The ethics committee of Anhui Medical University of Animal Welfare approved these experiments. Wistar rats (30 healthy males; weight, 200±20 g; 3–4 months old; food and water available ad libitum) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and bred in a modified specific pathogen free facility (temperature 22±2°C; humidity 55±2%) with a 12 h light/dark cycle). To induce lung injury, 3–4-month-old rats (male; weight, 200±20 g; 6 groups of 5 rats per group) were injected by caudal vein with one dose (5 mg/kg) of LPS (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). No treatment control (NT) rats were treated with physiological saline. After 24 h, bronchoalveolar lavage (BAL) fluid was collected and the lung was harvested to assess injury. In certain groups, rats received supplementation of 1, 5 or 25 mg/kg calcitriol (1086301; Sigma-Aldrich; Merck KGaA) by means of an intragastric injection 3 days prior to being challenged with LPS, while the control rats were administered physiological saline.

Pulmonary barrier permeability was assessed by using Evans blue dye. Evans blue dye (20 mg/kg) was injected via the caudal vein into rats 30 min prior to anesthesia, and was extracted from the lung lobes by incubation at room temperature for 24 h in formamide (3 ml/100 mg). The density of the supernatant was assessed spectrophotometrically at 620 nm. The total amount of Evans blue was determined against standard absorbance curves.

Suture clamps were placed on the right main bronchus resulting in the alveolus lavage being forced into the left lung. A tracheal injection of 3 ml physiological saline was injected and the lavage fluid was recovered; this was repeated three times. The lavage fluid was centrifuged for 10 min at 100 × g in 4°C to form a precipitant. The supernatant was removed and the precipitant was diluted to 1 ml by PBS containing 1% bovine serum albumin (Cat. #5003; TBD Science, Tianjin, China) and 0.1 µl transferred to a blood cell count plate. The number of cells were then counted under light microscopy at ×100 magnification.

Isolation of microvascular endothelial cells was performed according to methodology described in our previous study (19). Briefly, the fresh lungs, isolated from the sacrificed rats, were washed with 50 ml serum-free DMEM (FI101-01; Beijing Transgen Biotech Co., Ltd., Beijing, China). The pleura was carefully excised and discarded. The outer edges of the remaining lung tissue, which did not contain large blood vessels, were harvested and again rinsed in serum-free DMEM. This tissue was decreased further, using scissors, and was inserted into a glass pellet (Costar, Cambridge, MA) and washed in DMEM containing 20% fetal calf serum (SH40007-10; GE Healthcare Life Sciences. Logan, UT, USA), 100 U/ml penicillin/streptomycin and 2.5 g/ml amphotericin B. Tissue cultures were incubated at 37°C in saturated air containing 5% CO₂. After 60 h, the tissue was removed and thereafter, the culture medium was replaced every 3 days. Following reaching confluence, the cells were harvested by treating with a 0.25% trypsin. Cells were identified as endothelial cells by cobblestone morphology. A total of 2–3 drops of cell suspension were added to a coverslip and were allowed time to adhere. Coverslips were then washed with PBS 3 times prior to being air dried and fixed in cold acetone for 5–10 min. Coverslips were dried again and 0.5 ml fluorescein isothiocyanate labeled phytohemagglutinin (FITC-PHA; 25 mg/l; Sigma-Aldrich; Merck KGaA) was added and incubated for 30 min at 37°C. Finally, coverslips were observed by fluorescence microscopy at ×400 magnification. The cells used for experiments were between passages 2 and 5.

