Dynamic expression of CyclinD1 and p21CIP1 during lung development in rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>CyclinD1 and p21CIP1 are major proteins to regulate lung cell proliferation and involved in lung development and lung injury reparation. This study aimed to explore the expression manners of CyclinD1 and p21CIP1 at canalicular, saccular and alveolar stages during lung development in Sprague-Dawley rats.</p><p><b>METHODS</b>Lung tissues were obtained from fetal rats of 20 and 21 days gestational ages, and neonatal rats at 0, 3, 7, 14 and 21 days (n=6). Lung tissues were used for histopathology and the protein analysis of CyclinD1 and p21CIP1 (immunohistochemistry and Western blot).</p><p><b>RESULTS</b>The strongest expression of CyclinD1 and the weakest expression of p21CIP1 occurred at 20-21 days gestation (canalicular stage). At the canalicular stage, CyclinD1 was mainly expressed in epithelial cells, and the expression of p21CIP1was negative. At the saccular stage, the expression of CyclinD1 decreased significantly and the p21CIP1 expression increased significantly. Positive expression of CyclinD1 and p21CIP1 was found in epithelial cells and interstitial cells. At the alveolar stage, the CyclinD1 expression was the lowest and the p21CIP1 expression was the highest. The positive expression of CyclinD1 was found in interstitial cells and that of p21CIP1 was found in epithelial cells.</p><p><b>CONCLUSIONS</b>The location and quantity of CyclinD1 and p21CIP1 expression are different at various stages during lung development in rats. A strongest CyclinD1 expression found in the canalicular stage may be associated a high lung cell proliferation. A strongest p21CIP1 expression found in the alveolar stage may be associated with alveolar maturity.</p>

Retinoic acid diminished the expression of lung tissue matrix metalloproteinase-2 and matrix metalloproteinase-9 in hyperoxia-exposed premature rats through regulating mitogen-activated protein kinases / 中华儿科杂志

<p><b>OBJECTIVE</b>To further investigate the protective effect of retinoic acid (RA) on hyperoxia induced lung injury and the role of RA as a modulator on mitogen-activated protein kinases (MAPKs).</p><p><b>METHODS</b>Establishment of hyperoxia (85%) induced lung injury model of premature Sprague-Dawley (SD) rats: 21 d gestational age SD rat's fetuses (term = 22 d) were delivered by hysterectomy. Within 12 - 24 h after birth, the premature rat pups were randomly divided into 4 groups: Group I, air-exposed control group; Group II, hyperoxia-exposed group; Group III, air plus RA-exposed group, Group IV, hyperoxia plus RA-exposed group. Group I and III were remained in room air, and group II and IV were placed in 85% oxygen. The pups in Group III and IV were injected with RA (500 microg/kg, every day) intraperitoneally. The entire lung tissues of premature rat pups were collected at 4 d, 7 d and 14 d. The mRNA levels of MMP-2 and MMP-9 were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-2 and MMP-9 activities were measured by zymography. Western blot was used to detect phosphorylated and total nonphosphorylated form of ERKs, JNKs and p38.</p><p><b>RESULTS</b>Exposure to oxygen for 4 d, 7 d, and 14 d resulted in increased mRNA levels of MMP-2 and MMP-9 compared with air-exposed control group (P < 0.01 for all). The mean protein levels of active MMP-2 and pro/active MMP-9 after exposure to O2 were higher than air control groups on each experimental day (P < 0.01 or < 0.05). The phosphorylated ERK1/2, JNK1/2 and p38 proteins in hyperoxia-exposed group increased markedly compared with air-exposed control group (P < 0.01 for all). The pups treated with RA in the hyperoxic environment expressed significantly lower mRNA levels of MMP-2 and MMP-9 than the hyperoxic control pups on each experimental day (P < 0.05 for all). The levels of active MMP-2 and pro/active MMP-9 decreased to a different degree after RA treatment in hyperoxia exposure rat pups. In addition, RA treatment led to a decrease of p-JNK1/2 and p-38 (P < 0.01 for all) protein levels and a further elevation of p-ERK1/2 compared with hyperoxia-exposed group.</p><p><b>CONCLUSION</b>Hyperoxia exposure elevated the expression of MMP-2 and MMP-9 markedly, which played a role in oxygen-induced lung injury. RA could have a protective effect on hyperoxia induced lung injury by decreasing active levels of JNK and p38, which subsequently reduce the expression and activation of MMP-2 and MMP-9.</p>

