Vitamin D protects intestines from liver cirrhosis-induced inflammation and oxidative stress by inhibiting the TLR4/MyD88/NF-κB signaling pathway

2.1 Animals and clinical blood samples

A total of 30 adult male SD rats (200–220 g, 6 weeks old) were obtained from Shanghai Model Organisms Center. We have successfully constructed the liver cirrhosis model using male rats according to a previous report [17]. The same results are expected in female animals.

1. Ethics approval and consent to participate: All procedures were approved by the Ethics Committee of Chengdu Public Health Clinical Center (Approval No: PJ-K2020-51-01, Chengdu, China). All of the blood samples were collected after written informed consent and the clinical characteristics are listed in Table 1.

2.2 Liver cirrhosis model and vitamin D therapy

Briefly, the rats were treated with CCl₄ (Cat No. C07315202, Nanjing Reagent, China) by oral gavage at a dose of 2 mL/kg body weight twice a week for 8 weeks to generate a liver cirrhosis model. The cirrhosis rats were randomly divided into five groups (n = 5 per group). The low-dose vitamin D (VD-L) and high-dose vitamin D (VD-H) rats were injected daily of vitamin D injection (Zhejiang Xianju Pharmaceutical Co., Ltd, Taizhou, China) intraperitoneally [18,19,20]. Besides, GSK1795091 (HY-111792; MedChemExpress, Princeton, NJ, USA) and diallyl tetrasulfide (Dat, ab143603; Abcam, Shanghai, China) were administered by tail vein injection to induce TLR4 and ROS levels, respectively. All rats were allowed free access to water and standard rat chow. Rats were anesthetized with isoflurane inhalation (induction dose of 5% and maintenance dose of 1.5%) and 3 mL of blood collected from the inferior vena cava in an EDTA tube, which was centrifuged at 1,500 rpm for 10 min to get the serum. After sacrificing the rats, the liver and ileum tissues of every rat were clipped and stored at −80℃ for the follow-up experiments.

Control group: Rats received oral administrations of the same volume of saline.

Cirrhosis group: Rats received CCl₄ by oral gavage at a dose of 2 mL/kg for 8 weeks.

Cirrhosis + VD-L group: Rats received vitamin D (500 IU/kg) on the fourth week of CCl₄ treatment.

Cirrhosis + VD-H group: Rats received vitamin D (2,000 IU/kg) on the fourth week of CCl₄ treatment.

Cirrhosis + VD-H + GSK1795091 group: Rats received vitamin D (2,000 IU/kg) and GSK1795091 (20 mg/kg) on the fourth week of CCl₄ treatment.

Cirrhosis + VD-H + Dat group: Rats received vitamin D (2,000 IU/kg) and Dat (25 mg/kg) on the fourth week of CCl₄ treatment.

2.3 Liver function and histological evaluation

Liver function was assessed by the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and albumin (ALB). The blood serum samples were obtained and subjected to analysis using an automatic biochemical analyzer (HITACHI 7600, Japan). Moreover, after being fixed with 10% formalin and embedded with paraffin, the liver tissues were cut into 4 µM slices for hematoxylin and eosin (HE) staining and Masson staining analyses according to the methods described previously to evaluate the development of liver fibrosis [21]. In addition, the ileum tissues of each group were stained with HE to examine the effect of liver cirrhosis on the intestinal morphological structure. The sections were photographed under a light microscope (Olympus BX51, Japan).

2.4 Immunohistochemistry

The clinical ileum tissues were collected and fixed with 4% paraformaldehyde. Then, the tissues were dehydrated, cleared, and paraffin-embedded. Four-micrometer paraffin sections were incubated with primary antibodies against ZO-1 (dilution ratio 1:1,000, ab221546; Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit horseradish peroxidase (dilution ratio 1:50, A0208; Beyotime, Shanghai, China) for 1 h at room temperature. Subsequently, the slides were stained with DAB and counterstained with hematoxylin for 30 s, followed by conventional treatments. Finally, photographs were photographed under observation by a light microscope (Olympus BX51, Japan).

2.5 Detection of the levels of 25(OH)VD₃, VDR, CYP24A1, and DEFA5

Serum 25(OH)VD₃ levels were analyzed through enzyme immunoassay using the 25-Hydroxy Vitamin D3 ELISA kit (Cat No. LS-F5643; LifeSpan Biosciences, USA). Serum VDR concentrations were detected using Enzyme-linked Immunosorbent Assay Kit (Cat No. MBS022460; MyBioSource, USA). The levels of CYP24A1 were determined by Cytochrome P450 24A1 (CYP24A1) ELISA kit (Cat No. abx508211; Abbexa, USA). The Defensin 5 (Sandwich ELISA) kit (Cat No. LS-F33773; LifeSpan Biosciences) was applied to measure the levels of DEFA5 in human blood samples. The DEFA5 level of rat intestinal tissues was determined using the reverse transcription polymerase chain reaction (RT-PCR) analysis.

