Pravastatin alleviates oxidative stress and decreases placental trophoblastic cell apoptosis through IL-6/STAT3 signaling pathway in preeclampsia rats.

OBJECTIVE: The aim of this study was to explore the effects of pravastatin on oxidative stress and placental trophoblastic cell apoptosis in preeclampsia rats via the interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway. MATERIALS AND METHODS: Experimental rats were randomly assigned into three groups, including control group (C group), model group (M group) and pravastatin group (P group). The rat model of preeclampsia was successfully established. Blood pressure, urinary protein and nitric oxide (NO) as well as oxidative stress indicators in rats were detected at 7, 14 and 21 d, respectively. The content of serum IL-6 was determined via enzyme-linked immunosorbent assay (ELISA). The messenger ribonucleic acid (mRNA) expression of IL-6 in the placenta of rats in each group was detected using quantitative polymerase chain reaction (qPCR). Western blotting (WB) was used to determine the protein expression level of STATs in the placental tissues of rats. In addition, cell counting kit (CCK)-8 assay was conducted to detect the proliferation of rat placental trophoblasts. RESULTS: The content of serum NO was (14.32±2.32) µM in M group, (28.37±3.32) µM in C group and (22.54±3.12) µM in P group, respectively. It was significantly elevated in P group compared with M group (p<0.05). Blood pressure in M group was evidently higher than that in C group at 14 and 21 d (p<0.05). However, P group exhibited distinctly lower blood pressure than M group (p<0.05). No statistically significant differences were observed in the urinary protein of rats among all the three groups at 7 d (p>0.05). At 14 and 21 d, the content of urinary protein in M group was considerably higher than that in C group (p<0.05). However, P group had distinctly lower urinary protein content than M group (p<0.05). Compared with C group, the content of malondialdehyde (MDA) and advanced oxidation protein products (AOPP) rose significantly in M group, whereas the content of superoxide dismutase (SOD) declined remarkably (p<0.05). In comparison with M group, P group exhibited declined MDA and AOPP content and increased SOD content, with statistically significant differences between the two groups (p<0.05). The expression level of serum IL-6 in rats in M group was markedly higher than that in C group (p<0.05). Meanwhile, the expression level of serum IL-6 evidently declined in P group compared with M group (p<0.05). Compared with C group, the protein expressions of phosphorylated STAT1 (p-STAT1) and p-STAT3 were considerably up-regulated in M group (p<0.01). However, they decreased prominently in P group in comparison with M group (p<0.01). C group exhibited a remarkably worse proliferation ability of rat placental trophoblasts than C group (p<0.01). In comparison with M group, the proliferation ability of rat placental trophoblasts was evidently enhanced in P group (p<0.05). Flow cytometry results indicated that the apoptosis of trophoblastic cells increased significantly in M group compared with that in C group (p<0.01). However, it significantly declined in P group in comparison with M group (p<0.05). CONCLUSIONS: Pravastatin can repress the IL-6/STAT3 signaling pathway to alleviate oxidative stress, improve preeclampsia and decrease the apoptosis of placental trophoblastic cells in preeclampsia rats.

[Different effects of pravastatin on sFlt-1, PlGF and VEGF in different preeclampsia-like mouse models].

