calcipotriol) with 8 mice per group randomly. Model group were administrated topically on the back skin with 0 % Sauchinone cream daily for 7 d. Drug treated groups were administrated topically on the back skin with 4 % or 2 % Sauchinone cream (62.5 mg) or 0.5 % Calcipotriol (62.5 mg) daily for 7 d. The hair on dorsal skins were removed with grainer and depilatory creams 2 d ahead of the experiment. Psoriasis-like skin lesion was induced by topically receiving IMQ cream (62.5 mg) on the back skin daily for 7 d. To verify the pharmacological effects of Sauchinone on psoriasis is dependent on TGR5, WT C57BL/6 mice were divided into 3 groups (WT-Normal, WT-Model, WT-4 % Sauchinone) and Tgr5^-/- mice (C57BL/6 background) were divided into 2 groups (Tgr5^-/--Model, Tgr5^-/--4% Sauchinone) with 6 mice per group randomly. Model group were administrated topically on the back skin with 0 % Sauchinone cream daily for 6 d. Sauchinone treated groups were administrated topically on the back skin with 4 % Sauchinone cream (62.5 mg) for 6 d. The hair on dorsal skins were removed with grainer and depilatory creams 2 d ahead of the experiment. Psoriasis-like skin lesion was induced by topically receiving IMQ cream (62.5 mg) on the back skin daily for 6 d. The Psoriasis Area and Severity Index (PASI), including scales, thickness, and erythema were recorded daily. The criteria of the calculated scores of PASI is defined as follows: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. At the end point of the experiment, mice were euthanatized, then serum and dorsal skin were collected for the following analysis.

Histological analysis

Mice dorsal skins were cut and fixed in 4 % formaldehyde. Paraffin embedding, slice cutting and H&E staining were performed by Servicebio Company (Wuhan, China). Images were captured using Olympus VS200 Slide scanner (Tokyo, Japan). Epidermal thickness was recorded by measuring the average thickness of the interfollicular epidermis of each skin slice with Image-Pro Plus software (Silver Springs, MD, USA). Briefly, two curve lines named T1 and T2 were draw closely along the upside and downside of the epidermal layer. Then the average distance between the epidermal layer was automatically calculated by the software after clicking the CT button.

Immunofluorescence

For the detection of macrophage polarization in skin infiltrated macrophages, the skin tissue frozen sections were prepared with Leica CM1950 freezing microtome (Wetzlar, Germany), followed by fixing with acetone. Then the slices were blocked with 2 % BSA and stained with FITC-F4/80 antibody plus APC-CD86 antibody, PE-F4/80 antibody plus AF488-INOS antibody and FITC-F4/80 antibody plus APC-CD206 antibody respectively at room temperature. Finally, the nuclei were stained with DAPI. Images were subsequently captured using Zeiss LSM 710 confocal microscope.

FACS

To detect innate immune cells infiltration in skin, mice dorsal skins were cut into small pieces and then digested with Collagenase IV (1 mg/ml) for 2 h at 37 °C. Then the tissues were grinded mechanical followed by centrifuging and passing through 70 μm cell filter. Single cell suspensions were stained with Fixable Viability stain 780 to distinguish the live cells and dead cells, followed by fixing with IC Fixation Buffer. Cells were stained with FITC-Gr-1/PE-F4/80/Percp-Cy5.5-CD11b/APC-CD11c/BUV395-CD45/FITC-CD86/APC-CD206/FITC-CD11b/Percp-Cy5.5-F4/80 antibody after blocking with anti-CD16/CD32 (2.4G2). To detect the polarization of macrophages in vitro, the trypsin detached BMDM were stained with AF488-INOS or BV421 MHC-Ⅱ or FITC-CD86 or PE-CD301 antibody after blocking with anti-CD16/CD32 (2.4G2).The FACS data was collected on LSR Fortessa (BD), and the analysis of FACS data were performed on Flowjo 10 software (Treestar, Ashland, OR, USA).

Statistical analysis

All experiments performed in this study have been repeated for three or more times and data were shown as mean ± SEM. Significant difference analysis was performed by using one-way ANOVA in GraphPad Prism 8.0 software (La Jolla, CA, USA). p < 0.05 was considered as statistically significant.