2.6 Measurement of inflammatory cytokines and oxidative stress

The levels of inflammatory cytokines, including IL-6, CXCL1, IL-1β and TNF-α, in the ileum tissues were determined with ELISA kits (Beyotime). And the catalog numbers were listed as follows: IL-6 (PI335), CXCL1 (PC175), IL-1β (PI303), and TNF-α (PT516). Besides, the oxidative stress markers were detected in the ileum tissues of the rats. Ileum samples were homogenized in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, and the supernatants were used for the detection. The malondialdehyde (MDA) content and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were measured using MDA, CAT, SOD, and GSH-Px ELISA kits ((Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s protocol. And the catalog numbers were listed as follows: MDA (A003-1-2), CAT (A007-2-1), SOD (A001-3-2), and GSH-Px (A005-1-2).

2.7 Western blot

The ileum tissues were lysed in RIPA buffer (Cat No. P0013C; Beyotime) and the total protein concentrations were detected through a BCA kit (Cat No. P0012S; Beyotime). Immunoblotting was carried out as previously reported. Proteins (50 μg/lane) were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. After blocking the PVDF membrane (Cat No. IPVH00010; Millipore, USA), they were subsequently incubated overnight with primary antibodies against TLR4 (dilution ratio 1:1,000, AF7017; Affinity, China), MyD88 (dilution ratio 1:500, AF5192; Affinity), p-NF-kB (dilution ratio 1:1,000, ab194726; Abcam), NF-kB (dilution ratio 1:1,000, ab16502; Abcam), and GAPDH (dilution ratio 1:3,000, AF7021; Affinity). Then, the membranes were incubated with secondary HRP-conjugated antibodies (dilution ratio 1:3,000, S0001; Affinity) at room temperature for 2 h. The immunoreactive bands were then analyzed using the Western ECL reagent (Cat No. RPN2106V1, GE Healthcare, UK) according to the protocol of the manufacturer.

2.8 RT-PCR analysis

Total RNA was isolated by TRIzol reagent (Cat No. 10296010; Invitrogen, USA) according to the manufacturer’s instructions. cDNA was derived from 1 μg RNA using SuperScript III Reverse Transcriptase (Cat No.18080044; Invitrogen). Quantitative PCR was performed with an SYBR-Green Realtime PCR Master Mix (Cat No. FSK-101; ToyoBo Life Sciences, Japan) in a CFX Real-Time System (Bio-Rad, Germany). The sequences of primers for DEFA5 were forward, ATCCACTCCTGCTCTCCCTC, and reverse, AGAAAGACACAAGGTACACAGAGT. The sequences of primers for TLR4 were forward, ACTGGGTGAGAAACGAGCTG, and reverse, GTCCACAGCAGAAACCCAGA. The primers for MyD88 were forward, GTTTGTGCTTCCGGGAACAC, and reverse, GCAAAGAGGCCTCCATTCCT. The primers for NF-kB were forward, ACGGTGGGATTGCATTCCAT, and reverse, GCCAAGTGCAAAGGTGTCTG. GAPDH was used as an internal control. The primers for GAPDH were forward, ATGCCATCACTGCCACTCA, and reverse, CCTGCTTCACCACCTTCTTG. The quantification of the relative gene levels was calculated using the 2^−ΔΔCt method [22].

2.9 Statistics analysis

All the experiments were repeated at least three times, and the data were represented as mean  ±  standard deviation (SD). p-Value was calculated by either a two-tailed unpaired Student’s test or one-way analysis of variance followed by Tukeys’s post hoc test. Statistical analysis was carried out by GraphPad prism 7.0 version (GraphPad Software, CA, USA). p < 0.05 was considered statistically significant.

