EZH2 plays a crucial role in ischemia/reperfusion-induced acute kidney injury by regulating p38 signaling.

OBJECTIVE AND DESIGN: Renal ischemia-reperfusion (IR)-induced acute kidney injury (AKI) remains a major challenge in clinic. The histone methyltransferases enhancer of zest homolog-2 (EZH2) is associated with the development of renal injury. However, the molecular mechanism has not been fully elucidated. MATERIALS: AKI in C57BL/6 mice was generated by renal IR. TREATMENTS: The 3-deazaneplanocin A (DZNeP), a selective EZH2 inhibitor, or vehicle was administrated in mice after IR. HK-2 cells were exposed to hypoxia-reoxygenation (H/R) stress. METHODS: Apoptosis was detected by TUNEL assay or flow cytometry. EZH2, caspase-3, p38, F4/80+ macrophages, and CD3+ T cells were examined by immunohistochemistry or Western blot. Tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, IL-6, and IL-18 were measured using RT-PCR. RESULTS: Mice treated with DZNeP exhibited less severe renal dysfunction and tubular injury following IR. EZH2 inhibition decreased apoptotic cells while reducing activation of caspase-3 in kidneys under IR condition. Moreover, EZH2 inhibition impaired the recruitment of CD3+ T cells and F4/80+ cells in kidneys with IR. Administration of DZNeP suppressed the production of TNF-α, MCP-1, IL-6, and IL-18 in IR-treated kidneys. Of note, EZH2 inhibition reduced p38 phosphorylation in kidneys after IR. In H/R-treated HK-2 cells, DZNeP treatment or EZH2 knockdown reduced apoptosis. EZH2 inhibition inactivated p38 resulting in reduction of active caspase-3 and proinflammatory molecules. By contrast, EZH2 overexpression induced p38 phosphorylation, caspase-3 activation, and production of proinflammatory molecules, which was reversed by SB203580. CONCLUSIONS: EZH2 plays a crucial role in IR-induced AKI via modulation of p38 signaling. Targeting EZH2/p38 signaling pathway may offer novel strategies to protect kidneys from acute kidney injury induced by ischemia-reperfusion.

Pharmacological inhibition of Rac1 exerts a protective role in ischemia/reperfusion-induced renal fibrosis.

Acute kidney injury induced by renal ischemia-reperfusion (IR) is a prominent risk factor in the development towards renal fibrosis. Ras-related C3 botulinum toxin substrate 1(Rac1) has been involved in the pathophysiology of fibrotic disorders. But the role of Rac1 in the pathogenesis of IR-induced renal fibrosis is still unknown. Here, we examined the effects of NSC23766, an inhibitor of Rac1, on the progression of renal fibrosis after IR injury. In mice, IR induced Rac1 activation in kidneys. Rac1 inhibition alleviated renal damage and dysfunction. Mice treated with NSC23766 displayed diminished collagen area in the kidneys compared with IR controls, which was associated with reduction of extracellular matrix (ECM) proteins and α-SMA. Furthermore, Rac1 inhibition reduced profibrotic molecules levels in the kidneys of mice with IR. Finally, Rac1 inhibition impaired the accumulation of bone marrow-derived M2 macrophages and the transition of M2 macrophages to myofibroblasts. In cultured mouse monocytes, IL-4 treatment activated Rac1, which was abrogated by NSC23766. Moreover, application with IL-4 induced polarization of monocytes to M2 phenotype and increased the levels of ECM proteins and α-SMA, which was abolished by NSC23766. In summary, Rac1 plays a crucial role in the pathogenesis of renal fibrosis after IR via regulation of expressions of profibrotic molecules, bone-marrow derived M2 macrophages recruitment, and M2 macrophages-myofibroblasts transition.

Interleukin-33 signaling contributes to renal fibrosis following ischemia reperfusion.

