Vitamin D Ameliorates High-Fat-Diet-Induced Hepatic Injury (PDF)

Archives of Biochemistry and Biophysics 705 (2021) 108894 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics...

Archives of Biochemistry and Biophysics 705 (2021) 108894 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi Vitamin D ameliorates high-fat-diet-induced hepatic injury via inhibiting pyroptosis and alters gut microbiota in rats Xiaolei Zhang, Xueying Shang, Shi Jin, Zhuoqi Ma, He Wang, Na AO, Jing Yang, Jian Du * Department of Endocrinology, The Fourth Affiliated Hospital of China Medical University, Shenyang, China A R T I C L E I N F O A B S T R A C T Keywords: Accumulating evidence suggests that vitamin D (VD) has a therapeutic effect on non-alcoholic fatty liver disease Vitamin D (NAFLD). Pyroptosis and gut microbiota have been recognized as critical factors of the progression of NAFLD. 1,25(OH)2D3 However, the effect of VD on the pyroptosis and gut microbiota in NAFLD remains inconclusive. Herein, rats Pyroptosis were fed high fat diet (HFD) for 12 weeks and concurrently treated with 5 μg/kg 1,25(OH)2D3 twice a week. BRL- Gut microbiota Non-alcoholic fatty liver disease 3A cells were stimulated with 0.4 mmol/L palmitic acid (PA) and 1 μg/ml lipopolysaccharide (LPS) for 16 h and treated with 10− 6 mol/L 1,25(OH)2D3. Effect of VD on the hepatic injury, lipid accumulation, activation of NLRP3 inflammasome and pyroptosis was determined in vivo and in vitro. Next, gasdermin D N-terminal (GSDMD-N) fragment was overexpressed in BRL-3A cells to investigate the role of pyroptosis in the therapeutic effect of VD on NAFLD. In addition, gut microbiota in NAFLD rats was also analyzed. Results showed that VD attenuated HFD-induced hepatic injury in vivo and PA-LPS-induced impairment of cell viability in vitro, and inhibited lipid accumulation, activation of NLRP3 inflammasome and pyroptosis in vivo and in vitro. GSDMD-N fragment overexpression suppressed the protective effect of VD on PA-LPS-induced activation of NLRP3 inflammasome, impairment of cell viability and lipid accumulation, indicating that VD might attenuate NAFLD through inhibiting pyroptosis. Additionally, VD also restored HFD-induced gut microbiota dysbiosis by increasing the relative abundance of Lactobacillus and reducing that of Acetatifactor, Oscillibacter and Flavoni­ fractor. This study provides a novel mechanism underlying VD therapy against NAFLD. 1. Introduction activation of NLRP3 inflammasome could ameliorate liver inflammation and fibrosis in NAFLD mice, which provides a novel insight for the Non-alcoholic fatty liver disease (NAFLD) is a disorder encompassing treatment of NAFLD. a broad spectrum of pathologies ranging from deposition of adipose Pyroptosis is an inflammatory form of programmed cell death. tissue in liver to steatosis, steatohepatitis, fibrosis, cirrhosis and even The canonical pathway during pyroptosis is initiated by inflammasomes hepatocellular carcinoma. NAFLD has become a public health and in turn, activates caspase-1. The activated caspase-1 cleaves problem, which is affecting 25% of the adult population worldwide. pro-interleukin-1β (pro-IL-1β) and pro-IL-18 into their active forms Notably, the increasing incidences of NAFLD will continue for the next mature IL-1β and IL-18 and gasdermin D (GSDMD) into a N-terminal and decade. a C-terminal fragment. GSDMD and its N-terminal fragments were NOD-like receptor pyrin domain containing 3 (NLRP3) inflamma­ highly expressed in NAFLD or non-alcoholic steatohepatitis patients and some is a multiprotein complex consisting of NLRP3, apoptosis associ­ GSDMD-knockout mice exhibited a lower steatosis , indicating the ated speck like protein containing a CARD (ASC) and serine protease significant role of pyroptosis in NAFLD progression. caspase-1. NLRP3 inflammasome is the most well-characterized Vitamin D3 (VD) is of vital importance in calcium homeostasis and inflammasome and its activation is implicated in the