Objective      To investigate the effect of lipopolysaccharide (LPS) tolerance and glycogen synthase kinase 3 (GSK-3) inhibitor on GSK-3 functional activity.        Methods      A total of 36 male healthy SD rats were randomly divided into control group (NC)

LPS tolerance group (ET) and LiC1 treatment group (LiC1)

with 12 rats in each group. ET group was established through intraperitoneal injection at 0.015

0.030 and 0.060 mg/kg on the first

second and third day. LiC1 group was also received intraperitoneal injection of 0.25 ml LiCl of 2.5 mol/L at the same time point

while the drinking water contained 3 mmol/L LiCl. NC group was intraperitoneally injected PBS 0.25 ml every day. Then each group was divided into the  LPS  after attack group and the  LPS  before attack group  on the basis that whether lethal dose LPS (10 mg/kg) was accepted. In the  LPS after attack group

the rats received 10 mg/kg LPS intraperitoneal injection

and the  LPS  before attack group  took the same volume of PBS intraperitoneal injection. After 12 h of last injection

the rats were killed to collect the liver tissues. Total activity of GSK-3 in the liver tissue was detected by [γ-32P] incorporation. mRNA expression of GSK-3α and GSK-3β was detected by RT-PCR

and expression of GSK-3α/β and pGSK-3α/β was measured by Western blotting. Calpain activity were analyzed by Coomassie brilliant blue staining. The distribution of GSK-3 in the sub-cells RAW264.7 were observed by laser scanning confocal microscopy.       Results      Attack of LPS resulted in a significant decrease in GSK-3 kinase activity of liver tissue in ET-group (1 311±57

P=0.023 5)

while there was no significant difference in other groups before and after the attack of LPS (P0.05). The mRNA expression and the total protein expression of GSK-3α and β had no statistically significant effect on LPS tolerance group and LiCl pretreatment group before and after the attack of LPS (P0.05). Before the LPS attack

GSK-3α phosphorylation in the NC

ET and LiCl groups had no significant increase (P0.05). But after LPS attack

GSK-3α phosphorylation in LiCl group was significantly increased (3.07±0.62

P0.05). Before the LPS attack

GSK-3β phosphorylation of ET and LiCl groups was significantly increased compared with the NC group (ET: 1.49±0.26

LiCl: 2.05±0.43

P0.05). But after LPS challenge

only GSK-3β phosphorylation of the LiCl group was increased (1.54±0.32

P0.05). Meanwhile

the GSK-3α in the ET group was seriously degraded after large dose of LPS attack. LPS attack obviously enhanced the Calpain activity in each group（NC:2.78±0.53;ET:2.45±0.22;LiCl:2.39±0.86

P0.05 when compared with the  LPS  before attack group）

but there was no outstanding difference between groups（P0.05）. The GSK-3 subcellular positioning analysis showed that the GSK-3 gathered in the cell nucleus decreased during the tolerance

which mainly focused in the cytoplasm. With the stimulation of LiCl at different concentrations

the cell nucleus GSK-3 was also declined

but not substantially. With large dose of LPS attack

t GSK-3 was decreased in the cell nucleus

and mainly concentrated in the cytoplasm and surrounding the cell nucleus.       Conclusion      LPS tolerance and LiCl pretreatment may change the functional activity of GSK-3

but the influence and mechanism are not totally equivalent. The mechanism of LPS tolerance may be connected with restraining GSK-3 kinase activity

inducing GSK-3 phosphorylation

promoting GSK-3α cracking

and inducing GSK-3 to transfer from cell nucleus to cytoplasm.