Total RNA was extracted from rat lung tissue and cultured rat PMVECs using TRIzol reagent (Sigma-Aldrich; Merck KGaA). cDNA was synthesized from 2 µg total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. qPCR was performed using the SYBR Premix DimerEraser kit (Takara Bio, Inc.) in an ABI 7300 Real-Time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reactions were performed under the following thermocycling conditions: 95°C for 60 sec, followed by 40 cycles at 95°C for 15 sec, 61°C for 15 sec and 72°C for 45 sec. The relative expression level was determined by the 2^−ΔCq method and normalized against GAPDH (20). The primers used are as follows: GAPDH, 5′-GTGCTGAGTATGTCGTGGAG-3′ (forward) and 5′-CGGAGATGATGACCCTTTT-3′ (reverse); ACE, 5′-CGGTTTTCATGAGGCTATTGGA-3′ (forward) and 5′-TCGTAGCCACTGCCCTCACT-3′ (reverse); ACE2, 5′-ACCCTTCTTACATCAGCCCTACTG-3′ (forward) and 5′-TGTCCAAAACCTACCCCACATAT-3′ (reverse); AT1R, 5′-GAAGCCAGAGGACCATTTGG-3′ (forward) and 5′-CACTGAGTGCTTTCTCTGCTTCA-3′ (reverse); renin, 5′-TTACGTTGTGAACTGTAGCCA-3′ (forward) and 5′-AGTATGCACAGGTCATCGTTC-3′ (reverse); and Ang II, 5-GTGGAGGTCCTCGTCTTCCA-3′ (forward) and 5′-GTTGTAGGATCCCCGAATTTCC-3′ (reverse); AT2R, 5-GCCAACATTTTATTTCCGAGATG-3′ (forward) and 5′-TTCTCAGGTGGGAAAGCCATA-3′ (reverse).

Renin activity and angiotensin II concentration in the bronchoalveolar lavage fluid or culture medium of rat PMVECs were determined using a Rat Renin ELISA kit (cat. no. CSB-E08702r; Flarebio Biotech LLC, College Park, MD, USA) or a Rat Ang II ELISA kit (cat. no. CSB-E04494r; Flarebio Biotech LLC), according to manufacturer's protocol.

Rat lung tissue or cultured rat PMVECs were lysed in ice-cold radioimmunoprecipitation assay cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (Beijing Transgen Biotech Co., Ltd.). Protein concentration of samples were estimated by BCA. The proteins (20 µg/lane) were separated with a SDS-PAGE (4–12% gel; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was put in glass bottle containing 20 ml sealing liquid (1 g skimmed milk powder, 20 ml PBS-Tween 20) and then was placed on the horizontal shaker and left to agitate at 25°C for 2 h. Probing was performed with specific primary antibodies (4°C overnight) and horseradish peroxidase-conjugated secondary antibodies (25°C for 2 h). The primary antibodies used were ACE (1:1,000; ab11734), ACE2 (1:500; ab108252), AT1R (1:500; ab124734) and AT2R (1:100; ab92445) from Abcam (Cambridge, UK) and GAPDH (1:1,000; HC301) from Beijing Transgen Biotech Co., Ltd. The secondary antibodies were horseradish peroxidase-conjugated affinity-purified goat anti-rabbit IgG (1:3,000; HS101-01) from Beijing Transgen Biotech Co., Ltd. Signals were detected using SuperSignal West Femto Trial kit (Thermo Fisher Scientific, Inc.; 34094; Lot#QE218149). The relative amount of proteins was quantified using gel analysis software UNSCAN-IT gel (version 5.3; Silk Scientific, Orem, UT, USA) and normalized to GAPDH internal loading control.

Data are presented as the mean ± standard deviation from at least three independent experiments. Statistical comparisons were performed by one-way analysis of variance followed by Tukey's test for multiple comparisons using SPSS (version 10.01; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

As previously described in our previous study (19), PMVECs were isolated from the outer periphery of the rat lung tissue and grew in monolayers with morphology consistent with endothelial cells, examined by phase-contrast microscopy. The cells grew initially as capillary-like structures and assumed cobblestone morphology, typical of endothelial cells at confluence. These cells were characterized as endothelial cells by factor VIII-associated Ag expression, and combined with FITC-PHA to demonstrate yellow green fluorescence. (Fig. 1A). To investigate the effects of calcitriol and LPS on the expression of ACE and ACE2 in PMVECs, rat PMVECs were incubated with 100 µg/ml LPS in the presence or absence of different concentrations of calcitriol (5, 20 or 100 nM). As demonstrated in Fig. 1B, compared with the NT group, LPS significantly induced ACE mRNA expression (8.1967±0.5749 vs. 4.7257±0.4767; P<0.01), and inhibited ACE2 mRNA expression (0.7553±0.0723 vs. 1.3931±0.0714; P<0.01; Fig. 1B). ACE and ACE2 have opposing functions in ALI; ACE2 counterbalances the deleterious effects of ACE and prevents lung injury (21,22). Therefore, the results demonstrated that LPS successfully induced ALI in the present study.

