Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function. Renal ischemia-reperfusion injury (RIRI) is one of the main causes of AKI with the underlying mechanism incompletely clarified. The liver X receptors (LXRs), including LXRα and LXRβ, are members of the nuclear receptor superfamily. It has been shown that LXRs play an important role in regulating glucose and lipid metabolism, cholesterol efflux, and inflammation. The purpose of this study was to explore the role and mechanism of LXRs in RIRI. We determined the effects of LXR activation on renal function and histological changes in a mouse RIRI model and a cellular model of hypoxia/reoxygenation (H/R). In vivo results showed that LXRs agonist GW3965 significantly inhibited the increase of serum creatinine and urea nitrogen levels induced by RIRI. Both HE and PAS staining of kidney tissues revealed that GW3965 alleviated the morphological damages caused by RIRI. Immunohistochemical staining showed that GW3965 mitigated 4-HNE and GRP78 levels induced by RIRI. Furthermore, TUNEL assay indicated that GW3965 reduced RIRI-induced renal cell apoptosis. Quantitative real-time PCR (qPCR) analysis revealed that GW3965 attenuated RIRI-induced IL-6 and IL-1β mRNA expression. Compared with wild-type group, LXRα gene deficiency had little effect on RIRI-associated renal functional decline and morphological damages. Additionally, in vitro study demonstrated that GW3965 alleviated H/R-induced decrease of HK-2 human renal proximal tubule cell viability and restored the activity of superoxide dismutase (SOD) after H/R. Western blot results showed that GW3965 mitigated the increase of 4-HNE and GRP78 protein expression levels after H/R; However, knockdown of LXRβ using the small interfering RNA (siRNA) technique reduced cell viability compared to GW3965-treated group. Taken together, the LXRs agonist GW3965 significantly alleviates RIRI in mice possibly by reducing apoptosis, oxidative stress, endoplasmic reticulum stress and inflammation. These results also preliminarily confirm that the renal protective effects of LXRs agonists are dependent on LXRβ.
Animals ; Liver X Receptors/genetics* ; Reperfusion Injury/prevention & control* ; Mice ; Benzoates/pharmacology* ; Benzylamines/pharmacology* ; Male ; Endoplasmic Reticulum Chaperone BiP ; Mice, Inbred C57BL ; Apoptosis ; Acute Kidney Injury/prevention & control* ; Kidney/pathology* ; Humans

OBJECTIVE: To examine the expression of liver X receptor-β (LXR-β) in human gastric cancer tissue, and to explore the effect of GW3965, an agonist of LXRs, on proliferation of gastric cancer cell line SGC-7901.
 METHODS: The immunohistochemical assay was used to detect the expression of LXR-β, activating transcription factor 4 (ATF4) in gastric cancer tissues and the corresponding pericarcinoma tissues in 114 patients. Real-time quantitative PCR and Western blot were used to determine mRNA and protein levels of ATF4 and ATP-binding cassette 1 (ABCA1), one of the downstream target genes of LXRs, in SGC-7901 cells with or without GW3965 treatment. Cell counting kit-8 (CCK-8) assay was performed to detect cell proliferation. The expression of ATF4 was silenced by short hairpin RNA (shRNA).
 RESULTS: The expressions of LXR-β and ATF-4 were obviously down-regulated in the gastric cancer tissues than that in the corresponding pericarcinoma tissues (both P<0.05). Compared with the control cells, GW3965 treatment inhibited proliferation of SGC-7901 cells and up-regulated ATF4 and ABCA1 expressions (both P<0.05). Knockdown of ATF4 can reverse the antiproliferative effect of GW3965 on SGC-7901 cells.
 CONCLUSION The expression of LXR-β is decreased in human gastric cancer tissues, and activation of LXRs by GW3965 could inhibit the proliferation of SGC-7901 cells via ATF4.
Activating Transcription Factor 4 ; genetics ; metabolism ; Benzoates ; pharmacology ; Benzylamines ; pharmacology ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Liver X Receptors ; Orphan Nuclear Receptors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Stomach Neoplasms ; pathology ; Up-Regulation

