BACKGROUND Esophageal squamous cell carcinoma (ESCC) is a worldwide concern. This study looked at the relationship between the expression of differential proteins and the clinicopathological data and survival rate of ESCC patients to identify potential tumor markers for the growth and metastasis of ESCC. MATERIAL AND METHODS This study included 162 patients who underwent surgical excision for management of ESCC. Fresh ESCC tissue and adjacent normal tissue specimens were collected. Protein expressions were detected by western blotting. The expression of Hsp27 and P38MAPK were detected by immunohistochemistry in formalin-fixed paraffin embedded primary tissue specimens. RESULTS The rate of positive Hsp27 and P38MAPK expression in ESCC tissue were higher than in normal esophageal tissue (p<0.05). The expression of P38MAPK was related to the depth of infiltration (p<0.05). The expression of Hsp27 was correlated with lymph node metastasis (p<0.05), but not with age, depth of infiltration, or tumor size. ROC were plotted to estimate the significance of the diagnosis: for Hsp27, AUC=0.735 (p<0.05), for P38MAPK, AUC=0.882 (p<0.05). CONCLUSIONS The expression of Hsp27 and P38MAPK plays a role in ESCC development. Hsp27 and P38MAPK could be used as prognostic factors in ESCC.

Mitochondrial biogenesis is crucial for the maintenance of mitochondrial function and cellular homeostasis. C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that owns multiple functions on metabolic and cardiovascular diseases. However, whether CTRP3 affects mitochondrial biogenesis in cardiomyocytes remains unknown.

The main aim of this study was to evaluate the neuroprotective effect of aspirin combined with ginkgolide injection on cerebral ischemic stroke model rats and its effect on extracellular regulated protein kinase 1/2 (REK1/2) signaling pathway, and to clarify the possible mechanism of aspirin combined with ginkgolide injection on neuroprotective mechanism. Experimental rats were randomly divided into sham group, model group, aspirin group, ginkgolide group and combination group (aspirin + ginkgolide injection) (n = 20). The results revealed scores of neurological dysfunction and infarct volume in aspirin group, ginkgolide group and combination group rats were lower than those in model group (P < 0.05). Score of neurological dysfunction and the volume of cerebral infarction in combination group rats were lower than those in aspirin group and ginkgolide group (P < 0.05). Combination of aspirin and ginkgolide injection could better reduce brain water content, reduce apoptosis rate of cortical cells P < 0.05, reduce expression levels of caspase-3, Bax and p-REK1/2 proteins in ischemic brain tissue P < 0.05, and increase expression level of Bcl-2 protein than aspirin and ginkgolide injection alone P < 0.05). In conclusion, the synergistic neuroprotective effect of aspirin and ginkgolide injection on cerebral ischemic stroke rats is better than that of aspirin and ginkgolide injection alone. The mechanism of action may be that the two compounds can play a synergistic role and inhibit the activation of REK1/2 signaling pathway, thus inhibiting apoptosis of nerve cells and exerting neuroprotective effect.

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy. The mycotoxin of T-2 toxin is extensively accepted as a major etiological contributor to KBD. However, its function and mechanism in KBD remains unclearly elucidated. Here, T-2 toxin treatment induced chondrocyte injury in a time- and dose-dependent manner by repressing cell viability and promoting cell necrosis and apoptosis. Importantly, T-2 suppressed the transcription of type II collagen and aggrecan, as well as the release of sulphated glycosaminoglycan (sGAG). Furthermore, exposure to T-2 enhanced the transcription of matrix metalloproteinases (MMPs), including MMP-1, -2, -3 and -9. In contrast to control groups, higher expression of insulin-like growth factor binding protein 2 (IGFBP2) was observed in chondrocytes from KBD patients. Interestingly, T-2 toxin caused a dramatical elevation of IGFBP2 expression in chondrocytes. Mechanism analysis corroborated that cessation of IGFBP2 expression alleviated T-2-induced damage to chondrocytes. Simultaneously, transfection with IGFBP2 siRNA also attenuated matrix synthesis and catabolism-related gene expressions of MMPs. Together, this study validated that T-2 toxin exposure might promote the progression of KBD by inducing chondrocyte injury, suppressing matrix synthesis and accelerating cellular catabolism through IGFBP2. Therefore, this research will elucidate a new insight about how T-2 toxin participate in the pathogenesis of KBD.

