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Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota, improving intestinal mucosal barrier function

Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,... Objective The orphan nuclear receptor Nur77 is an important factor regulating metabolism. Nur77 knockout mice become obese with age, but the cause of obesity in these mice has not been fully ascertained. We attempted to explain the cause of obesity in Nur77 knockout mice from the perspective of the gut microbiota and to investigate the inhibitory effect of calcipotriol combined with BRD9 inhibitor (iBRD9) on obesity. Methods Eight-week-old wild-type mice and Nur77 knockout C57BL/6J mice were treated with calcipotriol combined with iBRD9 for 12 weeks. Mouse feces were collected and the gut microbiota was assessed by analyzing 16S rRNA gene sequences. The bacterial abundance difference was analyzed, and the intestinal mucosal tight junction protein, antimicrobial peptide, and inflammatory cytokine mRNA levels of the colon and serum LPS and inflammatory cytokine levels were measured. Results Calcipotriol combined with iBRD9 treatment reduced the body weight and body fat percentage in Nur77 knockout mice. In the gut microbiota of Nur77 knockout mice, the relative abundances of Lachnospiraceae and Prevotellaceae decreased, and Rikenellaceae increased; while Rikenellaceae decreased after treatment (p < 0.05). Correspondingly, the mRNA levels of intestinal mucosal tight junction proteins (occludin (Ocln), claudin3 (Cldn3)) in the colons of Nur77 knockout mice were significantly decreased, and they increased significantly after treatment (p < 0.001). The mRNA levels of inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β)) were sig- nificantly increased in Nur77 knockout mice, and TNF-α and IL-6 levels were significantly decreased after treatment (p < 0.05, <0.01, or <0.001). The levels of serum LPS, TNF-α, and IL-1β in Nur77 knockout mice were significantly increased (p < 0.05). Serum LPS, TNF-α, and IL-6 levels were significantly decreased after treatment (p < 0.05 or <0.01). Conclusions Calcipotriol combined with iBRD9 can regulate the gut microbiota, improve intestinal mucosal barrier func- tion, reduce LPS absorption into the blood, and alleviate obesity in Nur77 knockout mice. Introduction The immune response is essential to protect the body from These authors contributed equally: Qingqing Lv, Aolin Yang physical, chemical, and biological damage. However, per- Supplementary information The online version of this article (https:// sistent low-grade inflammatory conditions can cause doi.org/10.1038/s41366-020-0564-0) contains supplementary damage to the body tissues and is the cause of metabolic material, which is available to authorized users. diseases such as obesity, diabetes, and other chronic non- * Difei Wang communicable diseases [1]. Nuclear receptor subfamily 4, dfwang@cmu.edu.cn group A (NR4A) is an important regulator of the inflam- matory response, which can directly promote the expression Nutrition Department, The First Hospital of China Medical University, Shenyang, Liaoning, China of FoxP3 transcription factor and is related to the produc- tion, differentiation, and maintenance of regulatory T (Treg) Department of Geriatric Endocrinology, The First Hospital of China Medical University, Shenyang, Liaoning, China cells [2], and T-cell-specific deletion of all NR4A family members causes significant multiorgan inflammation [3, 4]. Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning, China NR4A1 (also called Nur77) has no effect on FoxP3 1234567890();,: 1234567890();,: Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1053 transcription, and Nur77 knockout mice produce Treg cells kept in a controlled environment (12-h light-dark cycle, and cannot develop autoimmune diseases [5, 6]. Nur77 22 ± 1 °C), and provided with unlimited food (GB 14924.3- knockout mice become obese with age and have low-grade 2010 feed formula) and deionized water. All experimental inflammation [7, 8]. However, the cause of the systemic protocols were approved by the Animal Care and Use low-grade inflammation and obesity has not been fully Committee of China Medical University. explored. Seven-week-old wild-type C57BL/6J and Nur77 knockout The influence of the gut microbiota on energy metabo- male mice were randomly divided into two groups of seven lism has attracted considerable attention [9]; it can regulate mice each. The mice were adapted at 8 weeks of age. One metabolic homeostasis, and gut microbiota dysbiosis has group of the wild-type mice was separately injected intra- been proven to be associated with obesity [10, 11]. Studies peritoneally with the vehicle (30% hydroxypropyl- have shown that there is a significant difference in the β-cyclodextrin), and the other group was injected with calci- composition of the gut microbiota between hereditary potriol combined with iBRD9. The Nur77 knockout mice obese (ob/ob) mice and wild-type mice [12], and additional were treated in the same way. The four groups were the wild- studies have found that the composition of the gut micro- type mouse vehicle control group (WT-V), wild-type mouse biota is affected by genotype [13–15]. Therefore, we sus- treatment (calcipotriol combined with iBRD9) group (WT-T), pect that Nur77 deficiency may cause changes in the gut Nur77 knockout mouse vehicle control group (KO-V), and microbiota and attempted to elucidate the cause of obesity Nur77 knockout mouse treatment group (KO-T). Calcipotriol in Nur77 knockout mice from the perspective of the gut combined with iBRD9 was administered three times a week microbiota. for a total of 12 weeks. The doses were [23] calcipotriol at Cross-sectional studies show that vitamin D deficiency is 60 µg/kg, and iBRD9 at 10 mg/kg. positively associated with obesity [16], but vitamin D sup- Fresh feces were collected before the mice were sacri- plementation does not effectively reduce obesity [17–19]. ficed, frozen in liquid nitrogen, stored in a freezer at Mechanistic research shows that calcitriol increased lipolysis −80 °C, and taken out before sequencing. After the mice and energy consumption and reduced lipid content in adi- were euthanized, blood and colon tissues were obtained. pocytes in vivo in part through regulation of β-oxidation and The blood was centrifuged at 3000 × g for 25 min at 4 °C, UCP expression regulated by vitamin D receptor (VDR) and the supernatant serum was collected and stored in a [20–22]. In addition, interest in the anti-inflammatory poten- −80 °C freezer for use. tial of vitamin D continues to grow. An interesting study showed that VDR shuttles between BAF and PBAF com- Body weight and fat mass measurement plexes in a ligand-dependent manner, and iBRD9 cooperates with VDR ligand to favor PBAF complex binding, which Body weights were monitored weekly. Fat mass and lean enhances chromatin accessibility at consensus VDR binding mass were determined in the last week using a Bruker elements to modulate the expression of key inflammatory Minispec LF50. response genes [23]. The anti-inflammatory