Results

Sauchinone possesses TGR5 agonistic activity

Although the anti-inflammatory activity of Sauchinone has been reported, the potential target still remains to be uncovered. TGR5 is a member of GPCR superfamily that has been reported to mediate anti-inflammatory reaction in macrophages. Here, we discovered the TGR5 agonistic activity of Sauchinone in both mouse and human TGR5/CRE reporter gene assay (Fig. 1B). Meanwhile, in the parental cell line-HEK293 cells stably expressed with CRE-driven luciferase reporter but without TGR5. Sauchinone showed no activity compared with adenylate cyclase activator Forskolin (Fig. 1C). These results indicate Sauchinone indeed activates TGR5 but not through other endogenously express GPCRs. Also, Sauchinone did not activate farnesoid X receptor (FXR), which is another common bile acid receptors (Fig. 1D). In mouse or human macrophages (BMDM and THP-1 respectively) that naturally expressing TGR5, pre-incubation of Sauchinone (30 μM) increased the thermostability of TGR5 protein to retarded its heat-degradation between 55 and 58 ℃ (Fig. 1E), confirming that Sauchinone could bind to TGR5 protein directly. In response to its ligands, TGR5 signals by increasing intracellular cAMP concentrations and subsequent phosphorylation of downstream kinases such as CREB. At the same time, Sauchinone treatment induced the phosphorylation of CREB in both BMDM and THP-1 macrophages (Fig. 1F), indicating the activation of TGR5 downstream signaling pathway. Therefore, the above results suggested that Sauchinone possesses TGR5 agonistic activity.

Sauchinone inhibits macrophage M1 polarization and promotes macrophage M2 polarization in BMDM

Recently, a connection between TGR5 and macrophage polarization has been illustrated [[35], [36], [37]]. We therefore sought to study the influence of Sauchinone on macrophage polarization. We used LPS plus IFN-γ to stimulated BMDM M1 polarization. Under non-toxicity dose (Fig. 2A), Sauchinone decreased the transcription levels of M1 markers including Tnf-α, Il-6, Il-12, Inos, Il-1β, Cox-2 and Nlrp3 (Fig. 2B). Also, Sauchinone inhibited the release of TNF-α, IL-6, IL-12 and NO in cell medium (Fig. 2C), which are the M1-like cytokines and inflammatory mediators. In addition, Sauchinone inhibited the phosphorylation of STAT-1 and the expression of INOS (Fig. 2D). What’s more, Sauchinone suppressed the percentages of INOS⁺, MHCⅡ⁺ and CD86⁺ BMDM (Fig. 2E). All these data indicated that Sauchinone could inhibit macrophage M1 polarization. We then stimulated BMDM with IL-4 and IL-13 to induce M2 polarization and investigated whether Sauchinone could promote macrophage M2 polarization. Sauchinone strongly enhanced the transcription level of M2 markers including Arg-1, Fizz1, Ym-1, Cd206, Cd301 and Dectin-1 (Fig. 2F). Meanwhile, Sauchinone increased the protein level of p-STAT6, which is M2-like transcription factor (Fig. 2G). In accordance with the result of PCR, the result of FACS presented that Sauchinone unregulated the percentages of CD301⁺ BMDM (Fig. 2H). These results reflected that Sauchinone could promote macrophage M2 polarization. Combining all the above results, we concluded that Sauchinone restrains M1 macrophage polarization but facilitates M2 macrophage polarization in macrophages.

Fig. 2.

Sauchinone inhibits M1 macrophage polarization and promotes M2 macrophage polarization in BMDM. (A) The cytotoxicity of Sauchinone on BMDM was detected by CCK-8 kit. (B-G) BMDM were stimulated with LPS plus IFN-γ to induce M1 polarization in the presence or absence of different doses of Sauchinone. The transcription levels of Tnf-α, Il-6, Il −12, Inos, Il-1β, Cox-2 and Nlrp3 (B) were detected by RT-qPCR. TNF-α, IL-6, IL-12 and NO levels in culture supernatants (C) were measured by ELISA or Griess assay. The protein levels of p-STAT-1, STAT-1, and INOS in cell lysates (D) were detected by western blot. The percentages of INOS⁺, MHCⅡ⁺ and CD86⁺ BMDM (E) were measured by FACS. (F-H) BMDM were stimulated with IL-4 plus IL-13 to induce M2 polarization in the presence or absence of different doses of Sauchinone. The transcription levels of Arg-1, Fizz1, Ym-1, Cd206, Cd301, and Dectin-1 (F) were detected by RT-qPCR. The protein levels of p-STAT-6 and STAT-6 in cell lysates (G) were detected by western blot. The percentages of CD301⁺ BMDM (H) were measured by FACS. Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05, **P < 0.01 compared as indicated.