pathological pro­ metabolism. VD is modified and activated by hydroxylases existed in cess of multiple diseases [4–6]. Aberrant activation of NLRP3 inflam­ liver and kidney and then its active form 1,25(OH)2D3 (calcitriol) per­ masome has been observed in the development of liver diseases and can forms its functions through binding to vitamin D receptor. VD ex­ lead to hepatocyte pyroptosis, inflammation and fibrosis. Inhibiting erts anti-fibrotic and anti-inflammatory potentials in liver [12,13]. * Corresponding author. Department of Endocrinology, The Fourth Affiliated Hospital of China Medical University, No.4 Chongshan East Road, Huanggu District, Shenyang, 110032, China. E-mail address: [email protected] (J. Du). https://doi.org/10.1016/j.abb.2021.108894 Received 18 January 2021; Received in revised form 16 April 2021; Accepted 25 April 2021 Available online 6 May 2021 0003-9861/© 2021 Elsevier Inc. All rights reserved. X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Insufficient VD levels were found in the serum of NAFLD patients. 2.6. Western blotting Moreover, VD could attenuate hepatic fibrosis in NAFLD rats. The precursor of 1,25(OH)2D3 could alleviate colonic damage in colitis mice Total protein was extracted with Cell lysis buffer for Western and IP by inhibiting pyroptosis. However, it remains ambiguous whether (Beyotime, China) containing 1 mmol/L PMSF (Beyotime). The protein VD alleviates NAFLD via regulating pyroptosis. Hence, the function of was separated on SDS-PAGE gel and transferred onto PVDF membranes pyroptosis in VD-mediated effects on NAFLD was explored. NAFLD was (Millipore, USA). After blocking with 5% skim milk, the membrane was constructed in vivo and in vitro to investigate the effect of VD on lipid incubated with primary antibodies, including NLRP3 rabbit polyclonal accumulation, NLRP3 inflammasome activation and pyroptosis. antibody (1:1000, ABclonal, China), apoptosis associated speck like Furthermore, the function of VD-induced pyroptosis in lipid accumula­ protein containing a CARD (ASC) rabbit polyclonal antibody (1:1000, tion was verified in vitro. In addition, VD has been widely reported to ABclonal), caspase-1 rabbit polyclonal antibody (1:500, Wanleibio, alter gut microbiota [17,18]. Gut microbiota dysbiosis contributes to China), pro-IL-1β rabbit monoclonal antibody (1:1000, ABclonal), IL-1β NAFLD pathogenesis. Therefore, the effect of VD on intestinal rabbit polyclonal antibody (1:1000, Affinity, China) and GSDMD N- microbiome in NAFLD rats was also investigated. This study provides an terminal (GSDMD-N) rabbit monoclonal antibody (1:1000, Abcam, underlying mechanism of VD in NAFLD treatment. USA) and β-actin mouse monoclonal antibody (1:1000, Santa cruz, USA) at 4 ◦ C overnight. Then the membrane was incubated with HRP- 2. Materials and methods conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibody (1:5000, Beyotime) at 37 ◦ C for 45 min. Data of target proteins were 2.1. Animals normalized to β-actin. Male 5-week-old SD rats were used in this study. Animal experiments 2.7. Enzyme linked immunosorbent assay (ELISA) were conducted according to the Guide for Care and Use of Laboratory Animals, and approved by the ethics committee of China Medical Uni­ ELISA was performed to measure the IL-1β and IL-18 contents in liver versity (No. 2018162). tissues using respective assay kits (USCN KIT, China) following the manufacturer’s instructions. 