Effect of inhaled nitric oxide on aquaporin expression in newborn rat lung in acute hyperoxic lung injury

AIM:To investigate the effect of inhaled nitric oxide on aquaporin expression and alveolar epithelial fluid transport in newborn rats with acute hyperoxic lung injury. METHODS:32 newborn SD rats were randomized to breathe for 48 h room air (C), >95%O₂ (O), >95%O₂+10^-5 NO (NO only in the first 24 h, ONO), room air + NO (CN). Then, the rats were killed, the lung wet-to-dry weight ratio (Q_W/Q_D), the histology, and AQP1, AQP5, α₁-NKA, α-ENaC mRNA expressions in the lungs were measured. RESULTS:Compared with C group, the Q_W/Q_D in O group significantly increased (P<0.01), and AQP1 mRNA expression decreased significantly (P<0.01). Compared with O group, ONO group had a lower level of Q_W/Q_D (P<0.05), and AQP1 mRNA expression increased (P<0.05). AQP5 mRNA expression in all groups remained unchanged. CONCLUSION:In newborn rats with acute hyperoxic lung injury, inhaled 10-5 nitric oxide for 24 h may attenuate lung edema and increase AQP1 mRNA expression, suggesting that inhaled 10^-5 nitric oxide for 24 h may promote the AQP1 expression in lung in this model of acute lung injury.     

Aquaporins in lung

Aquaporins (AQPs) is a novel family of membrane protein whichis in charge of the transportation of water across the membranes. It has been testified that AQPs may be related to many physiologicalfunctions and pathophysiological disorders. In lung, 6 subtypes of AQPs were found, 4 of which have been located in certain tissue. AQP1, AQP3, AQP4 and AQP5 are located in upper respiratory tract and may contribute to the mucus production, bronchial moisturizing and gland secretion. AQP1 and AQP5 are located in lower respiratory tract and may participate in directing the water movement across the blood-gas barrier. AQPs in natal lung may be an important factor to accelerate the reabsorption of the lung liquid. In adult, some diseases such as asthma, pulmonary edema, adult respiratory distress syndrome, which have been considered unbalanced water movement, may be also concerned with AQPs. The relationship between lung edema and AQPs have not been elucidated in details. It should be verified how AQPs work in more organs and tissues as well as in some diseases.     

Alteration of AQP2 expression in kidney tissues of emphysema model rats

AIM: To observe the expressions of aquaporin 2 (AQP2) in kidney tissues and the contents of endotoxin (ET), interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α) in serum in emphysema model rats, and to investigate the relationship between lungs and kidney in humoral metabolism. METHODS: The rats of emphysema were treated by injecting lipopolysaccharide into the trachea with cigarette smoking. Immunohistochemistry and Western blotting analysis were used to observe the expression of AQP2 in kidney tissues. RT-PCR was applied to detect the expression of AQP2 mRNA in kidney tissues. Blood sample and lung tissue were taken and the levels of ET, IL-1β and TNF-α were measured by radioimmunoassay. RESULTS: AQP2 expression in the kidney tissue in model group was greater than that in control group, and the expression of AQP2 mRNA showed the same results (P<0.01). ET, IL-1β and TNF-α levels in serum and lung tissue in model group were markedly higher than those in control group (P<0.01). CONCLUSION: In the emphysema model rats, AQP2 expression is up-regulated in the kidney tissue. The mechanism of emphysema may be related to increasing the levels of ET, IL-1β and TNF-α in the serum and lung tissue obviously.     

Changes of aquaporin 4 expression after hepatic ischemia-reperfusion injury in rats

AIM: To study the relationship between the changes of aquaporin 4 (AQP4) expression and the liver functions in the process of hepatic ischemia-reperfusion (I/R) injury in rats. METHODS: Forty-eight Wistar rats were used to establish the animal model of hepatic I/R injury. The rats were subject to ischemia for 30 min and were randomly divided into 4 groups according to the time of reperfusion: 2 h group, 1 day group, 3 days group and 7 days group. The corresponding control animals were also set up. The serum was collected for detecting direct bilirubin (DB), indirect bilirubin (IB) and alanine transaminase (ALT). The pathological changes of the liver tissues were observed under microscope with HE staining. The protein expression of AQP4 was measured by the method of immunohistochemistry and the mRNA expression of AQP4 was detected by RT-PCR. RESULTS: Under microscope, degeneration and necrosis of the hepatic cells were observed in the liver tissues in I/R injury groups. Compared with sham operation group, the concentrations of DB, IB and ALT activity in I/R injury groups increased obviously, peaking on the first day after operation, then declining continuously and restoring to the normal levels on the 7th day after operation. The expression of AQP4 were significantly decreased in I/R injury animals in 2 h group, 1 day group and 3 days group, and reached the minimum level on the first day. The mRNA expression levels of AQP4 were also deceased in hepatic I/R injury rats in 2 h group, 1 day group and 3 days group, and reached the minimum level on the first day after operation, then increased slowly and restored to the normal levels on the 7th day after operation. CONCLUSION: Hepatic ischemia-reperfusion induces a decrease in AQP4 expression and impairs the liver functions, indicating an important role of AQP4 in the pathogenesis of liver ischemia-reperfusion injury.     

