Objective To investigate the changes of renal medulla aquaporin 2 expression and morphological changes of epithelia of collecting tube after bile duct recanalizaiton operation. Methods Thirty rats were divided into two groups randomly. Common bile duct ligation was performed on 20 experimental rats with silicon tubes 2 mm in extre-diameter, and sham operation on the other 10 rats. Seven days later, bile duct recanalizaiton was performed on obstructive jaundice group and sham operation on contrast group. Experimental rats were divided into two subgroups randomly. Half of them were killed immediately and the others would be killed 24 hours later. Serum of each rat was collected to detect hepatic function and renal function. Renal medulla was fixed for microscopic examination and was kept in the -80 ℃ refrigerator for aquaporin 2 expression measurement by Western blot technique. Results All of the animals accomplished the experiment smoothly. Golden ascites were found in the rats of obstructive jaundice group. Twenty-four hours after recanalization, serum bilirubin levels decreased 〔(45.95±8.39) μmol/L〕, P＜0.01, and there was no significant change in blood urine and creatine level. Compared with sham operation group (21 966.20±1 544.70), expression of aquaporin 2 decreased significantly after common bile duct ligation in obstructive jaundice group (15 665.30±1 181.85), P＜0.01. After recanalizaion, the expression of aquaporin 2 in obstructive jaundice group increased (19 490.80±4 239.32), P＜0.01. Conclusion Common bile duct obstruction would lead to epithelium injury of renal collecting tube, and down regulate the aquaporin 2 expression.
ObjectiveTo observe the effects of aquaporin 1 (AQP1) on the proliferation and migration of endothelial progenitor-endothelial progenitor cells (EPC).MethodsBone marrow cells of AQP1 wild-type (WT) (n=6) and knockout-type (KO) mice (n=6) were isolated and differentiated into EPC in vitro. Immunofluorescence was used to detect cell surface antigens to identify EPC. Live cell kinetic imaging and quantification technology, transwell migration assays, as well as scratch test were used to compare the function of EPC between AQP1 WT and KO mice.ResultsEPC culture showed that cells were initially suspended and gradually adhered to typical mesenchymal stem cells within 7 days. After cultured on special medium for endothelial cells they were adhered and differentiated, and fusiform or polygonal, paving stone-like EPC were observed around 14 days. When cultured by special medium of EPC, CD133 and CD31 were positively detected after 7 days, and CD34 and Flk-1 were positively detected after 14 days. Positive expression of AQP1 was only detected in EPC of AQP1 WT mice. Functional studies of EPC revealed there was no significant difference in the proliferation of EPC between AQP1 WT and KO group mice. Transwell assay showed that EPC migration ability of AQP1 KO mice was significantly weaker than that of WT mice. The scratch healing ability of EPC in AQP1 KO mice was significantly lower than that of WT mice.ConclusionsEPC initially shows the characteristics of stem cells and with the prolongation of culture time, EPC gradually shows the characteristics of endothelial cells. AQP1 affects the EPC migration rather than proliferation.
Objective Aminoguanidine (AG) can reduce brain edema and increase the recovery of neuron functions in surgical brain injury and stroke. To investigate the effect of AG on spinal cord injury (SCI) in rats and its mechanism. Methods A total of 150 adult male Sprague Dawley rats (weighing, 230-255 g) were divided into control group (group A, 25 rats without treatment), the sham-operated group (group B, 25 rats undergoing laminectomy), SCI group (group C, 25 SCI rats with injection of 5%DMSO), SCI + AG groups (groups D, E, and F, 25 SCI rats and AG injection of 75, 150, and 300 mg/kg, respectively). The optimal dosage of AG was screened by dry-wet weight method with the percentage of water content at 0, 12, 24, and 48 hours after injury. The blood-spinal cord barriar permeability was further detected by Evans blue (EB) method, aquaporins 4 (AQP4) mRNA expression by RT-PCR, AQP4 protein expression by immunohistochemistry and Western blot. Results AG injection at dosage of 150 mg/kg can significantly reduce edema of spinal cords at 12, 24, and 48 hours after SCI (P lt; 0.05), so 150 mg/kg was the optimal dosage. The EB content in group E was significantly lower than that in group C at 12, 24, and 48 hours after SCI, and the permeability of blood-spinal cord barrier was significantly decreased compared with group C (P lt; 0.05). The AQP4 mRNA expressions in groups B and E were significantly lower than that in group C at 12, 24, and 48 hours after SCI (P lt; 0.05). AQP4 protein expressions in groups B and E were significantly lower than that in group C at 24 and 48 hours after SCI (P lt; 0.05) by Western blot. Immunohistochemical staining revealed that AQP4 protein expression in group C was significantly higher than that in groups B and E (P lt; 0.05) at 48 hours after SCI, but no significant difference was found between group B and group E (P gt; 0.05). Conclusion AG injection at dosage of 150 mg/kg can induce spinal cord edema and injury in rats, which could be correlated with the down-regulation of AQP4 expression.