Protective effects of amygdalin on hyperoxia-exposed type II alveolar epithelial cells isolated from premature rat lungs in vitro / 中华儿科杂志

<p><b>OBJECTIVE</b>To analyze the effect of hyperoxia on the proliferation and surfactant associated protein messenger RNA levels of type II alveolar epithelial cells (AECIIs) of premature rat, and to investigate the effect of amygdalin on the change resulted from hyperoxia in AECIIs isolated from premature rat lung in vitro.</p><p><b>METHODS</b>The lung tissue of 20-day fetal rat was digested by trypsin and collagenase. AECIIs and lung fibroblasts (LFs) were isolated and purified at different centrifugal force and different adherence, then cultured. The nature of the cultures was identified by cytokeratin staining, vimentin staining and transmission electron micrography. For establishing hyperoxia-exposed cell model, purified AECIIs were cultured for 24 hours after culture flasks were filled with 95% oxygen-5% CO2 at 3 L/min for 10 min, and then sealed. Oxygen concentrations were tested in CYS-1 digital oxygen monitor after 24 hours of exposure. A sample was discarded if its oxygen concentration was < 90%. Cell proliferating vitality was examined by MTT assay after treatment with amygdalin at various concentrations. DNA content, protein expression of proliferating cell nuclear antigen (PCNA) and mRNA levels of SPs of AECIIs were analyzed with flow cytometric assay, Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively after 24 hours of air or hyperoxia exposure in the presence or absence of 200 micromol/L amygdalin.</p><p><b>RESULTS</b>Excellent yields of highly purified, culturable AECIIs could be obtained from 20-day fetal lungs. The expression of cytokeratin in AECIIs was positive and that of vimentin negative by immunocytochemistry. Those, however, in LFs were just opposite. Lamellar bodies in purified AECIIs were revealed by transmission electron micrography. The established hyperoxia-exposed cell model assured the oxygen concentrations of culture flasks more than 90%. Amygdalin at the concentration range from 50 micromol/L to 200 micromol/L stimulated the proliferation of AECIIs in a dose-dependent manner; however, at the concentration of 400 micromol/L inhibited the proliferation of AECII. Flow cytometric analysis showed that the apoptosis rate and G0/G1 phase percentage increased significantly (P < 0.01), S phase and G2/M phase percentage decreased significantly (P < 0.01), in hyperoxia group compared with that of air group. The apoptosis rate of air plus 200 micromol/L amygdalin group, compared with air group, was not significantly different (P > 0.05); however, G0/G1 phase percentage decreased markedly, S phase percentage increased significantly, G2/M phase percentage did not significantly change (P > 0.05). The apoptosis rate of hyperoxia plus 200 micromol/L amygdalin group was not significantly different (P > 0.05) from that of hyperoxia group, S phase and G2/M phase percentage increased significantly (P < 0.01), G0/G1 phase percentage decreased significantly (P < 0.01). Western blot analysis showed that the protein expression levels of PCNA in all group was significantly different, in turn, hyperoxia group < hyperoxia plus 200 micromol/L amygdalin < air group < air puls 200 micromol/L amygdalin (P < 0.01). SPs mRNA levels were significantly decreased in hyperoxia group, as compared with air group (P < 0.01). After amygdalin was added, SPs mRNA levels were elevated in air plus amygdalin group and hyperoxia plus amygdalin group, as compared with hyperoxia group (P < 0.01, P < 0.05, respectively), but compared with air group, SP mRNA levels were not significantly elevated (P > 0.05).</p><p><b>CONCLUSION</b>AECIIs of premature rats were isolated, purified and cultured successfully. Hyperoxia-exposed cell model was established in AECIIs of premature rat in this experiment. Amygdalin promotes the proliferation of premature rat AECII exposed to air or hyperoxia, the concentration of amygdalin with the best effect was 200 micromol/L. Hyperoxia inhibited the proliferation and decreased SPs mRNAs levels in AECIIs in vitro, which may contribute to hyperoxia-induced lung injury in premature rats. Amygdalin could inhibit the changes of SPs mRNAs levels and cell proliferation of AECIIs resulted from hyperoxia and may play partial protective role in hyperoxia-induced premature lung injury.</p>