Effect of rhSOD on pulmonary nuclear factor-kappa B and MIP-1α expression in meconium-induced acute lung injury

AIM: To evaluate the role and mechanisms of recombinant human superoxide dismutase (rhSOD) in meconium-induced acute lung injury (ALI) by evaluating pulmonary MIP-1α and NF-κB expression. METHODS: 24 health male Sprage-Dawley rats were randomized to 3 groups (8, each group), followed by intratracheal (IT) administration with (1) saline at 1 mL/kg (control group); (2) 20% human newborn meconium suspension at 1 mL/kg, followed by saline at 1 mL/kg (Mec/saline group); (3) 20% human newborn meconium suspension at 1mL/kg, followed by rhSOD at 20 mg/kg (Mec/rhSOD group). The animal was killed 24 h after treatment. The measurements included the bronchoalveolar lavage (BAL) cell count, RT-PCR analysis of pulmonary MIP-1α mRNA expression, Western blotting analysis of pulmonary NF-κB expression. RESULTS: Meconium-induced ALI was characterized by increased BAL cell count, increased expressions of pulmonary MIP-1α mRNA and NF-κB protein ［(4.68±1.40)×109 cells/L vs (0.53±0.19)×109 cells/L, 3.60±0.75 vs 1.56±0.33, 0.72±0.31 vs 0.23±0.12, respectively in control rats, all P<0.01］. IT administration of rhSOD early in the ALI rat significantly decreased meconium-induced BAL cell count ［(3.13±0.77)×109 cells/L vs (4.68±1.40)×109 cells/L in Mec/saline rats, P<0.01］, inhibited the expression of pulmonary MIP-1α mRNA (2.20±0.39 vs 3.60±0.75, in Mec/saline rats, P<0.01) and NF-κB protein (0.44±0.21 vs 0.72±0.31 in Mec/saline rats, P<0.05). CONCLUSION: The early IT administration of rhSOD in ALI rat following meconium aspiration protects lung from inflammatory injury through inhibiting meconium-induced pulmonary MIP-1α mRNA and NF-κB protein expression.     

Effects of left renal vein partial ligation on rat renal aquaporin 1 and aquaporin 2 mRNA expression

AIM:Changes of rat renal aquaporin (AQP1) and AQP2 mRNA expression after partially ligating left renal vein were observed to explore molecular mechanism of renal tubular reabsorption decrease resulted by increasing renal vein pressure.METHODS: Adult male rats were randomly divided into left renal vein partial ligation group(ie. VCG, varicocele group)and sham operation group(SOG), left renal mRNA expression was analyed Northen blot two months later. RESULTS:Intensity of left renal AQP1 mRNA expression in VCG was weaker than that in SOG, there was not any difference in left renal AQP2 mRNA expression between two groups, no hybridization signal was found in testes.CONCLUSION:Decrease of renal tubular reabsorption resulted by partially ligating renal vein might be related to decrease of AQP1 mRNA expression; "hormone escape" might occur in collecting tubules.     

Effects of intratracheal administration of aflatoxin G1 on the expression of CC-10 in rat lung tissues

AIM: To explore the putative effects of single intratracheal administration of aflatoxin G1 (AFG1) on the expression of CC-10 of lung tissues in SD rats. METHODS: Male SD rats were intratracheally administrated with AFG1 (30 μg/kg body weight) and the animals were respectively sacrificed at 1, 3, 7 and 14 d after AFG1 treatment. Bronchial alveolar lavage fluid (BALF) was centrifuged and the supernatants were collected for LDH release assay using Detection Kit with biochemical method. The expression of clara cell 10 kD protein (CC-10 protein) in lung tissues was determined by FCM analysis and Western blotting respectively. The expression of CC-10 at mRNA level was analyzed by RT-PCR. RESULTS: LDH activity in BALF after AFG1 treatment for 1, 3 and 7 d was significantly increased as compared to that in their corresponding control group (P<0.01) and restored to control level at 14 d after AFG1 treatment. FCM, Western blotting and RT-PCR results showed that no significant changes in CC-10 expression at both protein and mRNA levels were found between AFG1 group and control group 1 day after AFG1 treatment. While the CC-10 expressions of lung tissues at both protein level and mRNA levels in AFG1 treated group were significantly decreased as compared to those in their corresponding control group at 3, 7 and 14 d after AFG1 treatment (all P<0.01). CONCLUSION: Intratracheal administration of AFG1 may cause injuries and decrease the expression of CC-10 in lung tissues of SD rats in vivo.     