2.2. Treatments 2.8. Cell culture and treatment Twenty-four rats were randomly divided into 4 groups (6 rats per Normal rat hepatocytes BRL-3A cell was obtained from iCell group): ND and ND + VD group, rats were fed normal diet; HFD and Bioscience Inc (China) and cultured in DMEM containing 10% FBS, 1% HFD + VD group, rats were fed HFD. Concurrently, rats in ND + VD and penicillin streptomycin at 37 ◦ C and 5% CO2. Cells were stimulated with HFD + VD group were treated with 1,25(OH)2D3 (5 μg/kg, intraperi­ 0.4 mmol/L palmitic acid (PA, Sigma, USA) and 1 μg/ml lipopolysac­ toneal injection, Cayman, USA) twice a week, while rats in ND and HFD charide (LPS, Sigma, USA) in the presence/absence of different levels of group were intraperitoneally injected with an equivalent volume of 1,25(OH)2D3 (0, 10− 8, 10− 7, 10− 6 mol/L, Cayman) for 16 h. GSDMD-N vehicle. HFD was prepared as previously described. After 12 weeks, in BRL-3A cells was overexpressed via transfection with plasmids all rats were euthanized and the peripheral blood and the liver tissues overexpressing GSDMD-N fragment using Lipofectamine 2000 (Invi­ were collected for further analyses. trogen, USA) according to the manufacturer’s instructions. 2.3. Biochemical parameters 2.9. Cell viability assay The triglyceride (TG) contents in liver tissues and cells, the aspartate Cell viability was analyzed using cell counting kit-8 (CCK-8) assay. aminotransferase (AST) and alanine aminotransferase (ALT) activities in Cells (3 × 103 cells or 4 × 103 cells per well) were seeded into a 96-well liver tissues, and the lactic dehydrogenase (LDH) release of liver cells plate. After treatment, culture medium was replaced by 100 μl complete were determined using respective assay kits (Nanjing Jiancheng, China) medium. Then cells were incubated with CCK-8 solution (10 μl per well, following the manufacturer’s instructions. Sigma-Aldrich) for 1 h at 37 ◦ C and 5% CO2. The optical density was measured at 450 nm. 2.4. Histological analysis 2.10. Immunofluorescence (IF) staining Histological changes in liver tissues were detected using The fixed cell slides were permeabilized with 0.1% Triton X-100, hematoxylin-eosin (H&E) staining. The fixed liver tissues were blocked in goat serum and then incubated with NLRP3 antibodies (1:50, embedded in paraffin and cut into 5-μm-thick slices. After xylol depar­ Abclonal) at 4 ◦ C overnight. Next, the cells were incubated with Cy3- affinization and rehydration, the slice was stained with hematoxylin and conjugated goat anti-rabbit IgG (1:200, Beyotime) for 60 min and then eosin. Images were acquired with a microscope (200x magnification, the nucleus was counterstained using DAPI. Images were acquired with Olympus, Japan). The NAFLD activity score was scored as previously a fluorescence microscope (400x magnification, Olympus). described. 2.11. Gut microbiota analysis 2.5. Oil red O staining Changes in gut microbiota were analyzed using high-throughput 16s Lipid droplets in cells and tissues were assayed using Oil red O ribosomal DNA (rDNA) sequencing. DNA was isolated from fecal sam­ staining. The fixed liver tissues were embedded in optimal cutting ples using a DNA extraction kit (Omega Bio-tek, USA) according to the temperature compound and cut into slices with a thickness of 10 μm. manufacturer’s protocols. The V3–V4 region of 16s rRNA gene was Then the slices were stained with Oil red O (Sigma-Aldrich, USA) and amplified by PCR (98 ◦ C for 30 s, 32 cycles of denaturation at 98 ◦ C for hematoxylin. Liver cells were fixed with 4% paraformaldehyde. Then 10 s, annealing at 54 ◦ C for 30 s and extension at 72 ◦ C for 45 s, and the cells were stained with 0.5% Oil red O solution. The stained cells and finally extension at 72 ◦ C for 10 min) with primers (341F: 5′ - slices were observed under a microscope (200x or 400x magnification, CCTACGGGNGGCWGCAG-3′ ; 805R: 5′ -GACTACHVGGGTATCTAATCC- OLYMPUS). 3′ ). The products were purified on a 2% agarose gel using AxyPrep PCR 2 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Clean-up Kit (Beckman Coulter, USA) and then quantified using Qubit histological changes in liver tissues, the serum levels of AST, ALT and TG (Invitrogen). The amplicon libraries were prepared and sequenced on as well as lipid accumulation in liver tissues were analyzed. Histological NovaSeq PE250 platform. The sequences whose similarity reached 97% analysis was performed using H&E staining. As shown in Fig. 1A, hepatic were assigned to the same operational taxonomic units (OTUs) using lobules arranged neatly in ND and ND + VD group. HFD resulted in Vsearch. Taxonomic composition was generated using the Ribosomal blurred structure of hepatic lobules and some vacuoles of fat in the liver Database Project classifier. Microbial diversities including α-diversity tissues, while VD administration restored the HFD-triggered production and β-diversity were obtained using the QIIME software. of fat vacuoles and disordered structure of hepatic lobules in some parts of liver tissues (Fig. 1A). Subsequently, the NAFLD activity score was 2.12. Statistical analysis scored. The NAFLD activity scores in the HFD group were significantly higher than those in the ND group, while VD administration restored the Data were analyzed using GraphPad Prism 8.0.1 software. Results HFD-triggered increases in NAFLD activity scores (Fig. 1B). The serum were expressed as means ± standard deviation (SD). Data are analyzed levels of AST, ALT and TG were measured using commercial assay kits. using Kruskal-Wallis test followed by Dunn’s test and one-way analysis HFD was observed to increase the AST and ALT activities and TG con­ of variance (ANOVA) or two-way ANOVA followed by Tukey’s multiple tents in serum, while VD administration significantly suppressed the comparisons test. Statistical significance was set at the level of p < 0.05. HFD-induced increases in AST and ALT activities and TG contents (Fig. 1C–E). Lipid accumulation was evaluated by staining with Oil Red 3. Results O. It was shown that HFD triggered the accumulation of lipid droplets yet VD administration significantly restored the HFD-induced lipid 3.1. VD protects against the HFD-induced hepatic injury and lipid accumulation in liver tissues (Fig. 1F). These findings suggested that VD accumulation in NAFLD rats administration alleviated the HFD-induced hepatic injury and lipid accumulation in NAFLD rats. In order to investigate the effect of VD on NAFLD rats, the Fig. 1. Effect of VD on hepatic injury and lipid accumulation in HFD-fed rats. (A) Histological changes in liver tissues were observed using H&E staining (Scale bar, 100 μm). (B) NAFLD activity score was graded according to HE staining. (C, D) AST and ALT activities in liver tissues. (E) TG contents in liver tissues. (F) Accumulation of lipid droplets in liver tissues was detected using Oil red O staining (Scale bar, 100 μm). Data were Mean ± SD (n = 6). **p < 0.01 vs. ND group. ##p < 0.01 vs. HFD group. ND: normal diet. HFD: high fat diet. VD: 1,25(OH)2D3. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 3.2. VD restrains the activation of NLRP3 inflammasome and pyroptosis contents in BRL-3A cells were significantly decreased in the presence of in the liver tissues from NAFLD rats 10− 8 mol/L (p < 0.05), 10− 7 mol/L (p < 0.01) and 10− 6 mol/L (p < 0.001) VD (Fig. 3A). Furthermore, the accumulation of lipid droplets To explore the effect of VD on the activation of NLRP3 inflamma­ was also markedly reduced in the presence of 10− 7 mol/L (p < 0.001) some and pyroptosis in the liver tissues from NAFLD rats, the expression and 10− 6 mol/L (p < 0.001) VD (Fig. 3B). Subsequently, CCK-8 assay of NLRP3 inflammasome-related and pyroptosis-related proteins was was performed to measure the viability of BRL-3A cells. PA-LPS stimu­ detected using western blotting and the contents of IL-1β and IL-18 using lation significantly decreased cell viability (Fig. 3C), while 10− 6 mol/L commercial assay kits. The results of western blotting showed that HFD VD significantly reversed the PA-LPS-induced loss of cell viability. facilitated the NLRP3, ASC, cleaved-caspase-1, pro-IL-1β, IL-1β and Moreover, there is no significant difference between the viability of GSDMD-N expressions in liver tissues, while VD administration inhibi­ control cells and that of the cells treated with PA-LPS and 10− 6 mol/L 1, ted the HFD-induced expressions of these proteins (Fig. 2A and B). 