The protective effects of glutamine against bronchopulmonary dysplasia are associated with MKP-1/MAPK/cPLA2 signalingmediated NF-kappaB pathway.

Glutamine is proven to have potential therapeutic effects on decreasing hyperoxia-induced acute pulmonary injury. The aim of this study is to investigate the effects and mechanism of glutamine on bronchopulmonary dysplasia (BPD) induced by hyperoxia in rat alveolar type II epithelial cells (AECIIs) RLE-6TN. Following hyperoxia induction and glutamine treatment, ROS levels were detected by DCFH-DA assay and TUNEL staining was performed to detect cell apoptosis. The levels of inflammatory indicators and expression of apoptosis-related proteins were detected through ELISA and Western blot, respectively. Besides, the expression of related proteins in mitogen-activated protein kinase phosphatase-1 (MKP-1)/mitogen-activated protein kinases (MAPK)/cytoplasmic phospholipase A2 (cPLA2) signaling was also detected by Western blot. To further analyze the role of MKP-1/MAPK/cPLA2 signaling, MKP-1 was silenced and anisomycin was used to treat cells, respectively. It was shown that glutamine significantly decreased inflammation, oxidative stress and apoptosis in hyperoxia-induced cells while MKP-1 interference and anisomycin were able to reverse these effects, suggesting that the protective effects of glutamine on BPD induced by hypoxia were related to MKP-1/MAPK/cPLA2 signaling. To sum up, glutamine protected against BPD by decreasing inflammation, oxidative stress and apoptosis via MKP-1/MAPK/cPLA2 signaling.

Protective effects of glaucocalyxin A on the airway of asthmatic mice.

The aim of this study is to investigate the protective effects of glaucocalyxin A (GLA) on airways in mouse models of asthma, concerning the inflammatory mediators, Th1/Th2 subgroup imbalance, and Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. Hematoxylin and eosin/periodic acid-Schiff staining was used to observe the pathological changes in lung tissues. Inflammatory cytokine contents in the bronchoalveolar lavage fluid were detected by enzyme-linked immunosorbent assay. Protein expression levels were detected with Western blot, immunohistochemistry, and immunofluorescence. In vivo studies showed that, in ovalbumin (OVA)-induced asthmatic mouse models, the GLA treatments reduced the airway hyperresponsiveness and the secretion of inflammatory cells, declined the proliferation of goblet cells, decreased the levels of IL-4, IL-5, and IL-13, and increased the contents of interferon-γ and IL-12. Moreover, GLA inhibited the protein expression levels of TLR4, MyD88, TRAF6, and NF-κB in OVA-induced asthmatic mouse models. Further in vitro studies showed that GLA inhibited the expression of NF-κB, p-IκBα, tumor necrosis factor-α, IL-6, and IL-1ß and blocked the nuclear transfer of NF-κB in lipopolysaccharide-stimulated RAW264.7 macrophages. Conclusively, GLA can inhibit the inflammatory responses in OVA-induced asthmatic mice and inhibit the release of inflammatory factors in LPS-induced RAW264.7 macrophages, which may be related to the inhibition of TLR4/NF-κB signaling pathway.

Salidroside Mitigates Airway Inflammation in Asthmatic Mice via the AMPK/Akt/GSK3ß Signaling Pathway.

INTRODUCTION: This study aimed to explore the effects and mechanisms of salidroside (SAL) in airway inflammation in asthmatic mice. METHODS: Mice were sensitized with ovalbumin (OVA) to establish an asthma model. They were divided into the control group, OVA group, SAL low-dose group (SAL-L), SAL high-dose group (SAL-H), and dexamethasone (DXM) group. The airway reactivity of the mice was measured, and the total cells, neutrophils, eosinophils, and lymphocytes were counted, respectively. The levels of IL-4, IL-5, IL-13, and IFN-γ in bronchoalveolar lavage fluid (BALF) were detected by ELISA. Immunohistochemistry was used to detect the expression levels of p-AMPK, p-Akt, and p-GSK3ß. Western blot was used to detect cytokine levels in lung tissue and p-AMPK, p-Akt, and p-GSK3ß levels in LPS-induced 16HBE cells. RESULTS: The airway hyperresponsiveness of asthmatic mice in the SAL-H group decreased (p < 0.05), and the total number of cells, neutrophils, eosinophils, and lymphocytes decreased significantly (p < 0.05). In addition, the airways of mice showed airway inflammatory infiltration and goblet cell proliferation, and the corresponding cellular inflammatory factors IL-4, IL-5, and IL-13 were significantly decreased. However, the expression of IFN-γ in BALF and lung tissues was increased (p < 0.05). Moreover, after the mice were treated with SAL, the phosphorylation level of AMPK was significantly increased, which further reduced the phosphorylation levels of Akt and GSK3ß (p < 0.05). Both SAL and AMPK inhibitors exerted effects on LPS-induced 16HBE cells, consistent with in vivo results. CONCLUSION: SAL can inhibit bronchial hyperresponsiveness and reduce tracheal inflammation by increasing AMPK phosphorylation and inhibiting Akt and GSK3ß signaling pathways.