effect of vitamin D and iBRD9 combined is worthy of further research. Biochemical analyses Therefore, we intended to use the VDR ligand calcipo- triol and iBRD9 to intervene in Nur77 knockout mice to Mouse serum triacylglycerol (TG) and total cholesterol explore the effects of vitamin D on obesity in Nur77 (TC) were measured by an enzyme colorimetric assay using knockout mice and investigated changes in the gut a commercial assay kit (Jiancheng Bioengineering Research microbiota. Institute Co., Ltd, Nanjing, China). Mouse serum leptin (LEP), TNF-α, IL-6, and IL-1β concentrations were deter- mined using an enzyme-linked immunosorbent assay kit Materials and methods (Xinfan Technology Co., Ltd, Shanghai, China). A TM Pierce Color Rendering Endotoxin Quantitation Kit Animals and treatments (88282, Thermo) was used to detect lipopolysaccharide (LPS) levels in serum by using the limulus amebocyte Nur77 knockout mice with C57BL/6J as the background lysate assay. A blood glucose meter (NC, Roche, Germany) were produced in the Jackson Laboratory (Bar Harbor, was used to measure tail vein blood glucose after fasting ME). Fourteen male wild-type (C57BL/6J) mice that were overnight. Serum calcium (Ca) and phosphorus (P) were 3 weeks old were purchased from Changsheng Bio- detected using the methyl thymol blue method and the technology Co., Ltd. (Liaoning, China), housed in trans- phosphomolybdic acid method, respectively (Xinfan Tech- parent plastic feeding cages (seven mice per feeding cage), nology Co., Ltd, Shanghai, China). 1054 Q. Lv et al. Fecal 16S rRNA analysis synthesized from 500 ng of total RNA using an iScript cDNA Synthesis Kit (BIO-RAD, USA). Real-time quanti- Total genome DNA from samples was extracted using the tative PCR (qPCR) was performed using a Power Up SYBR CTAB/SDS method. DNA concentration and purity were Green master mix (Applied Biosystems, USA) and an monitored on 1% agarose gels. According to the con- LC480 II (Roche) qPCR instrument. The qPCR results were −ΔΔCt centration, DNA was diluted to 1 ng/µL using sterile water. calculated using the 2 method. Primer sequences are The extracted DNA from each sample was used as template shown in Supplementary Table 1. to amplify the V3 + V4 region of 16S rRNA genes of distinct regions (16S V3 + V4) with specific primers (341F: Statistical analysis 5′-CCTAYGGGRBGCASCAG-3′, 806R: 5′-GGACTA CNNGGGTATCTAAT-3′). All PCR reactions were carried Experimenters were blind to the groups during data analy- out in 30 µL reactions with 15 µL of Phusion High-Fidelity sis. No animals were excluded from the analyses. Statistical PCR Master Mix (New England Biolabs). PCR products analysis was performed using SPSS v17.0 (Chicago, IL, were mixed with the same volume of 1× loading buffer USA), and p < 0.05 was considered significant in all cases. (containing SYBR green) and detected with electrophoresis Data are expressed as the mean ± standard error of the mean on a 2% agarose gel. PCR products were mixed in equi- (mean ± SEM). For comparisons among multiple groups, density ratios. Then, the mixture of PCR products was one-way analysis of variance (ANOVA) and the Bonferroni TM purified with a GeneJET Gel Extraction Kit (Thermo post hoc test was used to compare the two groups. For 16S Scientific). Sequencing libraries were generated using Ion rRNA sequencing data, statistical tests were performed in Plus Fragment Library Kit 48 rxns (Thermo Scientific) the R programming environment [24]. following the manufacturer’s recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific). Finally, the library was sequenced on Results TM an Ion S5 XL platform and 600 bp single-end reads were generated. Nur77 knockout mice gain weight, and weight loss occurs after treatment with calcipotriol combined Quantification of genes expression in colon tissue with iBRD9 Total RNA was isolated from ~40 mg of colon tissue using After 17 weeks of age, Nur77 knockout mice showed a TRIzol reagent according to the manufacturer’s instructions significant increase in body weight, which decreased sig- (Life Technologies, CA) and quantified by a Nano nificantly after treatment to a level that was close to the Photometer-N50 (Implen, Germany). cDNA was weight of the wild-type mice (p < 0.05) (Fig. 1a). However, A B Fig. 1 The effects of 33 WT-V 4.0 calcipotriol and iBRD9 on WT-T # # body weight, food intake, and # KO-V ** ** body composition of Nur77 ** KO-T 3.5 knockout mice. a Weight gain curves; b food intake curves; c the percentage of body fat 3.0 mass; d the percentage of body lean mass (n = 7/group). Data are expressed as the mean ± 2.5 SEM. *p < 0.05; **p < 0.01 8 10 12 14 16 18 20 8 101214161820 versus WT-V; p < 0.05 versus Week Week KO-V. C D 18 80 ** 9 40 0 0 WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T Body weight (g) Fat mass/body weight (%) Food intake (g) Lean mass/body weight (%) Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1055 A B Fig. 2 Calcipotriol and iBRD9 treatment effect on the gut microbiota structure in Nur77 knockout mice. Alpha diversity analysis: the ACE estimator (a), Chao1 estimator (b), Shannon index (c), and Simpson index (d) 450 were used for evaluation. The results are the means ± SEM (n = 7). Data were analyzed by 1-factor ANOVA, followed by the Tukey–Kramer multiple comparison test. Venn diagram (e) showing the unique and WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T shared OTUs in the gut microbiota between groups. C D Plots were generated using a 0.98 weighted UniFrac distance- based PCoA (f). 6.5 0.96 0.94 6.0 0.92 5.5 0.90 WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T EF PCoA - PC1 VS PC2 KO-V KO-T WT-V WT-V WT-T WT-T 80 69 0.1 KO-V KO-T 20 7 24 28 22 24 0.0 10 8 -0.1 17 19 -0.2 0.0 0.2 0.4 PC1 (60.15%) there was no significant difference in mouse food intake difference in fasting blood glucose (FBG), TG, TC, Ca, and (Fig. 1b). The difference in body weight was reflected P (Supplementary Table 2). mainly in body fat, as shown in Fig. 1c; Nur77 knockout mice showed a significant increase in body fat percentage The diversity of gut microbiota in calcipotriol and that significantly decreased after treatment (p < 0.05 or iBRD9-treated Nur77 knockout mice <0.01), and there was no significant difference in the lean mass ratio in mice (Fig. 1d). Community richness was determined using the ACE esti- mator and the Chao1 estimator (Fig. 2a, b), and community Serum biochemical parameters of calcipotriol and diversity was estimated using the Shannon index and the iBRD9-treated Nur77 knockout mice Simpson index (Fig. 2c, d). Community richness and diversity were not significantly different between groups The LEP level was significantly increased in Nur77 according to the Wilcoxon rank sum test. knockout mice and was significantly reduced after treatment For 99.88% of the operational taxonomic units (OTUs), (p < 0.05 or <0.01). In addition, there was no significant based on the common and unique OTUs between the four ACE Shannon Simpson Chao1 PC2 (7.48%) 1056 Q. Lv et al. A Cladogram B Cladogram Fig. 3 Specific biomarkers of calcipotriol and iBRD9-treated a: f_Prevotellaceae