Attenuation of Remifentanil-Induced Hyperalgesia by Betulinic Acid Associates with Inhibiting Oxidative Stress and Inflammation in Spinal Dorsal Horn.

Remifentanil-induced hyperalgesia (RIH) is known to be associated with oxidative stress and inflammation. Betulinic acid (BA) was reported to reduce visceral pain owing to its anti-oxidative and anti-inflammatory potential. Here, we -explored whether BA can attenuate RIH through inhibiting oxidative stress and inflammation in spinal dorsal horn. Sprague-Dawley rats were randomly divided into 4 groups: Control, Incision, RIH, and RIH pre-treated with BA. After pretreated with BA (25 mg/kg, i.g.) for 7 days, rats were subcutaneously infused with remifentanil (40 µg/kg) for 30 min during right plantar incision surgery to induce RIH. The paw withdrawal mechanical threshold (PWMT), paw withdrawal thermal latency (PWTL), spinal oxidative stress and inflammatory mediators were determined. Intraoperative remifentanil infusion induced postoperative hyperalgesia, as evidenced by the significant decrease in PWMT and PWTL (p < 0.01), and the significant increase in oxidative stress and inflammation evidenced by up-regulations of malondialdehyde, 3-nitrotyrosine, interleukin-1ß and tumour necrosis factor-α (p < 0.01) in spinal dorsal horn and matrix metalloproteinase-9 (MMP-9) activity (p < 0.01) in dorsal root ganglion, as well as a decrease in manganese superoxide -dismutase activity (p < 0.01) compared with control and -incision groups. All these results mentioned above were markedly reversed by pre-treatment with BA (p < 0.01) compared with RIH group. These findings demonstrated that BA can effectively attenuate RIH, which associates with potentially inhibiting oxidative stress and subsequently down-regulating MMP-9-related pro-inflammatory cyokines in spinal dorsal horn.

[Effect of AT1 receptor on changes of tyrosine hydroxylase-immunoreactivity in rostral ventrolateral medulla induced by brain cholinergic stimuli in rats].

OBJECTIVE: To investigate the effect of AT1 receptor on the changes of tyrosine hydroxylase-immunoreactivity (TH-IR) in rostral ventrolateral medulla (RVLM) induced by brain cholinergic stimuli in rats. METHODS: Male SD rats were randomly divided into 4 groups: NS + CBC group, Los + CBC group, Los + NS group and NS + NS group. AT1 was blocked by pretreatment of 20 µg losartan in Los + CBC and Los + NS groups; intracerebroventricular injection of 0.5 µg carbachol was used for cholinergic stimuli in NS + CBC and Los + CBC groups; normal saline (NS) was used for control. The output amount of natrium in kidney, glomerular filtration rate (GFR) and renal plasma flow (PRF) were observed. The changes of TH-IR in the RVLM were observed by immunohistochemistry. RESULT: In NS + CBC group carbachol induced potent natriuresis, after pretreatment of losartan the natriuretic effect was partially inhibited in Los + CBC group. Both the number and optical density of TH-IR positive neurons in NS + CBC group were markedly increased than those in NS + NS group (P < 0.05); while those in Los + CBC group were significantly lower than those in NS+CBC group (P < 0.05). Intracerebroventricular injection of carbachol and losartan had no effect on GFR and RPF(P > 0.05). CONCLUSION: The results suggest that cholinergic stimuli can induce potent natriuresis and increase the activity of adrenergic neurons in the RVLM; the above effects can be down regulated by blockade of brain AT1 receptor.

[Activation of mitochondrial aldehyde dehydrogenase 2 and inhibition of mitochondrial permeability transition pore involved in cardioprotection of ethanol postconditioning].

