Effect of Substance P on type II alveolar epithelial cells exposed to hyperoxia and its regulation of the Sonic hedgehog signaling pathway

Abstract

Oxidative stress injury and cell death in alveolar epithelial cells may lead to abnormal repair, further resulting in acute and chronic pulmonary diseases. Substance P (SP) has multiple biological activities. The Sonic hedgehog (SHH) pathway is important in lung development and decreasing epithelial injury. To investigate the effects of SP on alveolar epithelial type II cells (AEC IIs), AEC IIs were exposed to 95% oxygen and the SHH signaling pathway was examined. Primary AEC IIs were isolated and purified from premature rats. The cells were divided into four groups: The air (21% oxygen) group, hyperoxia (95% oxygen) group, hyperoxia + SP group and hyperoxia + SP + L703.606 group. The activity of AEC IIs was examined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. The apoptotic rate of AEC IIs was analyzed by flow cytometry. The oxidative damage was evaluated by flow cytometry and reactive oxygen species (ROS) were detected using a 2',7'‑dichlorodihydrofluorescein diacetate probe. Quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression of the SHH signaling molecule Smoothened (SMO). The results demonstrated that exposure to 95% oxygen for 24 h significantly increased the level of ROS, contributed to apoptosis and markedly decreased the proliferation of AEC IIs. Compared with hyperoxia exposure, SP treatment decreased the level of ROS, reduced AEC II apoptosis and improved the cell survival sequentially. SMO was found to be expressed in AEC IIs and its expression increased when the cells were in hyperoxic conditions. These effects were enhanced by treatment with SP, which was able to significantly increase the expression of SMO. The aforementioned protective effect was weakened following treatment with L703.606. These findings suggested that SP was a protective regulatory factor that was able to decrease the hyperoxia‑induced cell injury and death, and improve the survival of AEC IIs exposed to hyperoxia, which may be associated with the activation of the SHH signaling pathway.

Regulation of the angiotensin II-p22phox-reactive oxygen species signaling pathway, apoptosis and 8-oxoguanine-DNA glycosylase 1 retrieval in hyperoxia-induced lung injury and fibrosis in rats

“…In rats, alveolar development occurs only in the uesicae stage at birth, which corresponds to 25–35 gestational weeks in humans (1). Thus, continuous hyperoxic exposure of newborn rats might induce features such as alveolar maldevelopment and fibrosis, which are similar to those of ALI in human newborns (13,14,16). In the present study, the untreated hyperoxic model group exhibited the typical intolerance to lack of oxygen on day 7, which manifested mainly as breathing difficulties following deoxygenation, cyanosis and convulsions.…”

“…The bilateral lung tissues were preserved for microscopy, electron microscopy, and tissue-associated molecular examinations (13,14). …”

“…The optical density ratio (OD) to the GAPDH amplified fragment was used to express the relative amount of the above two mRNAs (13,14). …”

“…ROS levels were determined as previously described (13,14). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) presents green fluorescence following ROS oxidation in cells.…”

“…Positive cells were observed under a light microscope at ×400 magnification and exhibited brownish yellow-stained nuclei. Ten fields in each section were randomly observed to calculate the percentage of positive cells and the average number was considered as the apoptosis index (AI) (13,14). …”