Claudins are found in junctional complexes mediating cell adhesion and are involved in the attachment of tight junctions to the underlying cytoskeleton. Abnormal claudin-6 expression has been observed for a variety of malignant solid tumors, but the expression of claudin-6 in non-small cell lung cancer (NSCLC) has not yet been characterized.

Cytochrome P450 17A1 (P450c17) is the key enzyme required for the production of androgenic sex steroids by converting progestogens to androgens. 5α-reductases are enzymes that convert testosterone (T) to dihydrotestosterone (DHT), which has a greater affinity for androgen receptors (AR) and stronger action than T. Our previous studies revealed that the scented glands of male muskrats expressed AR during the breeding and nonbreeding seasons. To further seek evidence of the activities of androgens in scented glands, the expression patterns of P450c17 and 5α-reductase 2 were investigated in the scented glands of male muskrats during the breeding and nonbreeding seasons. The weight and size of scented glands in the breeding season were significantly higher than those of the nonbreeding season. Immunohistochemical data showed that P450c17 and 5α-reductase 2 were presented in the glandular cells and epithelial cells of scented glands in both the seasons. The protein and mRNA expression of P450c17 and 5α-reductase 2 were significantly higher in the scented gland during the breeding season than those during the nonbreeding season. In addition, the levels of DHT and T in the scented gland were remarkably higher during the breeding season. Taken together, these results suggested that the scented glands of male muskrats were capable of locally synthesizing T and DHT, and T and DHT might play an important role in the scented glandular function via an autocrine or paracrine manner.

Background: Momordica charantia is used in China for its jianghuo (heat-clearing and detoxifying) effects. The concept of shanghuo (the antonym of jianghuo, excessive internal heat) in traditional Chinese medicine is considered a type of stress response of the body. The stress process involves internal organs, especially the liver. Objective: We hypothesized that Momordica charantia water extract (MWE) has a hepatoprotective effect and can protect the body from stress. The aim of this study was to investigate the possible effects of MWE against liver injury in restraint-stressed mice. Design: The mice were intragastrically administered with MWE (250, 500 and 750 mg/kg bw) daily for 7 days. The Normal Control (NC) and Model groups were administered distilled water. A positive control group was intragastrically administered vitamin C 250 mg/kg bw. After the last administration, mice were restrained for 20 h. Results: MWE reduced the serum AST and ALT, reduced the NO content and the protein expression level of iNOSin the liver; significantly reduced the mitochondrial ROS content, increased the mitochondrial membrane potential and the activities of mitochondrial respiratory chain complexes I and II in restraint-stressed mice. Conclusions: The results indicate that MWE has a protective effect against liver injury in restraint-stressed mice. Abbreviations: MWE: Momordica charantia water extract; M. charantia: Momordica charantia L.; ROS: reactive oxygen species; NO: nitric oxide; iNOS: inducible nitric oxide synthase; IL-1β: interleukin-1 beta; TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; IFN-γ: interferon gamma; VC: vitamin C; ALT: alanine transaminase; AST: aspartate aminotransferase; GSH: glutathione; GSH-PX: glutathione peroxidase; MDA: malondialdehyde; BCA: bicinchoninic acid; TBARS: thiobarbituric acid reactive substances; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; JC-B: Janus Green B; DW: dry weight; FC: Folin-Ciocalteu; GAE: gallic acid equivalents; bw: body weight; NC: normal control group; Model: restraint stress model group; VC: positive control vitamin C group, 250 mg/kg bw; MWEL: Momordica charantia water extract low-dose group, 250 mg/kg bw; MWEM: Momordica charantia water extract middle-dose group, 500 mg/kg bw; MWEH: Momordica charantia water extract high-dose group, 750 mg/kg bw; HE: hematoxylin and eosin; ORAC: total oxygen radical absorbance capacity; ABAP: dihydrochloride; ATP: adenosine triphosphate.