OBJECTIVE: To investigate whether activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) and inhibition of mitochondrial permeability transition pore (mitoPTP) were involved in the cardioprotection of ethanol postconditioning in isolated rat heart. METHODS: Hearts isolated from male Sprague-Dawley rats were perfused on a langendorff apparatus and subjected to 30 min of regional ischemia (occlusion of left anterior descending artery) followed by 120 min of reperfusion. The ventricular hemodynamic parameters and lactate dehydrogenase (LDH) release during reperfusion were measured. Infarct size was measured by TTC staining method and the expression of ALDH2 at mRNA level of left anterior myocardium was detected by RT-PCR. RESULT: In contrast to ischemia and reperfusion, ethanol postconditioning improved the recovery of left ventricular developed pressure, maximal rise/fall rate of left ventricular pressure during reperfusion, reduced LDH release and infarct size. The expression of ALDH2 mRNA level was increased. Administration of mitoPTP activator atractyloside attenuated the effect of ethanol postconditioning, LDH release and infarct size were increased, and the recovery of hemodynamic parameters was inhibited. The expression of ALDH2 mRNA was decreased. CONCLUSION: Ethanol postconditioning has cardioprotection effect, which may be associated with upregulating mitochondrial ALDH2 mRNA expression and inhibiting the opening of mitochondrial permeability transition pore.

[Hemodynamics and aortic tension induced by two septic shock models in rats].

OBJECTIVE: To compare the ventricular-dynamic parameters and thoracic aorta tension induced by two septic shock models in rats. METHODS: Septic shock models were induced by cecal ligation or puncture (CLP) and intraperitoneal injection of lipopolysaccharide (LPS) in rats. The carotid artery was cannulated and connected to a pressure transducer to determine mean arterial blood pressure (MABP). Ventricular dynamic parameters, including heart rate (HR), left ventricular developed pressure (LVDP) and maximal rise/fall velocity of ventricular pressure (± dP/dtmax) were determined. Isolated thoracic rings were mounted on an organ bath and the tension of the vessel was recorded. RESULT: The mortality was 65.2% in CLP shock rats, but no death in LPS shock rats. The MABP and HR of CLP rats were decreased more prominently than those of LPS rats (P < 0.01). Contraction induced by high K(+) (60 mmol/L) or 10⁻6 mol/L phenylephrine (PE) in endothelium-intact and endothelium-denuded aortic rings was all attenuated, but in LPS rats it was more prominent (P < 0.01). CONCLUSION: Two rat septic shock models can decrease ventricular-dynamic parameters and vasoconstriction responsiveness of aorta. The ventricular-dynamic parameters decrease more prominently in CLP model, while vasoconstriction responsiveness of aorta changes more in LPS model.

[Betulinic acid ameliorates impairment of endothelium-dependent relaxation induced by oxidative stress in rat aorta].

OBJECTIVE: To investigate the effect of betulinic acid (BA) on relaxation in isolated rat aortic rings and its antioxidant property on oxidative stress of blood vessels. METHODS: Aortic rings were isolated and BA was cumulatively added into organ bath. Isometric tension of endothelium intact or endothelium denuded thoracic aortic rings previously contracted by phenylephrine (PE) was recorded. Then aortic rings were randomly divided into normal control group, BA control group, H(2)O(2) group and BA+H(2)O(2) group, after being previously contracted by PE, isometric tension of endothelium-dependent relaxation induced by Ach was recorded. RESULT: Exposure of intact endothelium rings previously contracted by PE to BA at the concentrations of 10(-7) mol/L-10(-4) mol/L evoked a significant concentration dependent relaxation, which was inhibited by pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4)mol/L), but not by indometacin (10(-5)mol/L). The pD2 value of BA was 5.24 ± 0.04, and the EC(50)value was 2.45 x 10(-6)mol/L. Exposure of endothelium denuded rings previously contracted by PE to BA did not affect the relaxation in isolated aortic rings. ACh induced a dose-dependent relaxation that was weakened by pretreatment with H(2)O(2) (5 10(-4) mol/L) for 15 min. The EC(50) of BA markedly attenuated the inhibition of relaxation induced by H(2)O(2). CONCLUSION: BA can evoke a concentration-dependent relaxation in aortic rings previously contracted by PE, which may be mediated by NO. And the decrease of endothelium-dependent relaxation in rat aortic rings exposed to H(2)O(2) can be markedly attenuated by BA, which may be mediated by reducing oxidative stress and maintaining the activity of NO in aortic rings.