a: f_Rikenellaceae b: f_Rikenellaceae KO-V KO-V Nur77 knockout mice. c: f_Lachnospiraceae WT-V KO-T a, b Cladogram representation of the differentially abundant families and genera. The root of the cladogram denotes the domain bacteria. The taxonomic levels of the phylum and class are labeled, while family and genus are abbreviated, with the colors indicating the greatest abundance. The size of each node represents their relative abundance. c, d LEfSe analysis shows differentially abundant genera as biomarkers determined using the Kruskal–Wallis test (p < 0.05) CD with an LDA score > 4. KO-V WT-V KO-V KO-T Correlation between the relative f_Lachnospiraceae abundance of Rikenellaceae and g_Parvibacter biological parameters after g_Alistipes g_Alistipes calcipotriol and iBRD9 f_Rikenellaceae f_Rikenellaceae treatment. e Fat mass (kg) and f_Prevotellaceae f body fat (%) exhibited significant correlations with levels of Rikenellaceae in the LDA SCORE (log 10) LDA SCORE (log 10) fecal microbiota. p < 0.05 based EF on Spearman rank correlation 0.15 0.15 analysis, n = 28. r =0.419 r =0.398 P=0.027 P=0.036 0.10 0.10 0.05 0.05 0.00 0.00 0 5 10 15 20 Fat mass (kg) Body fat (%) groups, a Wayne diagram is shown in Fig. 2e. The micro- Prevotellaceae (Fig. 3a, c), and the biomarker of KO- bial community composition of different samples was V–KO-T was Rikenellaceae (Fig. 3b, d). Alistipes was the compared by principal coordinate analysis (PCoA) (Fig. 2f). genus of Rikenellaceae. Rikenellaceae is positively corre- A significant difference was found between KO-V and WT- lated with body fat mass (r = 0.419, p = 0.027) and fat V, and there was a significant difference between KO-T and percentage (r = 0.398, p = 0.036) (Fig. 3e, f). KO-V(p < 0.05 or <0.01). Alteration of Lachnospiraceae and Akkermansiaceae LEfSe analysis in calcipotriol and iBRD9-treated abundance in calcipotriol and iBRD9-treated Nur77 Nur77 knockout mice knockout mice Linear discriminant analysis (LDA) effect size (LEfSe) Figure 4a, b shows gut microbiota constituents with the top analysis was used to detect species abundance data between ten relative abundances at the phylum and family levels. At groups by the rank sum test to detect different species the phylum level, Firmicutes and Bacteroidetes together within different groups, and the magnitude of the effects of accounted for a major portion of the bacterial population in the different species biomarkers was assessed by LDA. At all samples (93.24–97.43%). The Nur77 knockout mouse the family level, we found that the biomarkers of WT- had an increased relative abundance of Bacteroides that V–KO-V were Lachnospiraceae, Rikenellaceae, and decreased after treatment (Fig. 4a). The changes in the Rikenellaceae (%) Rikenellaceae (%) Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1057 A B Family Phylum 1.0 1.0 Others Others Chlamydiae Tannerellaceae Tenericutes Marinifilaceae Verrucomicrobia Lactobacillaceae Deferribacteres Rikenellaceae 0.5 Actinobacteria 0.5 Helicobacteraceae Melainabacteria Ruminococcaceae Proteobacteria Bacteroidaceae unidentified_Bacteria Prevotellaceae Firmicutes Lachnospiraceae Bacteroidetes Muribaculaceae 0.0 0.0 WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T Family Phylum Anaeroplasmataceae Tenericutes 0 Akkermansiaceae Verrucomicrobia Deferribacteraceae Deferribacteres -5 Nocardiaceae Bifidobacteriaceae Actinobacteria Eggerthellaceae Atopobiaceae Melainabacteria Melainabacteria Woeseiaceae Burkholderiaceae Moraxellaceae Proteobacteria Enterobacteriaceae Desulfovibrionaceae Rhizobiaceae Helicobacteraceae unidentified_Bacteria Clostridiales Veillonellaceae Erysipelotrichaceae Ruminococcaceae Peptostreptococcaceae Firmicutes Peptococcaceae Lachnospiraceae Christensenellaceae Streptococcaceae Lactobacillaceae Sphingomonadaceae Tannerellaceae Rikenellaceae Prevotellaceae Bacteroidetes Muribaculaceae Marinifilaceae Bacteroidaceae Fig. 4 Relative abundance distribution of gut microbiota con- relative abundances at the family level. c Relative abundances at the stituents at the phylum and family levels. a The gut microbiota family level associated with the top ten greatest abundances at the constituents with the top ten greatest relative abundances at the phy- phylum level that were altered in Nur77 knockout mice and reversed lum level. b The gut microbiota constituents with the top ten greatest by interventions. biomarker strains Lachnospiraceae, Rikenellaceae, and mucosal tight junction proteins Ocln and Cldn3 was sig- Prevotellaceae found by LEfSe analysis are shown in Fig. nificantly decreased in Nur77 knockout mice (Fig. 5a), and 4b. The relative abundance of Lachnospiraceae in Nur77 the expression of Ocln and Cldn3 was significantly knockout mice was low, and the relative abundance of increased after treatment (p < 0.001). Rikenellaceae and Prevotellaceae was high; the relative For the antibacterial peptides (Fig. 5b), namely, lyso- abundance of Lachnospiraceae increased after treatment, zyme C (Lyz1), regenerating islet-derived IIIγ (Reg3γ), the relative abundance of Rikenellaceae decreased. The phospholipase A2 group II (Pla2g2), and angiopoietin 4 relative abundance of Akkermansiaceae in Nur77 knockout (Ang4). There were no significant differences in anti- mice was low and increased after treatment (Fig. 4c). bacterial peptides between the four groups of mice. Among the mRNA expression levels of colonic inflam- Calcipotriol combined with iBRD9 protected the gut matory cytokines, TNF-α, IL-6, and IL-1β were sig- intestinal barrier integrity and function of Nur77 nificantly increased in Nur77 knockout mice (p < 0.05, knockout mice <0.01, or <0.001). TNF-α and IL-6 expression was sig- nificantly reduced after treatment (p < 0.05 or <0.001). Then we examined the mRNA levels of tight junction To further verify changes in intestinal mucosal barrier proteins, antimicrobial peptides, and inflammatory cyto- function, we measured serum LPS and inflammatory cyto- kines in the colon. The mRNA expression of the intestinal kine levels in mice. The serum LPS levels of Nur77 WT-V1 WT-V2 WT-V3 WT-V4 WT-V5 WT-V6 WT-V7 WT-T1 WT-T2 WT-T3 WT-T4 WT-T5 WT-T6 WT-T7 KO-V1 KO-V2 KO-V3 KO-V4 KO-V5 KO-V6 KO-V7 KO-T1 KO-T2 KO-T3 KO-T4 KO-T5 KO-T6 KO-T7 Relative abundance Relative abundance 1058 Q. Lv et al. A B Fig. 5 The effects of WT-V calcipotriol combined with iBRD9 on the expression of WT-T colon tight junction proteins, 3 KO-V ### colon antimicrobial peptides, KO-T *** and colon inflammatory ### cytokines in Nur77 knockout *** mice. The mRNA expression of tight junction proteins (a), antimicrobial peptides (b), and inflammatory cytokines (c)in 0 0 the colon (n = 7/group). Data Cldn3 Ocln ZO-1 Reg3γ Pla2g2 Lyz1 Ang4 are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; Tight junction proteins Antimicrobial peptides ***p < 0.001 versus WT-V; # ### p < 0.05; p < 0.001 versus KO-V. ** # 6 * *** ### TNF-α IL-6 IL-1β Inflammatory cytokines A B ## Fig. 6 The serum levels of 20 * 25 # lipopolysaccharide and inflammatory cytokines in 16 20 calcipotriol- and iBRD9- 12 15 treated Nur77 knockout mice. Serum levels of