KCNJ2/Kir2.1, a member of the classical inwardly rectifying potassium channel family, is commonly expressed in a wide range of tissues and cell types. Previous studies indicated that Kir2.1 may be associated with SCLC multidrug resistance (MDR). However, whether Kir2.1 can regulate MDR and its underlying mechanisms remain poorly understood in SCLC.

Methane is reported to have antioxidant, anti-inflammatory and anti-apoptotic properties. We investigated the potential neuroprotective effects of methane-rich saline (MS) on spinal cord ischemia-reperfusion injury and determined that its therapeutic benefits are associated with the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Rats received 9min of spinal cord ischemia induced by occlusion of the descending thoracic aorta plus systemic hypotension followed by a single MS treatment (10ml/kg, ip) and 72h reperfusion. MS treatment attenuated motor sensory deficits and produced high concentrations of methane in spinal cords during reperfusion, which increased Nrf2 expression and transcriptional activity in neurons, microglia and astrocytes in the ventral, intermediate and dorsal gray matter of lumbar segments. Heme oxygenase-1, superoxide dismutase, catalase and glutathione were upregulated; and glutathione disulfide, superoxide, hydrogen peroxide, malondialdehyde, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine were downregulated in MS-treated spinal cords. MS treatment reduced neuronal apoptosis in gray matter zones, which was consistent with the suppression of cytochrome c release to the cytosol from the mitochondria and the activation of caspase-9 and -3. Throughout the gray matter, the activation of microglia and astrocytes was inhibited; the nuclear accumulation of phosphorylated nuclear factor-kappa B p65 was reduced; and tumor necrosis factor α, interleukin 1β, chemokine (C-X-C motif) ligand 1, intercellular adhesion molecule 1 and myeloperoxidase were decreased. MS treatment attenuated blood-spinal cord barrier dysfunction by preventing the expression and activity of matrix metallopeptidase-9 and disrupting tight junction proteins. Consecutive intrathecal injection of specific siRNAs targeting Nrf2 at 24-h intervals 3 days before ischemia reduced the beneficial effects of MS. Our data indicate that MS treatment prevents IR-induced spinal cord damage via antioxidant, anti-inflammatory and anti-apoptotic activities that involve the activation of Nrf2 signaling. Thus, methane may serve as a novel promising therapeutic agent for treating ischemic spinal cord injury.

Rupture of vulnerable atherosclerotic plaque is the major pathological cause of luminal thrombosis in acute coronary syndromes. Since foamy macrophages have been identified as a prominent component in vulnerable atherosclerotic lesions and osteopontin (OPN) is reported to be highly expressed in foamy macrophages, OPN could be a potential target for vulnerable atherosclerotic plaque imaging. The current study designed an OPN-specific MRI/optical dual-modality probe to detect vulnerable plaques. Fluorescence imaging revealed that 24 h after injection of the Cy5.5-OPN-DMSA-MNPs (COD-MNPs), the atherosclerotic plaques in carotid artery exhibited significant higher signals in high fat diet (HFD) fed mice in comparison to the group injected with Cy5.5-IgG-DMSA-MNPs (CID-MNPs) or normal diet fed group injected with COD-MNPs (1.87 ± 0.19 × 1010 vs. 0.74 ± 0.04 × 1010, 0.73 ± 0.03 × 1010 p/sec/cm2/sr, P < 0.05). Meanwhile, MRI displayed stronger T2 contrast enhancement 24 h post-injection at the area of atherosclerotic plaques in the carotid of HFD fed group injected with COD-MNPs than group injected with CID-MNPs or normal diet fed group injected with COD-MNPs (post/pre signal ratio: 0.64 ± 0.04 vs. 0.95 ± 0.02, 0.98 ± 0.01, P < 0.05). As a dual-modality molecular probe, the resulting COD-MNPs conjugates exhibit promising potentials for noninvasive detection of vulnerable atherosclerotic plaque in vivo.

