Caffeic acid phenethyl ester extends survival of a mouse model of amyotrophic lateral sclerosis.

There is currently very limited effective pharmacological treatment for amyotrophic lateral sclerosis. Recent evidence suggests that caffeic acid phenethyl ester has strong anti-inflammatory, anti-oxidative, and anti-neuronal death properties; thus, the present study tested the effects of caffeic acid phenethyl ester in mice expressing a mutant superoxide dismutase (SOD1(G93A)) linked to human amyotrophic lateral sclerosis. Administration of caffeic acid phenethyl ester after symptom onset significantly increased the post-onset survival and lifespan of SOD1(G93A) mice. Moreover, immunohistochemical analysis detected less activation of microglia and astrocytes and higher motor neuron counts at an early symptomatic stage (7 days following onset) in the spinal cords of SOD1(G93A) mice given caffeic acid phenethyl ester treatment. Additionally, lower levels of phosphorylated p38, a mitogen-activated protein kinase that is involved in both inflammation and neuronal death, were observed in the spinal cords of SOD1(G93A) mice treated with caffeic acid phenethyl ester for 7 days. These results indicate that caffeic acid phenethyl ester may represent a novel and effective therapeutic for the treatment of amyotrophic lateral sclerosis, and these significant neuroprotective effects observed in a commonly used amyotrophic lateral sclerosis mouse model validate the therapeutic potential of caffeic acid phenethyl ester for slowing disease progression by attenuating the neuroinflammation and motor neuron cell death associated with clinical amyotrophic lateral sclerosis pathology.

Caffeic acid phenethyl ester prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration.

Parkinson's disease is associated with the loss of dopaminergic neurons in the substantia nigra and decreased striatal dopamine levels. We now report that caffeic acid phenethyl ester (CAPE), an active component of propolis, attenuated dopaminergic neurodegeneration and dopamine loss in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease. The neuroprotective effect of CAPE was associated with marked reductions in inducible nitric oxide synthase (iNOS) and caspase 1 expression. Additionally, CAPE inhibited MPP+-induced neurotoxicity in vitro and directly inhibited MPP+-induced release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Thus, CAPE may have beneficial effects in slowing or preventing the progression of Parkinson's disease and other neurodegenerative disorders.

Caffeic acid phenethyl ester prevents cerebellar granule neurons (CGNs) against glutamate-induced neurotoxicity.

Caffeic acid phenethyl ester (CAPE) is an active component of propolis obtained from honeybee hives and is found to have the following properties: anti-mitogenic, anti-carcinogenic, anti-inflammatory, immunomodulatory, and antioxidant. Recent reports suggest that CAPE also has a neuronal protective property against ischemic injury. Since excitotoxicity may play an important role in ischemia, in this study, we investigated whether CAPE could directly protect neurons against excitotoxic insult. We treated cultured rat cerebellar granule neurons (CGNs) with excitotoxic concentrations of glutamate in the presence or absence of CAPE and found that CAPE markedly protected neurons against glutamate-induced neuronal death in a concentration-dependent fashion. Glutamate-induced CGNs death is associated with time-dependent activation of caspase-3 and phosphorylation of p38, both events of which can be blocked by CAPE. Treating CGNs with specific inhibitors of these two enzymes together exerts a synergistic neuroprotective effect, similar to the neuroprotective effect of CAPE exposure. These results suggest that CAPE is able to block glutamate-induced excitotoxicity by inhibiting phosphorylation of p38 and caspase-3 activation. This finding may further help understanding of the mechanism of glutamate-induced neuronal death and CAPE-induced neuroprotection against excitotoxicity.

Cellular immunity after ethanol exposure and burn injury: dose and time dependence.

Acute ethanol exposure prior to burn injury increases the immune dysfunction seen with burn alone, which has been partially attributed to increased circulating and splenic macrophage production of interleukin-6 (IL-6). The current studies examined the effect dose and timing of ethanol exposure prior to burn on cellular immunity. Mice with high (300 mg/dl) circulating levels of ethanol at the time of burn demonstrated further suppression of the delayed type hypersensitivity (DTH) and splenocyte proliferative responses in comparison to mice with moderate (100 mg/dl) ethanol levels. Interestingly, the increase in macrophage IL-6 secretion seen at the moderate dose was not augmented at the high dose; however, the circulating IL-6 levels did reveal a further increase at the high ethanol dose. There were no alterations in splenocyte subset populations and/or apoptosis at the moderate vs. the high ethanol dose. Moderate ethanol exposure 24 h, in comparison to 30 min, before injury resulted in similar decreases in the DTH. These results suggest that the dose-dependent effects of ethanol on immunity following burn injury are not the result of splenic macrophage IL-6 production as shown at the moderate dose and that the immune suppressive effects of ethanol in this model persist after it is cleared from the circulation.

Ethanol exacerbates T cell dysfunction after thermal injury.

To understand the mechanism of suppressed immunity following alcohol consumption and thermal injury, we analyzed T cell functions in a mouse model of acute alcohol exposure and burn injury. Mice with blood alcohol levels at approximately 100 mg/dl were given a 15% scald or sham injury. Mice were sacrificed 48 h after injury. Our data demonstrated a 20-25% decrease in Con A-mediated splenic T cell proliferation (p<0.01) and 45-50% decrease in interleukin-2 (IL-2) production (p<0.01) following burn injury compared to the T cells from sham animals. A further decrease in the proliferation (25-30%) and IL-2 production (40-45%) was detected in T cells derived from burned animals receiving alcohol as compared to burn alone. No significant change in the proliferation and IL-2 production was observed in splenic T cells derived from sham-injured mice regardless of alcohol exposure. Additionally, there was no demonstrable difference in splenocyte apoptosis in any treatment group. These results suggest that alcohol consumption prior to burn injury causes a greater decrease in T cell proliferation and IL-2 production compared to either burn or alcohol injury alone that may further attenuate the cell-mediated immunity and thus enhance susceptibility to infection.

