Anti-inflammatory effect of low intensity ultrasound (LIUS) on complete Freund's adjuvant-induced arthritis synovium.

OBJECTIVES: Arthritis with intra-articular inflammation was accompanied by joint pain, swelling, and stiffness leading to significant functional impairment. Thus, regulation of joint inflammation is a good therapeutic approach for patients with arthritis. In this study, the effect of low intensity ultrasound (LIUS) applied to an adjuvant-induced arthritic rat model on the synovium was investigated. DESIGN: Synovial inflammation was induced by complete Freund's adjuvant (CFA)-injection into the rat knee joint. LIUS (200 mW/cm(2)) was applied on the ipsilateral knee everyday for 10 min beginning 1 day after inflammation induction. The expression of proinflammatory factors and immunohistochemical staining pattern of the synovium were assessed. RESULTS: CFA induced an increase of the knee circumference that was significantly diminished by LIUS. Synovial membrane hyperplasia in the ipsilateral joint was also affected by LIUS. The inflammatory mediators, COX-1/2, IL-1ß, and iNOS, but not TNF-α, in the synovial membrane were induced after 3 days, and they closely correlated with the degree of edema. In the synovial membrane, the expression of inflammatory mediators was reduced by LIUS. The chemoattractant chemokine receptor CCR5 also was involved. On immunohistochemical analysis, CFA caused increased infiltration of CD11b-positive cells in the synovium. After 3 days, neutrophils, myeloperoxidase (MPO)-positive cells filled the inflammatory core; later, monocytes and macrophages, ionized calcium binding adaptor molecule 1 (Iba1)-positive cells in the periphery infiltrated the core by day 5. LIUS markedly reduced CFA-induced inflammatory cells infiltration. CONCLUSION: LIUS showed a potent anti-inflammatory effect in this animal arthritis model with reduced infiltration of inflammatory cells into the synovium.

Diazoxide acts more as a PKC-epsilon activator, and indirectly activates the mitochondrial K(ATP) channel conferring cardioprotection against hypoxic injury.

BACKGROUND AND PURPOSE: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection. EXPERIMENTAL APPROACH: In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. KEY RESULTS: Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD. CONCLUSIONS AND IMPLICATIONS: Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.

Rutaecarpine, a quinazolinocarboline alkaloid, inhibits prostaglandin production in RAW264.7 macrophages.

In order to delineate the mechanism involved in the anti-inflammatory activity of rutaecarpine, its effects on the production of prostaglandin (PG) and therein involved enzymes were examined. Rutaecarpine reduced the production of PGE(2) in RAW264.7 cells treated with lipopolysaccharide (LPS) in a dose dependent manner when added to the culture media at the time of stimulation. However, the inhibition of total cellular cyclooxygenase (COX) activity under the same experimental condition was observed only at high concentrations of rutaecarpine. Rutaecarpine did not affected the levels of COX-2 mRNA and protein in macrophages stimulated with LPS. Calcium ionophore A23187 induced-PG production and [(3)H]-arachidonic acid release were significantly decreased by the pretreatment of rutaecarpine for 30 minutes. With the same treatment schedule, however, rutaecarpine failed to alter the activities of cellular COX-1 and COX-2. Collectively, our data suggest that anti-inflammatory effect of rutaecarpine is, at least in part, ascribed to the diminution of PG production through inhibition of arachidonic acid release albeit the nature of its effects on PLA(2) activity remains to be elaborated.

Differential roles of cyclooxygenase isoforms after kainic acid-induced prostaglandin E(2) production and neurodegeneration in cortical and hippocampal cell cultures.