Objective: To explore the pathways of preeclampsia by investigating different effects of pravastatin (Pra) on and soluble FMS tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) in different preeclampsia (PE)-like mouse models. Methods: C57BL/6J mice were randomly subcutaneously injected with N-nitro-L-arginine methyl ester (L-NAME) or intraperitoneally injected with lipopolysaccharide (LPS) as PE-like mouse model, saline as normal pregnancy control (Con) respectively, daily at gestational 7-18 days. Pra was given daily at gestational 8-18 days in each model group and the mice were divided into Pra (L-NAME+Pra, LPS+Pra, Con+Pra) and saline (L-NAME+NS, LPS+NS, Con+NS) groups. Liver,placental tissue and blood of pregnant mice were collected on the 18th day of pregnancy. The levels of VEGF, PlGF and sFlt-1 in the liver, placenta and serum of mice in each group were compared by western blot, ELISA and real-time fluorescence quantitative PCR (RT-PCR). Results: (1) ELISA: Serum VEGF (205.70±3.43, 154.60±2.31) and PlGF (131.5±3.75, 101.50±4.31) levels were significantly increased in L-NAME+Pra group compared with L-NAME+NS group (all P<0.05). Serum VEGF (202.30±4.90, 144.50±6.71) and PlGF (121.50±3.86, 95.41±4.08) levels were significantly higher in LPS+Pra group than those in LPS+NS group (all P<0.05). Serum sFlt-1 level in LPS+Pra group was significantly lower than that in LPS+NS group (3.01±0.50, 776.60±80.06), serum sFlt-1 level in L-NAME+Pra group was significantly lower than that in L-NAME+NS group (2.60±0.06, 583.70±9.83; all P<0.05). (2) Western blot: the expression levels of PlGF (1.344±0.118, 0.664±0.143) and VEGF (1.34±0.12, 0.66±0.14) in the liver of mice in the L-NAME+Pra group were significantly higher than those in the L-NAME+NS group (all P<0.05), but the expression levels of PlGF and VEGF in the placenta of L-NAME+Pra group were not significantly different from those of L-NAME+NS group (all P>0.05). The expression levels of PlGF and VEGF in placenta and liver of pregnant mice in LPS+Pra group were not significantly different from those in LPS+N group (all P>0.05). (3) RT-PCR: the mRNA expression of PlGF and VEGF in placenta and liver of L-NAME+Pra group were not significantly different from those in L-NAME+NS group (all P>0.05). The mRNA expression levels of PlGF and VEGF in placenta and liver of LPS+Pra group were not significantly different from those of LPS+NS group (all P>0.05). Conclusions: Pra has different regulatory effects on vascular endothelial function in different PE-like models. It reveals that different pathogenesis and pathways exist in different PE-like changes.

[Changes of Rheb gene and protein expression in preeclampsia-like mouse model treated with pravastatin].

Objective: To explore whether pravastatin (Pra) inhibits mammalian target of rapamycin (mTOR) signal pathway by regulating Ras homolog enriched in brain (Rheb) protein through the comparison of gene and protein expression changes of Rheb in liver and placenta in preeclampsia (PE)-like mouse model treated with Pra. Methods: C57BL/6J pregnant mice were randomly divided into two groups. The PE group was established by injecting N-nitro-L-arginine methyl ester (L-NAME) daily at gestational 7-18 days, saline was injected as contol group (Con); then giving mice Pra (PE+Pra, Con+Pra group, n=8) or normal saline (PE+N, Con+N group, n=8) every day from the 8th gestational day of pregnancy. The maternal liver and placenta tissues were collected on the 18th day of pregnancy. Western blot, real-time quantitative PCR and immunohistochemistry were used to compare the levels of Rheb protein and mRNA expression in the liver and placenta. Results: (1)The results of western blot: there were no significant differences in Rheb protein expression between PE+N group (liver: 0.706±0.123; placenta: 0.866±0.128) and Con+N group (liver: 0.732±0.123; placenta: 0.909±0.097) , and the differences between PE+Pra group (liver: 0.669±0.134; placenta: 0.940±0.221) and PE+N group were not significant either in liver or in placenta (all P>0.05). (2) The results of real-time quantitative PCR: when PE+N group (liver: 1.026±0.480; placenta: 1.102±0.361) compared with Con+N group (liver: 1.058±0.389; placenta: 1.067±0.400) , PE+Pra group (liver: 0.735±0.356; placenta: 0.822±0.304) compared with PE+N group, there were no significant differences either in liver or in placenta (all P>0.05). (3) The results of immunohistochemistry: Rheb protein expression did not change significantly in maternal liver and placenta, there were no significant differences in protein expression levels between PE+N group and Con+N group, and between PE+Pra group and PE+N group (all P>0.05). Conclusion: The inhibition of Pra on mTOR signaling pathway in some PE-like model may be independent of the expression of Rheb gene and protein.

[Regulation of pravastatin on long-chain fatty acid oxidative enzyme in pre-eclampsia-like mouse model].