Acute kidney injury caused by ischemia-reperfusion injury (IRI) is a major risk factor for chronic kidney disease, which is characterized by renal interstitial fibrosis. However, the molecular mechanisms underlying renal fibrosis induced by IRI are not fully understood. Our results showed that interleukin (IL)-33 was induced markedly after IRI insult, and the kidneys of mice following IRI plus IL-33 treatment presented more severe renal fibrosis compared with mice treated with IRI alone. Therefore, we investigated whether inhibition of IL-33 protects against IRI-induced renal fibrosis. Mice were administrated with soluble ST2 (sST2), a decoy receptor that neutralizes IL-33 activity, or vehicle by intraperitoneal injection for 14 days after IRI challenge. We revealed that mice treated with sST2 exhibited less severe renal dysfunction and fibrosis in response to IRI compared with vehicle-treated mice. Inhibition of IL-33 suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidneys after IRI stress, which was associated with less expression of extracellular matrix proteins. Furthermore, inhibition of IL-33 also showed a significant reduction of F4/80+ macrophages and CD3+ T cells in the kidneys of mice after IRI treatment. Finally, Treatment with IL-33 inhibitor reduced proinflammatory cytokine and chemokine levels in the kidneys of mice following IRI insult. Taken together, our findings indicate that IL-33 signaling plays a critical role in the pathogenesis of IRI-induced renal fibrosis through regulating myeloid fibroblast accumulation, inflammation cell infiltration, and the expression of proinflammatory cytokines and chemokines.

Molecular Cloning, Recombinant Expression, and Funtional Characterization of APRIL (TNFSF13) in Cat (felis catus).

A proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn(124)), and two conservative cysteine residues (Cys(196) and Cys(211)). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species.

Sevoflurane suppresses hypoxia-induced growth and metastasis of lung cancer cells via inhibiting hypoxia-inducible factor-1α.

PURPOSE: Hypoxia promotes the progression of lung cancer cells. Unfortunately, anesthetic technique might aggravate hypoxia of lung cancer cells. Sevoflurane is a commonly used anesthetic. Its effect on hypoxia-induced aggressiveness of lung cancer cells remains unknown. The aim of the study is to investigate the effects of sevoflurane on hypoxia-induced growth and metastasis of lung cancer cells. As hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in mediating the adaptation and tolerance of cancer cells under hypoxic microenvironment, the role of HIF-1α in the effect of sevoflurane on hypoxia-induced growth and metastasis has also been elucidated. METHODS: A549 cells were treated with normoxia, hypoxia, co-treatment of sevoflurane and hypoxia, and dimethyloxaloylglycine (DMOG, a HIF-1α agonist) for 4 h, respectively. MTT assay and colony formation assay were used to evaluate cell growth. Transwell assay was performed to detect invasion and migration ability. The protein level of HIF-1α, X-linked inhibitor of apoptosis protein (XIAP), survivin, fascin, heparanase (HPA), and p38 MAPK were determined by Western blotting. RESULTS: Hypoxia enhanced proliferation and metastatic potential of cells. Sevoflurane could suppress hypoxia-induced growth and metastasis ability of cells. Furthermore, HIF-1α, XIAP, survivin, fascin and HPA were down-regulated significantly by the co-treatment of sevoflurane and hypoxia as compared to hypoxia treatment. DMOG abolished the inhibiting effects of sevoflurane on hypoxia-induced growth and metastasis ability of cells. In addition, sevoflurane partly reversed the increase of p38 MAPK activity that was induced by hypoxia. CONCLUSIONS: Sevoflurane could suppress hypoxia-induced growth and metastasis of lung cancer cells, which might be associated with modulating HIF-1α and its down-stream genes. Moreover, p38 MAPK signaling pathway was involved in the regulation of HIF-1α by sevoflurane.

Identification of interferon-γ-inducible-lysosomal thiol reductase (GILT) gene in goldfish (Carassius auratus) and its immune response to LPS challenge.