ObjectiveTo explore the value of Aquaporin-3 (AQP-3) on the detection of early renal function damage by investigating the expressions of renal AQP-3 mRNA and protein of rats with obstructive jaundice (OJ). MethodsForty mature male Wistar rats were divided into two groups randomly: experimental group (n=20) in which the model of OJ rats was established, and control group (n=20, sham operation group). The levels of serum total bilirubin (TBIL), direct bilirubin (DBIL), creatinine (Cr)， and blood urea nitrogen (BUN) were detected by fullautomatic biochemical analyzer on 7 d and 14 d after operation. The expressions of renal AQP-3 mRNA and protein of rats were detected by RT-PCR and Western blotting, respectively. ResultsThe levels of serum TBIL and DBIL were significantly higher on 14 d than those on 7 d after operation in experimental group (P=0.000), which were significantly higher than those at corresponding time point in control group (P=0.000), while the difference within control group was not significant (P=0.154). Thus, the OJ models of rats were established successfully. The difference of serum Cr levels of rats between inter-and intragroup were not significant (Pgt;0.05). Serum BUN level on 14 d after operation in experimental group was significantly higher than those on 7 d after operation in experimental group and on 14 d after operation in control group (P=0.001), although serum Cr levels were not different between 7 d and 14 d after operation in control group (P=0.288). The expressions of AQP-3 protein of rats on 7 d and 14 d after operation in experimental group were significantly lower than those at corresponding time point in control group (P=0.033, P=0.000), meanwhile on 14 d after operation in experimental group was significantly lower than those on 7 d after operation in experimental group (P=0.000). The expressions of AQP-3 mRNA of rats on 7 d and 14 d after operation in experimental group were significantly higher than those at corresponding time point in control group (P=0.000), but the difference at different time point in two groups was not significant (P=0.139, P=0.059). ConclusionsThe changes of renal AQP-3 protein and mRNA expressions are prior to the changes of serum Cr and BUN levels of rats suffered from OJ complicated renal function damage, which are promised to improve the early diagnosis rate of renal function damage in rats with OJ.
ObjectiveTo observe the ocular manifestations and the titer of aquaporin 4 antibody (AQP-4) in NMO patients, and to evaluate the BCVA prognosis in patients with different titers of AQP-4Ab.MethodsA retrospective case study. From September 2009 to March 2014, 132 NMO patients diagnosed in Department of Neurology and Ophthalmology in Huashan Hospital of Fudan University were included in the study. Among the patients, 74 patients (56.06%) were involved in optic nerve for the first time, among which 63 patients (47.72%) were involved in optic nerve alone, and 11 patients (8.33%) were involved in optic nerve and spinal cord at the same time. The recurrence rate was 62.88% (twice or more). All patients underwent BCVA, slit lamp microscope, fundus examination, thyroid function, sex hormones, and serum AQP-4Ab detection. BCVA was recorded at admission and before discharge from hospital, and worse BCVA was recorded in binocular patients. The BCVA of patients with different titers of AQP-4Ab were analyzed comparatively.ResultsAmong the 74 patients with optic nerve involved in the first onset, 50 patients with BCVA＜0.1 at the initial diagnosis (67.57%); AQP-4Ab positive was found in 56 patients, which including 13, 9 and 34 patients of AQP-4Ab titer 5 - 60, 61 - 100 and ＞100 RSRU/ml. After 2 weeks of treatment, BCVA improved in 40 patients (71.42%), including 11 (84.62%), 6 (66.67%) and 23 (67.64%) of AQP-4Ab titer 5 - 60, 61 - 100 and ＞ 100 RSRU/ml. Among 132 patients, 98 patients (74.24%) were AQP-4Ab positive. There were 73 patients (55.30%) with abnormal immune rheumatoid index.ConclusionsThe optic nerve is involved in 56.06% patients with NMO for the first time, and 67.57% of the patients had poor vision with BCVA＜0.1. BCVA prognosis is better in patients with serum AQP-4Ab titer of 5 - 60 RSRU/ml.
