Cell apoptosis and bile acid induced newborn lung injury

Background:

Neonate respiratory distress syndrome(NRDS) is a serious newborn disease results from pulmonary surfactant deficiency, which continue to be a leading causes of infant death in China, as well as America. Previous study suggested that the incidence of NRDS complicated from maternal intrahepatic cholestasis of pregnancy(ICP) is twice as control with unknown pathogenesis. And, this type of NRDS could happens in term or near term babies, different from the classic preterm NRDS, manifest severe respiratory failure and are not well response to pulmonary surfactant replacement therapy. Bronchoalveolar lavage analysis showed elevated bile acid concentration in ICP newborn's alveoli was the possible cause of NRDS, suggested this type of lung injury might be explained as “bile acid pneumonia”. However, the typical pathogenesis of NRDS dose not well match an acute pneumonia. There seems to be other pathways involved in the process of bile acid induced pulmonary surfactant deficiency. The effects of bile acid to pulmonary surfactant producing should be examined. Pulmonary surfactant are synthesized in alveolar epithelial type II cells (AECII), then secreted to alveolar surface by exocytosis. AECII are the most important epithelium in the lung, which act as a multifunctional center for lung development and repair. AECII death or injury decrease the number or activity of pulmonary surfactant.. Glycochenodeoxycholate(GCDC) is the main toxic component of bile acid in ICP patients, it could induce cell apoptosis and causes a series of liver diseases. Oxidative stress, mitochondrial damage, and cell membranes disruption through their detergent action on lipid components are found in bile acid induced hepatocyte death. We suppose that elevated GCDC in the amniotic fluid of ICP mother induces fetus AECII death and leads to surfactant deficiency, which cause NRDS after delivery. In this study we investigated the cytotoxicity of bile acid to rat AEC II in vitro, intend to find out the mechanism of GCDC-induced cell death, and discover the pathogenesis of newborns lung injury from ICP mothers.

Method and major results:

(1) GCDC induced cell death including apoptosis and necrosis

We performed annexin V/PI staining followed by fluorescence-activated cell sorting analysis to detect cell apoptosis. The subpopulations of live, early apoptotic, late apoptotic, and dead cells were separated and calculated by different cell staining. Our results show that the cell death was a concentration-dependen profile in the presence of bile acid. A majority of apopotic cells (annexin V-positive cells) was arise in AECII cells treated with 200µm GCDC. Furthermore, there were more death cells(only PI-positive cells) in the AECII cells treated with 1000µm GCDC(Figure 2B). Subsequently, ATP release and LDH release were analyzed in AECII after treatment of GCDC. The results showed GCDC produced a dose-dependent increase in cell cytotoxicity measured as ATP release and LDH release. These results suggest that GCDC may contribute to AECII necrosis. To determine the mechanism of cell death activated as a result of GCDC treatment, AECII cells were treated with 200 and 1000µM GCDC. After 24h treatment, cells were harvested and DNA was isolated. The result shows an agarose gel electrophoresis of DNA with the characteristic laddering pattern of fragmented DNA. Each band represents polynucleosomal DNA in its typical multiple 200-bp fragments. Moreover, we also find the active caspase3-positive cells after treatment of 200uM GCDC for 24h.

(2) GCDC inhibit surfactant secretion from isolated AEC II

Bile acides can induce lung injury in newborn infants because these compounds are detectable in the bronchoalveolar lavage fluid of newborns from mothers with ICP affected by respiratory distress syndrome. Deficiency of surfactant was associated with NRDS. In this study, surfactant secretion from AEC II was detected after exposed to low and high concentrations of GCDC. The result shows surfactant secretion was significant decreased in AECII treated with GCDC.

Conclusion:

In this study, we report that GCDC induces primary cultured rat AECII cell death via oxidative stress, mitochondrial dysfunction, and inhibits surfactant secretion. Antioxidants, apoptosis and necrosis inhibitors decrease rat AECII cell death from GCDC stimulation. These findings discovered the bile acid's toxicity to lung epitheliums, which suggest cell death plays important roles in bile acid-induced lung injury. These likely contributes to the pathogenesis of NRDS in neonates from mothers with ICP. Furthermore, these finding suggest alveolar epithelial cell therapy should be considered as an important therapy for bile acid induced NRDS beside pulmonary surfactant replacement.

Previous study suggested that bile acid induced lung injury is extremely important in NRDS, meconium aspiration syndrome, aspiration related lung injury and in transplanted patients. Bile acid aspiration produces a severe chemical pneumonia in a porcine lung model and intratracheally injected bile acid shows to produce severe pulmonary edema in rabbits. ICP is a common complication of pregnancy with an incidence of 0.68%˜3.25%. Pruritus is the major manifestation based on chronic cholestasis with unknown causes. However, ICP is very dangerous to the fetus, it causes a serious of newborn disease including respiratory and cardiac diseases, even intrauterine sudden fetal death. A clinical study found bile acids were measurable in the bronchoalveolar lavage fluid of all of the infants in the intrahepatic cholestasis of pregnancy group but were absent in the control groups. Infants in the intrahepatic cholestasis of pregnancy group had significantly higher serum bile acid levels compared with those in both of the control groups. A study demonstrated that secretory phospholipase A2 activity was increased in babies born to ICP mothers, which suggested surfactant catabolize might be enhanced by bile acid. These data strongly suggested a bile acid induced lung injury, while the molecular pathway of bile acid induced pulmonocyte death was not reported yet, which is obviously very important, and different from the other pathways in published data.

Keywords: bile acid, glycochenodeoxycholate, alveolar epithelial type II cell, apoptosis, cell death