Chemical constituents from the deep sea-derived Streptomyces xiamenensis MCCC 1A01570 and their effects on RXRα transcriptional regulation.

From the deep sea-derived Streptomyces xiamenensis MCCC 1A01570, eight cyclic dipeptides (1-8) and five phenolics (9-13) were obtained. Cyclo-(I-Pro-D-Leu) (4) could moderately promote the gene transcriptional function of nuclear receptor RXRα, while 2, 3, and 13 showed weak reduction in RXRα gene transcriptional activities induced by 9-cis-retinoid acid (RA). These compounds might have beneficial effects against intractable diseases with relation to RXRα, such as cancer and metabolic diseases, due to their potential activities on regulating the transcriptional activation function of RXRα. In addition, 1-6, 8, 10, and 12 (20 µM) showed mild in vitro cytotoxicity against three cancer cell lines of ECA-109, Hela-S3 and PANC-1 with the inhibition rates arranging from 50% to 65%.

Andrastone A From the Deep-Sea-Derived Fungus Penicillium allii-sativi Acts as an Inducer of Caspase and RXRα-Dependent Apoptosis.

Two new (1, 2) and one known (3) meroterpenoids were isolated from the deep-sea-derived fungus Penicillium allii-sativi. The relative structures of new compounds were determined on the basis of an extensive analysis of the NMR and MS data, and the absolute configurations were established by ECD calculations. Andrastone A (1) is a rare andrastin bearing an unusual cyclopentan-1,3-dione. It shows a selectively antiproliferative effect against HepG2 tumor cells with an IC50 value of 7.8 µM. Mechanism study showed that apoptosis via Caspase and RXRα pathways are responsible for the inhibitory effect.

Luteolin Attenuates Cardiac Ischemia/Reperfusion Injury in Diabetic Rats by Modulating Nrf2 Antioxidative Function.

Luteolin has been reported to attenuate ischemia/reperfusion (I/R) injury in the diabetic heart through endothelial nitric oxide synthase- (eNOS-) related antioxidative response. Though the nuclear factor erythroid 2-related factor 2 (Nrf2) is regarded as a key endogenous factor to reduce diabetic oxidative stress, whether luteolin reduces cardiac I/R injury in the diabetic heart via enhancing Nrf2 function needs to be clarified. We hypothesized that pretreatment with luteolin could alleviate cardiac I/R injury in the diabetic heart by affecting the eNOS/Nrf2 signaling pathway. The diabetic rat was produced by a single injection of streptozotocin (65 mg/kg, i.p.) for 6 weeks, and then, luteolin (100 mg/kg/day, i.g.), eNOS inhibitor L-NAME, or Nrf2 inhibitor brusatol was administered for the succedent 2 weeks. After that, the isolated rat heart was exposed to 30 min of global ischemia and 120 min of reperfusion to establish I/R injury. Luteolin markedly ameliorated cardiac function and myocardial viability; upregulated expressions of heme oxygenase-1, superoxide dismutase, glutathione peroxidase, and catalase; and reduced myocardial lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R heart. All these ameliorating effects of luteolin were significantly reversed by L-NAME or brusatol. Luteolin also markedly reduced S-nitrosylation of Kelch-like ECH-associated protein 1 (Keap1) and upregulated Nrf2 and its transcriptional activity. This effect of luteolin on Keap1/Nrf2 signaling was attenuated by L-NAME. These data reveal that luteolin protects the diabetic heart against I/R injury by enhancing eNOS-mediated S-nitrosylation of Keap1, with subsequent upregulation of Nrf2 and the Nrf2-related antioxidative signaling pathway.