Effect of rosiglitazone on expression of AQP1, VEGF-A and COX-2 in human peritoneal microvascular endothelial cells

AIM: To investigate the effect of rosiglitazone on the expression of aquaporin-1 (AQP1), vascular endothelial growth factor-A (VEGF-A) and cyclooxygenase-2 (COX-2) in human peritoneal microvascular endothelial cells.METHODS: Cultured peritoneal microvascular endothelial cells were divided into 4 groups. The morphological changes of the cells were observed under inverted microscope. The protein expression of AQP1, VEGF-A and COX-2 in human peritoneal microvascular endothelial cells was determined by Western blotting. The mRNA expression of AQP1, VEGF-A and COX-2 in the cells was measured by real-time PCR. RESULTS: Rosiglitazone stimulated the proliferation of the cells. Rosiglitazone up-regulated the expression of AQP1, and down-regulated the expression of VEGF-2 and COX-2 at mRNA and protein levels in the cells. The PPAR-γ antagonist GW9662 partly inhibited the up-regulation of AQP1 expression by rosiglitazone (P<0.05), but had no obvious effect on the expression of VEGF-A and COX-2 (P>0.05). CONCLUSION: Rosiglitazone up-regulates the expression of AQP1 and down-regulates the expression of VEGF-A and COX-2 in human peritoneal microvascular endothelial cells, thus promoting water transportation and attenuating peritoneal fibrosis during peritoneal dialysis.     

Effect of edaravone on expression of Aβ and AQP4 and activity of MMP2 and MMP9 in acute cerebral ischemia/reperfusion rats

AIM: To investigate the effect of edaravone on acute cerebral ischemia/reperfusion rats. METHODS: SD rats were randomly divided into sham operation group (saline), model group (modeling given saline), low dose group (edaravone at 6 mg/kg) and high dose group (edaravone at 10 mg/kg). The rat model was established by Zea Longa suture method. The nerve function scores were evaluated after operation, and the infarct volume was measured by TTC assay. The mRNA expression of aquaporin 4 (AQP4) and amyloid β-protein (Aβ) in the brain tissue was detected by RT-qPCR. The protein levels of APQ4 and Aβ were determined by Western blot. The activity of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9(MMP9) was detected by gelatin zymography. RESULTS: Compared with model group, edaravone administration markedly alleviated neurological deficits, histological damages and brain edema. The mRNA and protein levels of AQP4 and Aβ, and the activity of MMP2 and MMP9 were downregulated (P<0.05). Furthermore, the improvements in high dose group were significantly more effective than those in low dose group. CONCLUSION: Edaravone significantly reduces neurological deficits and brain edema in the rats with acute ischemic stroke, and the mechanisms may be related to the downregulation of AQP4 and Aβ, and the activation of MMP2 and MMP9.     

Impact of hydrogen sulfide donor on endothelin-1 and connective tissue growth factor expression in rats with pulmonary hypertension induced by high pulmonary blood flow

AIM: To explore the possible impact of hydrogen sulfide (H2S) donor-sodium hydrosulfide (NaHS) on endothelin-1 (ET-1) and connective tissue growth factor (CTGF) expressions in rats with pulmonary hypertension induced by high pulmonary blood flow. METHODS: Thirty-two male SD rats were randomly divided into 4 groups: shunt group, shunt+NaHS group, sham group and sham+NaHS group. Rats in shunt group and shunt+NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. After 11 weeks of experiment, rat systolic pulmonary artery pressure (SPAP), lung tissue H2S, plasma ET-1 concentration and lung tissue ET-1mRNA expression, as well as pulmonary artery CTGF protein expression were detected.RESULTS: After 11 weeks of experiment, SPAP, lung tissue ET-1mRNA, plasma ET-1 as well as pulmonary artery CTGF expressions were increased markedly, respectively, whereas H2S in lung tissue decreased significantly in rats of shunt group as compared with that in sham group (all P<0.05). After administration of NaHS for 11 weeks, H2S in lung tissue increased significantly, whereas SPAP, plasma ET-1 and lung tissue ET-1 mRNA expression as well as pulmonary artery CTGF protein expression decreased significantly, respectively, in rats of shunt+NaHS group as compared with that in shunt group (all P<0.05).CONCLUSION: NaHS might be involved in the development of pulmonary hypertension induced by high pulmonary blood flow by down-regulating vasoactive peptides ET-1 and CTGF expressions in lung tissues of rats.     