Asymmetric dimethylarginine (ADMA) induces vascular smooth muscle cells (VSMCs) migration. VSMC phenotype change is a prerequisite of migration. RhoA and Rho-kinase (ROCK) mediate migration of VSMCs. We hypothesize that ADMA induces VSMC migration via the activation of Rho/ROCK signal pathway and due to VSMCs phenotype change. ADMA activates Rho/ROCK signal pathway that interpreted by the elevation of RhoA activity and phosphorylation level of a ROCK substrate. Pretreatment with ROCK inhibitor, Y27632 completely reverses the induction of ADMA on ROCK and in turn inhibits ADMA-induced VSMCs migration. When the Rho/ROCK signal pathway has been blocked by pretreatment with Y27632, the induction of ERK signal pathway by ADMA is completely abrogated. Elimination of ADMA via overexpression of dimethylarginine dimethylaminohydrolase 2 (DDAH2) and L-arginine both blocks the effects of ADMA on the activation of Rho/ROCK and extra cellular signal-regulated kinase (ERK) in VSMCs. The expression of differentiated phenotype relative proteins was reduced and the actin cytoskeleton was disassembled by ADMA, which were blocked by Y27632, further interpreting that ADMA inducing VSMCs migration via Rho/ROCK signal pathway is due to its effect on the VSMCs phenotype change. Our present study may help to provide novel insights into the therapy and prevention of atherosclerosis.

Hypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects.

Cervical cancer is the second most common gynecological cancer worldwide and remains one of the leading causes of cancer-associated mortality among women. S100A6 has been reported to be associated with the development of many types of cancer. The aim of the present study was to investigate the effect of S100A6 on the proliferation, apoptosis and migration of cervical cancer cells and its underlying molecular mechanisms. Quantative polymerase chain reaction (qPCR) was used to detect the basic mRNA level of S100A6 in HeLa, SiHa and CaSki cells. Western blot analysis was used to detect the protein level of S100A6, epithelial cadherin, neuronal cadherin, phosphorylated protein kinase B (p-Akt), t-Akt, p-glycogen synthase kinase 3β (GSK3β), t-GSK3β and β-catenin. Semi-qPCR was used to detect the mRNA level of Snail, Twist and Vimentin. MTT and Hoechst staining assays were used to detect the proliferation and apoptosis of cells, and wound healing and Transwell assays were used to detect the migration of cells. The results of the present study demonstrate that the levels of S100A6 were decreased in HeLa cells compared with in SiHa and CaSki cells. Overexpression of S100A6 in HeLa and CaSki cells promoted the proliferative and migratory ability, and had no significant effect on cellular apoptosis. Whereas the knockdown of S100A6 in SiHa and CaSki cells inhibited the proliferative and migratory ability, it had no significant effect on apoptosis. The overexpression of S100A6 in HeLa cells increased the levels of neuronal (N)-cadherin, vimentin, Snail and Twist. Conversely, knockdown of S100A6 in SiHa cells decreased the levels of N-cadherin, vimentin, Snail and Twist and increased the levels of epithelial (E)-cadherin. Furthermore, overexpression of S100A6 in HeLa cells activated the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, and treatment with the PI3K inhibitor LY294002 partially repressed S100A6-enhanced proliferation and migration of cervical cancer cells. These results indicate that S100A6 facilitates the malignant potential of cervical cancer cells, particularly metastatic ability and epithelial-mesenchymal transition, which is mediated by activating the PI3K/Akt signaling pathway.

It is well known that liver cancer is a highly aggressive malignancy with poor prognosis. Andrographolide (AD), a major bioactive component of Andrographis paniculata (Burm. F.), is a potential anti-cancer pharmacophore and the synthesis of AD derivatives with better cytotoxicity to cancer cells has attracted considerable attentions. In the present study, we evaluated the in vivo inhibitory effects of ADN-9, a 15-benzylidene substituted derivative of AD, on the growth and metastasis of murine hepatoma H22 using an orthotopic xenograft model and a subcutaneous xenograft model, and we further studied the anti-angiogenic action and the related mechanisms of ADN-9 in vivo and in vitro. Importantly, ADN-9 remarkably suppressed the growth and metastasis of both orthotopic and subcutaneous xenograft tumors, and the serum AFP level in orthotopic hepatoma-bearing mice treated with 100mg/kg ADN-9 (ig.) was decreased to the normal level. We also found that ADN-9 showed stronger abilities than AD in shrinking tumors, suppressing the invasion and metastasis of H22 cells, decreasing the MVD and promoting tumor cell apoptosis in subcutaneous xenograft of mice. Additionally, ADN-9 exhibited stronger inhibitory activity than AD against the migration and VEGF-induced capillary-like tube formation in HUVECs, which was further proved to be associated with attenuating VEGF/VEGFR2/AKT signaling pathway. The present research provides the first evidence that a 15-substituted AD derivative is more promising than the parent compound in therapeutic treatment of liver cancer.