25(OH)2D3 (Fig. 3C). Taken together, VD at a concentration of 10− 6 Additionally, the IL-1β and IL-18 contents in liver tissues were deter­ mol/L could not only reduce lipid accumulation but also protect BRL-3A mined by ELISA. HFD significantly increased IL-1β and IL-18 contents in cells against loss of cell viability induced by PA and LPS. Hence, 10− 6 liver tissues, while VD administration inhibited the HFD-induced in­ mol/L 1,25(OH)2D3 was used for further analyses. creases of IL-1β and IL-18 contents (Fig. 2C). These findings suggested that VD administration suppressed the HFD-induced activation of 3.4. VD inhibits the activation of NLRP3 inflammasome and pyroptosis in NLRP3 inflammasome and pyroptosis in liver tissues. the BRL-3A cells stimulated by PA and LPS 3.3. VD restores the loss of cell viability and lipid accumulation in the To further explore the effect of VD on the activation of NLRP3 BRL-3A cells stimulated by PA and LPS inflammasome and pyroptosis in the BRL-3A cells stimulated by PA and LPS, the expression of NLRP3 inflammasome-related and pyroptosis- To select an appropriate concentration of VD for further analyses, related proteins was detected using western blotting or IF staining and BRL-3A cells were stimulated with PA and LPS to mimic NAFLD in vitro the contents of LDH using commercial assay kits. The results of western and treated with 0, 10− 8, 10− 7 and 10− 6 mol/L VD according to a pre­ blotting showed that PA-LPS stimulation remarkably facilitated the ex­ vious study. TG contents and lipid droplets were measured to pressions of NLRP3, ASC, cleaved-caspase-1, IL-1β and GSDMD-N, while evaluate the lipid accumulation in BRL-3A cells. PA-LPS stimulation VD significantly inhibited the PA-LPS-induced expressions of these significantly increased the TG contents and promoted the accumulation proteins in BRL-3A cells (Fig. 4A and B). Additionally, PA-LPS and PA- of lipid droplets in BRL-3A cells (Fig. 3A and B). However, the TG LPS + VD treatment had no significant effect on pro-caspase-1 and pro- Fig. 2. Effect of VD on the NLRP3 activation and pyroptosis in HFD-fed rats. (A) The expression of NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, pro-IL-1β and IL-1β in liver tissues was detected using western blotting. (B) The expression of GSDMD-N in liver tissues was detected using western blotting. (C) The content of IL-1β and IL-18 in liver tissues was measured using ELISA. Data were Mean ± SD (n = 6). **p < 0.01 vs. ND group. #p < 0.05 and ##p < 0.01 vs. HFD group. ND: normal diet. HFD: high fat diet. VD: 1,25(OH)2D3. 4 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Fig. 3. Effect of VD on the TG contents, lipid accumulation and viability of PA-LPS-stimulated liver cells. BRL-3A cells were treated with PA (0.4 mmol/L), LPS (1 μg/ml) and 1,25(OH)2D3 (0, 10− 8, 10− 7, 10− 6 mol/L) for 16 h. (A) TG contents in BRL-3A cells. (B) Accumulation of lipid droplets in BRL-3A cells was detected using Oil red O staining (Scale bar, 50 μm). (C) Cell viability was measured by CCK-8 assay. Data were Mean ± SD (n = 3). *p < 0.05. **p < 0.01. ***p < 0.001. Ns: no significance. PA: palmitic acid. LPS: lipopolysaccharide. VD: 1,25(OH)2D3. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) IL-1β expressions. However, PA-LPS treatment exhibited a tendency to 3A cells stimulated by PA and LPS, indicating that VD inhibited TG enhance pro-IL-1β expression and VD showed a tendency to inhibit the contents and accumulation of lipid droplets in BRL-3A cells stimulated PA-LPS-induced pro-IL-1β expression in BRL-3A cells. The results of IF by PA and LPS via suppressing pyroptosis. staining exhibited that VD obviously inhibited the PA-LPS-induced NLRP3 expression in BRL-3A cells (Fig. 4C). Besides, PA-LPS stimula­ 3.6. VD regulates the gut microbiota in NAFLD rats tion significantly stimulated LDH release yet VD significantly inhibited the PA-LPS-induced LDH release of BRL-3A cells (Fig. 4D). These find­ To explore the effect of VD on the gut microbiota in NAFLD rats, the