[Involvement of nitric oxide in negative inotropic effect of interferon-alpha in rat cardiac muscle].

OBJECTIVE: To investigate the effects and underlying mechanisms of interferon-alpha (IFN-alpha) on the isolated Langendorff perfused rat hearts and the isolated papillary muscles. METHODS: The left ventricular developed pressure (LVDP), maximal rise/fall rate of left ventricular pressure (+/-dP/dt(max)), left ventricular end-diastolic pressure (LVEDP), heart rate (HR) and coronary flow (CF) were recorded in isolated Langendorff perfused rat hearts. The average contractile force was measured in the isolated papillary muscles of rat right ventricle. RESULT: IFN-alpha (10 - 10,000 U/ml) induced a concentration-dependent decrease of LVDP and +/-dP/dt(max), and increase of LVEDP and CF in the isolated perfused rat heart (P < 0.05), and decrease of the average contractile force of the papillary muscle (P <0.05). Pretreatment with L-NAME (10(-4) mol/L), an inhibitor of nitric oxide synthase, attenuated the effect of IFN-alpha in the isolated rat hearts and the isolated papillary muscles (P <0.05). Isoproterenol (ISO, 10(-9) - 10(-6)mol/L) increased the contractile force of the rat papillary muscles in a concentration-dependent manner. Perfusion for 10 min with IFN-alpha at 1,000 U/ml attenuated the enhancing effect of ISO. Pretreatment with L-NAME reduced the effects of IFN-alpha on the isolated papillary muscles. CONCLUSION: IFN-alpha may induce a negative inotropic effect in normal and beta-adrenergic activated cardiac muscles and this effect at least partly be mediated by nitric oxide.

[Vascular effect of extract from mulberry leaves and underlying mechanism].

OBJECTIVE: To investigate the vascular activity of extract from mulberry leaves (EML) on rat thoracic aorta and the underlying mechanism. METHODS: Isolated thoracic rings of Sprague-Dawley rats were mounted on the organ bath and the tension of the vessel was recorded. RESULT: (1) EML produced a concentration-dependent vasorelaxation of aorta preconstricted by high K(+) (60 mmol/L) or 10(-6) mol/L phenylephrine (PE) in endothelium-intact and endothelium-denuded arteries. (2) EML at EC(50) concentration reduced the calcium dose-response curve. (3) After incubation of aorta with verapamil, EML induced vasocontraction of aorta preconstricted by PE, which was abolished by ruthenium red. CONCLUSION: The vascular effect of EML is biphasic, the vasorelaxation is greater than the vasocontraction. The vasorelaxation induced by EML may be mediated by inhibition of voltage-and receptor-dependent calcium channels in vascular smooth muscle cells, while the vasocontraction is via activation of ryanodine receptor in endoplasmic reticulum.

[Comparison of vasodilatation effect between quercetin and rutin in the isolated rat thoracic aorta].