Objective: To investigate the modulation of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) expression by pravastatin in pre-eclampsia-like mouse model. Methods: C57BL/6J mice were randomly injected with N-nitro-L-arginine methyl ester (L-NAME) as pre-eclampsia-like model group (PE) or saline as normal pregnancy control group (Con) respectively, from gestational the 7th to 18th day. For each group, pravastatin (PE+Pra, Con+Pra group) or saline (PE+N, Con+N Group) was given from the 8th to 18th day of gestation, respectively. Liver and placenta of pregnant mice were collected on gestational day 18. The LCHAD protein expression and mRNA levels of liver and placenta were detected through western blot, immunohistochemistry and real-time quantitative PCR. Results: (1) The average arterial pressure of pregnant mice increased gradually from the 8th to 18th day in PE+N group, but decreased in PE+Pra group from gestational 10th day, 24 hour urinary protein levels in PE+N group [(1 494 ± 201) µg] were significantly higher than that in Con+N group [(935±128) µg, P<0.01], and also higher than that in PE+Pra group [(981±116) µg, P<0.01].(2) The results of western blot: the expression of LCHAD was significantly lower in PE+N group (liver: 0.64±0.11, placenta: 0.48±0.06) than that in Con+N group (liver: 1.06±0.10, placenta: 0.60±0.10), and lower than that in PE+Pra group (liver: 0.99±0.04, placenta: 0.60±0.08; all P<0.01).(3)The results of real-time quantitative PCR: the levels of LCHAD mRNA in liver and placenta in PE+N group (liver: 0.621±0.128, placenta: 0.646±0.129) were significantly decreased compared with Con+N group (liver: 1.007±0.130, placenta: 1.004±0.103; all P<0.01), but there was no significant difference between PE+Pra group (liver: 0.693±0.678, placenta: 0.662±0.183; P>0.05). (4) LCHAD protein was expressed widely and evenly in liver. The expression in placental cytotrophoblast and syncytial trophoblast cells located in outer layer of villous in labyrinth layer was the most. The expression of LCHAD was significantly lower in PE+N group (liver: 0.062±0.016, placenta: 0.147±0.018) than that in Con+N group (liver: 0.126±0.013, placenta: 0.183±0.024), and lower than that in PE+Pra group (liver: 0.111±0.017, placenta: 0.174±0.027; all P<0.05). Conclusion: Pravastatin could upregulate the LCHAD protein expression of liver and placenta in the pre-eclampsia-like mouse, which may be a mechanism to improve the clinical manifestations of pre-eclampsia.

Functional Characterization of a Missing Branch Component in Haematococcus pluvialis for Control of Algal Carotenoid Biosynthesis.

Cyclization of acyclic lycopene by cyclases marks an important regulatory point in carotenoid biosynthesis. Though some algal lycopene epsilon cyclases (LCYEs) have been predicted computationally, very few have been functionally identified. Little is known about the regulation mechanisms of algal LCYEs. Recent comparative genomic analysis suggested that Haematococcus pluvialis contained only the ß type cyclase (HpLCYB). However, in this study, carotenoid profiling found trace α-carotene in the salt-treated cells, indicating the in vivo activity of HpLCYE, a missing component for α-branch carotenoids. Thus, genes coding for HpLCYB and HpLCYE were isolated and functionally complemented in Escherichia coli. Substrate specificity assays revealed an exclusive cyclization order of HpLCYE to HpLCYB for the biosynthesis of heterocyclic carotenoids. Expression pattern studies and bioinformatic analysis of promoter regions showed that both cyclases were differentially regulated by the regulatory cis-acting elements in promoters to correlate with primary and secondary carotenoid biosynthesis under environmental stresses. Characterization of the branch components in algal carotenoid biosynthesis revealed a mechanism for control of metabolic flux into α- and ß-branch by the competition and cooperation between HpLCYE and HpLCYB; and supplied a promising route for molecular breeding of cyclic carotenoid biosynthesis.

An exploration on the radiological features associated with motor deficits in patients with metastatic epidural spinal cord compression.