Prostaglandins, which are cyclooxygenase (COX) products, are pathologically up-regulated, and have been proven to be closely associated with neuronal death. In this study, we investigated a role of COX isoforms (COX-1 and COX-2) in kainic acid-induced neuronal death in cultured murine cortical or hippocampal neurons. In primary cortical neurons, both indomethacin (COX-1/-2 nonselective inhibitor) and aspirin (COX-1 preferential inhibitor) reduced basal and kainic acid-induced PGE(2) production significantly and prevented neuronal cell death after kainic acid treatment. In contrast, NS398 (COX-2 selective inhibitor) had no effect on kainic acid-induced neuronal cell death. In hippocampal neurons, however, COX-2 inhibitors prevented both kainic acid-induced neuronal death and PGE(2) production. COX-2 expression was remarkably up-regulated by kainic acid in hippocampal neurons; whereas in cortical neurons, COX-2 expression was comparatively less significant. Astrocytes were unresponsive to kainic acid in terms of PGE(2) production and cell death. In conclusion, we suggest that the release of PGE(2) induced by kainic acid occurred through COX-1 activity rather than COX-2 in cortical neurons. The inhibition of PGE(2) release by COX-1 inhibitors prevented kainic acid-induced cortical neuronal death, while in the hippocampal neurons, COX-2 inhibitors prevented kainic acid-induced PGE(2) release and hippocampal neuronal death.

High glucose enhances IL-1beta-induced cyclooxygenase-2 expression in rat vascular smooth muscle cells.

The changes in vascular prostaglandin production are implicated in the derangement of vascular reactivity in diabetes. However, the mechanism of altered prostaglandin (PG) production in diabetes is largely unknown. In this study, we investigated the effect of high glucose on IL-1beta-induced PG production and the possible underlying mechanism in cultured vascular smooth muscle cell (VSMC). High glucose evoked an augmentation of IL-1beta-induced PG synthesis in a dose dependent manner and enhanced cyclooxygenase (COX) activity, which reached to maximum at 8-12 hours after stimulation. Western blot analysis supported the activity data. Protein kinase C (PKC) inhibitors, H-7 and chelerythrine, significantly inhibited the enhancement of IL-1beta-induced COX-2 expression by high glucose. The activation of PKC by PMA resulted in marked increase of PG production in low glucose group, whilst this was not the case in high glucose group. Furthermore, glucose-enhancing effect was significantly suppressed by zopolrestat, an aldose reductase inhibitor, and sodium pyruvate. These results suggest that the augmenting effect of high glucose on IL-1beta-induced PG production and COX-2 expression is, at least in part, due to increased glucose metabolism via sorbitol pathway following PKC activation.

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.

An animal model of neuropathic pain employing injury to the sciatic nerve branches.

The present study was conducted to develop a new animal model of neuropathic pain employing injury to the distal sciatic nerve branches. Under halothane anesthesia, the tibial, sural, and/or common peroneal nerves were injured and neuropathic pain behaviors were compared among different groups of rats. Different types of injury produced different levels of neuropathic pain. Rats with injury to the tibial and sural nerves showed the most vigorous mechanical allodynia, cold allodynia, and spontaneous pain. These neuropathic pain behaviors were not relieved by functional sympathectomy using guanethidine. The results suggested that injury to the tibial and sural nerves, while leaving the common peroneal nerve intact, can be used as a new animal model of neuropathic pain and that this model represents sympathetically independent pain (SIP). The present animal model is very simple to produce injury and can produce profound and reliable pain behaviors. These features enable the new animal model to be a useful tool in elucidating the mechanisms of neuropathic pain, especially SIP.

Protein kinase C inhibitors attenuate protective effect of high glucose against hypoxic injury in H9c2 cardiac cells.

In this study, we demonstrated that PKC inhibitors significantly attenuated the cardioprotective effect produced by high-glucose (22 mM) treatment for 48 h against hypoxic injury in H9c2 cardiac cells. PKC activators mimicked the cardioprotective effect of high glucose. These results suggest a possible role of PKC activation in high-glucose--induced protection.

Protein kinase C inhibitors abolish the increased resistance of diabetic rat heart to ischemia-reperfusion injury.

