Activation of autophagy is involved in the protective effect of 17ß-oestradiol on endotoxaemia-induced multiple organ dysfunction in ovariectomized rats.

Oestrogens have been reported to attenuate acute inflammation in sepsis. In this study, the effects of long-term oestrogen replacement with 17ß-oestradiol (E2 ) on endotoxaemia-induced circulatory dysfunction and multiple organ dysfunction syndrome were evaluated in ovariectomized (Ovx) rats. E2 (50 µg/kg, s.c., 3 times/week) was administered for 8 weeks, followed by the induction of endotoxaemia by intravenous infusion of lipopolysaccharides (LPS; 30 mg/kg/4 hrs). Oestrogen deficiency induced by ovariectomy for 9 weeks augmented the LPS-induced damage, including endotoxic shock, myocardial contractile dysfunction, renal dysfunction and rhabdomyolysis. Cardiac levels of NF-κB p65, iNOS and oxidized glutathione, free radical production in skeletal muscles, myoglobin deposition in renal tubules, and plasma levels of plasminogen activator inhibitor-1, TNF-α, and IL-6 were more pronounced in the Ovx + LPS group than in the Sham + LPS group. Long-term treatment of E2 prevented this amplified damage in Ovx rats. Six hours after LPS initiation, activation of the autophagic process, demonstrated by increases in Atg12 and LC3B-II/LC3B-I ratios, and induction of haem oxygenase (HO)-1 and heat-shock protein (HSP) 70 protein expression in myocardium were increased significantly in the Ovx + E2  + LPS group. These results suggest that activation of autophagy and induction of HO-1 and HSP70 contribute to the protective effect of long-term E2 replacement on multiple organ dysfunction syndrome in endotoxaemia.

Role of AMP-activated protein kinase in α-lipoic acid-induced vasodilatation in spontaneously hypertensive rats.

BACKGROUND: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) has recently emerged as an attractive and novel target for the regulation of vascular smooth muscle contraction. The present study investigated the vasodilatory effects of α-lipoic acid (α-LA) and the possible mechanism of its action on aortic rings from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Aortae were removed from WKY and SHR, and contractile responses to acetylcholine and α-LA studied in organ chamber. Phosphorylated AMPK (pAMPK), phosphorylated Peutz-Jeghers syndrome kinase LKB1 (pLKB1) and calcium/calmodulin-dependent protein kinase (CaMKK) protein level were measured in SHR, WKY, and aortic smooth muscle (A10) cells. RESULTS: α-LA (1-500 µmol/l) produced a concentration-dependent relaxation of precontracted aortic rings from 8- and 16-week-old SHR, but not in those from WKY rats. This vasodilatory effect of α-LA did not change after preincubation with N(G)-nitro-L-arginine methyl ester (100 µmol/l), but significantly suppressed by an AMPK inhibitor, compound C (40 µmol/l). The expression of pAMPKα, pLKB1, and CaMKK were also significantly reduced in endothelium-denuded arteries from 16-week-old SHR compared with those from younger SHR or age-matched WKY rats. After incubation with α-LA (100 µmol/l), the expression of pAMPKα and pLKB1 was significantly increased in the endothelium-denuded aortas from 16-week-old SHR, the expression of CaMKK was more reduced in the endothelium-denuded aortas of 8-week-old SHR, but this was not observed in WKY rats. α-LA also activated AMPKα phosphorylation in A10 cells. CONCLUSIONS: The effects of α-LA on vascular relaxation in SHR result from the enhanced phosphorylation of LKB1-AMPK in aortic smooth muscle.

Baicalein, an active component of Scutellaria baicalensis Georgi, improves cardiac contractile function in endotoxaemic rats via induction of heme oxygenase-1 and suppression of inflammatory responses.

AIM OF THE STUDY: To evaluate the protective effect of baicalein on myocardial dysfunction caused by endotoxaemia in rats and to explore the possible mechanisms. MATERIALS AND METHODS: Baicalein (10mg/kg, intravenous) was administered to conscious Wistar rats 30min after lipopolysaccharide (LPS; 10mg/kg, intravenous) challenge. Six hours after LPS administration, the contractile function of the isolated heart was examined using the Langendorff technique. Cardiac protein expression related to inflammatory responses, superoxide anion production and caspase-3 activity were measured. RESULTS: Post-treatment with baicalein significantly attenuated the LPS-induced hypotension with accompanying tachycardia. The contractile function of isolated heart was significantly preserved 6h after LPS administration, following treatment with baicalein. Furthermore, baicalein induced the expression of heme oxygenase-1 protein and reduced superoxide anion formation in the myocardium of LPS-treated rats. Cardiac levels of inducible nitric oxide synthase, monocyte chemoattractant protein-1, phospho-IκBα and phospho-p65 protein and caspase-3 activity significantly increased 6h after LPS challenge but baicalein significantly attenuated these LPS-induced changes. CONCLUSIONS: Baicalein improves myocardial contractility in LPS-induced sepsis, which may be related to reductions in oxidative stress, myocardial inflammatory responses and apoptosis.

