[Hesperetin Alleviates Doxorubicin-Induced Cytotoxicity in H9c2 Cells by Activating SIRT1/NRF2 Signaling].

Objective: To investigate whether hesperetin (Hes) alleviates doxorubicin (DOX)-induced cardiomyocytotoxicity by reducing oxidative stress via regulating silent information regulator 1 (SIRT1)/nuclear transcription factor E2-related factor 2 (NRF2) signaling in H9c2 cells. Methods: H9c2 cells were treated with DOX to establish the cardiotoxicity model and were randomly assigned to four groups, a control group (Control) and three treatment groups, receiving respectively DOX (the DOX group), Hes+DOX (the DOX+Hes group), and Hes+SIRT1 inhibitor EX527+DOX (the DOX+Hes+EX527 group). Cellular morphology was observed by the light microscope. Cell viability was evaluated by CCK-8. DOX-induced apoptosis in H9c2 cells was examined by flow cytometry. The levels of reactive oxygen species (ROS) in the H9c2 cells of the four groups were determied with 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and SIRT1 as well as the malondialdehyde (MDA) content were measured using ELISA kits. The expressions of cleaved caspase-3, cytochrome c, SIRT1, Ac-FOXO1, NRF2, and heme oxygenase 1 (HO-1) were determined by Western blot. Results: Compared with the Control group, the DOX group showed swollen cellular morphology, decreased cell density and viability, and increased LDH activity in the medium ( P<0.01); both apoptosis and the expression of cleaved caspase-3 and cytochrome c increased ( P<0.01); the activities of CAT and SOD decreased while the contents of MDA and ROS increased ( P<0.01); the expression of SIRT1, NRF2, and HO-1 decreased, the activity of SIRT1 decreased, and the expression of Ac-FOXO1 increased ( P<0.01). Compared with the DOX group, the DOX+Hes group showed improved cellular morphology, increased cell density and viability, and decreased LDH activity in the medium ( P<0.01); the apoptosis and the expression of cleaved caspase-3 and cytochrome c decreased ( P<0.01); the activities of CAT and SOD increased while the levels of MDA and ROS decreased ( P<0.01); the expression of SIRT1, NRF2, and HO-1 increased, the activity of SIRT1 increased, and the expression of Ac-FOXO1 decreased ( P<0.01). Comparison of the findings for the DOX+Hes group and the DOX+Hes+EX527 group showed that EX527 could block the protective effects of Hes against DOX-induced cell injury, oxidative stress, and SIRT1/NRF2 signaling. Conclusion: Hes inhibits oxidative stress and apoptosis via regulating SIRT1/NRF2 signaling, thereby reducing DOX-induced cardiotoxicity in H9c2 cells.

Lipopolysaccharide inhibits GPR120 expression in macrophages via Toll-like receptor 4 and p38 MAPK activation.

Free fatty acid receptor G protein-coupled receptor 120 (GPR120) is highly expressed in macrophages and was reported to inhibit lipopolysaccharide (LPS)-stimulated cytokine expression. Under inflammation, macrophages exhibit striking functional changes, but changes in GPR120 expression and signaling are not known. In this study, the effects of LPS treatment on macrophage GPR120 expression and activation were investigated. The results showed that LPS inhibited GPR120 expression in mouse macrophage cell line Ana-1 cells. Moreover, LPS treatment inhibited GPR120 expression in mouse alveolar macrophages both in vitro and in vivo. The inhibitory effect of LPS on GPR120 expression was blocked by Toll-like receptor 4 (TLR4) inhibitor TAK242 and p38 mitogen-activated protein kinase inhibitor LY222820, but not by ERK1/2 inhibitor U0126 and c-Jun N-terminal kinase inhibitor SP600125. LPS-induced inhibition of GPR120 expression was not attenuated by GPR120 agonists TUG891 and GW9508. TUG891 inhibited the phagocytosis of alveolar macrophages, and LPS treatment counteracted the effects of TUG891 on phagocytosis. These results indicate that pretreatment with LPS inhibits GPR120 expression and activation in macrophages. It is suggested that LPS-induced inhibition of GPR120 expression is a reaction enhancing the LPS-induced pro-inflammatory response of macrophages.

Erythropoietin attenuates vascular calcification by inhibiting endoplasmic reticulum stress in rats with chronic kidney disease.

