25(OH)-D3 Alleviate Liver NK Cytotoxicity in Acute but not in Chronic Fibrosis Model of BALB/C Mice due to Modulations in Vitamin D Receptor

Research Article

Austin J Gastroenterol. 2019; 6(2): 1102.

25(OH)-D3 Alleviate Liver NK Cytotoxicity in Acute but not in Chronic Fibrosis Model of BALB/C Mice due to Modulations in Vitamin D Receptor

Salhab A, Amer J* and Safadi R

Research Center for Liver Diseases, Liver & Gastroenterology Units; Division of Medicine, Hadassah– Hebrew University Hospital- Jerusalem, Israel

*Corresponding author: Johnny Amer, Research Center for Liver Diseases, Liver & Gastroenterology Units; Division of Medicine, Hadassah–Hebrew University Hospital- Jerusalem, Israel

Received: July 10, 2019; Accepted: August 27, 2019; Published: September 03, 2019


Background: Low 25-Hydroxy-vitamin-D; “25(OH)-D3” serum and vitamin D receptor (VDR) levels were recently correlated to advanced fibrosis. However, VDR mechanism in liver fibrosis modulations is not well understood.

Aim: To evaluate associations of VDR with NK cell modulations during fibrogenesis.

Methods: Carbon-tetrachloride (CCl4) hepatic-fibrosis was induced in BALB/c mice for 1 and 4 weeks as an acute and chronic fibrosis model, respectively. Along 1st to 4th weeks; vitamin D were i.p injected/2x week. Liver were assessed histologically and for proteins quantification for VDR and fSMA expressions. In vitro, potential killing of NK cells were evaluated following coculture with primary-hepatic-stellate-cells (pHSCs) obtained from BALB/c WTmice.

Results: Hepatic-fibrosis increased along 4 weeks of CCl4 as indicated by serum ALT, aSMA expressions (P‹0.02) and histological assessments. These results were associated with increased NK1.1 activations and hypercalcemia. While vitamin D administrations delayed fibrosis of early stages, vitamin D worsen hepatic-fibrosis of late stages of CCl4. In week 4, no further activations of NK cells were seen following vitamin D injections and were associated with down-expressions of VDR (1.7 Fold, P‹0.004) indicating the inability of vitamin D to ameliorate hepatic fibrosis. In vitro, NK cells from the chronic model of CCl4 did not affect pHSCs killing and fail to reduce fibrosis.

Conclusion: Vitamin D alleviate liver NK cytotoxicity in acute but not in chronic fibrosis model due to modulations in vitamin D receptor and calcium. Hypercalcemia associated with late fibrosis may inhibited VDR levels, however, may not explain the profibrogenic effects of vitamin D.

Keywords: Hepatic fibrosis; NK cells; Vitamin D receptor; Hypercalcemia


Vitamin D receptor- VDR, Hepatic stellate cells-HSCs, Carbon tetrachloride-CCl4, 25-(OH) D3 -Vitamin D.


Vitamin D is an important prohormone with known effect on calcium homeostasis [1], but recently there is increasing recognition that vitamin D also is involved in cell proliferation and differentiation, it has immunomodulatory and anti-inflammatory properties [2]. The effects of vitamin D are mediated through the vitamin D receptor (VDR) [3]. VDR is a member of the nuclear receptor super-family of ligand-inducible transcription factors, which are involved in many physiological processes, including cell growth and differentiation, embryonic development and metabolic homeostasis [4]. The transcriptional activity of this receptor is modulated by several ligands, such as steroids, retinoids and other lipidsoluble compounds, and by nuclear proteins acting as co-activators and co-repressors [5]. The liganded VDR heterodimerizes with the retinoid X receptor and binds to vitamin D response elements in the promoter of target genes, thereby affecting their transcription. The genomic organization of the VDR at locus 12q13.1 shows that the VDR gene itself is quite large (over 100kb) and has an extensive promoter region capable of generating multiple tissue-specific transcripts [6].