OBJECTIVE: This study was designed to explore the radiological features associated with the motor deficits in patients with metastatic epidural spinal cord compression (MESCC). PATIENTS AND METHODS: The patients with MESCC admitted in our department from July 2006 to December 2008 were included in this analysis. The data on the radiological features of affected vertebrae showed by computed tomography or magnetic resonance imaging scan and the motor deficits level assessed as paralysis or non-paralysis according to Frankel Classification Grading System were collected. A multiple regression model was established to indentify the radiological features associated with the paralysis status of MESCC patients. RESULTS: A total of 56 patients with MESCC were included in this study. All patients had invasion of vertebral body, main affected vertebrae was consecutive in 75% (8/12) of patients with lesions of upper thoracic spine and/or cervicothoracic junction and 18.18% (8/44) of patients with lesions of cervical spine, middle thoracic spine, lower thoracic spine, and lumbar spine. The paralysis status was consisted with the finding of epidural space involvement. A linear relationship between paralysis status and the radiological features including lamina involvement, retropulsion of posterior wall, and location in upper thoracic spine and/or cervicothoracic junction was detected by the "optimal" regression equation, of them lamina involvement has the greatest impact on paralysis status. CONCLUSIONS: The radiological features including lamina involvement, retropulsion of posterior wall, and location in upper thoracic spine and/or cervicothoracic junction were significantly associated with the motor deficits of patients with MESCC, which might be helpful to identify these patients who were susceptible to motor deficit, especially lamina involvement.

Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes.

Apoptotic stimuli have been shown to trigger lysosomal membrane permeability (LMP), leading to the release of cathepsins, which activate death signaling pathways in the cytosol. However, it is unknown whether this process is an initiating or amplifying event in apoptosis. In this study, we used fibroblasts and monocytes exposed to etoposide, ultraviolet light, FasL or deprived of interleukin-3 (IL-3) to show that LMP and the cytosolic release of cathepsins B, L and D consistently depends on Bax/Bak and components of the apoptosome. Neither Bax nor Bak resided on the lysosomes, indicating that lysosomes were not directly perforated by Bax/Bak but by effectors downstream of the apoptosome. Detailed kinetic analysis of cells lacking cathepsin B or L or treated with the cysteine protease inhibitor, E64d, revealed a delay in these cells in etoposide- and IL-3 deprivation-induced caspase-3 activation and apoptosis induction but not clonogenic survival, indicating that cathepsins amplify rather than initiate apoptosis.

An ephrin-A-dependent signaling pathway controls integrin function and is linked to the tyrosine phosphorylation of a 120-kDa protein.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection. Here, glycosylphosphatidylinositol-anchored A-ephrins are not only expressed in the tectum but also on retinal axons, raising the possibility that they function in this context as receptors. We now show that activation of ephrin-A2 or ephrin-A5 by one of their receptors, ephA3, results in a beta 1-integrin-dependent increased adhesion of ephrin-A-expressing cells to laminin. In the search for an ephrin-A-dependent signaling pathway controlling integrin activation, we identified a 120-kDa raft membrane protein that is tyrosine-phosphorylated specifically after ephrin-A activation. Tyrosine phosphorylation of this protein is not seen after stimulating ephrin-A2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor, or fetal calf serum containing a large set of different growth factors. The role of p120 as a mediator of an ephrin-A-integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of p120 correlates with an abolishment of the beta 1-dependent cell adhesion.

The EphA4 receptor tyrosine kinase is necessary for the guidance of nasal retinal ganglion cell axons in vitro.

The retinotectal projection serves as a model system for the study of topographic projections. It has been shown in the past few years that members of the Eph family are strongly involved in establishing this projection. The analysis so far has focused on a characterization of Ephrin ligands which are expressed in a gradient in both the tectum and the retina. Here we investigate the role of one of the multiple EphA receptors expressed on retinal ganglion cell axons, EphA4, which is uniformly expressed on nasal and temporal axons. We have adopted both a dominant negative approach and a method using neutralizing monoclonal antibodies in order to inactivate this receptor. The results of these in vitro experiments suggest that EphA4 is crucially involved in the repulsive guidance of nasal but not of temporal axons.

