In this study, the rat model of BPD was developed by exposing neonatal rats to a hyperoxic environment. Hyperoxia exposure increased oxidative stress and apoptosis levels in the lung tissue of neonatal rats and arrested the alveolar development of neonatal rats. We further investigated the expression of Cx26, Cx32, Cx43 and Cx46 in neonatal rat lung tissue. The results of real-time PCR showed that the expression levels of Cx26, Cx32, Cx43, and Cx46 genes in the hyperoxia group were significantly higher than those in the normoxia group. Immunohistochemical results showed that Cx26, Cx32, Cx43, and Cx46 were expressed in lung tissues of neonatal rats with normoxic and hyperoxic exposure. Immunofluorescence double-staining results confirmed that ATI cells expressed Cx26, Cx43 and Cx46, while ATII cells expressed Cx26, Cx32, Cx43 and Cx46, with Cx32 being expressed only in ATII cells. In addition, the Cx32 mRNA level was closely correlated with ROS level, AI index, and RAC value. Therefore, we believe that hyperoxia exposure increases the expression of Cx26, Cx32, Cx43 and Cx46, and that Cx32 may be closely related to oxidative stress, apoptosis and alveolar development, suggesting that Cx32 is likely involved in the development of BPD and could be a novel target for BPD management.
Each year, 15 million infants are born prematurely worldwide, which is approximately one-tenth of all births [30]. In the last 50 years, there has been tremendous progress in the management strategies for preterm infants, and the survival rate of extremely and very low birth weight infants has been on the rise, albeit the incidence of BPD remains high. Infants with BPD suffer from not only the significantly poor pulmonary function but also other systemic conditions, such as cardiac insufficiency, retinopathy, and delayed neurological development [31–32]. Therefore, BPD has become a major contributor to the poor prognosis of extremely and very low birth weight infants, putting heavy health and economic burdens on the patients' families and the society. Therefore, a more in-depth understanding of the molecular regulatory mechanisms underlying the development of BPD can help identify new targets and provide a new theoretical basis for BPD prevention and treatment.
The development of fetal lungs can be divided into five different periods, namely the embryonic, pseudoglandular, canalicular, saccular, and alveolar periods [33]. Most preterm infants with BPD are born in the saccular period and develop typical histopathological changes of BPD lungs during the alveolar period [34]. The primary reason for the occurrence of alveolar dysplasia in BPD infants is that the lungs of preterm infants are still in the saccular phase, with immature anatomical structures and physiological functions, making them highly susceptible to infection, hyperoxia, mechanical ventilation, and other influences, resulting in abnormal sequences and orientations of lung development and difficulties in completing normal growth and development through the alveolar period. Therefore, identifying the key molecular mechanisms underlying alveolar dysplasia has long been a topical research topic in the pathogenesis of BPD. An animal model of BPD is essential to developed a better understanding of the pathogenesis of BPD. Oxygen toxicity is an important trigger for BPD in preterm infants. Even when preterm infants are given the minimum required oxygen supplementation while a lung ventilation protection strategy is implemented, it is still strong enough to induce oxidative stress and cause lung injury [35–36]. The rodent BPD model established by hyperoxia exposure can well mimic the pathological changes of BPD in human preterm infants, which can help shape a better understanding of the pathogenesis of BPD and examine the therapeutic efficacy of anti-BPP approaches [37–40]. In this study, we placed neonatal rats within 12 hours after their birth in a hyperoxic environment (85% O2) until day 14 after birth. HE staining confirmed that the histopathological changes in their lungs were consistent with alveolar dysplasia in BPD, as demonstrated by a radical decrease in the number of alveoli, a dramatic increase in alveolar volume, the absence of secondary alveolar septa, the simplification of alveolar structure, and a significant decrease in the RAC value.
Clinical studies have confirmed that the levels of oxidative stress markers 8-OHDG and MDA in plasma or bronchoalveolar lavage fluid of preterm infants ultimately diagnosed with BPD were significantly higher than those of non-BPD preterm infants within 1 week after birth, in contrast to a significantly lower level of the antioxidant GSH than that in non-BPD preterm infants [41–43]. The results of this study also demonstrate that the levels of ROS and MDA in lung tissues of hyperoxia-exposed rats were significantly higher, while the GSH level was much lower, indicating that oxidative stress was induced in lung tissues of BPD rats. Therefore, antioxidant therapies may be effective in preventing and treating BPD. However, studies on antioxidant therapy against BPD have not provided a definite answer. The administration of ROS scavengers or antioxidant enzyme supplements in preterm infants in the early postnatal period has failed to reduce the incidence of BPD significantly, although it alleviated the clinical symptoms of BPD in preterm infants to a certain extent [44–47]. In this regard, people seek to improve the current antioxidant treatment regimen for preterm infants and identify new therapeutic strategies for preventing and treating BPD by gaining insight into the molecular regulatory network underlying the development of oxidative stress-induced BPD. Oxidative stress-induced apoptosis has been proven for its involvement in the onset and progress of several diseases. Apoptosis is essential in normal cell turnover, tissue development, and injury repair. A significant decrease or an excessive increase in apoptosis can lead to developmental disorders or abnormal damage repair in organs or tissues. In this study, we have assessed the apoptosis in the lung tissue through TUNEL staining and AI, and confirmed that the AI in lung tissues of group H was significantly higher than that of group N. To sum up, this study confirms that oxidative stress is triggered while apoptosis is significantly increased in lung tissues of hyperoxia-exposed neonatal rats.