Clinical observations have recently demonstrated that 25-OH D serum levels were significantly lower in patients with chronic hepatitis C than in controls and that low 25-OH D serum levels were associated with more severe fibrosis and lower responsiveness to interferonbased therapy in those patients [7,8]. Other studies have shown that low 25-OH D serum levels are associated with poor liver function and more advanced stages of liver fibrosis in hepatitis C virus patients [9]. Moreover, a possible role for vitamin D in liver fibrosis has gained further support from the finding that VDR is expressed in human as well as in rat liver nonparenchymal cells, such as Hepatic stellate cells (HSCs) [10]. It has recently been suggested that VDR polymorphism is associated with primary biliary cirrhosis [11]. Vitamin D deficiency is a common phenomenon in chronic liver disease, particularly in advanced fibrosis and cirrhosis [12]. Whether this association reflects the cause of accelerated fibrosis progression or the consequence of impaired liver function in advanced disease is still unclear.

Materials and Methods


Male mice on the BALB/c background, 12 weeks of age, weighing 22 U± 0.5 g, received care according to NIH guidelines. Mice were purchased commercially from Harlan Laboratories, Jerusalem-Israel. All animal protocols were approved by the institutional animal care ethical committee of the Hebrew University and housed in a barrier facility.

CCl4 Model of Liver Injury and Fibrosis

BALB/c mice were IP injected with 0.5ml/kg body weight CCl4 (1:10 v/v in corn oil from Sigma) or vehicle (corn oil) twice a week for 4 weeks. 25(OH) D3 (Biogems, Cat# 3220632-10mg) at the dose of 0.5 microgram/100g body was IP injected twice a week, commencing one day after the first dose of CCl4. The animals were terminated 72hr after the final CCl4 injection, and whole livers and serum were collected for histological, cytological and biochemical analyses.

Alanine aminotransferase (ALT)

Blood samples were collected from BALB/c male hearts at the volume of 1ml blood; the samples were centrifuged for 5min at 4000rpm. Serum samples were drawn to an Eppendorf tubes, after blood centrifuge. Serum samples of 32μl were dropped onto the strip of the ALT (Reflotron Company) and analyzed by Reflovet ® plus Roche.

Liver NK cells isolation

Under deep ether anesthesia, mice were euthanized by isoflurane, USP 100% (INH), then the liver was removed and a part of the liver was transferred to Petri dish that contains 5ml DMEM medium (Biological industries; Cat# 01-055-1A). The liver tissue was thoroughly dissected by stainless steel mesh, the cells were harvested with the medium and added to 50ml tubes that contain 10ml DMEM, and then carefully cells were transferred to new tubes that contain Ficoll (Abcam; Cat# AB18115269). Tubes were centrifuged for 20 minutes, at 1,600rpm at 20°C. The supernatant in each tube was transferred to a new tube, for another centrifuge for 10 min, at 1,600 rpm at 4°C. After the second centrifuge, the pellet in each tube was resuspended in 1ml of DMEM for NK isolation kit (Stem Cells; Cat# 19665).

Primary HSCs isolation

HSCs were isolated from 12-week-old male BALB/c mice by in situ pronase, collagenase perfusion, and single-step Histogenz gradient as previously reported (Hendriks et al., 1985; Knook et al., 1982). Isolated HSCs were cultured in Dulbecco’s modified Eagle’s medium (Mediatech) containing 20% fetal bovine serum (Hyclone) on six-well plates for 40hr prior to end-point assays.

Flow cytometry

Harvested mice liver NK cells were adjusted to 106/ml in buffer saline containing 1% bovine albumin (Biological Industries; Cat# 02-023-5A), and were stained for the following antibodies: Antimice NK1.1 (murine NK cell marker) (Biogems; Cat# 83712-70), Anti CD49a (MACS; Lot# 5150716246), and anti-mice Lysosomalassociated Lysosomalassociated membrane protein-1 (CD107a; NK1.1 cells cytotoxicity marker) (eBioscience; Cat# 48-1071). All antibodies were incubated for 45min at 4°C. The cells were washed with 0.5ml of staining buffer and fixed with 20ml of 2% paraformaldehyde. All stained cells analyzed with a flow cytometer (The BD LSRFortessa™, Becton Dickinson, Immunofluorometry systems, Mountain View, CA).