lipopolysaccharide (a), TNF-α (b), IL-6 (c), and IL-1β (d)(n = 7/group). Data are expressed as the mean ± SEM. *p < 0.05 0 # ## WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T versus WT-V; p < 0.05; p < 0.01 versus KO-V. C D 200 120 * WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T knockout mice were significantly increased compared with Discussion those of wild-type mice and decreased significantly after treatment (p < 0.05 or <0.01) (Fig. 6a). The serum levels of In this study, we used Nur77 knockout mice and assessed TNF-α and IL-1β in Nur77 knockout mice increased sig- the effects of calcipotriol combined with iBRD9; we found nificantly (p < 0.05) (Fig. 6b, d). After treatment, the TNF-α that Nur77 knockout mice showed a significant increase in and IL-6 levels were significantly decreased (p < 0.05) body weight after 17 weeks of age, consistent with the lit- (Fig. 6b, c). erature [7]. After treatment with calcipotriol combined with Relative expression of mRNA Relative expression of mRNA IL-6 (pg/mL) LPS (EU/mL) Relative expression of mRNA IL-1β (pg/mL) TNF-α (ng/L) Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1059 iBRD9, the body weight of Nur77 knockout mice was due to the increased intestinal permeability and destruction significantly reduced. This change in body weight was of tight junction proteins attached to epithelial cells, derived from changes in body fat rather than lean weight. increasing portal vein and systemic plasma LPS con- We explored changes in the gut microbiota by 16S rRNA centrations [43]. Therefore, we measured the level of serum sequencing. The results showed that Nur77 deficiency had a LPS, and the serum LPS level of Nur77 knockout mice was significant effect on the gut microbiota composition, while increased significantly but decreased significantly after calcipotriol combined with iBRD9 treatment had a sig- treatment. This finding supported the occurrence of damage nificant effect on the composition of gut microbiota in only of the intestinal mucosal barrier in Nur77 knockout mice Nur77 knockout mice but not wild-type mice. Bacteroidetes and the improvement of intestinal mucosal barrier function and Firmicutes accounted for the majority of the bacterial after treatment with calcipotriol combined with iBRD9. The population (93.24–97.43%), and the Firmicutes/Bacter- increase in LPS absorbed into the blood may be one of the oidetes ratio in Nur77 knockout mice was decrease. How- causes of low-grade inflammation leading to obesity [44– ever, there is currently no consensus on the relationship 46]. The serum levels of inflammatory factors in our between the Firmicutes/Bacteroidetes ratio and obesity experiments indicated that low-grade inflammation occurs [25, 26]. We were pleased to find that the Nur77 knockout in Nur77 knockout mice, and the level of inflammation mice had a low relative abundance of Lachnospiraceae and decreased after treatment, which corresponded to LPS levels Akkermansiaceae. After calcipotriol combined with iBRD9 and was consistent with colon mRNA expression levels. treatment, their abundances increased. Lachnospiraceae Studies have shown that obesity is positively associated belongs to Firmicutes and degrades dietary fiber to produce with low-grade inflammation and that obesity can be short-chain fatty acids, which enhance intestinal barrier effectively alleviated by reducing inflammation [47, 48]. function by regulating tight junction proteins and mucins We applied calcipotriol combined with iBRD9 to reduce [27, 28]. Akkermansiaceae plays an important role in body weight by reducing the level of inflammation in Nur77 maintaining intestinal homeostasis, and it has been exten- knockout mice. sively studied in diseases such as obesity and inflammatory Our results indicate that changes in the gut microbiota of bowel disease [29–31]. In addition, we detected four groups Nur77 knockout mice contribute to obesity. Furthermore, of differentially abundant bacteria, including Rikenellaceae, the expression of intestinal mucosal tight junction protein- and studies show that Rikenellaceae abundance increases in related genes is reduced, and serum LPS concentration and high-fat-fed mice [32, 33] and that Rikenellaceae is posi- inflammatory factor levels are increased. Calcipotriol com- tively correlated with body fat percentage and fat mass bined with iBRD9 can regulate the gut microbiota, improve (Fig. 3e, f), which is consistent with reports in humans [34]. intestinal mucosal barrier function, reduce LPS absorption Previous studies have shown that activation of the into the blood, and alleviate inflammation and obesity. This Nur77/RXR heterodimer is responsible for reducing study clarified the causes of low-grade inflammation and monocyte-mediated inflammation in the intestine [35] and obesity in Nur77 knockout mice and demonstrated the that Nur77 has an important protective effect on the therapeutic effect of calcipotriol combined with iBRD9 on development of inflammatory bowel disease [36, 37]. obesity. The specific mechanism of Nur77 knockout mice Therefore, it is speculated that Nur77 knockout mice may gut microbiota dysbiosis, and how calcipotriol combined have undetected intestinal damage. Next, we examined the with iBRD9 regulates the gut microbiota have not been mRNA levels of tight junction proteins, antimicrobial pep- explored in depth, but it is a subject worth studying. tides, and inflammatory cytokines in the colon. The mRNA expression levels of the tight junction proteins Cldn3, Ocln, Acknowledgements This work was supported by the local develop- ment foundation of science and technology guided by the central and ZO-1 can reflect intestinal mucosal barrier function commission (2016007024), the science and technology project of [30, 38–40], and the results showed that the intestinal Shenyang (Z18-5-104), and the National Natural Science Foundation mucosal barrier function of Nur77 knockout mice was of China (31570819). impaired and that there was improvement after treatment with calcipotriol combined with iBRD9. The increased Compliance with ethical standards mRNA expression of colonic inflammatory cytokines observed in Nur77 knockout mice decreased after calcipo- Conflict of interest The authors declare that they have no conflict of interest. triol combined with iBRD9 treatment, suggesting that similar changes may occur in serum inflammation factors. Publisher’s note Springer Nature remains neutral with regard to Studies in the literature have shown that increased LPS jurisdictional claims in published maps and institutional affiliations. levels can induce a large number of proinflammatory responses and inflammatory cytokine release by activating Open Access This article is licensed under a Creative Commons Toll-like receptors 2, 4, and 5 [41, 42]. This effect may be Attribution 4.0 International License, which permits use, sharing, 1060 Q. Lv et al. adaptation, distribution and reproduction in any medium or format, as 16. 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Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota, improving intestinal mucosal barrier function