To determine whether calcitriol has an effect on LPS-induced ALI, concentrations of 5, 20 and 100 nM calcitriol were used in LPS-treated PMVECs. The results indicate that calcitriol-only treatment exhibited no obvious effect on ACE and ACE2 mRNA expression levels, compared with NT cells (Fig. 1B). However, the highest concentration of calcitriol (100 nM) significantly reduced the effects of LPS on ACE and ACE2 levels (Fig. 1B), which indicates an important calcitriol-dependent pathway in attenuating ALI caused by LPS.

As AT1R is a downstream effector of ACE, the effects of LPS and calcitriol on AT1R expression were subsequently investigated. As demonstrated in Fig. 1C, consistent with results for the ACE expression pattern, LPS significantly upregulated AT1R mRNA expression compared with NT cells, while calcitriol reduced the effect of LPS on AT1R mRNA levels in a dose-dependent manner. The mRNA expression of Ang II type 2 receptor (AT2R), employed as a control (21,22), was not significantly altered (Fig. 1C). Furthermore, alterations in the expression levels of ACE, ACE2, AT1R and AT2R at the protein level were confirmed by western blot analysis (Fig. 1D and E). Therefore, these results indicate that calcitriol may prevent LPS-induced ALI in rat PMVECs.

Increased renin activity stimulates the conversion of Ang I and ultimately Ang II (23), and Ang II is a key factor in the ACE/AT1R axis. Therefore, the expression of renin and Ang II in LPS-treated rat PMVECs was also investigated in the current study. As presented in Fig. 2A, LPS addition significantly increased renin and Ang II expression compared with NT cells. Although calcitriol-only treatment exhibited no effect on their expression compared with NT cells, calcitriol inhibited renin and Ang II expression in LPS-treated PMVECs in a concentration-dependent manner (Fig. 2A). ELISA was also performed to determine renin and Ang II concentrations in the culture medium, and the results were similar to RT-qPCR results (Fig. 2B). These results indicate that LPS supplement may trigger the ACE/Ang II/AT1R axis, while calcitriol exhibits an opposing function.

To determine whether calcitriol may prevent LPS-induced ALI in vivo, LPS was used to induce ALI in rats, as previously reported (15), and the total number of rat bronchoalveolar lavage cells were counted. Compared with the NT group, a significant increase was observed in the LPS group (Fig. 3A). However, high concentrations of calcitriol (5 and 25 mg/kg) reduced the number of bronchoalveolar lavage cells compared with the LPS-only group (Fig. 3A). To assess the severity of the lung vascular leakage, a leak index was calculated using Evans blue dye. Compared with the NT group, the Evans blue leakage significantly increased in the LPS group, and was reduced in calcitriol groups in a dose-dependent manner compared with the LPS-only group (Fig. 3B). These results indicate that calcitriol reduces rat lung permeability damage induced by LPS.

The mRNA expression levels of ACE and ACE2 in NT, LPS and LPS + calcitriol-treated lung tissues were also determined, and the results demonstrated that LPS increased ACE expression and decreased ACE2 levels compared with the NT group. Calcitriol treatment inhibited the effects of LPS on ACE and ACE2 mRNA expression (Fig. 3C). Furthermore, mRNA expression of the downstream factor AT1R was also induced by LPS in rat lung tissue, which was suppressed when combined with calcitriol treatment (Fig. 3D). Western blot results confirmed alterations in expression levels at the protein level (Fig. 3E and F). These results indicate that calcitriol may attenuate LPS-induced ALI in vivo.

As previously stated, renin and Ang II are two key members of the RAS (23). Therefore, the mRNA expression of renin and Ang II in rat lung tissues was also determined. LPS treatment induced pulmonary renin and Ang II mRNA expression in rat lung tissues significantly compared with the NT group (Fig. 4A). However, this induction was suppressed when combined with calcitriol treatment (Fig. 4A). ELISA was also performed to confirm renin and Ang II concentrations in the bronchoalveolar lavage fluid (Fig. 4B), and the results were similar to RT-qPCR results (Fig. 4A). Together, these results indicate that calcitriol may inhibit LPS-induced renin and Ang II expression in vivo.