3.1 Liver cirrhosis affected the intestinal morphological structure

We established a liver cirrhosis rat model using CCl₄ treatment for 8 weeks. The morphological changes were detected through HE and Masson staining. As shown in Figure 1a and b, the results showed that in the control group, the hepatic lobule structure was clear, and hepatic cords were arranged radially around the central veins. However, the liver samples of the cirrhosis group represented the destroyed hepatic lobular structure and remarkable interstitial collagen deposition. Moreover, the data of serum biochemical indicators in rats showed that the levels of ALT, AST, and ALP in rat serum were remarkably increased in the cirrhosis group while the ALB level was lowered compared with the control group (Figure 1c–f). The results indicated that liver cirrhosis rat models were constructed successfully. We next sought to investigate the influence of liver cirrhosis on the structure of intestines using HE staining. As shown in Figure 1g, the control group had normal intestinal morphology. However, the liver cirrhosis group indicated severe intestinal mucosal damage with wider villi spacing and inflammatory cell infiltrations. Furthermore, the result of ZO-1 staining showed a significantly decreased expression of ZO-1 in intestinal tissues of the cirrhosis group compared to the control group (Figure 1h). The results demonstrated that liver cirrhosis triggered gut injury and intestinal barrier disruption.

Figure 1

Liver cirrhosis affected the intestinal morphological structure. The rat liver cirrhosis model was established by CCl₄ induction for 8 weeks. Histopathological characteristics in liver tissues with HE staining (a) and Masson Trichrome staining (b) (n = 5). Scale bar = 50 µm. Biochemical analysis of liver function between control and liver cirrhosis group: (c) ALT, (d) AST, (e) ALP, and (f) ALB (n = 5). (g) The histopathological changes in the intestines were evaluated by HE staining to investigate the impact of liver cirrhosis (n = 5). Scale bar = 50 µm. (h) Immunohistochemistry staining was applied to determine the expression of ZO-1. **p < 0.01 in 2-tailed t-test. Data are presented as mean ± SD.

3.2 Liver cirrhosis reduced 25(OH)-VD and decreased the levels of DEFA5 in intestinal tissues

The intestinal epithelium is the main target tissue of the biologically active form of vitamin D-25(OH)-VD₃, which regulates intestinal absorption of calcium, phosphate, and other biological effects [23]. 25(OH)-VD₃ predominantly acts through the VDR, a nuclear, ligand-dependent transcription factor. 25(OH)-VD₃ is inactivated through 24-hydroxylation by mitochondrial 24-hydroxylase (CYP24A1) to lose its normal physiological functions [24]. We measured the levels of 25(OH)-VD₃, VDR, and CYP24A1 in the ileum of rats and the serum of liver cirrhosis patients. As shown in Figure 2a–f, the concentrations of 25(OH)-VD₃ (Figure 2a and b) and VDR (Figure 2c and d) were remarkably attenuated in rat and human serum of liver cirrhosis, and in contrast, augmented levels of CYP24A1 (Figure 2e and f) were detected compared with the control group. Enteric defensins are antibacterial peptides secreted by Paneth cells in the small intestine. We further examined the levels of defensin 5 (DEFA5) in rat intestinal tissues using RT-PCR analysis and in human serum of liver cirrhosis using enzyme-linked immunosorbent assay (ELISA). The results revealed that a down-regulation of DEFA5 was determined in rat intestinal tissues (Figure 2g) and human serum of liver cirrhosis (Figure 2h). To summarize, we hypothesized that liver cirrhosis reduced hepatic 25(OH)-VD and inhibited the levels of DEFA5 in intestinal tissues.

Figure 2

Liver cirrhosis reduced 25(OH)-VD and decreased the levels of DEFA5 in intestinal tissues. The levels of 25(OH)-VD (a and b), VDR (c and d), and 24-hydroxylation by mitochondrial 24-hydroxylase (CYP24A1) (e and f) of rat and human serum of liver cirrhosis were measured through ELISA. (g and h) The concentrations of defensin 5 (DEFA5) in rat intestinal tissues and human serum of liver cirrhosis were detected using RT-PCR and ELISA, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 in 2-tailed t-test. Data are presented as mean ± SD.

3.3 Vitamin D inhibited liver cirrhosis-induced intestinal pathological injury and inflammation

Based on the above results, we concluded that liver cirrhosis caused severe injury and suppressed the concentrations of 25(OH)VD3 in intestinal tissues. Vitamin D acts a crucial role in safeguarding the intestinal barrier and preventing gastrointestinal mucosal inflammation. Ileum tissues of each group were stained with HE and the results are shown in Figure 3a. The intestinal injury was observed in liver cirrhosis rats with inflammatory cell infiltration and wider villi spacing compared to the control group. Rats with low-dose or high-dose vitamin D administration exhibited improved intestinal integrity. However, the treatment of GSK1795091 and Dat effectively reversed the vitamin D-mediated intestinal repair. Moreover, we next measured the levels of the inflammatory cytokines, including IL-6, CXCL1, IL-1β, and TNF-α, in the intestine to examine the role of liver cirrhosis on inflammatory response following vitamin D treatment. As shown in Figure 3b–e, the intestinal secretion levels of IL-6. CXCL1, IL-1β, and TNF-α were obviously increased in the liver cirrhosis group compared with the control group. And the treatment with low-dose or high-dose vitamin D significantly reduced inflammatory cytokine levels in the ileum tissues. Furthermore, the use of GSK1795091 and Dat obviously reversed the vitamin D-mediated inhibited inflammatory response. Hence, the intervention of vitamin D remarkably attenuated the secretion of inflammatory cytokines induced by liver cirrhosis.