Gap junction channels (GJCs) are one of the most common forms that constitute intercellular communication networks. GJCs are composed of gap junction proteins (connexins, Cxs) that can directly connect the cytoplasm of adjacent cells, allowing direct cell-to-cell transmission and exchange of various ions, nutrients, and second messengers, such as Ca2+, ATP, cAMP, IP3, amino acids, NO, and GSH. Cxs and GJCs serve as essential regulators in the respiratory system. They are indispensable in physiological conditions while playing an important role in pathological conditions such as the response of the organism to environmental stress [23]. The GJCs facilitate intercellular calcium wave transmission in bronchioles, which contributes to synchronized ciliary motility, thus allowing for targeted transport of mucus for the removal of toxins and microorganisms from the environment. In alveolar epithelial cells, intracellular calcium concentration is increased by mechanical stimulation affecting ATI cells, which in turn triggers the secretion of lung surface active substances by ATII cells by transmitting calcium waves to ATII cells via GJCs. In pulmonary capillaries, pulmonary endothelial cells can transmit pro-inflammatory signals via GJCs and upregulate the expression of P-selectin on the cytoplasmic membrane, which induces an inflammatory response.
Alveoli are the gas exchange units of the lung tissue. Disturbances in alveolar development can directly lead to abnormal gas exchange, which is one of the main pathological changes in BPD. Alveolar epithelial cells are the key target cells in BPD alveolar dysplasia. Alveolar epithelial cells comprise ATI and ATII cells. ATI cells are large and extremely flat, occupying more than 90% of the alveolar area and playing an essential role in the gas exchange process. However, AT II cells, the "progenitor" cells of alveolar development, are approximately twice as many as ATI cells. By means of freeze-fracture electron microscopy, the interconnectivity of alveolar epithelial cells through gap junction channels has long been demonstrated [48]. Each AT I cell shares gap junction channels with at least one AT I cell and one AT II cell. Among the 21 Cxs expressed in mammals, alveolar epithelial cells mainly express Cx26, Cx32, Cx43, and Cx46. Among them, AT I cells express Cx26, Cx43 and Cx46, with Cx43 being expressed predominantly. ATII cells, on the other hand, express Cx26, Cx32, Cx43 and Cx46, with Cx26 and Cx32 expressed predominantly and Cx32 expressed only in AT II cells. Cx43 is the most widely expressed Cx in mammals and is expressed in both AT I and AT II cells. However, in adult rat lung tissues, Cx32 is expressed only in ATII cells, and it may be involved in constituting inter-ATII GJCs. Interestingly, ATI cells cannot form GJCs with cells expressing only Cx32 [24]. Thus, ATII cells have two independent types of GJCs, Cx43-compatible GJCs and Cx32-compatible GJCs, whereas AT I cells have only Cx43-compatible GJCs. This suggests that in normal lung tissues, ATI-ATII intercellular communication mainly depends on Cx43-compatible GJCs; by contrast, ATII-ATII cells have independent intercellular channels, i.e., Cx32-compatible GJCs, which are not available for communication between ATII cells and ATI cells. However, in normal lung tissues, ATII cells can hardly connect directly with ATII cells to form inter-ATII GJCs. Therefore, Cx32 appears to play a smaller role than Cx43 in undamaged lung tissues. However, it has been reported that in damaged lung tissues, there is an increased chance of direct ATII-ATII connections, and Cx32-compatible GJCs may form a "priority" inter-ATII channel [23–24]. Thus, Cx32 plays a special part in the connection between ATIIcells and the outside world and may have unexpected effects, especially when alveolar epithelial cells are damaged.
To quantify the expression of Cx26, Cx32, Cx43 and Cx46 in lung tissues, we have performed real-time PCRs on neonatal rat lung tissues, and results revealed that Cx26, Cx32, Cx43 and Cx46 mRNA levels were significantly higher in neonatal rat lung tissue in the hyperoxia group than in the normoxia group. In this study, we have confirmed based on immunohistochemical results that Cx26, Cx32, Cx43 and Cx46 were expressed in both normoxia and hyperoxia exposed neonatal rat lung tissue. We used immunofluorescence double-staining to identify ATI and ATII on lung tissue sections and simultaneously detect the expression of Cx26, Cx32, Cx43, and Cx46. The immunofluorescence double-staining results confirmed that Cx26 was expressed in both ATI and ATII cells, with expression mainly in ATII cells; Cx32 was expressed only in ATII cells; Cx43 was expressed in both ATI and ATII cells; and Cx46 was expressed in both ATI and ATII cells, with expression mainly in ATI cells. These results align with those reported in past literature [23–24] that ATIcells predominantly express Cx43, while Cx32 is expressed only in ATII cells. We then analyzed Cx32 mRNA level with ROS level, AI level, and RAC value by the Pearson correlation coefficient methodology, and revealed a positive correlation between Cx32 mRNA level and ROS level, a positive correlation between Cx32 mRNA level and AI, and a negative correlation between Cx32 mRNA level and RAC value. The above results indicate that in hyperoxia-exposed neonatal rat lung tissue, Cx32 may be associated with oxidative stress and apoptosis and may be involved in the development of alveolar dysplasia in BPD.
This study has several limitations. First, BPD is a multi-factorial disease, and oxygen toxicity is only one of the many contributing factors; infection and ventilator-related volume/pressure injury can all fuel the development of BPD. In this study, we have only established a hyperoxia-exposed neonatal rat model to simulate BPD. Therefore, other BPD models are needed to further clarify the expression and effects of gap junction proteins in BPD. Second, this study lacks in vitro experiments on ATII cells, and the expression of Cxs in ATII cells has not been confirmed in vitro. In addition, the effects of Cx32 in BPD deserve further investigation, and the role that Cx32 plays in BPD has not been well defined in this study. Targeted intervention on Cx32 at the animal or cellular level may help to specify the effects of Cx32 in BPD and the mechanism underlying its actions.