OBJECTIVE: To determine the possible difference in vasodialtation effect of quercetin and rutin. METHODS: The isolated rat thoracic aorta was treated with phenylephrine (PE), and the effects of quercetin and rutin on the preconstricted aorta rings with or without endothelium were determined by organ bath technique. Nitric oxide synthase inhibitor L-N(G)-nitroarginine methyl-ester (L-NAME), guanylyl cyclase inhibitor methylene blue, cyclooxygenase inhibitor indomethacin were used to explore the mechanism. RESULTS: Quercetin (10-160 micromol/L) caused vasorelaxation of aorta rings preconstricted with PE in endothelium-intact and denuded aorta rings in a dose-dependent manner. Rutin(10-160 micromol/L) caused dose-dependent vasorelaxation in endothelium-intact rings preconstricted with phenylephrine, but not in denuded aorta rings. The maximal response (Rmax) values calculated from vasorelaxation curves of quercetin and rutin were (77.20+/-6.11)% and (44.28+/-7.48)%, respectively. There was no difference between median effective concentration (EC(50)) values of quercetin and rutin. Pretreatment with L-NAME (0.1 mmol/L) abolished the vasorelaxation by rutin,but did not influence the vasodilating effect of quercetin in endothelium-intact rings. Pretreatment with methylene blue (10 mmol/L) canceled the vasorelaxation both by quercetin and rutin. Pretreatment with indomethacin (10 micromol/L) attenuated the vasodilatation of quercetin, but did not affect the vascular effect of rutin. CONCLUSION: The vasodilatation effect of quercetin is more potent than rutin. The vasodilatation effect of quercetin might be mediated by guanylyl cyclase and cyclooxygenase-dependent pathway, while the vasodilatation by rutin might be via nitric oxide-guanylyl cyclase pathway.

[Treatment of thoracolumbar spinal tuberculosis with single posterior debridement, bone grafting, internal fixation and local chemotherapy].

OBJECTIVE: To evaluate the clinical effects of single posterior debridement, bone grafting, internal fixation and local chemotherapy in treating thoracolumbar spinal tuberculosis. METHODS: From February 2009 to September 2012,11 patients with thoracolumbar spinal tuberculosis were treated by single posterior debridement, bone grafting, internal fixation and local chemotherapy. There were 7 males and 4 females, aged from 27 to 65 years old with an average of 53.7 years. The courses of disease was from 3 months to 2 years with the mean of 9 months. According to ASIA standard of spinal cord injury, 3 cases were grade C and 8 cases D. After treatment, clinical effects were evaluated by ASIA grade, visual analogue score (VAS) and Oswestry Disability Index (ODI); kyphosis Cobb angle change was observed by X-rays. RESULTS: Eleven patients were followed up from 12 to 29 months with an average of 18 months. ASIA grade of spinal cord injury, 3 patients with grade C improved to grade D in 2 cases and grade E in 1 case 8 patients with grade D improved to grade E in 7 cases and unchanged in 1 case. VAS decreased from preoperative 6.10 ± 1.30 to 1.70 ± 0.80 at 3 d after operation (P < 0.05). ODI improved from preoperative (68.36 ± 10.41)% to (14.55 ± 8.99)% (P < 0.05) at 3 d after operation. Kyphotic Cobb angle was corrected from preoperative (22.64 ± 4.84)° to (4.27 ± 1.49)° (P < 0.05) on the 3rd day after operation, and angle loss was mild at final follow-up, there was no significant difference between postoperative at 3 d and final follow-up. CONCLUSION: Single posterior debridement, bone grafting, internal fixation and local chemotherapy for the treatment of thoracolumbar spinal tuberculosis can effectively remove the lesion, improve nerve function and correct deformity, has advantage of single incision, little trauma, and low recurrence rate. But it still need long-term and systemic treatment with anti-TB drugs.

Betulinic acid inhibits superoxide anion-mediated impairment of endothelium-dependent relaxation in rat aortas.

OBJECTIVES: To investigate the protective effect of betulinic acid (BA) on endothelium-dependent relaxation (EDR) in rat aortas exposed to pyrogallol-produced superoxide anion and its underlying mechanism. MATERIALS AND METHODS: The thoracic aorta of male Sprague-Dawley rats was isolated to mount in the organ bath system and the effect of BA on acetylcholine (ACh)-induced EDR, nitric oxide (NO) level, reactive oxygen species (ROS) level, nitric oxide synthase (NOS) activity, and superoxide dismutase (SOD) activity of aortic rings exposed to pyrogallol (500 µM) for 15 min were measured. RESULTS: BA evoked a concentration-dependent EDR in aortas, and pretreatment with EC(50) (2.0 µM) concentration of BA markedly enhanced ACh-induced EDR of aortas exposed to pyrogallol-produced superoxide anion (E(max) rose from 23.91 ± 5.41% to 42.45 ± 9.99%), which was markedly reversed by both N(w) -nitro-L-arginine methyl ester hydrochloride (L-NAME) and methylene blue, but not by indomethacin. Moreover, BA significantly inhibited the increase of ROS level, as well as the decrease of NO level, the endothelial NOS (eNOS) activity, and the SOD activity in aortas induced by pyrogallol-derived superoxide anion. CONCLUSION: These results indicate that BA reduces the impairment of EDR in rat aortas exposed to exogenous superoxide anion, which may closely relate to the reduction of oxidative stress and activation of eNOS-NO pathway.