The role of interleukin 6 in interferon-gamma production in thermally injured mice.

Following traumatic injury, patients suffer from compromised immunity increasing their susceptibility to infection. Previous studies from this laboratory demonstrated that female BALB/c mice subjected to a 15% total body surface area (TBSA) scald injury exhibit a decrease in cell-mediated immunity 10 days post-burn. Studies described herein revealed that concanavalin A (Con A; 2 microg/ml)-stimulated splenocytes from sham treated animals produced 3557+/-853 pg/ml of IFN-gamma while splenocytes from burn injured animals released two-fold more cytokine (P<0.05). To determine whether leukocyte production of IFN-gamma was under the influence of macrophages, splenic macrophage supernatants generated from burned animals were incubated with splenic lymphocytes from sham and burn animals. The amount of IFN-gamma released by lymphocytes from sham animals increased when cultured with macrophages from burned mice (P<0.05). This suggests that the increase in IFN-gamma production by unfractionated splenocytes in burned mice relative to sham treated animals is macrophage-dependent. Macrophage supernatants from burned mice released twice as much IL-6 as supernatants from sham animals (P<0.05), and when IL-6 was blocked in vivo, the amount of IFN-gamma production in burned mice decreased to sham levels (P<0.05). Thus, IL-6 mediates IFN-gamma production following burn.

Anti-interleukin-6 antibody treatment restores cell-mediated immune function in mice with acute ethanol exposure before burn trauma.

BACKGROUND: Previous studies from this laboratory reported that suppression of cell-mediated immune function was coincident with elevated interleukin (IL)-6 production after acute ethanol exposure before burn trauma, compared with either insult alone. The goal of this study was to investigate whether treatment with an anti-IL-6 antibody could restore immunocompetence in mice subjected to burn trauma with previous exposure to alcohol, as assessed by delayed-type hypersensitivity (DTH) and mitogen-induced splenocyte proliferative responses. METHODS: Mice given an ethanol treatment designed to reach a blood alcohol level of 100 mg/dl before a 15% total body surface area burn injury were treated with an anti-IL-6 antibody at 30 min and 24 hr postinjury. RESULTS: Burn/ethanol mice exhibited a 91% suppression of the DTH response ( < 0.01) and a 76% suppression of mitogen-induced splenocyte proliferation (p < 0.01) at 48 hr postinjury, along with increased levels of circulating and splenic macrophage-derived IL-6, compared with all other treatment groups. After anti-IL-6 antibody administration to burn/ethanol mice, there was a 25% (p < 0.05) and 63% (p < 0.01) recovery of the DTH and splenocyte proliferative responses, respectively. Addition of exogenous IL-6 to splenocyte cultures isolated from anti-IL-6 antibody-treated burn/ethanol mice resulted in a 70% inhibition of mitogen-induced proliferative responses (p < 0.03). CONCLUSIONS: These data confirm previous findings that burn in combination with acute ethanol exposure suppresses cell-mediated immune function compared with either insult alone. Furthermore, the ability of the anti-IL-6 antibody treatment to improve cellular immune responses in the burn/ethanol group suggests that blocking this cytokine may be beneficial for the ethanol-exposed, thermally injured individual.

Dose-dependent effect of ethanol on hepatic oxidative stress and interleukin-6 production after burn injury in the mouse.

BACKGROUND: Burned patients with detectable blood alcohol levels (BAL) show an elevated mortality rate. Interleukin (IL)-6 and reactive oxygen species (ROS) production is stimulated independently by alcohol and burn injury. The aim of the study was to determine whether increasing levels of alcohol differentially enhance the hepatic production of IL-6 and ROS after burn in a murine model of dorsal scald injury. Groups of mice received either saline or alcohol intraperitoneally to reach a BAL of 100 mg/dl or 300 mg/dl at the time of burn (15% total body surface scald) or sham injury. RESULTS: Burn injury alone resulted in a low mortality rate at 24 hr after injury as did the burn group with a BAL of 100 mg/dl (15%), whereas 57% of the mice burned with a BAL of 300 mg/dl did not survive (p = 0.02). Twenty-four hours after burn or sham injury, IL-6 levels were measured by enzyme-linked immunosorbent assay in serum and liver. In the saline-treated group, IL-6 circulating and hepatic levels rose after burn injury (p < 0.03). Circulating IL-6 levels in sham mice increased 1.5-fold in the group with a BAL of 100 mg/dl and 3-fold in those with a BAL of 300 mg/ml (p = 0.005 versus burn-injured, saline-treated). IL-6 hepatic production after burn injury was higher in the mice with a BAL of 300 mg/dl than in those with a BAL of 100 mg/dl and the saline-treated group (p = 0.001). Among the burned mice, alcohol exposure increased hepatic ROS production, measured by lipid peroxidation and protein oxidation, in a dose-dependent manner. CONCLUSIONS: Alcohol enhances in a dose-dependent manner the hepatic production of IL-6 induced by burn injury through the modulation of oxidative stress. The increased mortality rate of mice exposed to alcohol and burn injury may be due to the adverse effect on immune function induced by IL-6 elevation.