Sauchinone inhibits macrophage M1 polarization through targeting TGR5/PKA pathway

Next, we sought to determine whether Sauchinone regulates macrophage polarization through targeting TGR5. We found SBI-115, a TGR5 specific antagonist, reversed the protective effective of Sauchinone on BMDM M1 polarization, indicated by the transcription level of Tnf-α, Il-6, Il-1β and Inos (Fig. 3A) and the protein expression of p-STAT-1 and INOS (Fig. 3B). To further verify that Sauchinone inhibits M1 polarization through agonizing TGR5, we knocked down TGR5 on BMDM that derived from WT mice (Fig. S2B). Similarly, the influence of Sauchinone on the transcription level of Tnf-α, Il-6, Inos and Il-1β (Fig. 3C), the protein expression of p-STAT-1 and INOS (Fig. 3D), and the IL-6 and NO release (Fig. 3E) was weakened or even diminished. Moreover, we studied the influence of Sauchinone on macrophage polarization in BMDM that derived from Tgr5^-/- mice. Surprisingly, Sauchinone was not able to inhibit the M1 polarization induced IL-6 and NO release (Fig. 3F), the transcription of Tnf-α, Il-6, Inos and Il-1β (Fig. 3G), as well as the protein level of p-STAT-1 and INOS (Fig. 3H). To further verify that Sauchinone inhibits M1 polarization through TGR5 downstream pathway, PKA inhibitor H89 was also utilized. We observed that H89 also canceled the influence of Sauchinone on M1 polarization in BMDM (Fig. 4A-C). Combing all above, these data strongly proved that Sauchinone inhibits macrophage M1 polarization through targeting TGR5/PKA pathway. As for macrophage M2 polarization, however, inhibition of TGR5 by SBI-115 was not able to abolish the upregulated Irf-4, Cd206, Cd301 by Sauchinone (Fig. S2A). Simultaneously, knocking down of TGR5 was not able to cancel the upregulated Ym-1, Cd206, Cd301 and Dectin-1 by Sauchinone (Fig. S2C). Moreover, we found Sauchinone still could promote the transcription of Ym-1, Irf-4, Cd206 and Dectin-1 (Fig. S2D), and the phosphorylation of STAT6 (Fig. S2E) in BMDM that separated from Tgr5^-/- mice. All these results suggested that Sauchinone facilitates macrophage M2 polarization independent of TGR5.

Fig. 3.

Sauchinone inhibits macrophage M1 polarization in TGR5 dependent manner. (A-B) BMDM from WT mice were stimulated with LPS plus IFN-γ to induce M1 polarization in the presence or absence of Sauchinone (30 μM) or SBI-115 (5 μM). The transcription levels of Tnf-α, Il-6, Inos and Il-1β (A) were detected by RT-qPCR. The protein levels of p-STAT-1, STAT-1 and INOS in cell lysates (B) were detected by western blot. (C-E) BMDM from WT mice were stimulated with LPS plus IFN-γ to induce M1 polarization in the presence or absence of Sauchinone (30 μM) or TGR5 si-RNA. The transcription levels of Tnf-α, Il-6, Inos and Il-1β (C) were detected by RT-qPCR. The protein levels of p-STAT-1, STAT-1 and INOS in cell lysates (D) were detected by western blot. IL-6 and NO levels in culture supernatants (E) were measured by ELISA and Griess assay. (F-H) BMDM from Tgr5^-/- mice were stimulated with LPS plus IFN-γ to induce M1 polarization in the presence or absence of Sauchinone (30 μM). IL-6 and NO levels in culture supernatants (F) were measured by ELISA or Griess assay. The transcription levels of Tnf-α, Il-6, Inos and Il-1β (G) were detected by RT-qPCR. The protein levels of p-STAT-1, STAT-1 and INOS in cell lysates (H) were detected by western blot. Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05, **P < 0.01 compared as indicated.

Fig. 4.