ings demonstrated that VD inhibited the PA-LPS-induced activation of fecal samples were collected for further analyses. The average opera­ NLRP3 inflammasome and pyroptosis in BRL-3A cells. tional taxonomic units (OTUs) of the ND, ND + VD, HFD, and HFD + VD groups were 2296, 2325, 1639 and 2103 with 97% sequence similarity, 3.5. VD suppresses lipid accumulation in the BRL-3A cells stimulated by respectively. The shannon rarefaction curve of each sample tended to PA and LPS via inhibiting pyroptosis reach an asymptote (Fig. S1) and the results of goods coverage were all over 0.99, indicating that the sequencing depth was sufficient to To verify the role of pyroptosis in the protective effect of VD against represent the biological diversity. The results of unweighted pair group NAFLD in vitro, BRL-3A cells were transfected with GSDMD-N fragment method with arithmetic mean (UPGMA) cluster analysis revealed that overexpression vector or empty vector (EV). The expression of GSDMD- ND, ND + VD and HFD + VD groups clustered together while separated N, NLRP3 inflammasome-related proteins was detected using western from HFD group, indicating that HFD consumption strongly affected the blotting. The results showed that GSDMD-N fragment overexpression gut microbiota in rats and VD effectively relieved the HFD-induced ef­ reversed the VD-induced decreases in the expression of GSDMD-N, fect (Fig. 6A). That could also be verified by the principal coordinate NLRP3, ASC, cleaved-caspase-1, pro-IL-1β, IL-1β and GSDMD-N in analysis (PCoA, Fig. 6B). BRL-3A cells (Fig. 5A). Cell viability was measured using CCK-8 assay. The gut microbiota compositions in rats from each group were GSDMD-N fragment overexpression was observed to significantly compared at the phylum (Fig. 7A) and genus (Fig. 7B) levels. At the reverse the VD-induced decreases in loss of cell viability (Fig. 5B), phylum level, Firmicutes, Bacteroidetes and Proteobacteria made up more indicating that VD promoted the viability of the BRL-3A cells stimulated than 78% of the total microbial population in all groups. Fig. 7C showed by PA and LPS via the regulation of pyroptosis. Additionally, TG content that HFD consumption significantly decreased the relative abundance of and Lipid accumulation was also analyzed. GSDMD-N fragment over­ Bacteroidetes and VD administration significantly decreased the relative expression significantly reversed the VD-induced reduction in TG con­ abundance of Firmicutes and increased the relative abundance of Bac­ tents (Fig. 5C) and accumulation of lipid droplets (Fig. 5D) in the BRL- teroidetes in the rats fed HFD. However, both HFD consumption and VD 5 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Fig. 4. Effect of VD on the NLRP3 activation and pyroptosis in PA-LPS-stimulated liver cells. BRL-3A cells were treated with PA (0.4 mmol/L), LPS (1 μg/ml) and 1,25(OH)2D3 (10− 6 mol/L) for 16 h. (A) The expression of NLRP3, ASC, pro-caspase-1, cleaved- caspase-1, pro-IL-1β and IL-1β in BRL-3A cells was detected using western blotting. (B) The expression of GSDMD-N was detected using western blotting. (C) The expression of NLPR3 (Red) in BRL-3A cells was detected using IF staining (Scale bar, 50 μm). The nuclei was stained with DAPI (Blue). (D) LDH release of BRL-3A cells. Data were Mean ± SD (n = 3). **p < 0.01 vs. Control group. #p < 0.05 and ##p < 0.01 vs. PA + LPS group. PA: palmitic acid. LPS: lipopolysaccharide. VD: 1,25(OH)2D3. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 6 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Fig. 5. VD exhibited a protective effect on PA-LPS-stimulated liver cells via induction of pyroptosis. BRL-3A cells were transfected with plasmids overexpressing GSDMD-N fragment. After 24 h, the cells were treated with PA (0.4 mmol/L), LPS (1 μg/ml) and 1,25 (OH)2D3 (10− 6 mol/L) for 16 h. (A) The expression of GSDMD-N, NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, pro-IL-1β and IL-1β was detected using western blotting. (B) Cell viability was measured by CCK-8 assay. (C) TG contents in BRL-3A cells. (D) Accumulation of lipid droplets in BRL-3A cells was detected using Oil red O staining (Scale bar, 50 μm). Data were Mean ± SD (n = 3). *p < 0.05 and **p < 0.01 vs. PA + LPS group. #p < 0.05 and ##p < 0.01 vs. PA + LPS + VD + EV group. PA: palmitic acid. LPS: lipopolysaccharide. VD: 1,25(OH)2D3. EV: empty vector. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 7 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Fig. 6. Effect of VD on the β-diversity of the microbial community in HFD-fed rats. (A) UPGMA cluster analysis. (B) PCoA plot. ND: normal diet. HFD: high fat diet. VD: 1,25(OH)2D3. administration had no significant effect on the relative abundance of damage-associated molecular patterns (DAMPs) are able to stimulate the Proteobacteria (Fig. 7C). At the genus level (Fig. 7D), HFD consumption transcription of NLRP3, and then facilitate transcription-independent resulted in significant increases in the relative abundance of Acetati­ activation of caspase-1. Therefore, increases in pyroptosis pro­ factor, Oscillibacter and Flavonifractor, while VD administration signifi­ moted the expression of IL-1β that might function as a DAMP to affect cantly increased the relative abundance of Lactobacillus and inhibited NLPP3, ASC and cleaved-caspase-1 expressions. the HFD-induced increases in the relative abundance of Acetatifactor, Activation of NLRP3 inflammasome is implicated in the progression Oscillibacter and Flavonifractor. of NAFLD. Inflammasome-triggered pyroptosis is a canonical process mediated by caspase-1. The pore-forming effector protein GSDMD is 4. Discussion cleaved by caspase to liberate its N-terminal fragment, thereby resulting in pyroptosis. A previous research suggested that GSDMD knockout Low concentration of 1,25(OH)2D3 has been shown in the serum of markedly decreased hepatic TG content and steatosis in HFD-fed mice patients undergoing NAFLD. Furthermore, reduced concentration and overexpression of GSDMD-N fragment increased the TG content in of 1,25(OH)2D3 has been reported to be closely related to the histolog­ cultured hepatocytes. Furthermore, GSDMD knockout inhibited ical severity of hepatic steatosis and hepatic fibrosis. A long-term hepatic lipogenesis by restraining the mRNA expression of lipogenic intake of HFD is able to cause an oversupply of fat, which leads to gene SREBP-1C and promoting the expression of lipolytic genes excessive lipid accumulation in liver. VD deficiency could further including PPAR-α and its downstream target genes. Taken together, aggravate hepatic steatosis and inflammation and up-regulate the level GSDMD-driven pyroptosis is closely associated with the pathogenesis of of lipogenesis-related genes in HFD-fed mice [24,25]. Hence, VD sup­ hepatic steatosis. It was speculated that VD might alleviate hepatic plementation might be a therapeutic strategy for NAFLD patients. In steatosis in NAFLD rats via regulating pyroptosis. To confirm the hy­ recent studies, it was confirmed that oral administration of VD did pothesis, GSDMD-N fragment was overexpressed to augment pyroptosis reduce TG content, ALT and AST activities in patients undergoing in BRL-3A cells. GSDMD-N overexpression was observed to weaken NAFLD [26,27]. VD-induced inhibition of lipid accumulation in BRL-3A cells and in­ So far, great effort has been dedicated to verify the pharmaceutical creases in cell viability, impling that VD prevented lipid accumulation in effect of VD on the pathogenesis of NAFLD. However, only a few studies BRL-3A cells by suppressing pyroptosis. Due to the importance of lipid focused on the protective mechanism of VD on NAFLD. Liu et al. metabolism in NAFLD pathogenesis, the mechanism underlying the role suggested that VD supplementation attenuated the progression of of VD in lipid metabolism will be investigated in the future. NAFLD by inhibiting p53 pathway. Li et al. demonstrated that