Protein kinase C (PKC) has been implicated in ischemic preconditioning, but whether it plays a role in the cardioprotection observed in the diabetic heart is not known. We assessed the possible role of PKC by investigating whether the inhibition of PKC with staurosporine (Stau, 0.01 microM) or chelerythrine (Chel, 1 microM) can abolish the increased resistance to ischemia (25 min)-reperfusion (30 min) injury in Langendorff perfused hearts from streptozotocin-induced 4-week diabetic rats. In the diabetic heart, pre-ischemic left ventricular developed pressure (LVDP), double product (DP: LVDPxheart rate/1,000), +/- dP/dt(max) and coronary flow rate (CFR) were all reduced compared to the control. The pretreatment with Stau or Chel significantly improved these parameters. The post-ischemic contractile function was recovered to a greater extent in the diabetic heart (116.9 +/- 20.5% of pre-ischemic DP) than in the control (23.3 +/- 2.3% of pre-ischemic DP), indicating the increased resistance of the diabetic heart to ischemia-reperfusion injury. The treatment with Stau or Chel abolished the enhanced recovery in the diabetic heart (36.0 +/- 14.6 and 54.1 +/- 12.8% of pre-ischemic DP, respectively). The reduction in post-ischemic end diastolic pressure (EDP) and lactate dehydrogenase (LDH) release in diabetes (13.5 +/- 2.5 mmHg and 27.2 +/- 6.2 U/g heart) compared to the control (55.8 +/- 2.9 mmHg and 60. 3 +/- 5.7 U/g heart) was significantly (p<0.05) increased by pretreatment with Stau (39.0 +/- 4.9 mmHg and 53.1 +/- 7.6 U/g heart) or Chel (36.2 +/- 3.0 mmHg and 48.8 +/- 4.3 U/g heart). Neither Stau nor Chel had any influence on the post-ischemic values of LVDP, DP, +/- dP/dt(max), EDP and LDH release in the control heart. In the conclusion, the present results suggest that PKC activation may, at least in part, contribute to the increased resistance of the diabetic heart to ischemia-reperfusion injury.

Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus.

Cyclooxygenase-2 (COX-2) in the brain is expressed constitutively and also increased in pathological conditions such as seizure, cerebral ischemia, and inflammation. This study examined the role of COX-2 in kainic acid-induced seizure and in the following neuronal death by using selective inhibitors. Systemic kainate injection (50 mg/kg; i.p.) in mice evoked seizure within 15 min and led to 29% mortality within 2 h. TUNEL-positive neuronal death peaked at 3 days after injection and was prominent in CA(3a) regions of the hippocampus. NS-398 or celecoxib (10 mg/kg, COX-2 selective inhibitor) and indomethacin (5 mg/kg, nonselective inhibitor) exaggerated kainic acid-induced seizure activity and mortality. COX-2 selective inhibitors induced the seizure at earlier onset and more severe mortality within the first hour than indomethacin and aspirin. NS-398 also aggravated kainic acid-induced TUNEL positive neuronal death and decreased Cresyl violet stained viable neurons, and extended lesions to CA(1) and CA(3b). Kainic acid increased the levels of PGD(2), PGF(2a) and PG E(2) in the hippocampus immediately after injection. Indomethacin attenuated the production of basal and kainic acid-induced prostaglandins. In contrast, NS-398 failed to reduce until the first 30 min after kainic acid injection, during which the animals were severely seizured. It has been challenged the endogenous PGs might have anticonvulsant properties. Thus, COX-2 selective inhibitor, including nonselective inhibitor such as indomethacin, aggravated kainic acid-induced seizure activity and the following hippocampal neuronal death even with variable prostaglandin levels.

Pharmacokinetics of losartan and its metabolite, EXP3174, after intravenous and oral administration of losartan to rats with streptozotocin-induced diabetes mellitus.