Previous studies suggested that endoplasmic reticulum (ER) stress induced-apoptosis promoted vascular calcification (VC). Interestingly, erythropoietin (EPO), an endogenous glycoprotein, exerts multiple tissue protective effects by inhibiting ER stress and apoptosis. We investigated the role and potential mechanism of EPO on VC in chronic kidney disease (CKD) rats and cultured vascular smooth muscle cells (VSMCs). The calcification model was established by subtotal nephrectomy in vivo or phosphate overload in vitro. The protein level of EPO receptor (EPOR) was increased in the calcified aortas of CKD rats. EPO prevented the reduction of VSMC phenotypic markers, and reversed the increased calcium content and calcium salt deposition in the aortas of CKD rats and cultured calcified VSMCs. The protein levels of activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94) were upregulated in aortas and VSMCs under calcifying conditions, indicating ER stress activation. EPO treatment of CKD rats or calcified VSMCs downregulated the protein levels of ATF4 and GRP94. Furthermore, ER stress-mediated apoptosis, determined by the protein levels of CCAAT/enhancer-binding protein-homologous protein and cleaved caspase 12, was increased in tunicamycin or calcification media-treated VSMCs, but the increased effect was reversed in EPO-treated groups. The increased apoptotic cells in calcified VSMCs, as indicated by Hoechst staining and flow cytometry, were downregulated by the co-administration of EPO or 4-phenyl butyric acid. In conclusion, EPO might attenuate VC by inhibiting ER stress mediated apoptosis through EPOR signaling.

[Na+/Ca2+ exchanger mediates ischemia-reperfusion injury by activation of CaMKII in isolated rat heart].

OBJECTIVE: To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemia-reperfusion injury and the underlying mechanisms.
 Methods: Forty Sprague-Dawley rats were divided into 4 groups randomly: a control group, a KB-R7943 group, an ischemia-reperfusion group (IR group), and an IR plus KB-R7943 group (KB-R7943+IR group). Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion. The ratio of left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), the infarct size of myocardium, and the lactate dehydrogenase (LDH) activity in the coronary flow was determined. HE staining was used to assess the change of myocardial morphology. Western blot was used to determine the levels of cleaved caspase-3, cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the Thr17 site of phospholamban.
 Results: Compared with the control group, IR group significantly induced an enlarged infarct size, reduction of the ratio of LVDP, up-regulation of cytochrome c, cleaved caspase-3, p-CaMKII and p-phospholamban, and increased in the activity of LDH, the level of LVEDP (P<0.01) and the disordered myocardial morphology. These effects were significantly attenuated in the presence of KB-R7943 treatment (10 µmol/L).
 Conclusion: NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKII.

[Inhibition of CaMKII alleviates myocardial ischemia?reperfusion injury by reducing mitochondrial oxidative stress in isolated perfused rat heart].

OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in myocardial ischemia-reperfusion (IR) injury in isolated perfused rat heart and explore the underlying mechanisms. METHODS: An ischemia-reperfusion (IR) model was prepared using isolated rat hearts perfused with Krebs-Henseleit solution were randomly divided into control group, 2.5 µmol/L KN-93 group, IR (induced by ischemia for 45 min followed by reperfusion for 120 min) group and KN-93+IR group. The myocardial performance was evaluated by assessing the left ventricular pressure. Lactate dehydrogenase (LDH) activity and cTnI content in the coronary flow and the infarct size were determined to evaluate the myocardial injury. The phosphorylation of CaMKII (p-CaMKII) and PLN (p-PLN) and oxidation of CaMKII (ox--CaMKII) were measured with Western blotting. The activity of mitochondrial superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using ELISA. RESULTS: Compared with the control group, KN-93 treatment at 2.5 µmol/L produced no significant effects on cardiac function or performance in rat hearts without IR injury. Myocardial IR injury significantly decreased myocardial performance and mitochondrial SOD activity in the perfused hearts (P<0.01) and caused significantly increased infarct size, LDH activity, cTnI content, expressions of p-CaMKII, ox-CaMKII and p-PLN, and also increased mitochondrial MDA content (P<0.01). KN-93 treatment at 2.5 µmol/L administered before ischemia and before reperfusion markedly attenuated such changes induced by ischemia and reperfusion (P<0.01). CONCLUSION: CaMKII participates in myocardial IR injury in isolated rat heart, and inhibiting CaMKII can alleviate myocardial injury by relieving mitochondrial oxidation stress.