Abnormal enhanced transmural dispersion of repolarization (TDR) plays an important role in the maintaining of the severe ventricular arrhythmias such as torsades de pointes (TDP) which can be induced in long-QT (LQT) syndrome. Taking advantage of an in vitro rabbit model of LQT2, we detected the effects of KN-93, a CaM-dependent kinase (CaMK) II inhibitor on repolarization heterogeneity of ventricular myocardium. Using the monophasic action potential recording technique, the action potentials of epicardium and endocardium were recorded in rabbit cardiac wedge infused with hypokalemic, hypomagnesaemic Tyrode's solution. At a basic length (BCL) of 2000 ms, LQT2 model was successfully mimicked with the perfusion of 0.5 μmol/L E-4031, QT intervals and the interval from the peak of T wave to the end of T wave (Tp-e) were prolonged, and Tp-e/QT increased. Besides, TDR was increased and the occurrence rate of arrhythmias like EAD, R-on-T extrasystole, and TDP increased under the above condition. Pretreatment with KN-93 (0.5 μmol/L) could inhibit EAD, R-on-T extrasystole, and TDP induced by E-4031 without affecting QT interval, Tp-e, and Tp-e/QT. This study demonstrated KN-93, a CaMKII inhibitor, can inhibit EADs which are the triggers of TDP, resulting in the suppression of TDP induced by LQT2 without affecting TDR.
Action Potentials ; drug effects ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Arrhythmias, Cardiac ; etiology ; physiopathology ; prevention & control ; Benzylamines ; pharmacology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; antagonists & inhibitors ; metabolism ; Electrocardiography ; Electrophysiologic Techniques, Cardiac ; Endocardium ; drug effects ; physiopathology ; Heart ; drug effects ; physiopathology ; In Vitro Techniques ; Long QT Syndrome ; complications ; Pericardium ; drug effects ; physiopathology ; Piperidines ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Pyridines ; pharmacology ; Rabbits ; Sulfonamides ; pharmacology ; Torsades de Pointes ; etiology ; physiopathology ; prevention & control

Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related. The aim of this study was to determine the effects of the calmodulin-dependent protein kinase (CaMK) II inhibitor, KN-93, on L-type calcium current (I(Ca, L)) and early after-depolarizations (EADs) in hypertrophic cardiomyocytes. A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation (LVH group). The control group (sham group) received a sham operation, in which the abdominal aortic was dissected but not coarcted. Eight weeks later, the degree of left ventricular hypertrophy (LVH) was evaluated using echocardiography. Individual cardiomyocyte was isolated through collagenase digestion. Action potentials (APs) and I(Ca, L) were recorded using the perforated patch clamp technique. APs were recorded under current clamp conditions and I(Ca, L) was recorded under voltage clamp conditions. The incidence of EADs and I(ca, L) in the hypertrophic cardiomyocytes were observed under the conditions of low potassium (2 mmol/L), low magnesium (0.25 mmol/L) Tyrode's solution perfusion, and slow frequency (0.25-0.5 Hz) electrical stimulation. The incidence of EADs and I(ca, L) in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92 (KN-92 group) and KN-93 (KN-93 group). Eight weeks later, the model was successfully established. Under the conditions of low potassium, low magnesium Tyrode's solution perfusion, and slow frequency electrical stimulation, the incidence of EADs was 0/12, 11/12, 10/12, and 5/12 in sham group, LVH group, KN-92 group (0.5 μmol/L), and KN-93 group (0.5 μmol/L), respectively. When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group, the incidence of EADs was 10/12 and 2/12, respectively. At 0 mV, the current density was 6.7±1.0 and 6.3±0.7 PA·PF(-1) in LVH group and sham group, respectively (P>0.05, n=12). When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups, the peak I(Ca, L) at 0 mV was decreased by (9.4±2.8)% and (10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups, respectively (P>0.05, n=12). When the drug concentration was increased to 1 μmol/L, the peak I(Ca, L) values were lowered by (13.4±3.7)% and (40±4.9)%, respectively (P<0.01, n=12). KN-93, a specific inhibitor of CaMKII, can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I(Ca, L), which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.
Animals ; Benzylamines ; pharmacology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; antagonists & inhibitors ; metabolism ; Cardiomegaly ; metabolism ; physiopathology ; Female ; Myocytes, Cardiac ; drug effects ; metabolism ; physiology ; Rabbits ; Sulfonamides ; pharmacology

OBJECTIVETo explore the efficacy of inductible nitric oxide synthase (iNOS) inhibitor 1400W in vivo in blocking the death pathway of lipopolysaccharide (LPS)-induced activated-microglia to preoligodendrocytes (preOLs) in neonatal rats with infective-type periventricular leukomalacia (PVL) induced by LPS.