[Effects of diesel exhaust particles inhalation on immediate reaction in asthma rats].

OBJECTIVE: The role of air pollution on asthma can not be ignored, diesel exhaust particles (DEP) in the air is one of the most important pollutants. This study aimed to investigate the effect and mechanism of DEP inhaled on immediate reaction in the asthma rats. METHOD: Sixty male Wistar rats of "Clean" grade, 6 - 7 week-old, with an average weight of (140 +/- 20) g were used in this study. The rats were randomly divided into 6 groups, 10 in each. Group A was treated with normal saline attack as a negative control, Group B with ovalbumin attack as a positive control. After ovalbumin attack, groups C, D, E, F continued to inhale DEP for 1 week, 2 weeks, 3 weeks and 4 weeks, respectively. The concentration of DEP was 200 microg/ml, the animals were subjected to inhalation of ultrasound nebulized DEP for 30 min per day. One week after all the attacks were concluded, Group A was stimulated with normal saline for 30 min, other groups were stimulated with ovalbumin. Then the airway resistance was determined with multi-channel signal acquisition and processing system and compared. The changes in neutrophils, eosinophils, and other inflammatory cells of BALF and the pathological changes in lung tissue, including epithelial cells loss, the inflammatory cells infiltration around the airway, basement membrane fibrosis, goblet cell hyperplasia etc. were observed. The concentration of IL-5 and gamma-interferon in the lung tissues, and the changes of serum IgE etc. were determined. RESULT: Airway resistance values of group A, B, C, D, E, F after ovalbumin excitation for 30 min were (3.56 +/- 0.21), (7.06 +/- 0.63), (6.46 +/- 0.38), (7.47 +/- 0.33), (8.87 +/- 0.61), (11.00 +/- 0.69) cm H2O/(ml.s). No airway hyperresponsiveness occurred in group A, while Groups B, C, D, E, F had higher airway resistance than group A, group E and F had higher airway resistance than that of group B, the differences were statistically significant. And the airway resistance was different in each group among 0 min, 10 min, 20 min and 30 min (F = 160.646, 148.901, 162.204, 156.186, P < 0.01 for both). The time of DEP inhalation and the airway resistance was positively correlated (r = 0.948, P < 0.01); IgE concentrations of the serum between groups B, C, D, E, F was not significantly different (P > 0.05), but higher than that of group A (F = 2.639, P < 0.01). The infiltrated inflammatory cells included eosinophils and lymphocytes, etc. The percentages of neutrophil(%) were (4.3 +/- 2.0), (9.7 +/- 5.2), (10.3 +/- 5.6), (13.0 +/- 5.2), (42.6 +/- 18.3), (55.3 +/- 6.9). The groups E and F had higher percentage than Group A and Group B (F = 114.226, P < 0.01). The percentages of eosinophils(%) were 0, (11.9 +/- 3.8), (15.8 +/- 6.3), (13.0 +/- 4.9), (21.1 +/- 5.6), (27.1 +/- 4.8). The difference between Groups B, C, D, E, F and Group A was statistically significant. There was significant difference between groups C, D, E, F and group B (F = 46.462, P < 0.05); Lung tissue biopsy in group A showed that the epithelial cells were intact, no inflammatory cells infiltrations were found around the airways, instead, mainly ciliated columnar epithelial cells and only a small number of goblet cells were seen without basement membrane fibrosis. With the inhalation of DEP, the epithelial cells showed gradual necrosis, disruption and loss, goblet cells showed hyperplasia, and infiltrations with inflammatory cells were seen around the airway. In the lung tissue, concentrations of IL-5 in group B, C, and E were (12.8 +/- 2.8), (17.1 +/- 5.2), (18.6 +/- 4.2) pg/mg, the difference between groups C, E and group B was statistically significant (F = 4.236, P < 0.01), the difference in gamma-interferon concentration among all groups was not statistically significance (F = 1.185, P > 0.05). CONCLUSION: DEP inhalation increased the airway responsiveness of asthma rats in immediate reaction, promoted the lung epithelial cell loss, inflammatory cell infiltration, basement membrane fibrosis and goblet cell hyperplasia.

[Effects of recombinant human insulin-like growth factor-1 on the expression of Clara cell secretory protein in lung of hyperoxia-exposed newborn rats].