Figure 3

Vitamin D inhibited liver cirrhosis-induced intestinal pathological injury and inflammation. Vitamin D was intraperitoneally administered daily to 100 μL of vitamin D solution at different doses. The ileum tissues of each group were harvested for HE staining (a). n = 5, scale bar = 50 µm. VD-L: low-dose vitamin D group (500 IU/kg); VD-H: high-dose vitamin D group (2,000 IU/kg). The levels of inflammatory cytokines including IL-6 (b), IL-8 (c), IL-1β (d), and TNF-α (e) in the intestinal samples were determined by ELISA (n = 5). *p < 0.05, **p < 0.01 in Tukey’s post hoc comparisons test. Data are presented as mean ± SD.

3.4 Vitamin D treatment effectively suppressed oxidative stress activated by liver cirrhosis

It has been reported that the regulation of oxidative stress plays crucial roles in the maintenance of intestinal homeostasis [25]. The changes in oxidative stress levels influence the microbial environment and the intestinal barrier integrity [26]. In our study, the oxidative damage was evaluated through the levels of CAT, GSH-Px, SOD, and MDA in intestinal tissues using ELISA kits. As shown in Figure 4a–d, the liver cirrhosis resulted in oxidative stress in the ileum tissues, reducing CAT, GSH-Px, and SOD and promoting MDA concentrations compared with the control group. In addition, the treatment of low-dose or high-dose vitamin D effectively inhibited oxidative stress by increasing CAT, GSH-Px, and SOD and attenuating MDA levels compared with the cirrhosis model group. However, the treatment of GSK1795091 and Dat effectively reversed the vitamin D-regulated oxidative stress. Taken together, liver cirrhosis may lead to oxidative stress in intestinal and the treatment of vitamin D significantly protects intestinal from oxidative stress-induced injury.

Figure 4

Vitamin D treatment effectively suppressed oxidative stress activated by liver cirrhosis. The levels of oxidative stress factors including CAT (a), GSH-Px (b), SOD (c), and MDA (d) in intestinal tissues were evaluated using ELISA kits (n = 5). *p < 0.05, **p < 0.01 in Tukey’s post hoc comparisons test. Data are presented as mean ± SD.

3.5 The intervention of vitamin D inhibited TLR4/MyD88/NF-κB signaling in the intestinal tissues of rats

The role of the TLR4/MyD88/NF-κB signaling pathway in mediating oxidative stress and inflammatory responses has been reported previously [27]. Given the role of TLR4 activation and the downstream adapter MyD88 in activating the NF-κB signaling pathway, we investigated the influences of liver cirrhosis on TLR4/MyD88/NF-κB signaling in ileum tissues using RT-PCR and Western blot analysis. As shown in Figure 5a–h, the results showed that liver cirrhosis activated TLR4/MyD88/NF-κB signaling in ileum samples of rats compared with the control group. Conversely, rats received low-dose or high-dose treatment of vitamin D exhibited decreased expression levels of TLR4, MyD88, and phospho-NF-κB p65 (p-NF-κB) in the ileum tissues compared with the liver cirrhosis model group. The treatment of GSK1795091 effectively reversed the vitamin D-mediated suppression of the TLR4/MyD88/NF-κB pathway while Dat had no significant influence on this. These findings unveiled that vitamin D suppressed the TLR4/MyD88/NF-κB signaling pathway activated by liver cirrhosis to facilitate intestinal damage repair.

Figure 5

The intervention of vitamin D inhibited TLR4/MyD88/NF-κB signaling in the intestinal tissues of rats. (a–c) The mRNA expression of TLR4, MyD88, and NF-κB was validated using RT-PCR analysis (n = 3). GAPDH mRNA was used to normalize the relative gene expression. (d–h) Western blot analysis was performed to determine the protein levels of TLR4, MyD88, NF-κB, and p-NF-κB of rat intestinal samples (n = 3). GAPDH protein was used as the internal reference. ns, no significant difference, **p < 0.01 in Tukey’s post hoc comparisons test. Data are presented as mean ± SD.