The protective effects of PCPA against monocrotaline-induced pulmonary arterial hypertension are mediated through the downregulation of NFAT-1 and NF-κB

Abstract

Inflammation and remodeling play a role in the pathogenesis of pulmonary arterial hypertension (PAH). Nuclear factor-κB (NF-κB) and nuclear factor of activated T cells-1 (NFAT-1) participate in inflammation and remodeling in a number of diseases. As a tryptophan hydroxylase inhibitor, 4-chloro-DL-phenylalanine (PCPA) had been reported to exert anti-inflammatory and remodeling effects. Therefore, we hypothesized that PCPA may attenuate monocrotaline (MCT)-induced PAH through the NFAT-1 and NF-κB signaling pathways. In order to confirm our hypothesis, we divided 68 Sprague-Dawley male rats into 4 groups as follows: the control, MCT, MCT + P1 and MCT + P2 groups. MCT was administered at a dose of 60 mg/kg once via intraperitoneal injection. PCPA was administered via intraperitoneal injection at a dose of 50 or 100 mg/kg once daily for 21 consecutive days. We then measured the hemodynamic index and morphological analysis was carried out on the lung tissues. Western blot analysis and immunohistochemistry were used to examine the levels of NFAT-1 and NF-κB p-65. The expression levels of phosphorylated inhibitor of NF-κB kinase (p-IKK), IKK, phosphorylated extracellular signal‑regulated kinase (p-ERK), ERK, intercellular adhesion molecule-1 (ICAM-1) and inter-leukin-6 (IL-6) were examined by western blot analysis. MCT was found to significantly induce PAH, with inflammation and remodeling of the lung tissues. This was associatd with an increased expression of NFAT-1, p-IKK, p-ERK and nuclear p65. PCPA significantly attenuated MCT-induced inflammation and arterial remodeling, and decreased the expression of NFAT-1, as well as that of relevant proteins of the NF-κB signaling pathway. The above-mentioned findings suggest that the inhibitory effects of PCPA on MCT-induced inflammation and arterial remodeling are related to the downregulation of the NFAT-1 and NF-κB signaling pathways in rats with PAH.

Figure 1

Comparison of haemodynamic measurements between the different groups. (A) Pulmonary arterial pressure (PAP) and (B) systolic arterial pressure were compared among the different groups. Data are shown as the means ± SD. ^*P<0.05 and ^**P<0.01 represent indexes compared with the control group. ⁺P<0.05, ++P<0.01 represent indexes compared with the monocrotaline (MCT) group. P represents 4-chloro-DL-phenylalanine (PCPA); MCT + P1 represent MCT 60 mg/kg once plus PCPA 50 mg/kg body weight a day for 21 days; MCT + P2 represent MCT 60 mg/kg once plus PCPA 100 mg/kg body weight a day for 21 days.

Figure 2

Results of the effects 4-chloro-DL-phenylalanine (PCPA) on pulmonary arterial wall thickness ratio are compared. (A) Control group, (B) monocrotaline (MCT) group, (C) MCT + P1 group (PCPA at 50 mg/kg body weight) and (D) MCT + P2 group (PCPA at 100 mg/kg body weight); (E) percentage of medical wall thickness of pulmonary arteries. Original magnification, ×400; scale bars, 50 µm. Data are shown as the means ± SD (n=3 rats). ^*P<0.05, ^**P<0.01 represent indexes compared with the control group. ⁺P<0.05 and ⁺⁺P<0.01 represent indexes compared with the MCT group.

Figure 3

Comparison of elastin and collagen deposition between the different groups. (A) Double staining in elastin and collagen in lungs. (B) Double staining in elastin and collagen in main pulmonary arteries. (a) Control group, (b) monocrotaline (MCT) group, (c) MCT + P1 group (PCPA at 50 mg/kg body weight) and (d) MCT + P2 group (PCPA at 100 mg/kg body weight). Bluish green represents elastin and the red represents collagen. Original magnification, ×400; scale bars, 50 µm.

Figure 4

Immunohistochemical determination of nuclear factor of activated T cells-1 (NFAT-1) expression in lung vessels. (A) Control group, (B) monocrotaline (MCT) group, (C) MCT + P1 group (PCPA at 50 mg/kg body weight), (D) MCT + P2 group (PCPA at 100 mg/kg body weight) and (E) average optical density of NFAT-1. Original magnification, ×400; scale bars, 50 µm. Data are shown as the means ± SD (n=3 rats). ^*P<0.05, ^**P<0.01 represent indexes compared with the control group. ⁺⁺P<0.01 represents indexes compared with the MCT group.