Hepatitis B virus (HBV) infection causes major public health problems worldwide. The clinical limitation of current antiviral drugs for HBV, such as lamivudine, is the emergence of drug-resistant viral strains during prolonged antiviral therapy. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has been reported to possess potent activity against various viruses. The present study was performed to evaluate the in vitro activity of CH against clinical wild-type and lamivudine-resistant HBV isolates in transiently transfected cells. HBV DNA was extracted from serum samples collected both before lamivudine therapy and at the time of viral breakthrough and was amplified by polymerase chain reaction (PCR). The amplicons were cloned into a novel expression vector, pHY106, which can initiate the intracellular HBV replication cycle after cell transfection. Following transfection of the cloned amplicon into HepG2 cells, a drug susceptibility assay was performed. The level of viral antigen, HBeAg, was determined by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (Q-PCR) was used for determining the amount of intracellular HBV DNA. Heat stress cognate 70 (Hsc70), a host protein required for HBV replication, was also analyzed by reverse transcription PCR (RT-PCR) to explore the possible antiviral mechanism of CH. The results showed that CH inhibited replication and HBeAg production by either wild-type or lamivudine-resistant HBV clinical isolates in a dose-dependent manner. The Hsc70 mRNA was also downregulated significantly. In conclusion, CH is active against both wild-type and lamivudine-resistant HBV clinical isolates, and its activity may be associated with its inhibition of host Hsc70.

The aim of this study was to construct the eukaryotic expression vector pEGFP-N1-hPer2 and assess its expression in the human osteosarcoma cell line MG63. Total mRNA was extracted from human osteosarcoma MG63 cells, the human period 2 (hPer2) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the pEGFP-N1 vector, then the recombinant pEGFP-N1-hPer2 plasmid was constructed and transfected into MG63 cells using Lipofectamine 2000. The expression of hPer2 in MG63 cells was measured by quantitative RT-PCR and western blot analysis. The accurate construction of pEGFP-N1-hPer2 was verified by double enzyme digestion and DNA sequencing. hPer2 gene expression in the transfected cells was assessed by RT-qPCR and western blot analysis. In conclusion, the recombinant pEGFP-N1-hPer2 plasmid was constructed successfully, and expressed effectively in MG63 cells.

NSC67657 is a new steroid drug that induces monocytic differentiation of acute myeloid leukemia cells. Here, we demonstrate that NSC67657 has opposing effects on expression of downstream targets of inhibitor of β-catenin and TCF (ICAT) and Wnt signaling in HL60 cells. ICAT binds to β-catenin, and this interaction is further increased in NSC67657-differentiated cells. ICAT overexpression decreases expression of Wnt downstream targets and increases sensitivity of HL60 cells to NSC67657, while ICAT silencing increases Wnt signaling and delays the NSC67657-induced cell differentiation. In addition, pharmacological inhibition of Wnt/β-catenin signaling increases the NSC67657-induced cell differentiation, while activation of Wnt/β-catenin signaling inhibits the differentiation, indicating Wnt/β-catenin signaling inhibits NSC67657-induced monocytic differentiation of HL60 cells. Our data demonstrate the opposing roles of ICAT and Wnt signaling in the NSC67657-induced monocytic differentiation, and suggest that ICAT and Wnt signaling may serve as therapeutic targets for leukemia chemotherapy.