Sauchinone inhibits macrophage M1 polarization through TGR5/PKA pathway. (A-C) BMDM were stimulated with LPS plus IFN-γ to induce M1 polarization in the presence or absence of different doses of Sauchinone (30 μM) or PKA inhibitor H89 (10 μM). IL-6 and NO levels in culture supernatants (A) were measured by ELISA or Griess assay. The transcription levels of Tnf-α, Il-6, Inos and Il-1β (B) were detected by RT-qPCR. The protein levels of p-STAT-1, STAT-1 and INOS in cell lysates (C) were detected by western blot. Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05, **P < 0.01 compared as indicated.

Sauchinone alleviates IMQ induced murine psoriasis-like skin lesion

The psoriasis pathogenesis is associated with aberrant macrophage polarization [2,4]. We studied the protective effects of Sauchinone cream in IMQ-induced murine psoriasis model (Fig. 5A). Sauchinone treatment significantly increased the body weight (Fig. 5B), ameliorated the clinical symptoms (Fig. 5C), and decreased the PASI scores (Scales, thickness, erythema and total scores) (Fig. 5D). These effects were better than the positive drug Calcipotriol. The image of HE staining (Fig. 5E) shows that Sauchinone cream ameliorated epidermis thickening (acanthosis), retention of nuclei in the upper layers and stratum corneum (parakeratosis), stratum corneum thickening (hyperkeratosis), the acanthosis cell layer thickening and neutrophils infiltration in the stratum corneum (Munro’s microabscesses). Psoriasis is charactered with increased infiltration of leukocytes in skin, including monocytes (CD11b⁺), neutrophils (CD11b⁺, Gr1⁺), macrophages (CD11b⁺, F4/80⁺), dendritic cells (CD11b⁺, CD11c⁺). The infiltration of these cells was decreased following Sauchinone treatment (Fig. 5F). Disrupted basal keratinocytes terminal differentiation in epidermis is the manifestation of psoriasis [38]. Sauchinone treatment reversed the decreased mRNA levels of Flg,Hrnr, Ivl, and Lor in skin of psoriasis mice (Fig. 5G), which are critical genes of epidermal differentiation complex (EDC) that regulates late epidermal differentiation [39]. The activation of keratinocytes worsens the inflammatory micro-environment in psoriatic skins. We also found that Sauchinone inhibited the activation of TNF-α/IFN-γ-stimulated HaCaT cells, indicated by decreased gene expression of cytokines, chemokines and proliferation markers (Fig. S3A). Taken together, these results suggested that Sauchinone cream alleviates IMQ induced murine psoriasis-like skin lesion.

Fig. 5.

Sauchinone alleviates IMQ induced murine psoriasis-like skin lesion. BALB/c mice were topically treated with IMQ cream for 7 d to induce psoriasis-like skin lesion. Sauchinone cream (4 %/2%/0%) and Calcipotriol was applied on lesional skin daily. (A) The schematic diagram of experiment design. (B) Body weights were monitored daily and shown as the percentage of initial body weight (n = 8). Green asterisk marks: 4 % Sau vs Model; Purple asterisk marks: 2 % Sau vs Model; Black asterisk marks: Calcipotriol vs Model. (C) The representative manifestations of the back skins on d 7 (end point). (D) Scales, thickness, and erythema scores (0–4) of the lesional skin were evaluated daily and the total scores were calculated (0–12) (n = 8). Green asterisk marks: 4 % Sau vs Model; Purple asterisk marks: 2 % Sau vs Model; Black asterisk marks: Calcipotriol vs Model. (E) The representative images of H&E-stained back skin and the quantification of epidermal thickness (n = 7). Scale bar: 100 µm. (F) The infiltration of monocytes (CD11b⁺), macrophages (CD11b⁺, F4/80⁺), dendritic cells (CD11b⁺, CD11c⁺) and neutrophils (CD11b⁺, Gr-1⁺) in skin was analyzed by FACS. (G) The transcription levels of Ivl, Lor, Flg and Hrnr were detected by RT-qPCR (n = 5). Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05, **P < 0.01 compared as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Sauchinone normalizes TGR5/PKA/CREB pathway and the aberrant macrophage polarization in psoriatic skin