OBJECTIVE: The development of neonatology and the availability of pulmonary surfactant have been helpful in effective reduction of the mortality of very low birth weight infants at the expense of an increasing number of survivors with bronchopulmonary dysplasia (BPD) caused by lung immaturity. BPD is a common syndrome in newborns, especially in preterm infants, when treated with hyperoxia and mechanical ventilation. Unfortunately, there have been no effective measure for the prevention and treatment of BPD. The purpose of this study was to investigate the influence of recombinant human insulin-like growth factor-1 (rh-IGF-1) on cell apoptosis and Clara cell secretory protein (CCSP) expression during the lung injury induced by hyperoxia, so as to assess its effect on the inflammatory lung injury and its developmental repair. METHODS: Eighty full term neonatal Wistar rats under the same condition were divided randomly into four groups on the second day after birth. Group I was air control, group II was exposed to hyperoxia, group III air + rh-IGF-1, and group IV was treated with hyperoxia + rh-IGF-1. The pups in the control group were kept in room air, while pups in hyperoxia group were kept in a Plexiglas chamber and exposed to over 85% oxygen. Pups in group III were under the same raising condition except for exposure to room air and treated with intraperitoneal injection of rh-IGF-1 (1 microg/Kg) everyday from the third day. Pups in group IV were treated with intraperitoneal injection of rh-IGF-1 (1 microg/Kg) everyday from the third day of exposure to hyperoxia. Lung tissue sections of the neonatal rats were stained with hematoxylin and eosin (HE) after 7 d of hyperoxia exposure, expression of CCSP was examined by immunohistochemical method, and apoptotic cell index of lung tissue was calculated by using TUNEL method. RESULTS: It was observed from immunohistochemical examination that positive staining of CCSP was distributed mainly in distal and respiratory bronchioles. The percentage of Clara cells in distal and respiratory bronchioles epithelium decreased in hyperoxia group (32.17 +/- 3.19)% compared to that in air control group (68.32 +/- 2.04)%, P < 0.01. Statistically significant differences were found in intensity of positiveness of Clara cells between hyperoxia (29.45 +/- 5.56) and air control group (42.37 +/- 3.24), P < 0.01. TUNEL assay showed that most apoptotic cells were alveolar and bronchial epithelial cells. The apoptotic index increased significantly in the hyperoxia group (55.77 +/- 6.09)% compared to the air control group (16.41 +/- 4.01)%, (P < 0.01). The positive rate (52.98 +/- 2.68)% of Clara cells and the expression (41.22 +/- 6.36) of CCSP in hyperoxia + rh-IGF-1 group increased significantly when compared with hyperoxia group, and the differences between these two group were also statistically significant (P < 0.01). The apoptotic index increased significantly in the hyperoxia + rh-IGF-1 group (27.98 +/- 3.09)% compared to the hyperoxia group (P < 0.01). CONCLUSIONS: Hyperoxia exposure can promote the pneumocyte apoptosis and inhibit the expression of CCSP. Rh-IGF-1 can remove the block of the formation of lung alveoli, increase the secretion of CCSP, mitigate inflammatory responses in airway and alleviate lung injury via pneumocyte apoptosis. Therefore, the results of this study provide a theoretic and experimental evidence for clinical application of rh-IGF-1 in prevention and treatment of BPD.

[Effects of immunoglobulin on the neuronal expression of IL-1beta and IL-1ra and the neuronal death at hippocampus in rats with convulsion induced by pentylenetetrazol].

OBJECTIVE: To study the effects of immunoglobulin on the neuronal expression of IL-1beta and IL-1ra and the neuronal death at hippocampus in rats with convulsion induced by pentylenetetrazol. METHODS: The epilepsy model was established by injecting intraperitoneally pentylenetetrazol (PTZ) into Wistar rats. Forty-five rats were randomly divided into three groups, normal control group, PTZ plus intravenous immunoglobulin (PTZ-IVIG); PTZ plus normal saline (PTZ-NS). Neuronal death was assessed by light microscopy with the hematoxylin-eosin (HE) staining and with in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). IL-1beta and IL-1ra expressions were examined by histochemistry. RESULTS: The ratio of IL-1beta/IL-1ra at hippocampal CA(1) region in PTZ-IVIG group (0.5 +/- 0.1) was significantly lower than that in PTZ-NS group (1.9 +/- 0.5, t = 12.9, P < 0.05). Apoptotic cell numbers at the hippocampal CA(1) region were significantly decreased in the PTZ-IVIG group, compared to PTZ-NS group (t = 27.1, P < 0.05). The numbers of positive cells were 16.4 +/- 3.3/1000 microm(2) in the former and 41.7 +/- 3.5/1000 microm(2) in the latter. Necrotic cell numbers at the hippocampal CA(1) region were significantly decreased in the PTZ-IVIG group (19.0 +/- 2.6/1000 microm(2)), compared to PTZ-NS group (42.3 +/- 4.9/1000 microm(2), t = 20.9, P < 0.05). CONCLUSION: Immunoglobulin could inhibit neuronal death induced by convulsion and its possible mechanism might be the regulation of IL-1 system in neurons.