Localization and surgical treatment of pancreatic insulinomas guided by intraoperative ultrasound.

BACKGROUND: Approximately 20% to 60% of insulinomas cannot be localized preoperatively, and 10% to 20% cannot be found even during surgery. The operative complications associated with the blind surgical explorations are relatively high. METHODS: Between January 1987 and December 1995, intraoperative ultrasound was used to localize insulinomas and guide surgical procedures in 28 patients. RESULTS: Insulinomas were found by intraoperative systematic palpation in 24 patients (85.7%), while intraoperative ultrasound localized the tumors in 27 patients (96.4%). By the combination of these two techniques, all tumors were discovered. The surgical procedures were guided by intraoperative ultrasound. The operative complication rate was 14.3%. CONCLUSION: Intraoperative ultrasound can accurately localize insulinoma, and delineate the spatial relationship between tumor and vital structures, such as pancreatic duct, common bile duct, and critical blood vessels. It can thereby help to increase the successful rate of surgery and avoid unnecessary blind pancreatectomy.

Intraoperative ultrasound-guided transhepatic lithotomy. A new alternative surgical procedure for the management of residual hepatic stones.

BACKGROUND: The high incidence of residual stones has been a major problem in the treatment of hepatolithiasis. Although various imaging techniques have been used to locate the stones, and many different postoperative procedures have been used as remedial modalities to remove the residual calculi, results have been far from satisfactory. OBJECTIVES: To remove obstinate hepatic stones and reduce the incidence of residual calculi. DESIGN: Prospective clinical trial. SETTING: Medical university-affiliated hospital. PATIENTS AND METHODS: Ten patients who had residual intrahepatic stones after conventional operative procedures underwent intraoperative ultrasound (IOUS)-guided transhepatic lithotomy between July 1988 and July 1995. This surgical technique includes accurately locating stones with IOUS, choosing a surgical approach path under the guidance of IOUS while avoiding critical blood vessels and uninvolved biliary tracts, dividing hepatic parenchyma to the involved biliary ducts, and removing the obstinate calculi using the real-time image of IOUS, which is able to monitor the movement of the lithotomy instruments without interruption. MAIN OUTCOME MEASURES: Clinical practical value of IOUS-guided transhepatic lithotomy in the treatment of residual hepatic stones. RESULTS: Complete removal of the stones was achieved in all 10 patients. There were no severe complications or mortality at a median follow-up of 39 months. CONCLUSIONS: IOUS-guided transhepatic lithotomy can greatly decrease the incidence of residual hepatic stones. It is accurate and safe. As a new surgical procedure, IOUS-guided transhepatic lithotomy should be an alternative modality in the management of hepatolithiasis, although the long-term benefits still need to be observed.

Use of intraoperative ultrasonography during hepatolithectomy.

Complete clearance of intrahepatic stones has long been a major surgical challenge. To reduce the incidence of residual stones, we used intraoperative ultrasonography (IOUS) to localize them and guide lithectomy in 38 patients with hepatolithiasis between July 1988 and December 1993. All patients had multiple intrahepatic stones; 24 had accompanying extrahepatic calculi. Hepatic stones were confined to the left intrahepatic biliary tract in 13 patients, to the right in 9, and in both lobes in 16. Twenty-three patients underwent common bile duct exploration followed by T-tube drainage, 8 had transhepatic lithotomy with or without choledocholithotomy, 3 had choledocolithotomy and Roux-en-Y side-to-side choledochojejunostomy, and 2 had hepaticojejunostomy, left lobectomy was performed in the remainder. In 35 patients cholecystectomy was performed at the same time. Complete clearance of the stones was achieved in 36 patients (94.7%). The incidence of retained stone was decreased to 5.3%. No associated complications occurred. IOUS can accurately localize intrahepatic calculi, directly orient lithotomy instruments to approach the stones, demonstrate the spatial relation between stone and intrahepatic critical structure, and thereby choose an optical route for transhepatic lithotomy. Imaging can be repeated at any time with no radiation exposure to the patient or the medical staff.