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.

[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.

[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.

[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.

[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.

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.