Grifolic acid induces mitochondrial membrane potential loss and cell death of RAW264.7 macrophages.

Grifolic acid is a phenolic compound that was first extracted from the mushroom Albatrellus confluens; it acts as an agonist of the free fatty acid receptor (FFAR4). FFAR4 is expressed in macrophages and mediates the anti­inflammatory effects of n­3 unsaturated free fatty acids. In the present study, the effects of grifolic acid on macrophages were observed in mouse RAW264.7 cells. It was demonstrated that grifolic acid (2.5­20 µmol/l) treatment reduced RAW264.7 cell viability in a dose­ and time­dependent manner. The number of apoptotic cells significantly increased following grifolic acid treatment compared with the untreated control cells. Grifolic acid treatment resulted in a significant decrease in cellular adenosine 5'­triphosphate (ATP) content in RAW264.7 cells. Mitochondrial membrane potential (MMP), as measured by JC­1 staining, was significantly diminished by grifolic acid treatment in a dose­ and time­dependent manner. Treatment with cyclosporine A, a protector of MMP, attenuated grifolic acid­induced reduction of MMP and viability in RAW264.7 cells. FFAR4 knockdown did not significantly influence grifolic acid­induced reduction of cell viability, ATP levels or MMP. In conclusion, grifolic acid may induce macrophage cell death by reducing MMP and by inhibiting ATP production probably in an FFAR4­independent manner.

[Capsaicin Alleviate Myocardial Ischemia Reperfusion Injury Through Attenuating Mitochondrial Oxidative Stress].

OBJECTIVE: To investigate the role of capsaicin (CAP) in myocardial ischemia reperfusion injury and its underlying mechanisms. METHODS: Twentyfour adult male SD rats were randomized into 4 groups,namely the control group,ischemia reperfusion group,ischemia reperfusion with CAP group,and ischemia reperfusion with CAPZ and CAP group. Isolated rat hearts underwent Langendorff perfusion. Left ventricular enddiastolic pressure (LVEDP) andleft ventricular developed pressure (LVDP) was calculated to evaluate myocardial performance at 30 min of reperfusion.Triphenyltetrazolium chloride staining was used to measure the infarct size of myocardium at 120 min reperfusion. The morphological changes in myocardial fiber was analyzed by HE staining at the end of reperfusion. Lactate dehydrogenase (LDH) content in the coronary flow was determined during the first 5 min reperfusion. The myocardial mitochondria was isolated and extracted for measuring a series of indicators of mitochondrial oxidative stress,including superoxide dismutase (SOD),methane dicarboxylic aldehyde (MDA) at the end of reperfusion. Western blot was used to determine the expression of caspase3 and cytochrome c at 10 min reperfusion. RESULTS: Compared with the control group,IR group significantly decreased in cardiac function,the level of LVDP and SOD activity and induced an enlarged infarct size ( P<0.01),accompanied by the disordered arrangement of myocardial cells,the content of MDA was increased ( P<0.01),the content of caspase3 and cytochrome c were also upregulated ( P<0.01).10 µmol/L CAP significantly attenuated these effects induced by ischemia reperfusion injury,levels of LVDP and infarct size at the end of reperfusion were significantly improved( P<0.01),nevertheless levels of LVEDP and MDA at the end of reperfusion and LDH were down-regulated markedly ( P<0.01),the content of caspase-3 and cytochrome c were also decreased ( P<0.01). CONCLUSION: These results demonstrate that CAP can suppresses cell apoptosis and necrosis,and alleviate heart function and cell survival from ischemia reperfusion injury through attenuating mitochondrial oxidative stress.

[Role of melatonin in calcium overload-induced heart injury].

OBJECTIVE: To investigate the role of melatonin in calcium overload-induced heart injury.
 Methods: Thirty-two rats were divided into 4 groups: a control group (Control), a melatonin control group (Mel), a calcium overload group (CaP), and a calcium overload plus melatonin group (Mel+CaP). Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Triphenyltetrazolium chloride staining was used to measure the infarct size of myocardium. Lactate dehydrogenase (LDH) activity in the coronary flow was determined. The expressions of caspase-3 and cytochrome c were determined by Western blot. The pathological morphological changes in myocardial fiber were analyzed by HE staining.
 Results: Compared with the control group, calcium overload significantly induced an enlarged infarct size (P<0.01), accompanied by the disordered arrangement of myocardial fiber, up-regulation of cytochrome c and caspase-3 (P<0.01), and the increased activity of LDH (P<0.01). These effects were significantly attenuated by 10 µmol/L melatonin (P<0.01).
 Conclusion: Melatonin can alleviate calcium overload-induced heart injury.