Figure 5

Western blot analysis of the expression of nuclear factor of activated T cells-1 (NFAT-1) in lung tissues of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). Comparisons between the NFAT-1 and the control group, MCT-induced pulmonary arterial hypertension (PAH) group and 4-chloro-DL-phenylalanine (PCPA) treatment groups were made. Data are shown as the means ± SD (n=5 rats). ^*P<0.05, ^**P<0.01 represent indexes compared with the control group. ⁺P<0.05 and ⁺⁺P<0.01 represent indexes compared with the MCT group. P represents 4-chloro-DL-phenylalanine (PCPA); MCT + P1 represent MCT 60 mg/kg once plus PCPA 50 mg/kg body weight a day for 21 days; MCT + P2 represent MCT 60 mg/kg once plus PCPA 100 mg/kg body weight a day for 21 days.

Figure 6

Immunohistochemical determination of nuclear factor-κB (NF-κB) p65 in lungs. (A) Control group, (B) monocrotaline (MCT) group, (C) MCT + P1 group, (D) MCT + P2 group and (E) average optical density of p65. Original magnification, ×400; scale bars, 50 µm. Data are shown as the means ± SD (n=3 rats). ^**P<0.01 represents indexes compared with the control group. ⁺P<0.05 and ⁺⁺P<0.01 represent indexes compared with the MCT group. P represents 4-chloro-DL-phenylalanine (PCPA); MCT + P1 represent MCT 60 mg/kg once plus PCPA 50 mg/kg body weight a day for 21 days; MCT + P2 represent MCT 60 mg/kg once plus PCPA 100 mg/kg body weight a day for 21 days.

Figure 7

Comparison of nuclear factor-κB (NF-κB) p65 expression in nucleus and cytoplasm in rat lungs by western blot analysis. Comparisons between the NF-κB p65 among different groups, control, monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and 4-chloro-DL-phenylalanine (PCPA) treatment groups were made. Data are shown as the means ± SD (n=5 rats). ^**P<0.01 represents indexes compared with the control group. ⁺⁺P<0.01 represents indexes compared with the MCT group. P represents 4-chloro-DL-phenylalanine (PCPA); MCT + P1 represent MCT 60 mg/kg once plus PCPA 50 mg/kg body weight a day for 21 days; MCT + P2 represent MCT 60 mg/kg once plus PCPA 100 mg/kg body weight a day for 21 days.

Figure 8

Western blot analysis of the phosphorylation levels of extracellular signal-regulated kinase (ERK) in lungs. Comparisons of ERK phosphorylation among different groups, control, monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and 4-chloro-DL-phenylalanine (PCPA) treatment groups, were made. Data are shown as the means ± SD (n=5 rats). ^**P<0.01 represents indexes compared with the control group. ⁺⁺P<0.01 represents indexes compared with the MCT group. P represents 4-chloro-DL-phenylalanine (PCPA); MCT + P1 represent MCT 60 mg/kg once plus PCPA 50 mg/kg body weight a day for 21 days; MCT + P2 represent MCT 60 mg/kg once plus PCPA 100 mg/kg body weight a day for 21 days.

Figure 9

Western blot analysis of the phosphorylation levels of IκB kinase (IKK) in lungs. IKK phosphorylation in the different groups, control, monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and the 50 and 100 mg/kg 4-chloro-DL-phenylalanine (PCPA) treatment groups was examined. Data are shown as the means ± SD (n=5 rats). ^*P<0.05 and ^**P<0.01 represent indexes compared with the control group. ⁺⁺P<0.01 represents indexes compared with the MCT group. P represents 4-chloro-DL-phenylalanine (PCPA); MCT + P1 represent MCT 60 mg/kg once plus PCPA 50 mg/kg body weight a day for 21 days; MCT + P2 represent MCT 60 mg/kg once plus PCPA 100 mg/kg body weight a day for 21 days.

Figure 10

Western blot analysis of the expression of (A) intercellular adhesion molecule-1 (ICAM-1) and (B) interleukin-6 (IL-6) in rat lungs. Comparisons of ICAM-1 and IL-6 expression in different groups, control group, monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) group and 4-chloro-DL-phenylalanine (PCPA) treatment groups were made. Data are shown as the means ± SD (n=5 rats). ^*P<0.05 and ^**P<0.01 represent indexes compared with the control group. ⁺P<0.05 and ⁺⁺P<0.01 represents indexes compared with the MCT group.