VD VD has been involved in some physiological processes through supplementation ameliorated hepatic steatosis and inflammation in binding to VD receptor (VDR), which is widely expressed in liver. NAFLD mice by inducing autophagy. Ma et al. elucidated that VD Active form of VD can bind to VDR and forms a heterodimer with supplementation inhibited cell senescence to block the progression of retinoid-X receptor. Subsequently, the heterodimer binds to vitamin D NAFLD by impeding p53-p21 signaling pathway. In this study, we response element that is located in the promoter region of target genes, investigated a novel protective mechanism of VD in NAFLD. Consis­ mediating the transcriptional expression of target genes to exert its tently, VD was found to inhibit HFD-induced decreases of TG contents genomic functions. Except for the genomic pathway, VD also ex­ and lipid droplets in liver tissues in the present study. Moreover, VD also hibits its functions via the cross-talk among different signaling pathways could inhibit lipid accumulation in PA-LPS-stimulated BRL-3A cells. Of. The mechanism underlying the effect of VD on the NLRP3 note, we found that VD inhibited the NAFLD-induced activation of inflammasome and pyroptosis in the liver was not explored in the pre­ NLRP3 inflammasome and pyroptosis in vivo and in vitro, as evidenced sent study, which will be performed in the subsequent experiments. by the down-regulated expressions of NLRP3, ASC, cleaved-caspase-1, Gut microbiota affects hepatic lipid metabolism, and gut microbiota pro-IL-1β, IL-1β and GSDMD-N in liver tissues and BRL-3A cells and dysbiosis has been emphasized to contribute to the pathogenesis of the reduced LDH release of BRL-3A cells and contents of IL-1β and IL-18 NAFLD. Dietary plays a pivotal role in regulating the composition in liver tissues. However, GSDMD-N overexpression was observed to of gut microbiota. HFD has been regarded as a risk factor for gut reverse the VD-induced decreases in NLRP3, ASC, cleaved-caspase-1, microbiota dysbiosis. At the phylum level, HFD led to a reduction in the pro-IL-1β and IL-1β expressions in PA-LPS-treated BRL-3A cells. The relative abundance of Bacteroidetes, which were consistent with previous 8 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 Fig. 7. Effect of VD on the composition of gut microbiota in HFD-fed rats. (A, B) Relative abundance of gut microbiota at the phylum and genus levels. (C) Relative abundance of Firmicutes, Bacteroidetes and Proteobacteria. (D) Relative abundance of Lactobacillus, Acetatifactor, Oscillibacter and Flavonifractor. Data were Mean ± SD (n = 5). *p < 0.05 and **p < 0.01 vs. ND group. #p < 0.05 and ##p < 0.01 vs. HFD group. ND: normal diet. HFD: high fat diet. VD: 1,25(OH)2D3. works [35,36]. However, VD reversed the HFD-induced changes in the attenuate HFD-induced NAFLD [37–39]. Bile acids play an essential role relative abundance of Firmicutes and Bacteroidetes. At the genus level, VD in modulating lipid metabolism. Bile acids are synthesized in the liver was observed to increase the relative abundance of Lactobacillus and and transferred into digestive tract. Over 95% of bile acids can be restore HFD-induced increases in the relative abundance of Acetatifactor, reabsorbed in the distal ileum and returned to the liver, while only a Oscillibacter and Flavonifractor. Lactobacillus has been suggested to small proportion are excreted in feces. Lactobacillus could promote 9 X. Zhang et al. Archives of Biochemistry and Biophysics 705 (2021) 108894 the fecal excretion of bile acids. In addition, Acetatifactor has also A. Wree, A. Eguchi, M.D. McGeough, C.A. Pena, C.D. Johnson, A. Canbay, et al., NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver been reported to convert bile acids into secondary bile acids, lithocholic inflammation, and fibrosis in mice, Hepatology 59 (2014) 898–910, https://doi. acids, which is hepatotoxic [41,42]. 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