MicroRNA-101 in the ventrolateral orbital cortex (VLO) modulates depressive-like behaviors in rats and targets dual-specificity phosphatase 1 (DUSP1).

Long-term exposure to stress plays a key role in the pathogenesis of major depression. Recently, the ventrolateral orbital cortex (VLO) has received considerable attention for its role in the antidepressant response. However, the mechanisms underlying stress response in the VLO remain largely elusive. MiR-101 has been implicated in regulating multiple neurological processes. The present study used the chronic unpredictable mild stress (CUMS) rat model to investigate the expression of miR-101 in the VLOs of rat brains and the possible relevance of miR-101 to depression. Furthermore, an intra-VLO administration of a miR-101 mimic was performed to provide insights into the miR-101-mediated dysregulation mechanisms associated with depression. The results showed that chronic stress induced typical depressive-like behaviors in rats and decreased miR-101 levels in the VLOs of rat brains. Moreover, the dual specificity phosphatase 1 (DUSP1) protein levels were increased in the VLOs in CUMS rats, whereas the ERK phosphorylation and BDNF levels in the CUMS rats were decreased. Enhancing miR-101 expression via an intro-VLO microinjection of its mimic reversed the depressive-like behaviors in CUMS rats. Intra-VLO treatment with the miR-101 mimic also attenuated the upregulation of CUMS-induced DUSP1 expression and inhibited the downstream ERK phosphorylation and BDNF expression. These results suggest that miR-101 has functional significance in the pathophysiology of stress-induced dysfunctions in the VLO. MiR-101 may directly regulate DUSP1 expression, and the mechanism underlying the antidepressant effects of miR-101 may involve the negative regulation of DUSP1 expression, which in turn promotes downstream ERK/BDNF signaling.

[Effect of 2,3-butanedione monoxime on calcium paradox-induced heart injury in rats].

OBJECTIVE: To investigate the Effect of 2,3-butanedione monoxime (BDM) on calcium paradox-induced heart injury and its underlying mechanisms. METHODS: Thirty-two adult male SD rats were randomized into 4 groups, namely the control group, BDM treatment control group, calcium paradox group, and BDM treatment group. Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion and the left ventricular pressure (LVP) and left ventricular end-diastolic pressure (LVEDP) were monitored. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Lactate dehydrogenase (LDH) content in the coronary flow was determined. Triphenyltetrazolium chloride staining was used to measure the infarct size, and myocardial cell apoptosis was tested with TUNEL method. Western blotting was used to determine the expression of cleaved caspase-3 and cytochrome c. RESULTS: Compared with the control group, BDM at 20 mmol/L had no effect on cardiac performance, cell death, apoptotic index or the content of LDH, cleaved caspase-3 and cytochrome c at the end of perfusion under control conditions (P>0.05). Calcium paradox treatment significantly decreased the cardiac function and the level of LVDP and induced a larger infarct size (P<0.01), an increased myocardial apoptosis index (P<0.01), and up-regulated expressions of cleaved caspase-3 and cytochrome c (P<0.01). BDM (20 mmol/L) significantly attenuated these effects induced by calcium paradox, and markedly down-regulated the levels of LVEDP and LDH (P<0.01), lowered myocardial apoptosis index, decreased the content of cleaved caspase-3 and cytochrome c (P<0.01), increased LVDP, and reduced the infarct size (P<0.01). CONCLUSION: BDM suppresses cell apoptosis and contracture and improves heart function and cell survival in rat hearts exposed to calcium paradox, suggesting the value of BDM as an potential drug for myocardial ischemia reperfusion injur.

FoxO3α-mediated autophagy contributes to apoptosis in cardiac microvascular endothelial cells under hypoxia.