Real Time PCR

Tissue RNA isolation: Total cellular RNA was isolated from liver tissue, using 2ml TRI Reagent (Bio LAB; Cat# 90102331) for each cm3 of tissue. The samples were homogenized for 5min at room temperature of 25°C. Chloroform at volume of 0.2ml (Bio LAB; Cat# 03080521) were added to each sample, incubated for 15min at room temperature and centrifuged (1,400rpm) for 15min at 4°C. RNA precipitation: The supernatant in each sample was transferred to new Eppendorf, 0.5ml of isopropanol (Bio LAB; Cat# 16260521) left for 10min at 25°C and centrifuged (12,000rpm) for 10min at 4°C. The supernatants were removed and one ml of ethanol 75% were added to the pellet and centrifuged (7,500rpm) for 5min. The pellets were dried in air at room temperature for 15min, 50μl of DEPC were added, and the samples were heated for 10min at 55°C.

cDNA preparation: Liver RNA was extracted as described above. Preparing of c-DNA was performed using High Capacity cDNA Isolation Kit (R&D; Cat # 1406197).

Real time PCR: Real-time PCR is performed for the quantification of the expression of the gene that encoded alpha Smooth Muscle Actin (aSMA) and Vitamin D Receptot (VDR), compared to GAPDH as a housekeeping gene by using Taqman Fast Advanced Master Mix (Applied Biosystem; Cat # 4371130).

Western blot analysis

Immunoblot analysis of aSMA in liver extracts performed. Following isolation, whole protein extracts were prepared in liver homogenization RIPA buffer (Sigma; R0278-50ML) with protease phosphatase inhibitor cocktail (Roche; 1183617011). Next, proteins (30μg per lane) were resolved on a 10% (wt/vol) SDS-polyacrylamide gel (Novex, Groningen, The Netherlands) under reducing conditions. For immunoblotting, proteins transferred to a PVDF membrane. Blots incubated 1h at 4°C temperature at in a blocking buffer containing 5% skim milk and then incubated with either mouse Mouse Anti Human\Mice a-SMA (Novusbio; NB600-531), Rabbit Anti-Collagen I (ab34710), Rabbit Anti-VDR (ab109234) and Mouse Anti Human\Mice β-Actin (R&D System; 937215), diluted 1/1000, overnight in 4°C, and subsequently, with peroxidase-conjugated goat anti-mouse and Rabbit IgG (PARIS, Compiegne, France) diluted 1/5,000, for 1.5h at room temperature. Immunoreactivity revealed by enhanced chemiluminescence using an ECL Kit (Abcam; ab133406).

Histological assessments of liver injury

The posterior one third of the liver was fixed in 10% formalin for 24 hours and then paraffin-embedded in an automated tissue processor. Seven-millimeter liver sections were cut from each animal. Sections (15mm) were then stained in 0.1% sirius red F3B in saturated picric acid as well as Masson trichrome stain for connective tissue stain (both from Sigma). Sirius red staining assessed using the modified Histological Activity Index (HAI) criteria, incorporating semi quantitative assessment of periportal/periseptal interface hepatitis (0- 4), confluent necrosis (0-6), focal lytic necrosis/apoptosis and focal inflammation (0-4), portal inflammation (0-4), and architectural changes/fibrosis and cirrhosis (0-6).

Statistical analysis

The results were evaluated using the Student’s t-test, with statistical significance set at P‹0.05. Comparison between the mean values of different experiments was carried out. All data were reported as mean ± SE.

Results and Discussion

25-(OH) D3 (vitamin D) reduced liver fibrosis and inflammation in acute while worsen fibrosis in chronic model of CCl4

Hepatic fibrosis was induced in BALB/c mice by biweekly IP CCl4 injections for 1 week (acute model) and 4 weeks (chronic model) and was compared with naive vehicle-treated mice (WT). Histological staining, ALT levels and aSMA expressions were performed for fibrosis assessments. Figure 1A shows staining of Trichrome and Sirius red for the three major animal groups with and without IP administration of vitamin D as described in materials and methods. Acute and chronic model of CCl4 showed gradual extent of collagenous connective tissue fibers of red fibrosis septae with both Trichrome and Sirius red stains in consistent with CCl4 exposure time.