Objective To investigate the effects of extracts of Pinellia ( EP) on a rat model of airway mucus hypersecretion induced by LPS. Methods Thirty Wisatr rats were randomly divided into 5 groups, ie. a blank group, a model group, and three EP groups treated with different doses of EP. There were 6 rats in each group. Airway mucus hypersecretion model was established by intratracheally instillation of LPS in the model group and three EP groups. The rats in three EP groups were orally administered with EP at dosages of 10 g/kg, 30 g/kg and 60 g/kg respectively for 4 days. The expression of Mucin 5AC ( MUC5AC) protein in airway was assayed by immunohistochemistry. The mRNA expressions of MUC5AC and Aquaporin-5( AQP-5) in lung tissue were detected by RT-PCR. ELISA technique was performed to detect TNF-αin bronchoalveolar lavage fluid( BALF) . Results LPS significantly stimulated the mRNA and protein expression of MUC5AC in lung and TNF-αlevel in BALF, and inhibited the expression of AQP-5 mRNA in lung. The EP at dosages of 10 g / kg and 30 g/ kg had little effect on mucus hypersecretion. While 60 g/kg of EP could significantly inhibited the expression of MUC5AC, and decreased the release of TNF-α in BALF. The AQP-5 mRNA was also up-regulated by 60 g /kg of EP. The expression of MUC5AC mRNA was positively correlated with level of TNF-α( r = 0. 948, P lt;0. 05) ; AQP-5 mRNA was negatively correlated with MUC5AC mRNA and TNF-α( r = - 0. 955, P lt; 0. 05; r = - 0. 909, P lt; 0. 05) . Conclusion EP ( 60 g/ kg) can significantly attenuated airway mucus hypersecretion in rats.
Objective To investigate the expression of aquaporin-1（AQP-1） on pleura in rats with carrageenan-induced pleural effusion and explore the role of AQP-1 in effusion formation.Methods Fifty-six healthy Wistar rats were randomly divided into a normal control group and 6 pleuritis groups（6，12，24，36，48 and 72 h groups respectively）.The rat model of inflammatory pleurisy was induced by injecting l-Carrageenan into the pleural cavity.The expression of AQP-1 on pleura was detected with immunohistochemistry.The mRNA and protein expression of AQP-1 on visceral pleura and parietal pleura were measured by RT-PCR and Western blot assay respectively.The volume of pleural effusions were measured.Results The volume of pleural effusion was 2.10±0.22，4.10±0.15，4.40±0.36，3.20±0.27，2.60±0.18，0.12±0.02 mL in the 6，12，24，36，48 and 72 h pleuritis groups respectively.AQP-1 were mainly expressed on visceral and parietal pleural mesothelial cells and capillary endothelial cells，and significantly increased in all pleuritic rats The mRNA and protein expression of AQP-1 on parietal pleura increased after 6 h and reached peak level at 24 h in pleuritic groups.The mRNA and protein expression of AQP-1 on visceral pleura increased after 12 h and reached peak level at 24 h in pleuritic groups.The expression of AQP-1 on parietal pleura at 12 h and 24 h in pleuritic groups was correlated positively with the volume of pleural effusion（r=0.857，r=0.846，all Plt;0.01）.The expression of AQP-1 on visceral pleura at 24 h in pleuritic groups was positively correlated with the volume of pleural effusion（r=0.725，Plt;0.05）.Conclusion The expression of AQP-1 on pleura were increased in rats with e carrageenan-induced pleural effusion.AQP-1 may play a role in pleural fluid transportation in pleural effusion.
ObjectiveTo investigate the effect of saikosaponin a (SSa) on the levels of immune inflammation in rats with acute spinal cord injury and its possible mechanism.MethodsSeventy-two Sprague Dawley rats (weighing, 220-250 g) were randomly divided into sham operation group (group A), spinal cord injury group (group B), and SSa treatment group (group C) respectively, 24 rats in each group. The spinal cord injury model was induced by using the Allen’s method in groups B and C; the spinous process and vertebral plate at both sides were cut off by lamina excision to expose the spinal cord in group A. The rats were given intraperitoneal injection of 10 mg/kg SSa in group C and equal volume of normal saline in group B at immediate after injury. The spinal cord tissue was harvested from 18 rats of each group at 24 hours after operation to measure the levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by ELISA, to detect the expressions of nuclear factor κB (NF-κB) P65, NF-κB P-P65, and aquaporin 4 (AQP4) by Western blot and to observe the morphology of spinal cord by HE staining. The motor function of the lower limbs was evaluated by BBB score and tiltboard experiment in 6 rats at 1, 3, 7, 14, 21, and 28 days after injury.ResultsThe BBB score and tiltboard experiment maximum angle were significantly higher in group A than groups B and C at each time point (P<0.05) and in group C than group B at 14, 21, and 28 days after operation (P<0.05). ELISA test showed that the concentrations of TNF-α and IL-6 were significantly lower in group A than groups B and C, and in group C than group B (P<0.05). Western blot results showed that the protein expression levels of NF-κB P65, NF-κB P-P65, and AQP4 were significantly lower in group A than groups B and C, and in group C than group B (P<0.05). HE staining demonstrated normal neurons of the spinal cord and no obvious lesion in group A; neuronal cells were observed in the injured area of group B, with hemorrhage, neutrophil infiltration, and nerve cell edema in the injured area; the neuronal cells were visible in the spinal cord of group C, with microglia mild hyperplasia, and the pathological changes were improved when compared with group B.ConclusionSSa has neuroprotective effects on acute spinal cord injury in rats by inhibiting NF-κB signaling pathway and AQP4 protein expression and reducing inflammation response and edema.
Objective The changes of the aquaporins 1 (AQP-1) expression may be related to the chondrocyte apoptosis. To explore the correlation between the expression of AQP-1 and chondrocyte apoptosis by observing the expression of the AQP-1 and the Caspase-3, so as to provide experimental evidence for the further study in the pathogenesis of osteoarthritis (OA). Methods Seventy-two 8-week-old clean grade male Sprague Dawley rats, weighing 286-320 g (mean, 300 g), were randomly divided into the operated group (n=24), the sham-operated group (n=24), and the control group (n=24).OA models were made by amputating the anterior cruciate l igament and medial collateral l igament, and partial excision of medial meniscus in operated group; the articular cavity was exposed only in sham-operated group; and no treatment was given in control group. The general condition of the rat was observed after model was establ ished. At 1, 2, 4, and 8 weeks, the specimens of knee joints were harvested to perform the gross and histological observations; the mRNA expressions of AQP-1 and Caspase-3 were determined by real-time fluorescent quantitative PCR; and the activity of the Caspase-3 protease was detected. The correlations between the expression of AQP-1 mRNA and the expressions of Caspase-3 mRNA and protease were analyzed. Results Totally 6 rats died after operation, and the rats were suppl ied immediately; the other rats survived to the end of experiment. The appearance and structure of knee articular cartilage were normal in control group and sham-operated group. While in operated group, the cartilage had a rough surface with fissure and vegetation, and fibrosis and irregular cell arrangement were seen on the surface of cartilage. There were significant differences in the Mankin score between the operated group and sham-operated group, control group at 2, 4, and 8 weeks (P lt; 0.05). There was no significant difference in expressions of the AQP-1 mRNA and Caspase-3 mRNA, and the activity of the Caspase-3 protease among 3 groups at 1 week after operation (P gt; 0.05); while the expressions of the AQP-1 mRNA, Caspase-3 mRNA, and the activity of the Caspase-3 protease in operated group were significantly higher than those in sham-operated group and control group at 2, 4, and 8 weeks after operation (P lt; 0.05), andthere was an increased trend over time. There was significantly positive correlation (r=0.817, P=0.000) between the expressions of AQP-1 mRNA and Caspase-3 mRNA, and the regression equation was y=0.426 7x2+0.051 5x; meanwhile, there was also significantly positive correlation (r=0.945, P=0.000) between the expression of AQP-1 mRNA and the activity of Caspase-3 protease, and the regression equation was y=15.423 0x+4.392 8. Conclusion The up-regulation of AQP-1 expression in OA cartilage may be related to the chondrocyte apoptosis, and the changes of AQP-1 expression may involve in the pathogenesis of OA.