Hypoxic injury of cardiac microvascular endothelial cells (CMECs) is an important pathophysiological event in myocardial infarction, whereas, the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for normal cardiac function and also has been implicated in various cardiovascular diseases. Here we investigated the potential role of autophagy in CMEC dysfunction under hypoxia. CMECs were isolated from SD rats. Hypoxia (6-24h, 1% O2) induced autophagy in CMECs as evidenced by formation of punctate LC3, increased conversion of LC3-I to LC3-II and increased p62 degradation. Importantly, hypoxia-induced apoptosis in CMECs was attenuated by 3-Methyladenine (5mM), an autophagy inhibitor, and aggravated by rapamycin (1.0 µg/L), an autophagy inducer. Meanwhile, hypoxia increased the nuclear localization of FoxO3α, accompanying with the decreased phosphorylation of FoxO3α and Akt. FoxO3α silencing decreased hypoxia-induced autophagy and the resultant apoptosis. Furthermore, treatment with 3-Methyladenine (10mg/kg/day) improved the endothelial-dependent diastolic function of coronary artery in rats with myocardial infarction. These results indicated that hypoxia-induced autophagy formation in CMECs is mediated by FoxO3α and contributes to hypoxic injury of hearts.

[Ventrolateral periaqueductal gray metabotropic glutamate receptor subtypes 7 and 8 mediate opposite effects on cardiosomatic motor reflex in rats].

OBJECTIVE: To investigate the role of ventrolateral periaqueductal gray (VL-PAG) metabotropic glutamate receptors subtype 7 and 8 (mGluR 7/8) in descending modulation of cardiosomatic motor reflex (CMR) in rats. METHODS: AMN082 (agonist of mGluR 7) and DCPG (agonist of mGluR 8) were injected into the VL-PAG of a rat model of CMR to observe their effects in modulating CMR. The raphe magnus nucleus (NRM) or the gigantocellular reticular nucleus (Gi) was then damaged, and the changes in VL-PAG descending modulation were observed. RESULTS: Selective activation of mGluR 7 of the VL-PAG by AMN082 obviously facilitated capsaicin (CAP)-induced CMR (P<0.05), which was suppressed by DCPG-induced mGluR 8 activation (P<0.05). These facilitatory or inhibitory effects were completely reversed by group III mGluR antagonist MSOP. Damaging the NRM of VL-PAG main relay nucleus did not significantly affect the facilitatory effect produced by AMN082 microinjection (P>0.05), but partially attenuated the inhibitory effect of DCPG microinjection (P<0.05). Both the facilitatory effect of AMN082 and the inhibitory effect of DCPG were reduced obviously after bilateral Gi damage (P<0.05). CONCLUSION: VL-PAG mGluR 7 and mGluR 8 mediate biphasic regulation of CMR in rats probably through activation of different sub-nuclei and different neurons in the rostroventral medulla.

[The role of NMDA and NK1 receptors in cardiac nociceptive information transmissions in the spinal cords of rats].

OBJECTIVE: To determine the effects of NMDA and NK1 receptor agonist and antagonist on the EMG and the synaptic mechanism of nociceptive information transmissions in the spinal cords. METHODS: Male SD rats were randomly divided into seven groups, with intrathecal injection of the following chemicals respectively: control group (10 microL saline), NMDA group (0.147 microg/10 pL NMDA), MK801 group (6.8 microg/10 microL MK801), MK801+NMDA group (6.8 microg/10 pL MK801+0. 147 microg/10 pL NMDA), Sar-SP group (1.4 pg/10 microL Sar-SP), CP-96345 group (5 microg/10 pL CP-96345), and CP-96345+Sar-SP group (1.4 micro/10 microL Sar-SP+5 microg/10 microL CP-96345). A cardiac pain model in rats through intrapericardial injection of capsaicin was established. Intrapericardial injection of capsaicin was given to the rats 10 min after intrathecal injection of the tested chemicals. The spinotrapezius electromyography (EMG) activities as an index of cardiac-somatic motor reflex were recorded simultaneously. RESULTS: Compared with the pre-test controls (100%), saline did not make a significant change to the capsaicin-evoked EMG response (96. 9% +/- 12. 5%, P>0. 05); NMDA agonist increased the capsaicin-evoked EMG response (185. 2% +/- 24. 4%) significantly (P<0. 05); neither MK801 nor a combined administration of MK801 and NMDA made a significant change to the capsaicin-evoked EMG response (106. 6% +/- 10. 2%, P> 0.05); Sar-SP increased the capsaicin-evoked EMG response (145. 6% 10. 1%) significantly (P<0. 05); whereas neither CP-96345 nor a combined administration of CP-96345 and Sar-SP made a significant change to the capsaicin-evoked EMG response (102. 2% +/- 8. 4%, P>0.05). CONCLUSION: NMDA and NK1 receptors may have participated in the transmissions of cardiac nociception information in the spinal cords of rats.