METHODSTwo-day-old neonatal rats were randomly divided into: a sham-operated group, an untreated PVL group, and four 1400W-treated PVL groups that were subcutaneously administrated with 20 mg/kg of 1400W at 0 h, 8 hrs, 16 hrs, and 24 hrs after LPS induction, respectively. The brain specimens were obtained 5 days after LPS induction. The pathological assessment of cerebral white matter was performed under a light microscope. Concentrations of nitric oxide (NO) were measured by nitric acid-deoxidize colorimetry. Synthesis of iNOS was determined by Western blot analysis. Peroxynitrite (ONOO(-)) level and the amount of preOLs were determined by immunocytochemistry. RETHODS: The obvious injuries of periventricular white matter, massive loss of positive O4-labelled preOLs, and increased levels of NO, ONOO(-) and iNOS were observed in neonatal rats with PVL. Compared to the untreated PVL group, the use of 1400W at 0 h, 8 hrs and 16 hrs after LPS induction significantly improved white matter injuries, reduced the levels of NO, ONOO(-) and iNOS, and increased the amount of O4-labelled preOLs. However, the use of 1400W at 24 hrs after LPS induction did not result in the improvements.

CONCLUSIONSiNOS inhibitor 1400W can effectively block the toxicity of LPS-activated microglia to preOLs and protect cerebral white matter through inhibiting iNOS and reducing the production of NO and ONOO(-). The use of 1400W within 16 hrs after LPS induction may provide cerebral protections in neonatal rats with PVL.

Amidines ; pharmacology ; Animals ; Apoptosis ; drug effects ; Benzylamines ; pharmacology ; Brain ; drug effects ; pathology ; Enzyme Inhibitors ; pharmacology ; Lipopolysaccharides ; toxicity ; Microglia ; cytology ; drug effects ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Oligodendroglia ; cytology ; Peroxynitrous Acid ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

OBJECTIVETo explore the toxicity of LPS-induced activated microglia to preoligodendrocytes (preOLs) and the effect of 1400W, a selective inhibitor of inducible nitric oxide synthetase (iNOS), on the blockage of the toxicity.

METHODSCo-cultured microglia and preOLs obtained from two-day-old Sprague-Dawley (SD) rats were divided into three groups: co-culture control group, co-culture LPS group and co-culture LPS plus 1400W group. After cultured cells were induced by LPS (100 ng/ml) for 48 hours, the concentration of nitric oxide (NO) was measured by nitric acid-oeoxidize-colorimetry, the level of peroxynitrite (ONOO(-)) was determined by immunocytochemistry, and the synthetic level of iNOS was detected by Western blotting, respectively. The morphologic observation of apoptotic preOLs stained with Hoechst 33342/PI and the apoptotic rate of preOLs detected by flow cytometry were processed simultaneously. Data were analyzed with SPSS 11.0 software.

RESULTSCompared to co-culture control group, there was significant increase in levels of NO [(82.27+/-3.41) micromol/L vs. (167.86+/-9.87) micromol/L, t=8.593, P<0.01], ONOO(-)[(6.14+/-1.27) x 10(7)/L vs. (34.38+/-7.75) x 10(7)/L, t=5.892, P<0.01], and iNOS [(0.18+/-0.027) vs. (0.79+/-0.068), t=9.26, P<0.01] induced by LPS in co-culture LPS group, and with a higher apoptotic rate of preOLs [(6.73+/-1.39)% vs. (24.77+/-2.05)%, t=12.619, P<0.01]. However, all levels of NO [(69.55+/-5.07) micromol/L, t=8.896, P<0.01], ONOO(-) [(10.33+/-3.47) x 10(7)/L, t=14.96, P<0.01] and iNOS (0.35+/-0.042, t=5.506, P<0.01) decreased significantly with the use of 1400W at a dose of 10 micromol/L in co-culture LPS plus 1400W group, and the apoptotic rate of preOLs [(11.8+/-2.06)%, t=7.715, P<0.01] was also reduced evidently.

CONCLUSIONSNO, ONOO(-) and iNOS, etc. play important roles in the death pathway of preOLs induced by LPS. 1400W can block effectively the toxicity of LPS-activated microglia toxicity to preOLs through inhibiting iNOS selectively and reducing the production of NO and ONOO(-), and improve the survival rate of preOLs.

Amidines ; pharmacology ; Animals ; Benzylamines ; pharmacology ; Cells, Cultured ; Lipopolysaccharides ; toxicity ; Microglia ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; antagonists & inhibitors ; metabolism ; Oligodendroglia ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of KN-93, a calmodulin kinase II inhibitor, on ventricular arrhythmias in rabbits with cardiac hypertrophy.