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10.1038/s41366-020-0564-0
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Abstract

Objective The orphan nuclear receptor Nur77 is an important factor regulating metabolism. Nur77 knockout mice become obese with age, but the cause of obesity in these mice has not been fully ascertained. We attempted to explain the cause of obesity in Nur77 knockout mice from the perspective of the gut microbiota and to investigate the inhibitory effect of calcipotriol combined with BRD9 inhibitor (iBRD9) on obesity. Methods Eight-week-old wild-type mice and Nur77 knockout C57BL/6J mice were treated with calcipotriol combined with iBRD9 for 12 weeks. Mouse feces were collected and the gut microbiota was assessed by analyzing 16S rRNA gene sequences. The bacterial abundance difference was analyzed, and the intestinal mucosal tight junction protein, antimicrobial peptide, and inflammatory cytokine mRNA levels of the colon and serum LPS and inflammatory cytokine levels were measured. Results Calcipotriol combined with iBRD9 treatment reduced the body weight and body fat percentage in Nur77 knockout mice. In the gut microbiota of Nur77 knockout mice, the relative abundances of Lachnospiraceae and Prevotellaceae decreased, and Rikenellaceae increased; while Rikenellaceae decreased after treatment (p < 0.05). Correspondingly, the mRNA levels of intestinal mucosal tight junction proteins (occludin (Ocln), claudin3 (Cldn3)) in the colons of Nur77 knockout mice were significantly decreased, and they increased significantly after treatment (p < 0.001). The mRNA levels of inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β)) were sig- nificantly increased in Nur77 knockout mice, and TNF-α and IL-6 levels were significantly decreased after treatment (p < 0.05, <0.01, or <0.001). The levels of serum LPS, TNF-α, and IL-1β in Nur77 knockout mice were significantly increased (p < 0.05). Serum LPS, TNF-α, and IL-6 levels were significantly decreased after treatment (p < 0.05 or <0.01). Conclusions Calcipotriol combined with iBRD9 can regulate the gut microbiota, improve intestinal mucosal barrier func- tion, reduce LPS absorption into the blood, and alleviate obesity in Nur77 knockout mice. Introduction The immune response is essential to protect the body from These authors contributed equally: Qingqing Lv, Aolin Yang physical, chemical, and biological damage. However, per- Supplementary information The online version of this article (https:// sistent low-grade inflammatory conditions can cause doi.org/10.1038/s41366-020-0564-0) contains supplementary damage to the body tissues and is the cause of metabolic material, which is available to authorized users. diseases such as obesity, diabetes, and other chronic non- * Difei Wang communicable diseases [1]. Nuclear receptor subfamily 4, dfwang@cmu.edu.cn group A (NR4A) is an important regulator of the inflam- matory response, which can directly promote the expression Nutrition Department, The First Hospital of China Medical University, Shenyang, Liaoning, China of FoxP3 transcription factor and is related to the produc- tion, differentiation, and maintenance of regulatory T (Treg) Department of Geriatric Endocrinology, The First Hospital of China Medical University, Shenyang, Liaoning, China cells [2], and T-cell-specific deletion of all NR4A family members causes significant multiorgan inflammation [3, 4]. Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning, China NR4A1 (also called Nur77) has no effect on FoxP3 1234567890();,: 1234567890();,: Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1053 transcription, and Nur77 knockout mice produce Treg cells kept in a controlled environment (12-h light-dark cycle, and cannot develop autoimmune diseases [5, 6]. Nur77 22 ± 1 °C), and provided with unlimited food (GB 14924.3- knockout mice become obese with age and have low-grade 2010 feed formula) and deionized water. All experimental inflammation [7, 8]. However, the cause of the systemic protocols were approved by the Animal Care and Use low-grade inflammation and obesity has not been fully Committee of China Medical University. explored. Seven-week-old wild-type C57BL/6J and Nur77 knockout The influence of the gut microbiota on energy metabo- male mice were randomly divided into two groups of seven lism has attracted considerable attention [9]; it can regulate mice each. The mice were adapted at 8 weeks of age. One metabolic homeostasis, and gut microbiota dysbiosis has group of the wild-type mice was separately injected intra- been proven to be associated with obesity [10, 11]. Studies peritoneally with the vehicle (30% hydroxypropyl- have shown that there is a significant difference in the β-cyclodextrin), and the other group was injected with calci- composition of the gut microbiota between hereditary potriol combined with iBRD9. The Nur77 knockout mice obese (ob/ob) mice and wild-type mice [12], and additional were treated in the same way. The four groups were the wild- studies have found that the composition of the gut micro- type mouse vehicle control group (WT-V), wild-type mouse biota is affected by genotype [13–15]. Therefore, we sus- treatment (calcipotriol combined with iBRD9) group (WT-T), pect that Nur77 deficiency may cause changes in the gut Nur77 knockout mouse vehicle control group (KO-V), and microbiota and attempted to elucidate the cause of obesity Nur77 knockout mouse treatment group (KO-T). Calcipotriol in Nur77 knockout mice from the perspective of the gut combined with iBRD9 was administered three times a week microbiota. for a total of 12 weeks. The doses were [23] calcipotriol at Cross-sectional studies show that vitamin D deficiency is 60 µg/kg, and iBRD9 at 10 mg/kg. positively associated with obesity [16], but vitamin D sup- Fresh feces were collected before the mice were sacri- plementation does not effectively reduce obesity [17–19]. ficed, frozen in liquid nitrogen, stored in a freezer at Mechanistic research shows that calcitriol increased lipolysis −80 °C, and taken out before sequencing. After the mice and energy consumption and reduced lipid content in adi- were euthanized, blood and colon tissues were obtained. pocytes in vivo in part through regulation of β-oxidation and The blood was centrifuged at 3000 × g for 25 min at 4 °C, UCP expression regulated by vitamin D receptor (VDR) and the supernatant serum was collected and stored in a [20–22]. In addition, interest in the anti-inflammatory poten- −80 °C freezer for use. tial of vitamin D continues to grow. An interesting study showed that VDR shuttles between BAF and PBAF com- Body weight and fat mass measurement plexes in a ligand-dependent manner, and iBRD9 cooperates with VDR ligand to favor PBAF complex binding, which Body weights were monitored weekly. Fat mass and lean enhances chromatin accessibility at consensus VDR binding mass were determined in the last week using a Bruker elements to modulate the expression of key inflammatory Minispec LF50. response genes [23]. The anti-inflammatory effect of vitamin D and iBRD9 combined is worthy of further