[Descending modulation of cardiac nociception by the rostral ventromedial medulla in rats].

OBJECTIVE: To observe the descending modulation of cardiac nociception by the rostral ventromedial medulla (RVM) in rats. METHODS: A rat model of cardiosomatic motor reflex (CMR) was established by injecting capsaicin into the pericardial sac to induce cardiac nociception, and the electromyogram (EMG) response of the dorsal spinotrapezius muscle was studied. The RVM was electrically stimulated (25, 75 and 100 µA) or destroyed to examine whether RVM exerted descending modulation on cardiac nociception. RESULTS: Electrical stimulation of the RVM at 8 sites produced intensity-dependent inhibition of EMG responses to noxious cardiac stimulus (F[2,21]=43.188, P=0.001). Electrical stimulation at 3 sites caused facilitated EMG responses, but the increased magnitude of the EMG was not dependent on stimulation intensity (F[2,6]=0.884, P=0.461). Stimulation at 11 sites produced biphasic effects: at a low intensity (25 µA), the elicited EMG magnitude was significantly larger than baseline (P<0.05), and at greater intensities (75/100 µA), the stimulation caused suppression of the EMG magnitude to a level significantly lower than the baseline (P<0.05). Electrolytic lesion of the RVM resulted in significantly increased EMG responses compared with the baseline and sham lesion group. CONCLUSION: Cardiac nociception evoked by capsaicin stimulation is subjected to descending biphasic modulation by the RVM, which produces predominantly descending inhibition on heart pain.

The role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex on allodynia following spared nerve injury.

The present study examined the role of α2 adrenoceptor in mediating noradrenaline action in the ventrolateral orbital cortex (VLO) on allodynia induced by spared nerve injury (SNI) in the rat. The mechanical paw withdrawal threshold (PWT) was measured using von-Frey filaments. Microinjection of noradrenaline (1, 2, 4 µg in 0.5 µl) into the VLO, contralateral to the site of nerve injury, reduced allodynia; PWT increased in a dose-dependent manner. Similar to noradrenaline, microinjection of selective α2 adrenoceptor agonist clonidine into the same VLO site also reduced allodynia, and was blocked by selective α2 adrenoceptor antagonist yohimbine. Furthermore, administration of γ-aminobutyric acid A (GABAA) receptor antagonist bicuculline or picrotoxin to the VLO significantly enhanced clonidine-induced inhibition of allodynia, while GABAA receptor agonist muscimol or THIP (2,5,6,7-retrahydroisoxazolo(5,4-c)pyridine-3-ol hydrochloride) attenuated clonidine-induced inhibition. These results suggest that noradrenaline acting in the VLO can potentially reduce allodynia induced by SNI, and this effect is mediated by α2 adrenoceptor. Moreover, GABAergic disinhibition may participate in α2 receptor mediating effects in neuropathic pain in the central nervous system.

Lateral reticular nucleus modulates the cardiosomatic reflex evoked by intrapericardial capsaicin in the rat.

The current study examined the role of the lateral reticular nucleus (LRN) in modulating the cardiosomatic reflex (CSR) induced by intrapericardial capsaicin in the anesthetized rat. Intrapericardial capsaicin was administered, and the CSR was monitored via electromyogram responses of the dorsal spinotrapezius muscle. Electrical stimulation of the LRN (10, 20 and 30 µA) depressed the CSR induced by intrapericardial capsaicin in an intensity-dependent manner. Microinjection of glutamate (4, 10, 20 and 40 nmol, in 0.2 µL) into the LRN replicated the effects of electrical stimulation. Furthermore, bilateral transections of the dorsolateral funiculus (DLF) decreased the LRN electrical stimulation-induced inhibition of the electromyogram responses. Intrathecal administration of the α2 -adrenergic receptor antagonist yohimbine or the serotonergic receptor antagonist methysergide significantly attenuated the LRN electrical stimulation-induced inhibition of the electromyogram responses. However, intrathecal application of the opioid receptor antagonist naloxone had no effect on the LRN electrical stimulation-induced inhibition. These results suggest that the LRN-DLF-spinal cord pathway is involved in descending inhibition of the CSR, and spinal α2 -adrenergic and serotonergic receptors participate in this descending inhibition.