The pharmacokinetics of losartan and its active metabolite, EXP3174, were investigated after intravenous and oral administration of the drug, 5 mg/kg, to control rats and streptozotocin-induced diabetes mellitus rats (SIDRs). After 1-min intravenous infusion, the mean arterial plasma concentrations and the resultant area under the plasma concentration-time curve from zero to infinity (AUC) of both losartan and EXP3174 were not significantly different between control rats and the SIDRs. However, the renal clearance (CLR) of losartan (0.181 versus 0.0815 ml/min/kg) and EXP3174 (0.0677 versus 0.0277 ml/min/kg) were significantly faster in SIDRs than in control rats due to significant increase in glomerular filtration rate. After oral administration, the mean arterial plasma concentrations and the resultant AUC of losartan (97 versus 166 micrograms min/ml) and EXP3174 (244 versus 423 micrograms min/ml) were significantly lower in SIDRs than in control rats. The absolute extent of oral bioavailability of losartan, F, (32.5 versus 55.1%) decreased considerably in SIDRs and it was possibly due to the reduced gastrointestinal absorption of losartan by gastrointestinal disorders occurring in the diabetic state. The low F in both groups of rats was at least partially due to the increase in first-pass effects. The CLR of losartan (0.207 versus 0.101 ml/min/kg) and EXP3174 (0.0615 versus 0.0196 ml/min/kg) were significantly faster in SIDRs than in control rats after oral administration of losartan.

Effects of iontophoretically applied naloxone, picrotoxin and strychnine on dorsal horn neuron activities treated with high frequency conditioning stimulation in cats.

Transcutaneous electrical nerve stimulation(TENS), acupuncture-needling, and electroacupuncture are useful non-ablative methods in medical practice for relief of pain. These procedures appear to work by causing an increased discharge in afferent nerve fibers which in turn modifies the transmission of impulses in pain pathways. It is known that the mechanism of analagesic effect via these maneuvers are variable depending on the stimulating parameters. For example, the endogenous opioid system is profoundly related to the mechanism when a peripheral nerve stimulation is applied with parameters of low frequency and high intensity. However, when stimulated with parameters of high frequency and high intensity, the reduced activity of dorsal horn neurons is only slightly reversed by a systemic administration of naloxone, a specific opiate antagonist. Thus, the present study was performed to investigate the neurotransmitter that concerns the mechanism of peripheral nerve stimulation with parameters of high frequency and high intensity. We used an iontophoretic application of antagonists of possible related neurotransmitters. The dorsal horn neuron activity which was evoked by squeezing the peripheral cutaneous receptive field, was recorded as an index of pain with a microelectrode at the lumbo-sacral spinal cord. Naloxone, picrotoxin and strychnine were applied at 200nA during a period of conditioning nerve stimulation. We observed the effects of these drugs on the change of dorsal horn neuron activities. The main results of the experiment can be summarized as follows. The spontaneous activity of dorsal horn neurons increased in the presence of glutamate and decreased with GABA. It did not change with naloxone, picrotoxin or strychnine. When naloxone was applied iontophoretically during peripheral nerve stimulation, there was no statistically significant analgesic effect compared with that of the control group. When picrotoxin was applied iontophoretically during peripheral nerve stimulation, the analgesic effect was reduced. When strychnine was applied, the analgesic effect was reduced but did not show a statistically significant difference with the control group. These results suggested that the GABAergic system may have been partially related in the analgesic action of peripheral nerve stimulation with parameters of high frequency and high intensity.

Mechanism of transmission and modulation of renal pain in cats; effect of nucleus raphe magnus stimulation on renal pain.