Expression of aquaporin 3 in human prostate

AIM: To determine the expression of aquaporin 3 (AQP3) in transitional and peripheral zone of human prostate tissue.METHODS: AQP3 mRNA expression was analyzed in five prostate tissues by RT-PCR. AQP3 protein expression and localization were characterized by Western blotting and immuno-staining with polyclonal anti-AQP3 antibody.RESULTS: The results of RT-PCR and Western blotting showed that AQP3 was expressed in transitional and peripheral zone of prostate tissue. The AQP3 protein expression in transitional zone was higher than that in peripheral zone. The results of immuno-staining indicated that AQP3 protein was expressed in secretory cells of prostate.CONCLUSION: The AQP3 was expressed in prostate tissue, suggesting that AQP3 play an important role in the secretion of prostate liquid.     

Effect of panaxadiols on AQP1 expression in lungs of LPS shock rats

AIM：To investigate the alteration of aquaporin 1 (AQP1) expression in lung tissues in endotoxic shock rats and the effect of panaxadiols.METHODS：The histological changes were examined by HE staining.The expression of AQP1 was analyzed by immunohistochemical staining and Western blotting.RESULTS：(1) Significant inflammatory changes in pulmonary interstitial in rats of LPS group were observed.However,in dexamethasone (DEX) treatment group and panaxadiols (PDS) treatment group,the pulmonary pathologic changes were much slighter.(2) Positive AQP expression in the endothelium of capillary vessels on the wall of alveolus were seen in control (CTR) group,while merely very weak expression in LPS group was detected.The positive staining in PDS groups and DEX group were significantly stronger than that in LPS group,and close to CTR group.(3) The results of Western blotting showed that the quantity of AQP1 expression in LPS group was significantly lower than that in other groups,also the expression in PDS groups and DEX group was slightly lower than that in control group.CONCLUSION：LPS inhibits the expression of AQP1,while PDS increases the strength of AQP1 expression in lungs of endotoxic shock rats.     

Role of 78 kD glucose-regulated protein in development of hepatopulmonary syndrome induced by multiple pathogenic factors

AIM: To explore the role of 78 kD glucose-regulated protein (GRP78) in the development of hepatopulmonary syndrome (HPS) in rats and the relation to intestinal endotoxemia (IETM). METHODS: The experimental animals were randomly divided into HPS groups of the 4th week, the 6th week and the 8th week. Normal control groups at the corresponding time points were also set up. The Wistar rat model of HPS produced in the process of liver cirrhosis was induced by employing multiple pathogenic factors to the animals. The rats in normal control group were designed by feeding with standard diet and tap water. Histopathological changes of the lung and liver were observed under microscope with the staining of hematoxylin and eosin (HE). The concentrations of alanine amino transferase (ALT), endotoxin and tumor necrosis factor α (TNF-α) in plasma, the contents of TNF-α and malondialdehyde (MDA) in the lung tissues were detected. The expression of GRP78 at mRNA and protein levels in the lungs was measured by the methods of RT-PCR and Western blotting, respectively. RESULTS: The level of endotoxin in plasma was gradually increased with the HPS development. The expression of GRP78 at mRNA and protein levels was also gradually increased with the HPS development and was significant at every time point. The endotoxin in plasma was positively correlated with the expression of GRP78 protein in the lung tissues of the rats with HPS. With the HPS development, the levels of ALT and TNF-α in plasma and the contents of TNF-α and MDA in the lung tissues were gradually increased. The content of endotoxin in plasma and the protein expression of GRP78 in the lung tissues were positively correlated with the contents of TNF-α in plasma and TNF-α and MDA in the lung tissues. The contents of TNF-α in plasma and GRP78 at mRNA and protein levels and TNF-α in the lung tissues were higher in the rats with HPS at every time point than those in normal control group. At the 6th week and the 8th week, the contents of endotoxin and ALT in plasma and MDA in the lung tissues of the rats with HPS were significantly higher than those in normal control group. CONCLUSION: IETM originated from the liver cirrhosis acts as a critical stressor of endoplasmic reticulum (ER) stress and activates ER stress in the lung by oxidative stress, resulting in increased expression of GRP78. Therefore,the increased expression of GRP78 induced by ER stress may play an important role in the development of HPS in rats.     