We then evaluated whether Sauchinone is able to normalize TGR5/PKA/CREB pathway and modulate macrophage polarization in IMQ induced psoriasis. We found TGR5 level and CREB phosphorylation level were both decreased in the model group. Sauchinone treatment rectified TGR5 protein expression and CREB phosphorylation, indicating Sauchinone could normalize TGR5/PKA/CREB pathway in psoriasis (Fig. 6A). In serum, Sauchinone treatment reduced the levels of M1-like cytokines TNF-α, IL-6, IL-12, IL-1β and IFN-γ, and increased M2-like cytokine IL-10 (Fig. 6B). The similar results were also achieved in skin homogenates (Fig. 6C). Meanwhile, we observed that Sauchinone reserved the decreased transcription levels of M2-like markers including Ym-1, Cd301, Dectin-1, Arg-1, Irf-4 and Ppar-γ in skin tissues (Fig. 6D). In contrast, Sauchinone downregulated the protein levels of p-p65, p-IκB, p-STAT-1, NLPR3 and Pro Caspase-1 in skin homogenates (Fig. 6E), which are hallmarks of macrophage M1 polarization. Furthermore, FACS was used to precisely detect the influence of Sauchinone on the polarization of skin infiltrated macrophages. We found Sauchinone decreased the percentage of CD86⁺ macrophages (M1 markers), but increased the percentage of CD206⁺ or CD301⁺ macrophages (M2 markers) in skin (Fig. 7A). Likewise, through immunofluorescence assay, we could observe clearly that Sauchinone decreased the expression of M1 markers CD86 (Fig. 7B) and INOS (Fig. 7C) but increased the expression of M2 markers CD206 (Fig. 7D) that co-localized with F4/80⁺ macrophages in lesional skins. From all above, we verified the influence of Sauchinone on macrophages polarization in the mice model of IMQ-induced psoriasis.

Fig. 6.

Sauchinone normalizes TGR5/PKA/CREB pathway and regulates M1/M2 macrophages markers in skin of IMQ-induced psoriasis mice. (A) The protein levels of TGR5, PKA. p-CREB, CREB in skin tissues were detected by western blot. (B) Cytokines including IL-6, IL-12 TNF-α, IFN-γ, IL-17 and IL-10 levels in serum were measured by ELISA (n = 6). (C) Cytokines including IL-6, IL-12, TNF-α, IFN-γ, IL-1β and IL-10 levels in skin homogenates were measured by ELISA (n = 6). (D) The mRNA levels of M2 markers including Ym-1, Cd-301, Dectin-1, Arg-1, Irf-4 and Ppar-γ in skin tissues were measured by qPCR (n = 6). (E) The protein levels of M1 markers including p-STAT-1, STAT1, p-p65, p65, NLRP3, p-IκB, IκB, Pro Caspase-1 and ASC in skin tissues were detected by western blot. Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05, **P < 0.01 compared as indicated.

Fig. 7.

Sauchinone decreases the markers of M1 macrophages and increases the markers of M2 macrophages in skin infiltrated macrophages. (A) The percentages of M1-like (CD86⁺) macrophage and M2-like macrophage (CD206⁺ or CD301⁺) in skin infiltrated macrophages (CD11b⁺, F4/80⁺) were detected by FACS. (b-d) The expression of M1-like markers CD86 (B) and INOS (C) and M2-like marker CD206 (D) on skin infiltrated macrophages (stained with F4/80) were detected by immunofluorescence. Scale bar: 200 µm.

Sauchinone protectes against murine psoriasis in TGR5 dependent manner

To further verify our conjecture that the protective effects of Sauchinone on psoriasis are dependent on TGR5, we constructed IMQ induced murine psoriatic lesion model on Tgr5^-/- mice (Fig. 8A). Compared with WT mice, Tgr5^-/- mice manifested with similar weight changes (Fig. 8B), clinical symptoms (Fig. 8C) and PASI scores (Scales, thickness, erythema and total scores) (Fig. 8D) after IMQ stimulation. In accordance with the results in Fig. 4, Sauchinone cream exerted significant pharmacological actions on WT psoriasis mice. However, when it comes to Tgr5^-/- mice, the protective effects were all diminished. The images of HE staining slice also showed that Sauchinone cream failed to ameliorate histological changes and epidermal thickening in Tgr5^-/- mice (Fig. 8E). To unravel the influence of Sauchinone on macrophage M1 polarization in skin, we detected M1-like cytokines in skin homogenates. we found Sauchinone cream could reduce the levels of IL-6, IL-12, TNF-α and IFN-γ in WT mice with tendency but was not able to inhibit those in Tgr5^-/- mice (Fig. 8F). As for macrophage M2 polarization, globally, Tgr5^-/- mice exhibited lowered expression of M2 makers in response to IMQ challenge. However, Sauchinone cream could to some extent upregulate the transcription levels of Ym-1, Cd301, Cd206, Irf-4 and Ppar-γ in psoriatic skin tissues, though these effects were weaker than those in WT mice (Fig. 8G). Overall, these results supported that Sauchinone protected against murine psoriasis in TGR5 dependent manner.

