Determination of rBD-2 mRNA in rat lungs by RT-qPCR

Total RNA was isolated from the whole lung tissue using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Takara Bio, Inc., Otsu, Japan). The following primers were used for PCR amplification of rBD-2: Forward, 5′-GAACTTGACCACTGCCACACC-3′; and reverse, 5′-GCTCTAGATTATCATTTCATGTACTTGCACC-3′. β-actin was used as a reference to normalize rBD-2 mRNA expression levels using the 2^−Cq method (23). The RT-qPCR primers used for β-actin in the present study were: Forward, 5′-AACAGTCCGCCTAGAAGCAC-3′; and reverse, 5′-GGTTGACATCCGTAAAGACC-3′. Each RNA sample was run in triplicate. PCR parameters were set as follows: 39 cycles of denaturation at 95°C for 20 sec, annealing at 59°C for 30 sec and extension at 72°C for 30 sec.

Establishment of acute pneumonia model of rat, animal grouping and treatment

Rats were randomly divided into four treatment groups and one control group, with eight rats each. Rats were anesthetized with ether, 1 cm skin incisions were made along the middle of the mouse's neck, and muscle tissues were bluntly dissected exposing the trachea and thyroid. A total of 0.2 ml PA (6×10⁸ CFU/ml, Xinjiang Uygur Autonomous Region Food and Drug Administration) was injected into each rat trachea in treatment groups, while, the control group was injected with the same dose of sterile phosphate buffered saline, then incisions were sutured. After three days, treatment groups were divided into four different groups: PA infected group, group treated with ATP at 16 mg/kg or cephalosporins at 0.4 g/kg through the tail vein, group treated with both ATP at 16 mg/kg and cephalosporins at 0.4 g/kg through tail vein. Rats were sacrificed 24 h following treatment, lung tissues and serum were collected and stored in liquid nitrogen for subsequent experiments.

Pathological examination

The grouping and treatment were the same as described above, lung tissues were isolated from rats 24 h following ATP and cephalosporin intervention. Fresh lung specimens were fixed in 4% paraformaldehyde, embedded in paraffin, cut and heated, and subjected to hematoxylin and eosin (H&E) staining. The morphological changes of H&E-stained tissues were analyzed by Leica digital microscopy.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections of lung tissues were obtained from treatment and control groups as described above in animal grouping and treatment. The expression of rBD-2 was detected following the immunohistochemistry protocol of Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The sections were incubated with primary rabbit polyclonal antibody rBD-2 (YY3068R; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:200 dilution overnight at 4°C and secondary antibody [SP9000D; DAB Detection kit (Streptavidin-biotin); ZSGB-BIO, Beijing, China] for another 2 h at room temperature. Finally, visualization of the sections was conducted and images were taken with a Leica confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).

Determination of IL-6 and tumor necrosis factor (TNF)-α expression in lung tissues and peripheral blood. At 24 h following ATP and cephalosporin intervention, rats from control and treatment groups were sacrificed and lung tissues were isolated, centrifuged at 2,045 × g for 20 min at room temperature followed by collection of supernatants. The peripheral blood samples were also collected. IL-6 and TNF-α expression was evaluated using ELISA assay kits in accordance with the instructions (Invitrogen; Thermo Fisher Scientific, Inc.).

ATP increases expression of rBD-2 protein in rat lungs

As expression of rBD-2 mRNA was upregulated by ATP in rat lungs, the next question concerned whether ATP promotes the production of rBD-2 protein in vivo. In the present study, the authors reported that the expression of the rBD-2 protein was slightly increased in the PA infected group, an enhanced expression of the rBD-2 protein was observed in the ATP or cephalosporin treatment group and the cephalosporin and ATP treatment group, in comparison with that in PA infected group and control group, suggesting ATP could induce rBD-2 expression at both mRNA and protein levels in rat lungs (Fig. 2).

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Figure 2.

Expression of β-defensin-2 protein of rat lungs detected by immunohistochemistry in different groups (magnification, ×200): (A) Control group treated with normal saline, (B) Pseudomonas aeruginosa infected group, (C) group treated with ATP, (D) group treated with ceftazidime, and (E) group treated with ceftazidime and ATP.

ATP alleviates histological changes in rat lungs

Microcopy indicated that alveolar and interstitial lung structures were normal, alveolar inflatable was good, no exudation of red blood cells was observed, part of bronchial and alveolar walls were infiltrated with a small amount of inflammatory cells in the lung tissues of the control group. The PA infected group presented alveolar wall edema, infiltration of a large number of inflammatory cells (neutrophils and lymphocytes) on bronchial and alveolar walls, red blood cells and cellulose exudation in the alveolar space. ATP or ceftazidime intervention groups presented an improved lung edema, less inflammatory cell infiltration, thicker partial alveolar wall and a small amount of red blood cells as compared with the PA infected group. The results demonstrated that ceftazidime, ATP and ceftazidime + ATP treatment groups improved the histological destruction of lung tissues caused by PA (Fig. 3).

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Figure 3.

Histopathological changes of rat lungs in different groups (magnification, ×200): (A) Control group treated with normal saline, (B) Pseudomonas aeruginosa infected group, (C) group treated with ATP, (D) group treated with ceftazidime, and (E) group treated with ceftazidime and ATP.