脂多糖预处理对GSK-3功能活性的影响

目的探讨脂多糖(lipopolysaccharide,LPS)耐受和糖原合成酶激酶-3(glycogen synthase kinase 3,GSK-3)抑制剂对其功能活性的影响。方法雄性健康SD大鼠36只,随机数字表法分为对照组(NC)、LPS耐受组(ET)和LiCl处理组(LiCl),每组12只;LPS耐受组(ET)于第1、2、3天接受0.015、0.030和0.060 mg/kg LPS腹腔注射;LiCl处理组(LiCl)同样时间点腹腔注射2.5 mol/L LiCl 0.25 ml,饮水含3 mmol/L LiCl;对照组每天腹腔注射磷酸盐缓冲液(PBS)0.25 ml;处理结束后每组再按是否接受致死量LPS(10 mg/kg)攻击而分为LPS攻击前和LPS攻击后2个亚组,LPS攻击后组按10 mg/kg腹腔注射LPS,LPS攻击前亚组等体积的PBS腹腔注射,12 h后取材。按照γ-32P]放射性掺入法、用液体闪射仪检测肝组织GSK-3激酶总活性,RT-PCR法检测GSK-3α、GSK-3βmRNA表达,Western blot法分析GSK-3α/β、pGSK-3α/β蛋白表达,考马斯亮蓝法测定Calpain活性,激光共聚焦显微镜下观察GSK-3亚细胞定位状况。结果 LPS攻击后ET组GSK-3活性显著低于NC组LPS攻击前水平(1 311±57),P=0.023 5],其他各组在LPS攻击前后差异无显著性。LPS耐受、LiCl预处理和LPS攻击未导致GSK-3α和β的mRNA表达变化和GSK-3α和β的总蛋白表达变化(P>0.05)。LPS攻击前,NC、ET和LiCl组GSK-3α磷酸化无显著增加(P>0.05),但LPS攻击后,LiCl组GSK-3α磷酸化显著增加(3.07±0.62),P<0.05]。LPS攻击前,ET和LiCl组GSK-3β磷酸化比NC组显著增加ET(1.49±0.26);LiCl(2.05±0.43),P<0.05];但LPS攻击后,只有LiCl组GSK-3β磷酸化显著增加(1.54±0.32),P<0.05]。同时,LPS耐受动物经大剂量LPS攻击后肝组织GSK-3α严重降解。LPS攻击导致各组与LPS攻击前比较Calpain显著升高NC(2.78±0.53);ET(2.45±0.22);LiCl(2.39±0.86),P<0.05],但组间比较没有显著差异(P>0.05)。GSK-3亚细胞定位分析,LPS耐受时胞核内聚集的GSK-3减少,并主要集中于细胞质内;在不同浓度LiCl刺激下,胞核GSK-3也可减少,但不显著;在大剂量LPS攻击下,胞核GSK-3减少,主要集中于细胞质内和胞核周围。结论 LPS耐受和GSK-3抑制剂LiCl预处理均可导致GSK-3功能活性改变,但影响和机制并不完全相同,LPS耐受的机制可能与抑制GSK-3激酶活性、诱导GSK-3磷酸化、促进GSK-3α裂解并诱导GSK-3从胞核向细胞质转移有关。

Effect of LPS pretreatment on GSK-3 functional activity in rats

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 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(P>0.05).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(P>0.05).Before the LPS attack,GSK-3α phosphorylation in the NC,ET and LiCl groups had no significant increase(P>0.05).But after LPS attack,GSK-3α phosphorylation in LiCl group was significantly increased(3.07±0.62,P<0.05).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,P<0.05).But after LPS challenge,only GSK-3β phosphorylation of the LiCl group was increased(1.54±0.32,P<0.05).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,P<0.05 when compared with the LPS before attack group),but there was no outstanding difference between groups(P>0.05).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.