research. Biochemical analyses Therefore, we intended to use the VDR ligand calcipo- triol and iBRD9 to intervene in Nur77 knockout mice to Mouse serum triacylglycerol (TG) and total cholesterol explore the effects of vitamin D on obesity in Nur77 (TC) were measured by an enzyme colorimetric assay using knockout mice and investigated changes in the gut a commercial assay kit (Jiancheng Bioengineering Research microbiota. Institute Co., Ltd, Nanjing, China). Mouse serum leptin (LEP), TNF-α, IL-6, and IL-1β concentrations were deter- mined using an enzyme-linked immunosorbent assay kit Materials and methods (Xinfan Technology Co., Ltd, Shanghai, China). A TM Pierce Color Rendering Endotoxin Quantitation Kit Animals and treatments (88282, Thermo) was used to detect lipopolysaccharide (LPS) levels in serum by using the limulus amebocyte Nur77 knockout mice with C57BL/6J as the background lysate assay. A blood glucose meter (NC, Roche, Germany) were produced in the Jackson Laboratory (Bar Harbor, was used to measure tail vein blood glucose after fasting ME). Fourteen male wild-type (C57BL/6J) mice that were overnight. Serum calcium (Ca) and phosphorus (P) were 3 weeks old were purchased from Changsheng Bio- detected using the methyl thymol blue method and the technology Co., Ltd. (Liaoning, China), housed in trans- phosphomolybdic acid method, respectively (Xinfan Tech- parent plastic feeding cages (seven mice per feeding cage), nology Co., Ltd, Shanghai, China). 1054 Q. Lv et al. Fecal 16S rRNA analysis synthesized from 500 ng of total RNA using an iScript cDNA Synthesis Kit (BIO-RAD, USA). Real-time quanti- Total genome DNA from samples was extracted using the tative PCR (qPCR) was performed using a Power Up SYBR CTAB/SDS method. DNA concentration and purity were Green master mix (Applied Biosystems, USA) and an monitored on 1% agarose gels. According to the con- LC480 II (Roche) qPCR instrument. The qPCR results were −ΔΔCt centration, DNA was diluted to 1 ng/µL using sterile water. calculated using the 2 method. Primer sequences are The extracted DNA from each sample was used as template shown in Supplementary Table 1. to amplify the V3 + V4 region of 16S rRNA genes of distinct regions (16S V3 + V4) with specific primers (341F: Statistical analysis 5′-CCTAYGGGRBGCASCAG-3′, 806R: 5′-GGACTA CNNGGGTATCTAAT-3′). All PCR reactions were carried Experimenters were blind to the groups during data analy- out in 30 µL reactions with 15 µL of Phusion High-Fidelity sis. No animals were excluded from the analyses. Statistical PCR Master Mix (New England Biolabs). PCR products analysis was performed using SPSS v17.0 (Chicago, IL, were mixed with the same volume of 1× loading buffer USA), and p < 0.05 was considered significant in all cases. (containing SYBR green) and detected with electrophoresis Data are expressed as the mean ± standard error of the mean on a 2% agarose gel. PCR products were mixed in equi- (mean ± SEM). For comparisons among multiple groups, density ratios. Then, the mixture of PCR products was one-way analysis of variance (ANOVA) and the Bonferroni TM purified with a GeneJET Gel Extraction Kit (Thermo post hoc test was used to compare the two groups. For 16S Scientific). Sequencing libraries were generated using Ion rRNA sequencing data, statistical tests were performed in Plus Fragment Library Kit 48 rxns (Thermo Scientific) the R programming environment [24]. following the manufacturer’s recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific). Finally, the library was sequenced on Results TM an Ion S5 XL platform and 600 bp single-end reads were generated. Nur77 knockout mice gain weight, and weight loss occurs after treatment with calcipotriol combined Quantification of genes expression in colon tissue with iBRD9 Total RNA was isolated from ~40 mg of colon tissue using After 17 weeks of age, Nur77 knockout mice showed a TRIzol reagent according to the manufacturer’s instructions significant increase in body weight, which decreased sig- (Life Technologies, CA) and quantified by a Nano nificantly after treatment to a level that was close to the Photometer-N50 (Implen, Germany). cDNA was weight of the wild-type mice (p < 0.05) (Fig. 1a). However, A B Fig. 1 The effects of 33 WT-V 4.0 calcipotriol and iBRD9 on WT-T # # body weight, food intake, and # KO-V ** ** body composition of Nur77 ** KO-T 3.5 knockout mice. a Weight gain curves; b food intake curves; c the percentage of body fat 3.0 mass; d the percentage of body lean mass (n = 7/group). Data are expressed as the mean ± 2.5 SEM. *p < 0.05; **p < 0.01 8 10 12 14 16 18 20 8 101214161820 versus WT-V; p < 0.05 versus Week Week KO-V. C D 18 80 ** 9 40 0 0 WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T Body weight (g) Fat mass/body weight (%) Food intake (g) Lean mass/body weight (%) Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1055 A B Fig. 2 Calcipotriol and iBRD9 treatment effect on the gut microbiota structure in Nur77 knockout mice. Alpha diversity analysis: the ACE estimator (a), Chao1 estimator (b), Shannon index (c), and Simpson index (d) 450 were used for evaluation. The results are the means ± SEM (n = 7). Data were analyzed by 1-factor ANOVA, followed by the Tukey–Kramer multiple comparison test. Venn diagram (e) showing the unique and WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T shared OTUs in the gut microbiota between groups. C D Plots were generated using a 0.98 weighted UniFrac distance- based PCoA (f). 6.5 0.96 0.94 6.0 0.92 5.5 0.90 WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T EF PCoA - PC1 VS PC2 KO-V KO-T WT-V WT-V WT-T WT-T 80 69 0.1 KO-V KO-T 20 7 24 28 22 24 0.0 10 8 -0.1 17 19 -0.2 0.0 0.2 0.4 PC1 (60.15%) there was no significant difference in mouse food intake difference in fasting blood glucose (FBG), TG, TC, Ca, and (Fig. 1b). The difference in body weight was reflected P (Supplementary Table 2). mainly in body fat, as shown in Fig. 1c; Nur77 knockout mice showed a significant increase in body fat percentage The diversity of gut microbiota in calcipotriol and that significantly decreased after treatment (p < 0.05 or iBRD9-treated Nur77 knockout mice <0.01), and there was no significant difference in the lean mass ratio in mice (Fig. 1d). Community richness was determined using the ACE esti- mator and the Chao1 estimator (Fig. 2a, b), and community Serum biochemical parameters of calcipotriol and diversity was estimated using the Shannon index and the iBRD9-treated Nur77 knockout mice Simpson index (Fig. 2c, d). Community richness and diversity were not significantly different between groups The LEP level was significantly increased in Nur77 according to the Wilcoxon rank sum test. knockout mice and was significantly reduced after treatment For 99.88% of the operational taxonomic units (OTUs), (p < 0.05 or <0.01). In addition, there was no significant based on the common and unique OTUs between the four ACE Shannon Simpson Chao1 PC2 (7.48%) 1056 Q. Lv et al. A Cladogram B Cladogram Fig. 3 Specific biomarkers of calcipotriol and iBRD9-treated a: f_Prevotellaceae a: f_Rikenellaceae b: f_Rikenellaceae KO-V KO-V Nur77 knockout