Therapeutic modalities targeting vascular endothelial growth factor (VEGF) have been used to treat neovascularization and macular edema. However, anti-VEGF treatment alone may cause up-regulation of connective tissue growth factor (CTGF) in the retina, increasing the risk of fibrosis and tractional retinal detachment. Therefore, in this study, we employ a novel dual-target intervention that involves intravitreal injection of the VEGF inhibitor ranibizumab and a transfection reagent-treated non-viral vector carrying anti-CTGF short hairpin RNA (shRNA) driven by human RNA polymerase III promoter U6. The effects of the dual-target intervention on the expression of VEGF and CTGF and on microvessel ultrastructure were examined in retina of streptozocin-induced diabetic rats. CTGF was significantly up-regulated at week 8 after diabetic induction, whereas VEGF was not up-regulated until week 10. The high expression of both genes was maintained at week 12. Transmission electron microscopy also revealed progressive exacerbation of microvessel ultrastructure during the same period. In addition, ranibizumab significantly lowered VEGF but elevated CTGF mRNA, whereas CTGF shRNA significantly reduced the mRNA levels of both CTGF and VEGF in diabetic retinas. Importantly, dual-target intervention normalized the transcript levels of both target genes and ameliorated retinal microvessel ultrastructural damage better than either single-target intervention. These results suggest the advantages of dual-target over single-target interventions in diabetic retina and reveal a novel therapeutic modality for diabetic retinopathy.

Autologous submandibular gland transplantation is an effective treatment for severe dry eye syndrome. However, the protein secretion in transplanted gland is altered by a mechanism that remains to be elucidated. In the present study, we found that β1-adrenoceptor (β1-AR) and β2-AR expression and the phosphorylation of the downstream molecule protein kinase A (PKA) were elevated in transplanted submandibular glands obtained from epiphora patients. Synaptobrevin/vesicle-associated membrane protein 2 (VAMP-2) interacted with syntaxin-4 and actin in human submandibular gland. The contents of syntaxin-4 and actin interacting with VAMP-2 were increased in transplanted gland. Moreover, VAMP-2 and syntaxin-4 expression in the secretory granule fraction, and VAMP-2 expression in the membrane protein fraction were increased in isoproterenol-treated and transplanted glands. Isoproterenol increased F-actin polymerization in the apical and lateral regions of the cytoplasm in both control and transplanted glands. Inhibiting PKA activity and/or F-actin formation abolished the isoproterenol-enhanced expression of VAMP-2 and syntaxin-4 in the secretory granule fraction and the isoproterenol-enhanced expression of VAMP-2 in the membrane protein fraction. Taken together, these results indicate that the activation of β-ARs induces secretory granules and cell membrane fusion via the interaction of VAMP-2 and syntaxin-4 in a PKA- and F-actin-dependent manner in human submandibular gland. Up-regulated β-ARs might participate in altering protein secretion in transplanted submandibular gland by promoting the interaction of VAMP-2 with syntaxin-4.

Stress is a major risk factor for psychiatric disorders, such as depression, posttraumatic stress disorder, and schizophrenia. Early life stress, such as maternal separation, can have long-term effects on the development of the central nervous system and pathogenesis of psychiatric disorders. In the present study, we found that maternal separation increased the susceptibility to stress in adolescent rats, increased the expression of Na+/K+/2Cl- cotransporter 1 (NKCC1) on postnatal day 14, and increased the expression of K+/2Cl- cotransporter 2 (KCC2) and γ-aminobutyric acid A (GABAA) receptor subunits on postnatal day 40 in the hippocampus. NKCC1 inhibition by the U.S. Food and Drug Administration-approved drug bumetanide during the first two postnatal weeks rescued the depressive- and anxiety-like behavior that was induced by maternal separation and decreased the expression of NKCC1, KCC2 and GABAA receptor α1 and β2,3 subunits in the hippocampus. Bumetanide treatment during early development did not adversely affect body weight or normal behaviors in naive rats, or affect serum osmolality in adult rats. These results suggest that bumetanide treatment during early development may prevent the maternal separation-induced susceptibility to stress and impairments in GABAergic transmission in the hippocampus.