Fig. 8.

Sauchinone alleviates IMQ induced murine psoriasis-like skin lesion. WT or Tgr5^-/- C57BL/6 mice were topically treated with IMQ cream for 6 d to induce psoriasis-like skin lesion. Sauchinone cream (4 %) was applied on lesional skin daily. (A) The schematic diagram of experiment design. (B) Body weights were monitored daily and shown as the percentage of initial body weight (n = 6). (C) The representative manifestations of the back skins on d 6 (end point). (D) Scales, erythema and thickness scores (0–4) of the lesional skin were evaluated daily and the total scores were calculated (0–12) (n = 6). Green asterisk marks: WT 4 % Sau vs WT Model; (E) The representative images of H&E-stained back skin and the quantification of epidermal thickness (n = 6). Scale bar: 100 µm. (F) Cytokines including IL-6, IL-12, TNF-α and IFN-γ levels in skin homogenates were measured by ELISA (n = 6). (G) The mRNA levels of M2 markers including Ym-1, Cd-301, Cd-206, Irf-4 and Ppar-γ in skin tissues were measured by qPCR  (n = 6). Data were shown as Mean ± SEM from triplicate measurements. *P < 0.05, **P < 0.01 compared as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Discussion

Saururus chinensis, which is a perennial herb that distributed widely in the northeastern regions of Asian, has long been utilized for treating gonorrhea, edema jaundice as well as other inflammatory diseases in China, Korean and Japan. Illustrating the therapeutic material basis and the mechanism of action of Saururus chinensis would open up new opportunities to promote its application worldwide. Sauchinone is a kind of diastereomeric lignan that extracted from Saururus chinensis. The existing study mainly focused on the anti-inflammatory activity of Sauchinone, especially in LPS stimulated macrophages [[29], [30], [31],40]. However, the exact target and mechanism of action still under investigation.

In this work, we for the first time identified Sauchinone as a novel TGR5 agonist without human/mouse species selectivity in in vitro TGR5 agonistic activity screening system and macrophages. TGR5 is a class A GPCR transducing signal through Gs-protein mediated cAMP accumulation [41]. We tested the TGR5 agonistic activity in HEK293 cells stably expressed with h/mTGR5 and CRE-driven luciferase reporter. Activation of CRE (cAMP response elements) would induce the expression of luciferase, and the status of CRE pathway can be sensitively reflected by detecting luciferase activity. Sauchinone treatment stimulated luciferase activity in HEK293 cells expressing h/mTGR5/CRE but not in HEK293 cells only expressing CRE, suggesting that Sauchinone activates CRE signal through TGR5 but not through other endogenously expressed GPCRs. Moreover, the results of CETSA in human/mouse macrophages proved the direct binding of Sauchinone towards TGR5. Also, Sauchinone increased the p-CREB in BMDM and THP-1 macrophages, again reflecting activation of TGR5 downstream cAMP signaling. These data strongly supported the agonistic activity of Sauchinone on TGR5 and we could reasonably suppose Gs protein is used by Sauchinone for cell signal transduction after activating TGR5.