Wogonin suppresses arrhythmias, inflammatory responses, and apoptosis induced by myocardial ischemia/reperfusion in rats.

Wogonin is a flavonoid isolated from Scutellaria baicalensis Georgi, a traditional Chinese medicine, and it possesses antioxidant and anti-inflammatory effects. The aim of this study is to investigate the in vivo effect of wogonin on myocardial ischemia/reperfusion injury in an open-chest anesthetized rat model, which was induced by 45-minute left coronary artery occlusion and 2-hour reperfusion. Rats were treated with wogonin (5, 10, and 20 mg/kg, intraperitoneal) 40 minutes before ischemia or treatment with 10 mg/kg of wogonin 15 minutes after occlusion. Pretreatment with 10 mg/kg of wogonin significantly delayed the occurrence of ventricular premature contractions and tachycardia, and it suppressed the incidence of ventricular tachycardia and ventricular fibrillation, and mortality elicited by ischemia when compared with that in the control group, accompanied by reducing the arrhythmia scores. After 2-hour reperfusion, pretreatment and posttreatment with wogonin significantly reduced the infarct size and plasma levels of creatine kinase muscle-brain fraction and lactate dehydrogenase. Wogonin also significantly reduced the elevation of plasma tissue necrosis factor-α and superoxide anion production in the myocardium with ischemia/reperfusion. The expression of monocyte chemoattractant protein-1, phosphorylated p38 mitogen-activated protein kinase, p65 and IκBα, and active caspase-3 in ischemic myocardium pronouncedly increased in the control group; these were significantly attenuated by treatment with wogonin. In conclusion, wogonin demonstrated in vivo cardioprotective effects by the attenuation of the severity of ischemia-induced arrhythmias and irreversible ischemia/reperfusion injury, which is associated with its antioxidant capacity and anti-inflammatory effects. The suppression of nuclear factor-κB and p38 mitogen-activated protein kinase activation and the inhibition of monocyte chemoattractant protein-1 expression contribute to the beneficial effects of wogonin.

Quantitative DNA methylation analysis detects cervical intraepithelial neoplasms type 3 and worse.

BACKGROUND: DNA methylation may be used a potential biomarker for detecting cervical cancer. The authors of this report used quantitative methylation analysis of 4 genes in a full spectrum of cervical lesions to test its potential clinical application. METHODS: This hospital-based, retrospective, case-control study was conducted in 185 patients and included patients who had a normal uterine cervix (n = 53), cervical intraepithelial neoplasm type 1 (CIN1) (n = 37), CIN2 (n = 22), CIN3 (n = 24), carcinoma in situ (CIS) (n = 22), squamous cell carcinoma (SCC, n = 20), and adenocarcinoma (AC) (n = 7). Methylation levels of the genes sex-determining region Y, box 1 (SOX1); paired box gene 1 (PAX1); LIM homeobox transcription factor 1α (LMX1A), and NK6 transcription factor-related locus 1 (NKX6-1) were determined by using real-time methylation-specific polymerase chain reaction (PCR) amplification. Cutoff values of the percentage of methylation reference (PMR) for different diagnoses were determined to test the sensitivity and specificity and to generate receiver operating characteristic (ROC) curves. Two-sided Mann-Whitney U tests were used to test differences in PMR between groups. RESULTS: The PMRs of the 4 genes were significantly higher in CIN3 and worse (CIN3+) lesions than the PMRs in specimens of normal cervix and CIN1 or CIN2 (P < .001). ROC curve analysis demonstrated that the sensitivity, specificity, and accuracy for detecting CIN3+ lesions were 0.88, 0.82, and 0.95, respectively, for SOX1; 0.78, 0.91, and 0.89, respectively, for PAX1; 0.77, 0.88, and 0.90, respectively, for LMX1A; and 0.93, 0.97, and 0.97, respectively, for NKX6-1. CONCLUSIONS: The current results indicated that quantitative PCR-based testing for DNA methylation of 4 genes holds great promise for cervical cancer screening and warrants further population-based studies using standardized DNA methylation testing.

Methylation analysis of SFRP genes family in cervical adenocarcinoma.