Initially, when periaqueductal gray (PAG) is electrically stimulated, analgesia is induced, and this phenomenon is called stimulation-produced analgesia. Nucleus raphe magnus (NRM) as well as PAG are known to be the potent analgesic centers. NRM could modulate the nociceptive response of spinal cord neurons through spinally projecting fibers. However, as well as the above analgesic effects have been confined to the somatic pain, it was variable according to species, and the analgesic effect by NRM stimulation on the visceral pain was not yet clarified. In this study the analgesic effect by NRM stimulation on the visceral pain was examined through recording the activities of the dorsal horn neurons with renal input and renal pain, as a type of visceral pain. The renal pain was induced by ureteral occlusion or renal arterial occlusion, which in turn activated the renal mechanoreceptor or chemoreceptor. These cells had concomitant somatic input. In order to compare the effects of NRM stimulation on the renal pain with somatic pain, the somatic stimulation such as squeezing was conducted on the peripheral receptive field. The main results are summarized as follows: 1) After an electrical stimulation of NRM, spontaneous activities of dorsal horn neurons with renal input were reduced to 73.3 +/- 9.7% of the control value. 2) After an electrical stimulation of NRM, activities of dorsal horn neurons with renal input evoked by a brush, a type of non-noxious stimuli, did not change significantly. But the activities by a squeeze, a type of noxious stimuli, the activities were reduced to 63.2 +/- 7.2% of the control value. 3) After an electrical stimulation of NRM, activities of dorsal horn neurons with renal input evoked by occlusion of ureter or renal artery were reduced to 46.7 +/- 8.8% and 49.0 +/- 8.0% of the control value respectively. 4) The inhibitory effect of NRM on the dorsal horn neurons with renal input did not show any difference between renal A delta fiber and C fiber group. 5) By the electrical stimulation of NRM, the activities evoked by ureteral occlusion showed more reduction in the high threshold cell group than in the wide dynamic range cell group. These results suggest that activation of NRM can alleviate the renal pain as well as the somatic pain by modulating the dorsal horn neurons activities.

Mechanism of transmission and modulation of renal pain in cats; effects of transcutaneous electrical nerve stimulation on renal pain.

Transcutaneous electrical nerve stimulation (TENS) has widely been employed as a method of obtaining analgesia in medical practice. The mechanisms of pain relief by TENS are known to be associated with the spinal gate control mechanism or descending pain inhibitory system. However, most of the studies concerning the analgesic effects and their mechanisms for TENS have dealt with somatic pain. Thus, in this experiment, we investigated the analgesic effects of TENS on renal pain as a model of visceral pain, and the characteristics of the dorsal horn cells with renal inputs. The renal pain was induced by acute occlusion of the ureter or renal artery. The main results are summarized as follows: 1) The renal nerve was composed of A beta, A delta and C fiber groups; the thresholds for each group were 400-800 mV, 1.1-1.5 V, and 2.1-5.8 V, respectively. 2) The dorsal horn cells tested received A and/or C afferent fibers from the kidney, and the more C inputs the dorsal horn cells had, the greater was the response to the stimuli that elicited the renal pain. 3) 94.9% of cells with renal input had the concomitant somatic receptive fields on the skin; the high threshold (HT) and wide dynamic range (WDR) cells exhibited a greater responses than low threshold (LT) cells to the renal pain-producing stimuli. 4) TENS reduced the C-responses of dorsal horn cells to 38.9 +/- 8.4% of the control value and the effect lasted for 10 min after the cessation of TENS. 5) By TENS, the responses evoked by acute occlusion of the ureter or renal artery were reduced to 37.5 +/- 9.7% and 46.3 +/- 8.9% of the control value, respectively. This analgesic effects lasted 10 min after TENS. 6) The responses elicited by squeezing the receptive fields of the skin were reduced to 40.7 +/- 7.9% of the control value and the effects lasted 15 min after TENS. These results suggest that most of dorsal horn cells with renal inputs have the concomitant somatic inputs and TENS can alleviate the renal pain as well as somatic pain.

Effect of peripheral nerve stimulation on the dorsal horn cell activity in cats with cutaneous inflammation.

There are some reports showing that an experience of long-enduring pain causes a change in the pain transmission system, suggesting a plastic nature of the nociceptive system. However, most of the studies concerning the analgesic effect of peripheral nerve stimulation dealt with normal animal or human subjects. So, the present study was undertaken to investigate the effect of peripheral nerve stimulation on the dorsal horn cell activity using a tonic pain model, which was made by producing a cutaneous inflammation. The main results are summarized as follows. 1) The evoked activity by electrical or natural stimulation as well as spontaneous activity was enhanced, and the receptive field size was also expanded by the inflammation. 2) Peripheral nerve conditioning stimulation reduced the C-response of the dorsal horn cell in the normal and inflamed group, and the degree of inhibition between the two groups showed no significant difference. 3) Inhibition of the C-response of the dorsal horn cells by peripheral conditioning stimulation was completely reversed by naloxone in the inflamed group whereas there was a partial block in the normal group.