GABAergic modulation mediates antinociception produced by serotonin applied into thalamic nucleus submedius of the rat.

Our previous studies have indicated that the thalamic nucleus submedius (Sm) is involved in modulation of nociception as part of an ascending component of an endogenous analgesic system consisting of spinal cord-Sm-ventrolateral orbital cortex (VLO)-periaqueductal gray (PAG)-spinal cord loop and that microinjection of 5-hydroxytryptamine (5-HT) into Sm produces antinociception. The aim of the present study was to examine whether the gamma-aminobutyric acid (GABA)ergic modulation is involved in the Sm 5-HT-evoked antinociception. Experiments were carried out on lightly anesthetized rats with an implanted cannula targeting the Sm nucleus. The microinjection of GABA(A) receptor antagonist bicuculline dose-dependently depressed the tail flick (TF) reflex. A smaller dose (100 ng) of bicuculline enhanced the inhibition of TF reflex produced by 5-HT application into Sm, whereas application of GABA (2.5 microg) did not influence the TF reflex but significantly attenuated the 5-HT-evoked inhibition. These results indicate that GABA(A) receptor may be involved in mediating the 5-HT-induced antinociception in Sm possibly through a disinhibition mechanism.

5-hydroxytryptamine 1A (5-HT1A) but not 5-HT3 receptor is involved in mediating the nucleus submedius 5-HT-evoked antinociception in the rat.

Our previous studies have indicated that the thalamic nucleus submedius (Sm) is involved in modulation of nociception as part of an ascending component of an endogenous analgesic system consisting of spinal cord-Sm-ventrolateral orbital cortex (VLO)-periaqueductal gray (PAG)-spinal cord loop. Microinjection of 5-hydroxytryptamine (5-HT) into Sm produces antinociception and this effect is blocked by 5-HT(2) receptor antagonist. The aim of the present study was to examine whether the 5-HT(1) and 5-HT(3) receptors were also involved in the Sm 5-HT-evoked antinociception. Nociception was assessed in lightly anesthetized rats with radiant-heat-evoked tail flick (TF). 5-HT(1A) and 5-HT(3) receptor antagonists were microinjected into the Sm alone or in combination with a microinjection of 5-HT into the same Sm site. 5-HT(1A) receptor antagonist p-MPPI (0.87 nmol) facilitated the TF reflex; a lower dose (0.43 nmol) of p-MPPI significantly attenuated the Sm 5-HT-evoked inhibition of TF reflex. Microinjection of the 5-HT(3) receptor antagonist LY-278,584 (12 nmol) had no effect either on the TF reflex or on the Sm 5-HT-evoked inhibition. These results suggest that 5-HT(1A) receptor but not 5-HT(3) receptor is involved in mediating the 5-HT-evoked antinociception. Possible mechanisms of Sm 5-HT-induced descending antinociception are discussed.

Differential effects of opioid receptors in nucleus submedius and anterior pretectal nucleus in mediating electroacupuncture analgesia in the rat.

Previous studies have indicated that the thalamic nucleus submedius (Sm) and the anterior pretectal nucleus (APtN) are involved in the descending modulation of nociception. The aim of the present study was to examine whether the opioid receptors in the Sm and APtN mediated the electroacupuncture (EA)-produced analgesia. The latency of tail flick (TF) reflex induced by radiant heat was used as an index of nociceptive response. The effects of microinjection of opioid receptor antagonist naloxone (1.0 microg, 0.5 ml) into Sm or APtN on the inhibition of the TF reflex induced by EA of "Zusanli" point (St. 36) with high- (5.0 mA) and low- (0.5 mA) intensity were examined in the lightly anesthetized rats. Sm microinjection of naloxone blocked the high- but not low-intensity EA-induced inhibition of the TF reflex. In contrast, naloxone applied to APtN blocked the low- but not high-intensity EA-induced inhibition. When naloxone applied to other brain regions adjacent to Sm or APtN, the EA-induced inhibition was not influenced under either high- or low-intensity condition. These results suggest that opioid receptors in Sm are involved in mediating the analgesia by high-intensity EA for exciting small (A-delta and C group) afferent fibers, while opioid receptors in APtN are involved in mediating the analgesia induced by low-intensity EA for only exciting large (A-beta) afferent fibers.