Since TGR5 activation has been reported to remodel macrophage polarization [[12], [13], [14], [15], [16], [17]], we then studied the action of Sauchinone on macrophage polarization in BMDM and shown Sauchinone inhibits LPS plus IFN-γ induced M1 polarization and promotes IL-4 plus IL-13 induced M2 polarization. Next, we wonder whether Sauchinone modulates macrophage polarization through activating TGR5. With the use of Tgr5^-/- mice derived BMDM, and by introducing TGR5 specific inhibitor SBI-115 and TGR5 si-RNA, we verified that Sauchinone repressed macrophage M1 polarization in TGR5 dependent manner. However, blocking or knocking down TGR5 failed to reverse the promotion effect of Sauchinone on macrophage M2 polarization, suggesting Sauchinone facilitates macrophage M2 polarization in TGR5 independent manner. Indeed, it has only been proved that TGR5 activation could stabilize the M2 phenotype during differentiation of monocytes into macrophages [42] and promote the shift of macrophages from M1 to M2 phenotype in response to LPS stimulation [15]. Simultaneously, TGR5 deficiency or inhibition weakens IL-4 + IL-13 induced macrophage M2 polarization [17]. Though the above published data indicated the key role of TGR5 in monocyte differentiation and macrophage M2 polarization, it still lacks direct evidence to prove that activation of TGR5 would certainly promote macrophage M2 polarization triggered by IL-4 + IL-13. It is well known that natural products share the characteristics of multi-targets effects. Though we uncovered TGR5 as the target of Sauchinone in this work, we cannot rule out some other pathways that might also be involved in regulating macrophage M2 polarization. As a typical natural product derived from Saururus chinensis, works dealing with the target identification of Sauchinone is still in research. Sauchinone has been reported to activate AMPK and TGF-beta-activated kinase (TAK1) to increase macrophage phagocytosis of fluorescent Escherichia coli. [43]. Also, Sauchinone could inhibit IL-1β induced catabolism and hypertrophy in mouse chondrocytes to attenuate osteoarthritis via Nrf2/HO-1 and NF-κB pathways [25]. AMPK and Nrf2/HO-1 pathway have also been reported to participate in regulating macrophage polarization [[44], [45], [46], [47], [48], [49]]. Whereas, whether Sauchinone promotes M2 polarization through Nrf2/HO-1, AMPK or other undefined targets would be disclosed in our future work.

Previous studies revealed that activating TGR5 shows promising therapeutical actions on inflammatory diseases in mice [12,15,16,18]. The protective action of Sauchinone on inflammatory diseases models has also been reported [[22], [23], [24], [25], [26], [27], [28]]. However, studies dealing with the topical formulation of TGR5 agonist or Sauchinone in skin inflammatory diseases models is lacking. In this study, we prepared the cream formulation of Sauchinone and evaluated the pharmacological action of Sauchinone topical formulation in IMQ induced psoriasis, where aberrant macrophage polarization plays important pathological effects [[7], [8], [9], [10]]. We found Sauchinone cream relieved the clinical symptoms, reduced the PASI scores and decreased skin thickening in psoriatic mice. These effects was better than the positive drug Calcipotriol [50]. Monocytes and its derived macrophages, neutrophils and dendritic cells amplify inflammation in psoriasis by secreting cytokines and activating adaptive immune [51]. Increased expression of INOS and overproduction of NO, which is the hallmark of M1 macrophages, would increase the permeability of local tissue to induce immune cell infiltration [52]. Also, the chemokines secreted by M1 macrophages, including CXCL1, CXCL2, CCL1 and CCL2, would recruit neutrophils and dendritic cells through chemokines-chemokine receptors interaction [53]. On the other side, the activated M2 macrophage would secret anti-inflammatory cytokine IL-10 and exert phagocytosis to promote inflammation resolving [2]. Here we found Sauchinone prevented the infiltration of dendritic cells and neutrophils in skin tissue and we rationally considered the decreased infiltration of innate immune cells is closely related to the improved macrophage polarization. Apart from inflammation, psoriasis is charactered by abnormal differentiation and overactivation of keratinocytes [54]. We found that Sauchinone recovered the genes of EDC that regulate late epidermal differentiation. Also, Sauchinone inhibited the activation of TNF-α plus IFN-γ stimulated HaCaT cells. Whereas, since keratinocytes show limited TGR5 expression [55], how Sauchinone rectify keratinocytes activation and aberrant differentiation needs further exploration.

