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 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 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.
Objective To investigate the expression of aquaporin-1( AQP-1) in pulmonary tissues of asthma mice and the effects of acetazolamide( AZ) on AQP-1 expression. Methods Forty C57BL/6 mice were randomly divided into five groups. Group A was treated with phosphate buffer as a non-asthmatic group.The mice in group B, C, D, and E were sensitized with ovalbumin( OVA) and challenged with aerosol OVA to establish asthma model. The mice in group B, C, and D were interperitoneally injected with AZ at doses of 300, 200, 100 mg/kg, respectively during the challenge period. Results ①Wet/dry weight ratio of lung tissues in group E was significantly higher than that in group A( P lt;0. 05) , while it was lower in B, C and D groups than group E. ②The total number of cells, the number of eosinophils, and interleukin-5( IL-5) inBALF of group E were higher than those in group A( P lt;0. 05) , and interferon-γ( IFN-γ) level was lower in group E than in group A ( P lt; 0. 05) . After AZ treatment, the total number of cells, the number of eosinophils, neutrophils and lymphocytes were significantly decreased( P lt; 0. 05) , which were positively correlated with the dose of AZ. ③AQP-1 were expressed in tracheal epithelium, microvascular endothelial cell and bronchial peripheral vascular bed, and the expression in group E was significantly higher than that in group A( P lt;0. 01) . AQP-1 expression was significantly decreased after the intervention of AZ ( P lt;0. 05) .The decrease was positively correlated with the dose of AZ. The expression of AQP-1 mRNA showed no significant difference among these groups( P gt;0. 05) . Conclusions AQP-1 was over-expressed in the lung tissue of mice with asthma. AZ can inhibit the expression of AQP-1 and relieve lung inflammatory cells infiltrationin a dose-dependent manner. It is the protein expression of AQP-1 not the AQP-1 mRNA which were significantly different in different groups, suggesting that AZ affected AQP-1 in the post-transcriptional stage.
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.
ObjectiveTo observe the expression of vascular endothelial growth factor (VEGF) and aquaporin 4 (AQP4) in the inner limiting membrane (ILM) of diabetic retinopathy (DR) with macular edema, and analyze the correlation between VEGF and AQP4 expression. Methods A cross-sectional study. From September 2019 to September 2020, 38 eyes of 38 patients with DR and idiopathic macular hole (iMH) who underwent vitrectomy (PPV) combined with ILM stripping at the Hangzhou campus of The Affiliated Eye Hospital of Wenzhou Medical University at Hangzhou were included in the study. Among them, there were 25 males and 13 females who aged 37-76 years old, average age was 59±10 years old; All eye included 15 right eyes and 23 left eyes. iMH and DR included 9 eyes in 9 cases and 29 eyes in 29 cases, respectively, and they were divided into iMH group and DR group. The DR group was divided into DME group and no DME group according to whether it was accompanied by diabetic macular edema (DME), with 14 eyes and 15 eyes respectively. After the stripped ILM tissue was fixed, immunofluorescence analysis was performed to obtain a picture of the fluorescence mode of AQP4 and VEGF, and the fluorescence intensity value of VEGF and AQP4 was measured by Image J software. The differences of VEGF and AQP4 immunofluorescence values in the specimens between groups were compared by one-way analysis of variance. The correlation between the fluorescence intensity of AQP4 and the fluorescence intensity of VEGF was analyzed by Pearson correlation analysis. Results The average fluorescence intensity valuesof VEGF and AQP4 in ILM specimens of DME group, no DME group and iMH group were 38.96±7.53, 28.25±3.12, 30.07±4.84 and 49.07±8.73, 37.96±6.45, 38.08±5.04, respectively. The average fluorescence intensity of VEGF and AQP4 in the ILM specimens of the DME group was significantly higher than that of the no DME group and iMH group, and the difference was statistically significant (F=13.977, 9.454; P＜0.05). The average fluorescence intensity values of VEGF and AQP4 on IML specimens in the DR group were 33.80±7.91, 43.76±9.44, respectively. The results of Pearson correlation analysis showed that the fluorescence intensity of VEGF and AQP4 in the ILM specimens of the DR group was significantly positively correlated (r=0.597, P=0.003). ConclusionsThe expressions of VEGF and AQP4 in ILM of eyes with DR and DME are significantly increased compared with those without DME. The expression of VEGF and AQP4 in ILM of eyes with DR is positively correlated.
Objective To observe the correlation of serum aquaporin 4 (AQP4) antibodies and condition and visual prognosis in patients with severe neuromyelitis optica spectral disorders (NMOSD). Methods Fifty NMOSD patients with visual acuity of 20/200 or worse in at least one eye were enrolled in this retrospective analysis. There were 12 males and 38 females. The age ranged from 17 to 65 years, with the mean of (39.86±2.02) years. The patients were divided into two groups according to the serum AQP4-IgG status. The ophthalmologic examination, serum anti-nuclear antibodies (ANA), myelin oligodendrocyte glycoprotein (MOG) antibody detection and vision prognosis were compared and analyzed. Glucocorticoid therapy was delivered to 46 patients who were within 1 month of onset. The visual acuity of the patients after treatment was divided into complete recovery, partial recovery, stabilization and reduction, and the visual acuity of the two groups were analyzed. Results Among 50 patients, there were 30 (60%) seropositive patients (positive group), 20 (40%) seronegative patients (negative group). The positive group had significantly higher ratio of female to male (P=0.004), and more binocular optic neuritis (ON) (P=0.010) compared with the negative group. More recurrence ON were also found in the positive group, but without statistic difference between two groups (P=0.167). There was no difference of age, course, and vision damage degrees and abnormal orbital MRI scanning between two groups (P＞0.05). Among 24 patients who underwent serum ANA detection in the positive group, 8 patients were positive. All of 18 patients who underwent serum ANA detection in the negative group were negative. The difference of the ratio of serum ANA positive patients between two groups was significant (P=0.030). Serum MOG antibody detection in the positive group was negative (0/10). Sixteen patients who underwent MOG antibody detection in negative group, 4 patients were positive. After treatment, there were 23.3%, 23.3%, 53.3% patients with vision of complete recovery, partial recovery and reduction in the positive group; 25.0%, 30.0%, 25.0% patients with vision of complete recovery, partial recovery and reduction in the negative group, respectively. There was no difference in proportion of vision with complete recovery and partial recovery between two groups (P=0.163, 0.607), but significant difference was observed in proportion of vision with stabilization and reduction between two groups (P=0.021, 0.048). Conclusions The positive serum AQP4 antibody is common in patients with severe NMOSD. The patients with AQP4 antibody in the serum are more likely combined with immunological serological markers and poor vision prognosis.
Neuromyelitis optica (NMO) is an autoimmune inflammatory diseases of the central nervous systems (CNS) mainly affecting the optic nerves and spinal cord. It has the characteristics of high recurrence rate and poor prognosis. NMO related optic neuritis is a common neuro-ophthalmic disease which often results in permanent visual loss or even blindness. Aquaporin 4 (AQP4) antibody is a specific and pathogenic autoantibody in NMO patients. Although AQP4 is expressed in multiple tissues, NMO pathology is remarkably limited to the CNS. Corticosteroids and other immunosuppressive drugs are the standard managements for NMO patients, in order to reduce the relapses and the severity of the acute attack. Multiple avenues of investigation in the laboratory have significantly advanced our understanding of NMO pathophysiology, which is helpful for our understanding of immunologic and nonimmunologic mechanisms. Many offer significant means for NMO therapy by selectively targeting pathways. In the future, moving these agents from the bench to the bedside offers the opportunity to identify safe and effective therapies that limit CNS injury and preserve visual function.