Regulation of retinoid X receptor-mediated-autophagy pathway in rat pulmonary ischemia/reperfusion injury

AIM: To investigate the regulation of retinoid X receptor (RXR)-mediated autophagy pathway in rat pulmonary ischemia/reperfusion (I/R) injury. METHODS: The male SPF-grade SD rats were randomly divided into 7 groups (n=10):normal control (C) group, sham (S) group, sham plus 9-cis-retinoic acid (SRA) group, sham plus HX531 (SH) group, I/R group, I/R plus 9-cis-retinoic acid (RA) group and I/R plus HX531 (HX531) group. The model of pulmonary I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. The animals in C group didn't receive any treatment. Only sternotomy was performed for the rats in S group, the hilum of lung was not clamped, and the rats were mechanically ventilated for 210 min. The clamping of the left hilum of lung for 30 min followed by 180 min of reperfusion was performed in I/R group. In SRA, SH, RA and HX531 groups, 9-cis-re-tinoic acid (5 mg/kg) was intraperitoneally injected at 90 min before establishment of the model. In SH group and HX531 group, HX531 at 5 mg/kg was intraperitoneally injected at 30 min before establishment of the model. Left lung tissues were removed after 180 min of reperfusion for determing the index of quantitative assessment (IQA) of alveolar damage. The pathological changes of the lung were observed by HE staining, and immunofluorescence staining was used to observe the changes of RXRα in various lung tissues. The mRNA expression of autophagy-associated molecules LC3, beclin 1 and mTOR was detected by RT-PCR. The protein levels LC3-Ⅱ, beclin 1 and p-mTOR in each group were determined by Western blot. RESULTS: Compared with C group, the lung IQA, the mRNA expression of LC3 and beclin 1, and the protein levels of LC3 -Ⅱ and beclin 1 in I/R, RA and HX531 groups were increased significantly, the mTOR mRNA and p-mTOR protein levels were decreased (P<0.05), and the morphological structure of the lung was also impaired. Compared with I/R group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels were significantly decreased, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were increased in RA group (P<0.05), and the structural damage of the lung tissue was also significantly reduced. No statistically significant difference was observed between I/R group and HX531 group. Compared with RA group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels was increased significantly, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were decreased in HX531 group (P<0.05), and the morphological damage of the lung tissue was increased. CONCLUSION: The activation of RXR effectively alleviates the pulmonary I/R injury in rats. The protective role of RXR in lung tissue may be related to the inhibition of autophagy pathway.     

EGFR-p38 MAPK signaling pathway is involved in expression of HMGB1 in pulmonary tissues of rats with ventilator-induced lung injury

AIM: To investigate the role of epidermal growth factor receptor (EGFR)-p38 mitogen-activated protein kinase (MAPK) pathway in the expression of high mobility group box 1 protein (HMGB1) in the lung tissues of rats with ventilator-induced lung injury (VILI).METHODS: Thirty-two healthy Sprague-Dawley (SD) rats were randomly divided into 4 groups (n=8 each): group A, spontaneous breathing; group B, small tidal volume ventilation (VT=8 mL/kg); group C, high tidal volume ventilation (VT=40 mL/kg); group D, high tidal volume ventilation plus EGFR antagonist AG-1478. The rats in group B, group C and group D were mechanically ventilated for 4 h and then all animals were sacrificed.Total protein content and white blood cell (WBC) count in bronchoalveolar lavage fluid (BALF), the lung wet/dry weight ratio (W/D) and myeloperoxidase (MPO) activity were determined. The histological changes of lung tissues were observed by HE staining. The EGFR protein and mRNA expression, p38 MAPK activity and HMGB1 protein expression in the lung tissues were also detected.RESULTS: The inflammatory responses as evidenced by lung HE staining, total protein and WBC in BALF, the lung W/D and MPO activity were significantly higher in group C than those in group A (P<0.05). The mRNA expression of EGFR, EGFR activity, p38 activity and HMGB1 protein level also significantly increased in group C (P<0.05) as compared with group A. Significant decreases in the above indexes in group D were observed as compared with group C.CONCLUSION: High tidal volume ventilation induces acute lung injury, which may be related to up-regulation of HMGB1 expression through EGFR-p38 MAPK signal pathway.