The interferon-γ-inducible lysosomal thiol reductase (GILT) has been demonstrated to play an important role in the processing and presentation of MHC class II restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, we cloned a GILT gene homolog from goldfish (designated gGILT), a kind of precious freshwater fish with high market value. The open reading frame of gGILT consists of 756 bases encoding a protein of 251 amino acids with an estimated molecular mass of 27.8 kDa and a theoretical isoelectric point of 5.24. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 10 conserved cysteines. RT-PCR results showed that gGILT and gIFN-γ (goldfish IFN-γ) mRNA were expressed in a tissue-specific manner and obviously up-regulated in splenocytes and the cells from head kidney after induction with LPS. Recombinant gGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Further study revealed that gGILT was capable of catalyzing the reduction of the interchain disulfide bonds from intact IgG. This study shows that gGILT may be involved in the immune response to bacteria challenge and maintain first line of innate immune defense at basal level in goldfish. It also provides the basis for investigating on the role of GILT using goldfish as an animal model.

Dynamic alterations of connexin43, matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 during ventricular fibrillation in canine.

The aim of this study is to investigate the dynamic alterations of cardiac connexin 43 (Cx43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF) duration. In this study, thirty-two dogs were randomly divided into sham control group, 8-min VF group, 12-min VF group, and 30-min VF group. Cx43 and phosphorylated Cx43 (p-Cx43) in tissues were detected by western blot and immunofluorescence analysis. MMP-2 and TIMP-2 were detected by western blot and immunohistochemistry analysis. The results showed that Cx43 levels in three VF groups were significantly decreased compared with sham control group. p-Cx43 levels in 12-min and 30-min VF groups were significantly reduced compared with sham control group. The ratio of p-Cx43/Cx43 was also decreased in VF groups. Compared with sham controls, no significant difference was observed between the sham control group and 8-min VF group in MMP-2 level, but MMP-2 level increased in 12-min and 30-min VF groups. The ratios of MMP-2/TIMP-2 were higher in VF groups, and were correlated with the duration of VF. A remarkable correlation was observed between the ratio of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P < 0.01). In conclusion, the alteration of Cx43 and/or p-Cx43 levels and the imbalance of MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate prognosis of VF.

The effects of combined treatment with sevoflurane and cisplatin on growth and invasion of human adenocarcinoma cell line A549.

Sevoflurane, an inhalational anesthetic, and cisplatin (DDP)-based chemotherapy have been widely used during lung cancer surgery. However, the effect of sevoflurane on the sensitivity of lung cancer cells to DDP chemotherapy remains unclear. In this study, the effects of combined treatment with sevoflurane and cisplatin on the growth and invasion of human lung adenocarcinoma A549 cell line have been investigated. The underlying mechanism has also been explored. In our experiment, A549 cells were treated with 2.5% sevoflurane, 10µmol/L DDP, or the co-treatment of sevoflurane and DDP for 4h, respectively. Cell proliferation was evaluated by the MTT assay and colony formation assay. Apoptosis was assessed by flow cytometry. Cell invasion was detected by Transwell assay. The expressions of X-linked inhibitor of apoptosis protein (XIAP), Survivin, matrix metalloproteinase (MMP)-2 and MMP-9 were determined by western blotting. Our results showed that sevoflurane combined with DDP resulted in a more pronounced inhibition of tumor cells growth and invasion as compared with either drug alone. Besides, XIAP, Survivin, MMP-2, and MMP-9 were downregulated more significantly by the co-treatment of the two drugs as compared to sevoflurane treatment or DDP treatment alone. Taken together, the growth-inhibitory and invasion-inhibitory synergy between sevoflurane and DDP in human adenocarcinoma A549 cell line was found in this study. Furthermore, we showed that the growth-inhibitory synergy between sevoflurane and DDP might be associated with the downregulation of XIAP and Survivin, and the invasion-inhibitory synergy between sevoflurane and DDP might be involved in the downregulation of MMP-2 and MMP-9.

