Table 1.
Real-Time PCR primers used in this study.
Table 2.
Serum alcohol levels after the continuous infusion.
Figure 1.
Alcohol increased brain CIRP level in a time dependent manner.
WT mice were infused intravenously with alcohol as described in Materials and Methods. Whole brain tissue from WT mice was collected at 5, 10 and 15(A) CIRP mRNA expression was determined by real time RT-PCR analysis and expression levels were normalized to β-actin. (B) Tissue lysates were collected and analyzed for CIRP by Western blotting. Blots were scanned and quantified by densitometry. Band intensity of CIRP was normalized to the corresponding band intensity of β-actin. The ratio of the control group is designated as 1 for comparison. Data presented as means ± SE (n = 3–4/group) and compared by one-way ANOVA and SNK method; *p<0.05 vs. Sham.
Figure 2.
Clinical markers of organ injury were improved in CIRP−/− following alcohol exposure.
WT and CIRP−/− mice were infused intravenously with alcohol for 15 h. Serum was collected and analyzed for AST (A), ALT (B) and LDH (C) using standardized assays. Data are presented as means ± SE (n = 5/group) and compared by one-way ANOVA and SNK method; *p<0.05 vs. respective Sham and #p<0.05 vs. WT alcohol group.
Table 3.
Time-course changes in tissue injury markers and TNF-α after acute alcohol.
Figure 3.
Brain proinflammatory cytokines TNF-αand IL-1β were attenuated in CIRP−/− following alcohol exposure.
Whole brain samples from WT and CIRP−/− mice were collected at 15 h after alcohol and analyzed. TNF-α mRNA expression (A) was determined by real time RT-PCR analysis and expression levels were normalized to β-actin. Brain protein cytokine levels of TNF-α (B) and IL- 1β (C) were determined by subjecting cell lysates to standardized mouse ELISA (BD Biosciences). Data are presented as means ± SE (n = 3–5/group) and compared by one-way ANOVA and SNK method;*p<0.05 vs. Sham, #p<0.05 vs. WT alcohol groups.
Figure 4.
Alcohol upregulated gene expression of CIRP and cytokines TNF-α and IL-1β in BV2 cells.
BV2 Cells were exposed to ethanol at a concentration of 50(A), TNF-α (B), and IL-1β (C) mRNA expressions were determined by real time RT-PCR analysis and expression levels were normalized to β-actin. The ratio of the control group is designated as 1 for comparison. Data are presented as means ± SE (n = 3–5/group) and compared by one-way ANOVA and SNK method; *p<0.05 vs. Control.
Figure 5.
CIRP protein was secreted into the cell culture medium following alcohol exposure.
BV2 Cells were exposed to ethanol at a concentration of 50