• Tuberculosis and Respiratory Diseases
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• v.73 no.6
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• pp.312-319
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• 2012

Background: Muscle wasting in sepsis is associated with increased proteolysis. Interleukin-15 (IL-15) has been characterized as an anabolic factor for skeletal muscles. Our study aims to investigate the role of IL-15 in sepsis-induced muscle atrophy and proteolysis. Methods: Mice were rendered septic either by cecal ligation and puncture or by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg i.p.). Expression of IL-15 mRNA and protein was determined by reverse transcriptase polymerase chain reaction and Western blot analysis in the control and septic limb muscles. C2C12 skeletal muscle cells were stimulated in vitro with either LPS or dexamethasone in the presence and absence of IL-15 and sampled at different time intervals (24, 48, or 72 hours). IL-15 ($10{\mu}g/kg$) was intraperitoneally administered 6 hours before sepsis induction and limb muscles were sampled after 24 hours of sepsis. Cathepsin L activity was determined to measure muscle proteolysis. Atrogin-1 and muscle-specific ring finger protein 1 (MuRF1) expressions in limb muscle protein lysates was analyzed. Results: IL-15 mRNA expression was significantly lower in the limb muscles of septic mice compared to that of controls. Cathepsin L activity in C2C12 cells was significantly lower in presence of IL-15, when compared to that observed with individual treatments of LPS or dexamethasone or tumor necrosis factor ${\alpha}$. Further, the limb muscles of mice pre-treated with IL-15 prior to sepsis induction showed a lower expression of atrogin-1 and MuRF1 than those not pre-treated. Conclusion: IL-15 may play a role in protection against sepsis-induced muscle wasting; thereby, serving as a potential therapeutic target for sepsis-induced skeletal muscle wasting and proteolysis.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1343-1348
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• 2015

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to be over-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whether knockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirus-mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression and was confirmed to be over 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometry and Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910 PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibited the invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cells of UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase. The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr 15.

• Environmental Analysis Health and Toxicology
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• v.13 no.3_4
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• pp.55-61
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• 1998

Glutathione-S-transferases (GSTs) detoxify electrophilic xenobiotics and reactive metabolites. Recently benzene-fused heterocycles have been shown to increase the total amount of hepatic GSTs in rats. Primarily this study aimed to determine the induction of GSTs by benzoazoles (BAs) including benzoxazole (BX), 2-methylbenzoxazole (M-BX), 2,5-dimethyl benzoxazole (D-BX), benzothiazole (BT), aminobenzothiazole (A-BT) and 2-mercaptobenzothiazole (M-BT) in rats. Hepatic cytosol and poly(A)$^+$ mRNA were prepared from rats after oral administration of BX, BT, M-BX, D-BX, A-BT and M-BT for 5 consecutive days at doses of 1 mmol/kg. Western immunoblot and northern blot analysis were conducted with rabbit anti-GST Ya, Yb$_1$, Yb$_2$, Yc antibodies and cDNA probes containing = 500 bps in the specific coding regions of Ya, Yb$_1$, Yb$_2$, Yc$_1$, and Yc$_2$, respectively. All BAs increased the amount of enzymes and mRNA levels of GSTs. BT was the most effective inducer of GSTs among the compounds examined in this study. Although A-BT and M-BT, the derivatives of BT, induced GSTs, these chemicals had lesser effect on induction of GSTs than BT. The derivatives of BX also induced less GSTs than the parent compound and the addition of methyl group to the benzene ring of BX reduced the induction of GSTs. BAs had better inductive effects on the class $\alpha$(Ya, Yc) than class $\mu$ GSTs (Yb$_1$, Yb$_2$). BAs enhanced mRNA levels of GSTs in parallel with the protein levels. These results indicate that 1) most of BAs induced various isozymes of GSTs, 2) the induction of GSTs appears to be correlated with the chemical structure of the derivatives, and 3) the expression of GST by BAs is presumably under the transcriptional regulation.

• Journal of Veterinary Science
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• v.24 no.5
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• pp.69.1-69.11
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• 2023

Background: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Objectives: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. Methods: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Results: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca⁺⁺-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runtrelated transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca²⁺-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. Conclusions: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

• The Korean Journal of Pain
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• v.23 no.1
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• pp.1-10
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• 2010

Background: Recent studies indicate that reactive oxygen species (ROS) are involved in persistent pain, including neuropathic and inflammatory pain. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N-methyl-D-aspartate (NMDA) receptors in the dorsal horn after an exogenous supply of three antioxidants in rats with chronic post-ischemia pain (CPIP). This serves as an animal model of complex regional pain syndrome type-I induced by hindpaw ischemia/reperfusion injury. Methods: The application of tight-fitting O-rings for a period of three hours produced CPIP in male Sprague-Dawley rats. Allopurinol 4 mg/kg, allopurinol 40 mg/kg, superoxide dismutase (SOD) 4,000 U/kg, N-nitro-L-arginine methyl ester (L-NAME) 10 mg/kg and SOD 4,000 U/kg plus L-NAME 10 mg/kg were administered intraperitoneally just after O-ring application and on the first and second days after reperfusion. Mechanical allodynia was measured, and activation of the NMDA receptor subunit 1 (pNR1) of the lumbar spinal cord (L4-L6) was analyzed by the Western blot three days after reperfusion. Results: Allopurinol reduced mechanical allodynia and attenuated the enhancement of spinal pNR1 expression in CPIP rats. SOD and L-NAME also blocked spinal pNR1 in accordance with the reduced mechanical allodynia in rats with CPIP. Conclusions: The present data suggest the contribution of superoxide, produced via xanthine oxidase, and the participation of superoxide and nitric oxide as a precursor of peroxynitrite in NMDA mediated central sensitization. Finally, the findings support a therapeutic potential for the manipulation of superoxide and nitric oxide in ischemia/reperfusion related pain conditions.

• Molecular & Cellular Toxicology
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• v.4 no.4
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• pp.278-284
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• 2008

The X-linked inhibitor of apoptosis (XIAP) is a member of a novel family of inhibitors of apoptosis and has several BIR domains (BIR1, BIR2, and BIR3) and a carboxy-terminal RING zinc-finger. Since suppressionof apoptosis is fundamentally important for carcinogenesis and tumor growth, we investigated the expression and function of XIAP in DEN-induced carcinogenesis using rat model. Wistar rats were injected intraperitoneally with DEN at a dose of 50 mg/kg in twice a week for 12 weeks (Group II) and 16 weeks (Group III) followed by the recovery periods, respectively. The evaluation of DEN-induced carcinogenesis carried out the blood, RT-PCR, histopathological and western blot analysis. The level of blood chemistry including GOT/GPT, albumin, and total bilirubin were significantly exchanged comparing to control and Group I/Group II. The expression of albumin and collagen mRNA were significantly exchanged (P<0.05) in both groups. In addition, AFP mRNA expression decreased more after recovery periods than Group II. XIAP was expressed constitutively in normal rat liver as well as DEN-induced Groups I and Group II. In addition, XIAP expression increased more in Group I with 4 weeks recovery periods than Group I. However, XIAP expression shown to increase in Group lI, otherwise, it was decreased in Group II with 10 weeks repair periods. Taken together, these results suggest the alteration of XIAP expression could be involved in hepatocellular carcinogenesis.

• Molecules and Cells
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• v.37 no.3
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• pp.226-233
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• 2014

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson's disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium ($MPP^+$) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by $MPP^+$ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.

• Molecules and Cells
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• v.44 no.3
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• pp.146-159
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• 2021

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.