WITHDRAWN: Propofol reduces MMPs expression by inhibiting PI3K/AKT activity in human HepG2 cells.

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Dynamic changes of cardiovascular regulating factors in rats after aerobic exhaustive exercise.

OBJECTIVE: To study the changes of cardiovascular regulating factors in rats during recovery of aerobic exhaustive exercise. METHODS: Sixty male Wistar rats were randomly divided into control group, 1 h-exercise group, 3 h-exercise group, exhausted group, 2 h-recovery group and 12 h-recovery group. The rats were killed at corresponding times for each group after an 8-week-long treadmill training, and the levels of NO, ET, ANP and TXB2 in plasma were measured in each group. RESULTS: NO/ET ratio of 1 h-exercise group was significantly higher than that in control group (P < 0.01), while it was significantly decreased in 3 h-exercise group and exhausted group (P < 0.05). ANP contents in rat plasma were significantly higher in 3 h-exercise group, exhausted group and 2 h-recovery group than that in control group (P < 0.05 or P < 0.01). The concentration of TXB2 in plasma was significantly increased in 3 h-exercise group, exhausted group and 2 h-recovery group (P < 0.05). CONCLUSION: Changes in cardiovascular regulating factors after exhaustive exercise may lead to deficiency of coronary circulation blood/oxygen supply, which may cause exercise-induced fatigue.

Isolation and characterization of LIGHT (TNFSF14) gene homologue in zebrafish (Danio rerio).

The tumor necrosis factor superfamily (TNFSF) proteins are cytokines involved in many biological processes. In this study, the TNF superfamily member 14 (TNFSF14, LIGHT) has been isolated from zebrafish Danio rerio (designated zLIGHT). The full-length open reading frame (ORF) of zLIGHT cDNA consists of 708 bp encoding a protein of 235 amino acids. The zLIGHT open reading frame (ORF) genomic sequence consists of three introns and four exons, is about 9.9 kb in size. Real-time quantitative PCR (qPCR) analysis suggested that zLIGHT was predominantly expressed in zebrafish spleen. The soluble LIGHT (zsLIGHT) had been cloned into the pSUMO vector, SDS-PAGE and Western blotting analysis confirmed that the recombinant protein SUMO-zsLIGHT was efficiently expressed in Escherichia coli BL21 (DE3). Laser scanning confocal microscopy analysis showed that SUMO-zsLIGHT could bind to its receptors on T cells. LIGHT is involved in many important biological effects, including up-regulating proinflammatory chemokines, cytokines, inducing cell death, apoptosis, and enhancing T cell survival. Zebrafish may conduct as a model animal for further research on LIGHT.

[Different strength intermittent treadmill training of growth period rats and related bone metabolism of the hormone influence].