[Effect of nitric oxide synthase inhibitors on the changes of hemodynamic parameters and aortic tension induced by septic shock in rats].

AIM: To investigate the effect of three types of nitric oxide synthase inhibitors on the changes of hemodynamic parameters and thoracic aorta tension induced by septic shock in rats. METHODS: We used cecal ligation and puncture (CLP) method to establish septic shock in rats, and the three types of nitric oxide synthase inhibitors were injected after CLP. The carotid artery was cannulated and connected to a pressure transducer to determine mean arterial blood pressure (MABP). Ventricular dynamic parameters were determined following intraventricular cannulation via the carotid artery, including heart rate (HR), left ventricular developed pressure (LVDP), maximal rise/fall velocity of ventricular pressure (+/- dP/dt(max)). Isolated thoracic rings were mounted on an organ bath and the tension of the vessel was recorded. RESULTS: (1) After using L-NAME, AMG and 7-NI the mortality decreased to 50.0%, 37.5%, and 42.1%, respectively (from 65.2% in septic shock rats); (2) The MABP in septic shock rats partly recovered after using the NOS inhibitors, all ventricular dynamic parameters partly recovered after using the inhibitors; (3) The hyporeactivity of endothelium-denuded aortic rings to vasoconstrictors induced by septic shock was partly recovered by pretreatment with the inhibitors. However, only L-NAME or 7-NI could inhibit the decrease of vasoconstriction induced by septic shock in endothelium-intact aortic rings. CONCLUSION: The three types of nitric oxide synthase inhibitors can improve the hemodynamic parameters and vasoconstriction responsiveness of endothelium-denuded aorta of septic shock rats. Furthermore, L-NAME and 7-NI improve the responsiveness of endothelium-intact aorta.

Rutin-induced endothelium-dependent vasorelaxation in rat aortic Rings and the underlying mechanism.

The aim of the present study was to determine the possible mechanism by which rutin causes vasodilatation in isolated thoracic aorta rings from the rat. The effects of rutin on rings preconstricted with phenylephrine, with or without endothelium, were determined using an organ bath technique. The mechanism was explored by measuring the effects of the nitric oxide synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), the guanylyl cyclase inhibitor methylene blue, the cyclooxygenase inhibitor indomethacin, the ATP-sensitive K+channel blocker glibenclamide and the beta-adrenoceptor antagonist propranolol. Rutin at the range of 10-160 mumol/L caused dose-dependent vasorelaxation in preconstricted endothelium-intact rings, but had no effect on rings without endothelium. The maximal response calculated from the vasorelaxation curves of rutin was 44.28 plus or mines 7.48%. Pretreatment with L-NAME (0.1 mmol/L), methylene blue (10 mumol/L), glibenclamide (10 mmol/L) or indomethacin (10 mmol/L) attenuated the vasorelaxation induced by rutin in endothelium-intact rings. Glibenclamide (10 mmol/L) enhanced the vasorelaxation of rutin. Propranolol (10 mumol/L) did not block the effect of rutin. The results indicate that vasorelaxation is induced by rutin via the nitric oxide-guanylyl cyclase pathway and a prostaglandin-mediated mechanism, as well as activation of the ATP-sensitive potassium channel.