mice. c: f_Lachnospiraceae WT-V KO-T a, b Cladogram representation of the differentially abundant families and genera. The root of the cladogram denotes the domain bacteria. The taxonomic levels of the phylum and class are labeled, while family and genus are abbreviated, with the colors indicating the greatest abundance. The size of each node represents their relative abundance. c, d LEfSe analysis shows differentially abundant genera as biomarkers determined using the Kruskal–Wallis test (p < 0.05) CD with an LDA score > 4. KO-V WT-V KO-V KO-T Correlation between the relative f_Lachnospiraceae abundance of Rikenellaceae and g_Parvibacter biological parameters after g_Alistipes g_Alistipes calcipotriol and iBRD9 f_Rikenellaceae f_Rikenellaceae treatment. e Fat mass (kg) and f_Prevotellaceae f body fat (%) exhibited significant correlations with levels of Rikenellaceae in the LDA SCORE (log 10) LDA SCORE (log 10) fecal microbiota. p < 0.05 based EF on Spearman rank correlation 0.15 0.15 analysis, n = 28. r =0.419 r =0.398 P=0.027 P=0.036 0.10 0.10 0.05 0.05 0.00 0.00 0 5 10 15 20 Fat mass (kg) Body fat (%) groups, a Wayne diagram is shown in Fig. 2e. The micro- Prevotellaceae (Fig. 3a, c), and the biomarker of KO- bial community composition of different samples was V–KO-T was Rikenellaceae (Fig. 3b, d). Alistipes was the compared by principal coordinate analysis (PCoA) (Fig. 2f). genus of Rikenellaceae. Rikenellaceae is positively corre- A significant difference was found between KO-V and WT- lated with body fat mass (r = 0.419, p = 0.027) and fat V, and there was a significant difference between KO-T and percentage (r = 0.398, p = 0.036) (Fig. 3e, f). KO-V(p < 0.05 or <0.01). Alteration of Lachnospiraceae and Akkermansiaceae LEfSe analysis in calcipotriol and iBRD9-treated abundance in calcipotriol and iBRD9-treated Nur77 Nur77 knockout mice knockout mice Linear discriminant analysis (LDA) effect size (LEfSe) Figure 4a, b shows gut microbiota constituents with the top analysis was used to detect species abundance data between ten relative abundances at the phylum and family levels. At groups by the rank sum test to detect different species the phylum level, Firmicutes and Bacteroidetes together within different groups, and the magnitude of the effects of accounted for a major portion of the bacterial population in the different species biomarkers was assessed by LDA. At all samples (93.24–97.43%). The Nur77 knockout mouse the family level, we found that the biomarkers of WT- had an increased relative abundance of Bacteroides that V–KO-V were Lachnospiraceae, Rikenellaceae, and decreased after treatment (Fig. 4a). The changes in the Rikenellaceae (%) Rikenellaceae (%) Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1057 A B Family Phylum 1.0 1.0 Others Others Chlamydiae Tannerellaceae Tenericutes Marinifilaceae Verrucomicrobia Lactobacillaceae Deferribacteres Rikenellaceae 0.5 Actinobacteria 0.5 Helicobacteraceae Melainabacteria Ruminococcaceae Proteobacteria Bacteroidaceae unidentified_Bacteria Prevotellaceae Firmicutes Lachnospiraceae Bacteroidetes Muribaculaceae 0.0 0.0 WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T Family Phylum Anaeroplasmataceae Tenericutes 0 Akkermansiaceae Verrucomicrobia Deferribacteraceae Deferribacteres -5 Nocardiaceae Bifidobacteriaceae Actinobacteria Eggerthellaceae Atopobiaceae Melainabacteria Melainabacteria Woeseiaceae Burkholderiaceae Moraxellaceae Proteobacteria Enterobacteriaceae Desulfovibrionaceae Rhizobiaceae Helicobacteraceae unidentified_Bacteria Clostridiales Veillonellaceae Erysipelotrichaceae Ruminococcaceae Peptostreptococcaceae Firmicutes Peptococcaceae Lachnospiraceae Christensenellaceae Streptococcaceae Lactobacillaceae Sphingomonadaceae Tannerellaceae Rikenellaceae Prevotellaceae Bacteroidetes Muribaculaceae Marinifilaceae Bacteroidaceae Fig. 4 Relative abundance distribution of gut microbiota con- relative abundances at the family level. c Relative abundances at the stituents at the phylum and family levels. a The gut microbiota family level associated with the top ten greatest abundances at the constituents with the top ten greatest relative abundances at the phy- phylum level that were altered in Nur77 knockout mice and reversed lum level. b The gut microbiota constituents with the top ten greatest by interventions. biomarker strains Lachnospiraceae, Rikenellaceae, and mucosal tight junction proteins Ocln and Cldn3 was sig- Prevotellaceae found by LEfSe analysis are shown in Fig. nificantly decreased in Nur77 knockout mice (Fig. 5a), and 4b. The relative abundance of Lachnospiraceae in Nur77 the expression of Ocln and Cldn3 was significantly knockout mice was low, and the relative abundance of increased after treatment (p < 0.001). Rikenellaceae and Prevotellaceae was high; the relative For the antibacterial peptides (Fig. 5b), namely, lyso- abundance of Lachnospiraceae increased after treatment, zyme C (Lyz1), regenerating islet-derived IIIγ (Reg3γ), the relative abundance of Rikenellaceae decreased. The phospholipase A2 group II (Pla2g2), and angiopoietin 4 relative abundance of Akkermansiaceae in Nur77 knockout (Ang4). There were no significant differences in anti- mice was low and increased after treatment (Fig. 4c). bacterial peptides between the four groups of mice. Among the mRNA expression levels of colonic inflam- Calcipotriol combined with iBRD9 protected the gut matory cytokines, TNF-α, IL-6, and IL-1β were sig- intestinal barrier integrity and function of Nur77 nificantly increased in Nur77 knockout mice (p < 0.05, knockout mice <0.01, or <0.001). TNF-α and IL-6 expression was sig- nificantly reduced after treatment (p < 0.05 or <0.001). Then we examined the mRNA levels of tight junction To further verify changes in intestinal mucosal barrier proteins, antimicrobial peptides, and inflammatory cyto- function, we measured serum LPS and inflammatory cyto- kines in the colon. The mRNA expression of the intestinal kine levels in mice. The serum LPS levels of Nur77 WT-V1 WT-V2 WT-V3 WT-V4 WT-V5 WT-V6 WT-V7 WT-T1 WT-T2 WT-T3 WT-T4 WT-T5 WT-T6 WT-T7 KO-V1 KO-V2 KO-V3 KO-V4 KO-V5 KO-V6 KO-V7 KO-T1 KO-T2 KO-T3 KO-T4 KO-T5 KO-T6 KO-T7 Relative abundance Relative abundance 1058 Q. Lv et al. A B Fig. 5 The effects of WT-V calcipotriol combined with iBRD9 on the expression of WT-T colon tight junction proteins, 3 KO-V ### colon antimicrobial peptides, KO-T *** and colon inflammatory ### cytokines in Nur77 knockout *** mice. The mRNA expression of tight junction proteins (a), antimicrobial peptides (b), and inflammatory cytokines (c)in 0 0 the colon (n = 7/group). Data Cldn3 Ocln ZO-1 Reg3γ Pla2g2 Lyz1 Ang4 are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; Tight junction proteins Antimicrobial peptides ***p < 0.001 versus WT-V; # ### p < 0.05; p < 0.001 versus KO-V. ** # 6 * *** ### TNF-α IL-6 IL-1β Inflammatory cytokines A B ## Fig. 6 The serum levels of 20 * 25 # lipopolysaccharide and inflammatory cytokines in 16 20 calcipotriol- and iBRD9- 12 15 treated Nur77 knockout mice. Serum levels of lipopolysaccharide (a), TNF-α (b), IL-6 (c), and IL-1β (d)(n = 7/group). Data