OBJECTIVES: Aberrant activation of the Wnt/beta-catenin signaling pathway is common in human cancers. Recently, we have shown that secreted frizzled-related proteins (SFRPs) are frequently methylated in cervical squamous cell carcinoma (SCC). Furthermore, reexpression of SFRP1 and SFRP2 could suppress tumor cell transformation and invasion. Here, we want to further investigate the methylation status and function of SFRPs in adenocarcinoma of uterine cervix. METHODS: The methylation status of SFRPs was assessed in 23 adenocarcinomas (AC), and 45 normal control swabs by methylation-specific polymerase chain reaction and bisulfite sequencing. Then, we used reexpression of SFRP5 in cervical cancer cell lines, HeLa3rd and CaSki, to study the role of SFRP5 in cervical adenocarcinoma by colony formation and invasion assays. Finally, we checked whether SFRP5 could repress the expression of Wnt/beta-catenin downstream genes by quantitative reverse transcription-polymerase chain reaction. RESULTS: The frequency of SFRP genes promoter hypermethylation in adenocarcinoma of cervix samples was 52.2% (12/23), 82.6% (19/23), 65.2% (15/23), and 73.9% (17/23), for SFRP1, SFRP2, SFRP4, and SFRP5, respectively. The frequency of SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in adenocarcinoma was significantly higher than in normal control samples (P < 0.001). Restoration of SFRP5 suppressed colony formation and invasive ability and inhibited expression of Wnt/beta-catenin downstream genes. CONCLUSIONS: Our data suggest that promoter hypermethylation of SFRPs is associated with cervical adenocarcinoma, which could be used for molecular screening of cervical adenocarcinoma in the future. Moreover, SFRP5 inhibits cervical tumorigenesis through interfering Wnt pathway in vitro.

SFRP1 and SFRP2 suppress the transformation and invasion abilities of cervical cancer cells through Wnt signal pathway.

OBJECTIVES: Aberrant activation of the Wnt/beta-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as Wnt antagonists and play important implications in carcinogenesis. Recently, we have shown that SFRP1 and SFRP2 are frequently downregulated through promoter hypermethylation. However, the function of SFRP1 and SFRP2 in cervical cancer remains unclear. METHODS: To improve our understanding of the role of SFRP1 and SFRP2 in cervical cancer cells, we use overexpression or shRNA approach in cervical cancer cell lines. RESULTS: Restoration of the expression of SFRP1 and SFRP2 attenuated Wnt signaling in CaSki cells, decreased abnormal accumulation of free beta-catenin in the nucleus, and suppressed cancer cell growth. In addition, different statuses of beta-catenin accumulation in the cytoplasm of CaSki or HeLa3rd cells were observed, suggesting that different Wnt pathways are executed. Furthermore, we demonstrated that SFRP1 and SFRP2 enhance the expression of the epithelial marker E-cadherin, through inhibition of the expression of SLUG, TWIST and SNAIL, three transcription factors involved in the epithelial mesenchymal transition (EMT) program. Finally, in a xenograft animal model, we showed that SFRP1 suppresses tumorigenicity of cancer cells in vivo. CONCLUSIONS: Taken together, these data strongly suggest that epigenetic silencing of SFRP genes leads to oncogenic activation of the Wnt pathway and contributes to cervical cancer progression through the EMT program.

Promoter methylation of SFRPs gene family in cervical cancer.

OBJECTIVES: Oncogenic activation of the Wnt/beta-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and play an important role in carcinogenesis. Frequent promoter hypermethylation of SFRPs has been identified in human cancers; however, the precise role of SFRPs in cervical cancer is not clear. METHODS: The methylation status of SFRPs gene family was analyzed in two cervical cancer cell lines and a full spectrum of cervical neoplasia, including 45 low-grade squamous intraepithelial lesions (LSIL), 49 high-grade squamous intraepithelial lesions (HSIL), 109 squamous cell carcinomas (SCC), and 45 normal controls. RESULTS: The SFRP1 promoter was hypermethylated in 33.9% of SCC, 8.2% of HSIL, 2.2% of LSIL, but not in normal tissues. The SFRP2 promoter was hypermethylated in 80.7% of SCC, 16.3% of HSIL, 15.6% LSIL and 4.4% normal tissues. The SFRP4 promoter was hypermethylated in 67.9% of SCC, 36.7% of HSIL, 4.4% of LSIL, but not in normal tissues. The SFRP5 promoter was hypermethylated in 10.1% of SCC, 4.1% of HSIL, 13.3% of LSIL and 4.4% normal tissues. The frequency of SFRP1, SFRP2 and SFRP4 promoter methylation in tumors was significantly higher than in normal, LSIL, and HSIL samples (P<0.0001). SFRP5 methylation was significantly different in patients with or without lymph-node metastases (0% vs 15.2%, respectively, P<0.05). CONCLUSIONS: Our data suggest that promoter hypermethylation of SFRP1, SFRP2 and SFRP4 is associated with cervical carcinogenesis, which could be used for molecular screening of cervical neoplasias in future.