METHODSFemale New Zealand white rabbits were randomly divided into four groups (n = 10 each): Sham; LVH; LVH + KN-92 and LVH + KN-93 group. LVH was induced by partially constricting the abdominal aorta. In Sham group, the abdominal aorta was exposed without constriction. Eight weeks later, the arterially perfused left ventricular wedge preparations were made and transmembrane action potentials (TAP) from epicardium and endocardium and transmural ECG were simultaneously recorded. Incidence of early after depolarization (EAD) and torsade de pointes (Tdp), QT interval, action potential duration (APD) and transmural depolarization dispersion (TDR) at different cycle lengths were observed under slow stimulation (2000 - 4000 ms), hypokalemic (2 mmol/L) and hypomagnesaemic (0.25 mmol/L) Tyrode's solution perfusion.

RESULTSLeft ventricular hypertrophy was detected in LVH group by echocardiography and not affected by KN-92 and KN-93. Perfused with hypokalemic, hypomagnesaemic Tyrode's solution and under slow stimulation (2000 - 4000 ms), the incidences of EAD and Tdp in Sham group, LVH group, LVH + KN-92 group (0.5 micromol/L) and LVH + KN-93 group (0.5 micromol/L) were 0/10, 10/10, 9/10, 5/10 and 0/10, 5/10, 4/10, 1/10, respectively. With 1 micromol/L KN-92 and KN-93, the incidences of EAD and Tdp in LVH + KN-92 and LVH + KN-93 group were 9/10, 3/10 and 4/10, 1/10 respectively. The QT interval, APD and TDR were not affected by KN-93.

CONCLUSIONThe calmodulin kinase II inhibitor KN-93 can effectively suppress ventricular arrhythmias in rabbits with cardiac hypertrophy by decreasing EAD.

Animals ; Arrhythmias, Cardiac ; complications ; drug therapy ; Benzylamines ; pharmacology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; antagonists & inhibitors ; Cardiomegaly ; complications ; drug therapy ; Disease Models, Animal ; Male ; Protein Kinase Inhibitors ; pharmacology ; Rabbits ; Sulfonamides ; pharmacology

AIMTo search for some substituted alpha-amino phosphonates as leading compounds with the vasodilator effects.

METHODSTarget compounds were prepared from benzyl aldehyde, piperazine and diethyl phosphite using alcohol as solvent via Mannich-type reaction. In isolated rat aorta and in isolated guinea pig ileum, the vasodilator effects of compounds were investigated and evaluated whether they activated muscarine receptor.

RESULTSSeven compounds of substituted alpha-amino phosphonates have been synthesized and identified by IR, 1H NMR and elemental analysis. Three of them, compound 2a, 2b and 2c have vasodilator activity and do not activate M receptor.

CONCLUSIONTwo (2b and 2c) of them were found to have the notable vasodilator effect, and the rates of relaxing are (67 +/- 21) % and (82 +/- 18)%, separately. But they did not activate M receptors on ileum.

Animals ; Aorta ; drug effects ; Benzylamines ; chemical synthesis ; chemistry ; pharmacology ; Guinea Pigs ; Ileum ; drug effects ; Molecular Structure ; Muscle Contraction ; drug effects ; Organophosphonates ; chemical synthesis ; chemistry ; pharmacology ; Rats ; Vasodilation ; drug effects ; Vasodilator Agents ; chemical synthesis ; chemistry ; pharmacology

AIMTo investigate the influence and the mechanism of SK on the contractility of cultured cardiomyocytes of rats.

METHODSThe primary cultured single myocardial cell was treated with SK and the contraction frequency and size of cardiomyocyte were determined by a computer image analysis system. At the same time the effects of propranolol (a beta receptor antagonist), phentolamine (a alpha receptor antagonist), DSP (a tachykinin receptor antagonist) on the action of SK were investigated.

RESULTSSK increased contractive extend of the cardiomyocyte, in which a dose-response relationship of SK at 1.78 x 10(-8) - 1.78 x 10(-5) mol/L exists. But the frequency of contraction did not change, pretreatment with propranolol, phentolamine had no action on the effect of SK, but DSP markedly attenuated the effects of SK.

CONCLUSIONSK may directly enhance the contractility of single cardiomyocyte, which may be related with the tachykinin receptor.

Animals ; Animals, Newborn ; Benzylamines ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Myocardial Contraction ; drug effects ; Myocytes, Cardiac ; drug effects ; physiology ; Neurokinin A ; pharmacology ; Phentolamine ; pharmacology ; Propranolol ; pharmacology ; Rats ; Rats, Wistar ; Receptors, Tachykinin ; antagonists & inhibitors