OBJECTIVE: To explore the influence of different strength intermittent treadmill training of growth period rats on the bone metabolism, so as to provide the training intensity of teenagers to set theory support. METHODS: Select 70 male four weeks Wistar rats according to body weight randomly divided into seven groups (n = 10): the control group and the exercise group. According to the VO2max the exercise group was divided into 6 groups: 65%, 70%, 75%, 80%, 85% and 90% group. Nine weeks treadmill training, training six days a week, each group of training three times, each time not less than 10min, the interval was 30 min. The last movement after 24 h, took the femur and blood to measured the bone mineral density (BMD), bone mass (BMC) and alkaline phosphatase (AKP), resist tartaric acid acidic phosphatase (Str-ACP). RESULTS: 1. The femoral BMD (0.1393 +/- 0.0031), BMC (0.4525 +/- 0.0335) of 70% group were significantly higher than those in the control group (BMD: 0.1200 +/- 0.0095, BMC: 0.3238 +/- 0.0485) and the other sports group (65% BMD:0.1339 +/- 0.0062, BMC: 0.4058 +/- 0.0492, 75% BMD: 0.1296 +/- 0.0015, BMC: 0.3869 +/- 0.0254, 80% BMD: 0.1223 +/- 0.0082, BMC: 0.3454 +/- 0.0483, 85% BMD: 0.1250 +/- 0.0044, BMC: 0.3731 +/- 0.0381, 90% BMD: 0.1171 +/- 0.0047, BMC: 0.3051 +/- 0.0286) (P < 0.05), the femoral BMD, BMC of 90% group were lower than those of the control group, the other in the exercise group were higher than those in the control group; 2. Serum AKP in the exercise group were significantly higher than those in the control group, the group of 65% (41.015 +/- 2.114), 70% (46.035 +/- 2.611), 75% (43.834 +/- 3.155), and 80% (38.043 +/- 4.073) were very significantly higher than those in the control group (26.875 +/- 1.188) (P < 0.01); 70% group and 75% group were significantly higher than those in the 80% group , 85% group and 90% group, while 70% group serum AKP level were significantly higher than those in 65% group (P < 0.05), it showed that 70% of the VO2 max training intensity of osteoblasts was most active. The serum Str-ACP of exercise group were significantly higher than those in the control group, along with the increase of the training intensity, serum Str-ACP level was rising and the group of 80% (22.430 +/- 1.591), 85% (23.990 +/- 1.870), and 90% (28.009 +/- 1.839) serum of Str-ACP were significantly higher than those in the group of 65% (18.503 +/- 2.429), 70% (16.447 +/- 2.120) and 75%(17.769 +/- 1.642) ( P < 0.05), the group of 90% serum Str-ACP were significantly higher than those in the group of 80% and 85% (P < 0.05). CONCLUSION: The training of 70% of the VO2max, moderate intensity intermittent running, make the growth period rat bone mass and bone mineral density to increase obviously.

[Effects of the compound extract of Chinese medicine on free radical metabolism of the rat brain in different states].

OBJECTIVE: To investigate the effect of the "compound extract of Chinese medicine" on free radical metabolism and antioxidant enzyme systems of the rat brain. METHODS: 70 Wistar mice were randomly divided into two groups (n = 35): normal control group (N), taking medicine group (M). After a week of feeding, M group was taking medicine for 8 weeks. After 9 weeks, killed the two groups when they were at the rest state, the immediate ends of the fixed load, the immediate ends of exhaustive exercise and 12, 24 hour ends of exhaustive exercise, respectively. And the activity of malonaldehyde (MDA), glutathione peroxidase (GSH-PX), glutathione (GSH), superoxide dismutase (SOD), total anti oxidation capacity (T-AOC) of the rat brain were measured. RESULTS: At the five states, the content of MDA in M group was lower than that in N group in different degree, the activity of GSH-PX, GSH, SOD, T-AOC in M group were higher than those in N group in different degree. CONCLUSION: The "Compound extract of Chinese medicine" can reduce the MDA level of the rat brain and improve the enzyme activity of GSH-PX, GSH, SOD, T-AOC.

ZP123 reduces energy required for defibrillation by preventing connexin43 remodeling during prolonged ventricular fibrillation in swine.

In ventricular fibrillation, the uncoupling of gap junctions slows conduction velocity and increases action-potential dispersion, which slows and diminishes defibrillation. We studied how the peptide ZP123, a gap-junction enhancer, might lower defibrillation-energy requirements during ventricular fibrillation in live pigs. We randomly assigned 33 pigs into 3 groups: ZP123 (receiving a 1-µg/kg bolus and 10 µg/kg/hr of ZP123), control (receiving saline solution), and sham (undergoing a sham operation). After a 30-min administration of agents, ventricular fibrillation was induced and left untreated for 8 min. Biphasic defibrillation of 50 J was increased by 50-J increments as necessary. Defibrillation-energy requirements were defined as the lowest energy required to achieve defibrillation. Electrocardiographic values were obtained before and after the administration of agents. Western blot and immunofluorescence analyses were performed on ventricular myocardial samples. All but one pig survived. The ZP123 treatment did not alter electrocardiographic variables. In the ZP123 group, the average required defibrillation energy was lower than that in the control group (327.28±269.6 vs 610±192.64 J; P=0.015), and the cumulative percentage of successful defibrillation at upper energy levels was higher (P<0.05). Supraventricular rhythm occurred more often in the ZP123 group than in the control group (72.7% vs 50%, P=0.042). Western-blot and immunofluorescence results showed that ZP123 did not alter the total amount of connexin43 but did prevent its dephosphorylation. We conclude that ZP123 can reduce defibrillation-energy requirements by preventing connexin43 remodeling during prolonged ventricular fibrillation.

Imbalance between tissue inhibitor of metalloproteinase 1 and matrix metalloproteinase 9 after cardiopulmonary resuscitation.

AIMS: This study aimed to determine whether (a) there was an imbalance between matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) after cardiopulmonary resuscitation (CPR) in a canine model of prolonged ventricular fibrillation (VF); (b) with the duration of VF, the degree of the imbalance would be greater; and (c) there was a relationship between the level of MMP-9 or TIMP-1 and the cardiac function. METHODS AND RESULTS: Ventricular fibrillation was electrically induced in 24 dogs. The animals were randomly divided into 3 groups (sham control, n = 8; 8-minute VF, n = 8; 12-minute VF, n = 8). Echocardiographic measurement and hemodynamic variables were recorded before VF and after return of spontaneous circulation. Tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-9 were analyzed by Western blot and immunohistochemistry. Compared with sham controls, dogs under VF and CPR showed significantly decreased level of TIMP-1 (P < .001), and with the duration of VF, the level of TIMP-1 declined (P < .01). The level of MMP-9 did not achieve statistical significance in the 3 groups (P > .05); however, they were higher in VF and longer duration VF groups. The ratios of TIMP-1/MMP-9 were lower in VF groups (P < .05). There was a negative correlation between TIMP-1 and left atrium dimension and left ventricular diastolic dimensions (r = -0.83 and r = -0.96, respectively; P < .01) and a positive correlation between TIMP-1 and left ventricular ejection fraction (r = 0.85; P < .01). CONCLUSIONS: There was an imbalance between TIMP-1 and MMP-9 after CPR. It may partly contribute to the postresuscitation cardiac dysfunction.

Effect of ZP123, a gap junction modifier, on prolonged ventricular fibrillation in swine.

OBJECTIVES: It was the aim of this study to investigate the effect of ZP123 on prolonged ventricular fibrillation (VF) in swine. METHODS: VF was electrically induced in 20 pigs. The animals randomly received either ZP123 or saline control infusion before VF. After 8 min of untreated VF, cardiopulmonary resuscitation and biphasic defibrillation shocks were applied. VF mean frequency (VF(mf)) and mean amplitude (VF(ma)), hemodynamics, outcome of defibrillation and the rate of return of spontaneous circulation (ROSC) were analyzed. RESULTS: Compared with the control group, VF(mf) was higher but VF(ma) lower during the 8 min of VF in the drug group (11.8 ± 2.1 vs. 10.4 ± 2.0 Hz and 0.24 ± 0.10 vs. 0.31 ± 0.16 mV, respectively; p < 0.05). Hemodynamic variables in the 2 groups were comparable (p > 0.05). The defibrillation threshold was lower and the rate of successful defibrillation was higher in the drug group compared with the control group (92.2 ± 26.4 vs. 133.3 ± 28.9 J and 90 vs. 30%, respectively; p < 0.05). The rate of ROSC was not different between the 2 groups (40 vs. 30%; p > 0.05). CONCLUSION: In prolonged VF, ZP123 could decrease the defibrillation threshold and improve the rate of successful defibrillation. However, it could not improve the rate of ROSC - which may be due to its side effect of decreasing VF(ma).