are expressed as the mean ± SEM. *p < 0.05 0 # ## WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T versus WT-V; p < 0.05; p < 0.01 versus KO-V. C D 200 120 * WT-V WT-T KO-V KO-T WT-V WT-T KO-V KO-T knockout mice were significantly increased compared with Discussion those of wild-type mice and decreased significantly after treatment (p < 0.05 or <0.01) (Fig. 6a). The serum levels of In this study, we used Nur77 knockout mice and assessed TNF-α and IL-1β in Nur77 knockout mice increased sig- the effects of calcipotriol combined with iBRD9; we found nificantly (p < 0.05) (Fig. 6b, d). After treatment, the TNF-α that Nur77 knockout mice showed a significant increase in and IL-6 levels were significantly decreased (p < 0.05) body weight after 17 weeks of age, consistent with the lit- (Fig. 6b, c). erature [7]. After treatment with calcipotriol combined with Relative expression of mRNA Relative expression of mRNA IL-6 (pg/mL) LPS (EU/mL) Relative expression of mRNA IL-1β (pg/mL) TNF-α (ng/L) Calcipotriol and iBRD9 reduce obesity in Nur77 knockout mice by regulating the gut microbiota,. . . 1059 iBRD9, the body weight of Nur77 knockout mice was due to the increased intestinal permeability and destruction significantly reduced. This change in body weight was of tight junction proteins attached to epithelial cells, derived from changes in body fat rather than lean weight. increasing portal vein and systemic plasma LPS con- We explored changes in the gut microbiota by 16S rRNA centrations [43]. Therefore, we measured the level of serum sequencing. The results showed that Nur77 deficiency had a LPS, and the serum LPS level of Nur77 knockout mice was significant effect on the gut microbiota composition, while increased significantly but decreased significantly after calcipotriol combined with iBRD9 treatment had a sig- treatment. This finding supported the occurrence of damage nificant effect on the composition of gut microbiota in only of the intestinal mucosal barrier in Nur77 knockout mice Nur77 knockout mice but not wild-type mice. Bacteroidetes and the improvement of intestinal mucosal barrier function and Firmicutes accounted for the majority of the bacterial after treatment with calcipotriol combined with iBRD9. The population (93.24–97.43%), and the Firmicutes/Bacter- increase in LPS absorbed into the blood may be one of the oidetes ratio in Nur77 knockout mice was decrease. How- causes of low-grade inflammation leading to obesity [44– ever, there is currently no consensus on the relationship 46]. The serum levels of inflammatory factors in our between the Firmicutes/Bacteroidetes ratio and obesity experiments indicated that low-grade inflammation occurs [25, 26]. We were pleased to find that the Nur77 knockout in Nur77 knockout mice, and the level of inflammation mice had a low relative abundance of Lachnospiraceae and decreased after treatment, which corresponded to LPS levels Akkermansiaceae. After calcipotriol combined with iBRD9 and was consistent with colon mRNA expression levels. treatment, their abundances increased. Lachnospiraceae Studies have shown that obesity is positively associated belongs to Firmicutes and degrades dietary fiber to produce with low-grade inflammation and that obesity can be short-chain fatty acids, which enhance intestinal barrier effectively alleviated by reducing inflammation [47, 48]. function by regulating tight junction proteins and mucins We applied calcipotriol combined with iBRD9 to reduce [27, 28]. Akkermansiaceae plays an important role in body weight by reducing the level of inflammation in Nur77 maintaining intestinal homeostasis, and it has been exten- knockout mice. sively studied in diseases such as obesity and inflammatory Our results indicate that changes in the gut microbiota of bowel disease [29–31]. In addition, we detected four groups Nur77 knockout mice contribute to obesity. Furthermore, of differentially abundant bacteria, including Rikenellaceae, the expression of intestinal mucosal tight junction protein- and studies show that Rikenellaceae abundance increases in related genes is reduced, and serum LPS concentration and high-fat-fed mice [32, 33] and that Rikenellaceae is posi- inflammatory factor levels are increased. Calcipotriol com- tively correlated with body fat percentage and fat mass bined with iBRD9 can regulate the gut microbiota, improve (Fig. 3e, f), which is consistent with reports in humans [34]. intestinal mucosal barrier function, reduce LPS absorption Previous studies have shown that activation of the into the blood, and alleviate inflammation and obesity. This Nur77/RXR heterodimer is responsible for reducing study clarified the causes of low-grade inflammation and monocyte-mediated inflammation in the intestine [35] and obesity in Nur77 knockout mice and demonstrated the that Nur77 has an important protective effect on the therapeutic effect of calcipotriol combined with iBRD9 on development of inflammatory bowel disease [36, 37]. obesity. The specific mechanism of Nur77 knockout mice Therefore, it is speculated that Nur77 knockout mice may gut microbiota dysbiosis, and how calcipotriol combined have undetected intestinal damage. Next, we examined the with iBRD9 regulates the gut microbiota have not been mRNA levels of tight junction proteins, antimicrobial pep- explored in depth, but it is a subject worth studying. tides, and inflammatory cytokines in the colon. The mRNA expression levels of the tight junction proteins Cldn3, Ocln, Acknowledgements This work was supported by the local develop- ment foundation of science and technology guided by the central and ZO-1 can reflect intestinal mucosal barrier function commission (2016007024), the science and technology project of [30, 38–40], and the results showed that the intestinal Shenyang (Z18-5-104), and the National Natural Science Foundation mucosal barrier function of Nur77 knockout mice was of China (31570819). impaired and that there was improvement after treatment with calcipotriol combined with iBRD9. The increased Compliance with ethical standards mRNA expression of colonic inflammatory cytokines observed in Nur77 knockout mice decreased after calcipo- Conflict of interest The authors declare that they have no conflict of interest. triol combined with iBRD9 treatment, suggesting that similar changes may occur in serum inflammation factors. Publisher’s note Springer Nature remains neutral with regard to Studies in the literature have shown that increased LPS jurisdictional claims in published maps and institutional affiliations. levels can induce a large number of proinflammatory responses and inflammatory cytokine release by activating Open Access This article is licensed under a Creative Commons Toll-like receptors 2, 4, and 5 [41, 42]. This effect may be Attribution 4.0 International License, which permits use, sharing, 1060 Q. Lv et al. adaptation, distribution and reproduction in any medium or format, as 16. 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International Journal of Obesity – Springer Journals

Published: May 17, 2020

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