We then paid attention to the macrophage polarization in psoriatic skin. Briefly, Sauchinone reshaped the M1/M2 cytokines levels in both skin and serum. Moreover, Sauchinone downregulated the protein expression in M1 associated pathway and improved the expression of M2 related gene expression in skin homogenates. As reported, TGR5 mainly localized to dermal macrophages in skin [21]. To further precisely determine macrophage polarization in skin infiltrated macrophages, we sorted macrophages in skin tissue by FACS or labelled macrophages by immunofluorescence and indicated that Sauchinone decreased the percentage of M1 macrophages but increased the percentage M2 macrophages in psoriatic skin. This finding strongly supported that Sauchinone repressed the M1 polarization of skin infiltrated macrophage through targeting TGR5. In addition, activation of TGR5 has been proved to induce proliferation of cholangiocytes and result in hepatic cystogenesis in polycystic liver disease [56]. Also, high expression of TGR5 was monitored in intra- and extrahepatic cholangiocarcinoma and might promote the tumor progress in patients [57,58]. Therefore, topical administration of Sauchinone, a novel TGR5 agonist, is sufficient to reverse macrophage M1 polarization and its associated inflammation in skin without causing TGR5 overactivation induced side-effects in other organs.

To further explore whether the protective effects of Sauchinone cream on psoriasis are dependent on TGR5, we evaluated the therapeutic effects of Sauchinone cream on psoriasis model in Tgr5^-/- mice. We observed Tgr5^-/- mice showed similar symptoms compared to WT mice but Sauchinone cream exerted no benefit towards Tgr5^-/- mice. Simultaneously, Sauchinone cream failed to lower M1-like cytokines in skin homogenates. On the other hand, though Sauchinone could to some extent upregulate M2-like markers in psoriatic skin, the action was not comparable to that in WT mice. Notably, the action of Sauchinone on macrophage M2 polarization in Tgr5^-/- mice is in accordance with our in vitro results that Sauchinone promotes macrophage M2 polarization independent of TGR5. Nevertheless, it has been reported that the imbalance of macrophage M1/M2 polarization would promote psoriasis progression [2,3], we might speculate that the diminished pharmacological action of Sauchinone on psoriasis in Tgr5^-/- mice is owing to unrectified M1/M2 macrophage ratio.

Conclusion

Overall, this work identified Sauchinone as a novel TGR5 agonist that could inhibit macrophage M1 polarization dependent on TGR5 and promote macrophage M2 polarization independent on TGR5. In vivo, Sauchinone cream protected against IMQ induced psoriasis through activating TGR5 and regulated the polarization of skin infiltrated macrophages (Fig. 9). This work would lay the foundation for the potential use of Sauchinone topical formulations in multiple skin inflammatory diseases and help illustrate the therapeutic material basis of Saururus chinensis to expand its application worldwide.

Fig. 9.

Schematic diagram of how Sauchinone regulates macrophage polarization through targeting TGR5 and ameliorates IMQ induced psoriasis. Sauchinone inhibits TNF-α + IFN-γ induced macrophage M1 polarization in TGR5 dependent way and promotes IL-4 + IL-13 induced M2 polarization in TGR5 independent way. Mechanistically, Sauchinone agonizes TGR5 on macrophage and induces CREB phosphorylation to inhibit NF-κB activation induced M1 polarization. TGR5 antagonist SBI-115 or PKA inhibitor H89 abolishes the influence of Sauchinone on M1 polarization. Moreover, Sauchinone shows potential pharmacological activity on IMQ induced psoriasis by regulating macrophage polarization and inhibiting keratinocyte activation.

Compliance with Ethics Requirements

Tgr5^-/- mice with C57BL/6 background were gifted by Professor Xie Xin at Shanghai Institute of Materia Medica (SIMM) kindly. C57BL/6 and BALB/c mice (8-10 weeks, 20 g) were purchased from Shanghai Lingchang Biotechnology Co. Ltd. All animal care and experimental procedures were conducted in accordance with the ARRIVE guidelines, National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals, and were approved by the Bioethics Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (IACUC: 2024-01-TW-157).

CRediT authorship contribution statement

Hao-yu Wang: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology, Writing – original draft. Su-ling Huang: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology, Writing – original draft. Jing Ren: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Li-yan Peng: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Lin-rui Chen: Data curation, Investigation, Formal analysis, Visualization, Methodology. Lu-yao Qi: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Ke-han Zhu: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Chun-lan Feng: Resources, Supervision. Rong Zhou: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Yi-pei Gu: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Lu Cao: Conceptualization, Data curation, Investigation, Formal analysis, Visualization, Methodology. Ying Leng: Project administration, Supervision, Resources. Qin-shi Zhao: Project administration, Supervision, Resources. Wei Tang: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Resources, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: