objective To assess the effect of aldosterone on the production of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9)and collagen Ⅳ in culture supematants of podocytes and the possible molecular mechanisms involved in the influence of aldosterone on the synthesis and degradation of extracellular matrix produced by podocytes. Methods Podecytes were treated with aldosterone at the concentration of 10-11, 10-9, 10-7 mol/L respectively. Cultured podocytes were examined at 24, 48 and 72 hours respectively. Spironolactone, a receptor antagonist of aldosterone, was added to observe the blocking effect on aldosterone. An inhibitor of TGF-β1 receptor was used to determine whether the effect of aldosterone on podocytes were mediated through TGF-β1 system. The enzymatic activities of MMP-2 and MMP-9 were assayed by gehtin zymography. Collagen Ⅳ 0.5 chain and TGF-β1 proteins released into culture supematants were assessed by Western blot and ELISA analysis. The adhesion rate of podocytes was monitored by flow cytometry. Results Aldosterone increased the activities of MMP-2 and MMP-9 in a dose- and time-dependent manner (P<0.05). Aldosterone decreased the level of collagen Ⅳ or5 chain protein in culture supernatants (P<0.05). Meanwhile, the expression of TGF-β1 was also increased (P<0.05). Spironolactone completely abolished the above-mentioned changes(P< 0.05). Blockage of TGF-β1 signaling with SB431542 prevented the aldosterone-induced upregulation of MMP-2 and MMP-9 as well as the downregulation of the collagen Ⅳ α5 chain protein and the adhesion rate of podocytes (P<0.05). Conclusions Aldosterone increases the activities of MMP-2 and MMP-9 but decreases the expression of collagen Ⅳ α5 chain and the adhension rate of podocytes possibly via TGF-β1 signaling pathway. Such alterations may contribute to glomerular podocyte injury associated with the GBM abnormality caused by the imbalance between matrix synthesis and degradation.

Objective To evaluate the efficacy of stent-assisted coiling in the treatment of unruptured vertebral dissecting aneurysm. Methods We retrospectively reviewed 59 consecutive patients with unruptured vertebral dissecting aneurysms that underwent endovascular treatment. 31 patients received single stent-assisted coiling, 28 patients received multiple stent-assisted coiling. Results Clinical outcome was favorable in 56 of the 59 patients, the modified Rankin Scale score had no difference in both groups (P = 0.24). The immediate obliteration grade in multiple-stent group was higher than that in single-stent group (75.0% vs. 41.9%, P=0.010). What′s more, the recurrence rate was lower in multiple-stent group (0% vs. 19.4%, P = 0.043). Conclusions Stent-assisted coiling in the treatment of unruptured vertebral dissecting aneurysm is safe and effective , multilayer disposition of stents with coils will decrease the complications and facilitate the aneurysmal occlusion. Larger , prospective studies are necessary to explore the long-term outcomes of reconstruction therapy.

Objective To explore the effect of PGE1 on the cognitive impairment and the expression of VEGF and BDNF in the hippocampus after bilateral common carotid artery occlusion in adult rats. Methods Forty-eight rats were randomly divided into PGE1 group (10μg·kg-1·d-1, iv), PGE1+VEGFR antagonist group (PGE1, 10μg·kg-1·d-1, iv;SU5416, 25 mg·kg-1·d-1, ip), saline group and sham group (n=12 each). Morris Water Maze test (MWM) was used to examine cognitive function in rats. Drugs and saline were given to VD rats at 24 d for 7 consecutive days following opera?tion. Half of the rats in each group were sacrificed for Western Blot at 6 days after MWM test. Western Blot was conduct?ed to examine the relative expression levels of VEGF and BDNF in the hippocampus. Results Compared to saline and PGE1+VEGFR antagonist groups, the escape latency in PGE1 group was shorter (P<0.05), and the times that rats swam across the platform location and time percentage in previous platform quadrant in PGE1 group was longer (5.77±0.83 vs.2.88 ± 0.47 vs. 2.63 ± 0.44, P<0.01;32.28%± 4.56%vs. 20.42%± 5.50%, 23.08%± 5.06%, P<0.05). Compared with saline group and sham group, PGE1 group had higher levels of VEGF (0.057±0.005 vs. 0.038±0.002 vs. 0.027±0.002, P<0.05) and BDNF (0.481±0.049 vs. 0.339±0.021 vs. 0.224±0.04, P<0.05). but the increase in VEGF expression in PGE1 group was no significant (0.057±0.005 vs. 0.053±0.003, P>0.05) compared with PGE1+VEGFR antagonist group, while the aug?ment of BDNF in PGE1 group was remarkable (0.481±0.049 vs. 0.373±0.034, P<0.05). Conclusions PGE1 can upregu?late VEGF and BDNF expression and modify cognitive impairment in VD rats, while the effects of PGE1 on cognitive function and BDNF expression can be partially blocked by VEGFR antagonist SU5416.

Objective To determine equilibrium solubility and apparent partition coefficient of salicylic acid at 25 ℃,and to provide a theoretical basis for design and preparation of its formulation.Methods Equilibrium solubility and apparent partition coefficient (Papp) of salicylic acid were respectively investigated in water,hydrochloric acid solution (pH 1.0) and phosphate buffer solution system (pH 2.0,3.0,4.0,5.0,6.0,7.0,7.8) at 25 ℃.The shake flask method and HPLC were used.The column was Waters C18 (4.6 mm ×250 mm,5 μn) with the mobile phase as methanol-0.1％ phosphoric acid water (47:53).The column temperature was room temperature.The flow rate was 1.0 mL·min-1.The detection wavelength was 270 nm and injection volume was 20 μL.Results Equilibrium solubility of salicylic acid was (2.205 ±0.020) mg·mL-1 at 25 ℃ in Water and its Papp was (6.18 ±0.08).The solubility were (1.169 × 10-3 ±7.40 × 10-6),(2.250 ±0.010),(2.410±0.010),(2.694 ±0.003),(5.208 ±0.010),(5.826 ±0.006),(6.255 ±0.030),(3.353 ±0.070) mg·mL-1,respectively,at hydrochloric acid solution (pH 1.0) and phosphate buffer solution system (pH 2.0,3.0,4.0,5.0,6.0,7.0,7.8),and the corresponding Papp were (16.39 ±0.19),(4.23 ±0.07),(6.03 ±0.11),(5.56 ±0.10),(1.25 ±0.01),(0.27 ± 0.001),(0.08 ± 0.001) and (0.07 ± 0.002),respectively.Conclusion The solubility of salicylic acid increases and its oil-water partition coefficient declines with pH value increasing.Salicylic acid is slightly soluble in water and oil.It belongs to Class Ⅳ drug in Biopharmaceutics Classification System (BCS).

To investigate the effects of albumin on the production of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) in podocytes. Podocytes were treated with bovine serum albumin (BSA) at the concentration of 0.1, 0.5, 1, 2 g/L, respectively. Conditioned media were harvested 12, 24, 48 and 72 h after the treatment. The expression of MMP-2 and MMP-9 was assayed by gelatin zymography, RT-PCR and Western blotting analysis. Our results showed that in comparison with the control group, BSA increased the expression of MMP-2 and MMP-9 mRNA and protein in a dose- and time-dependent manner (P<0.05). Meanwhile, the enzymatic activities of MMP-2 and MMP-9 in the culture supernatants of podocytes were also increased (P<0.05). It is concluded that albumin up-regulated the expression of MMP-2 and MMP-9 at gene and protein levels in a time- and dose-dependent manner.

Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside (PAN)-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN treatment+shRNA transfection group, and PAN treatment+ negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.

To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved, 36 male Sprague-Dawley rats were randomly divided into control group, adriamycin nephropathy (AN) group and eplerenone-treated group (100 mg·kg(-1)·d(-1) eplerenone). Blood pressure, 24-h urinary protein, serum potassium, sodium and creatinine were measured 28 days after adriamycin injection (a single tail intravenous injection of 6.5 mg/kg adriamycin). The morphological changes of renal tissues were observed by light and electron microscopy. Immunohistochemistry and Western blotting were performed to examine the expression of TGF-β(1) and desmin in renal cortex. The results showed that 28 days after adriamycin injection, there were no significant changes in the level of serum potassium, sodium, creatinine concentrations and blood pressure values in the rats of the three groups. Meanwhile, the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group (P<0.01), but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group (P<0.05). Mild mesangial cell proliferation and matrix expansion, diffuse deformation and confluence of foot processes in podocytes were found in the AN group. By contrast, rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions. The protein expression of TGF-β(1) and desmin in the AN group was markedly up-regulated in contrast to that in the control group (P<0.01), whereas that in the eplerenone-treated group was much lower than in the AN group (P<0.05). It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release, and restore the morphology of podocytes independent of its blood pressure-lowing effects.

Objective To access the effects of aldosterone (ALD) and its receptor antagonist- spironolactone (SPI) on the production of reactive oxygen species (ROS) and apoptosis in podocytes and to explore the possible mechanism involved. Methods Conditionally immortalized mouse podocytes were divided into control group, ALD group, SPI group and SPI +ALD group. The level of ROS production and the expression of nephrin protein were assayed by fluorescent spectrophotometry and indirect immunofluorescence technology. The apoptosis rate of podocytes was monitored by flow cytometry. The expression of bax and bcl-2 mRNA and protein was detected by RT-PCR and Western blotting methods. The anti-oxidant N-acetylcysteine (NAC)was applied to determine whether the effects of ALD on podocytes were mediated by ROS pathway.Results Compared with the control group, ALD significantly increased ROS production in podocytes (P＜0.05). SPI completely abolished the above-mentioned effect of ALD (P＜0.05). ALD induced the down-regulation of the expression of nephrin and the up-regulation of podocytes apoptosis (P＜0.05), which was accompanied with the elevated expression of bax mRNA and protein and the reduced expression of bcl-2 mRNA and protein (P＜0.05). SPI or NAC prevented the above-mentioned changes induced by ALD (P＜0.05). Conclusion ALD increases theexpression of pro-apoptotic factor (bax) but decreases the expression of anti-apoptotic factor (bcl-2)to induce podocytes apoptosis through the mineralocorticoid receptor (MR) possibly via the mechanisms involving ROS pathway.

Objective To evaluate the clinical effect of endovascular embolization for the treatment of cerebellar arteriouvenous malformations (AVMs) associated with aneurysm, and to discuss its technical points. Methods The clinical data of 142 patients with cerebellar AVMs were retrospectively analyzed. Of the 142 patients, 42 had concomitant aneurysms. The patients were divided into concomitant aneurysm group and without aneurysm group. Using univariate and multivariate statistical models, the patient’s gender and age, the presence or absence of hemorrhage, the lesion’s location and size, and the presence or absence of deep venous drainage were analyzed. Results A total of 61 concomitant aneurysms were detected in the 42 patients, which were located within the malformation mass (n=14) or on the feeding artery (n=47). The concomitant aneurysms located in the malformation were closely related to AVM hemorrhage and the deep venous drainage. During the follow-up period after endovascular embolization, no recurrence of bleeding was observed in all the 42 patients. Conclusion In order to reduce the relapse rate of hemorrhage, the formulation of therapeutic measures should be based on the management of the concomitant aneurysm when endovascular embolization treatment is employed for AVM associated with aneurysm.

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.