抑制糖原合成酶激酶3活性对Toll样受体4介导肝脏炎症反应的调节作用

Inhibition of glycogen synthase kinase 3β activity regulates Toll-like receptor 4-mediated liver inflammation

目的研究抑制糖原合成酶激酶3p（GSK-3p）活性对Toll样受体4介导的炎症反应的调节作用。方法以C57BL／6小鼠为研究对象，分别建立不同灌注时间的小鼠热缺血再灌注损伤模型以及通过腹腔注射脂多糖（LPS）建立小鼠肝脏炎症模型；通过小鼠的股骨分离出骨髓干细胞，经过分化培养获得骨髓来源的巨噬细胞，随后通过LPS激活TLR4配体引发炎症细胞模型。通过SB216763抑制GSK-3D活性，分别干预小鼠肝脏炎症模型和炎症细胞模型。通过Westemblot检测肝脏丝裂原活化蛋白激酶的表达；实时定量PCR方法检测细胞炎症因子的基因表达。用SPSSl3．0统计软件进行单因素方差分析，用LSD-t检验进行组间比较。结果在肝脏缺血再灌注过程中，丝裂原活化蛋白激酶（包括ERK、JNK和p38）的磷酸化水平在灌注1h后增加，ERK、JNK和p38的活性升高，但在4h后，这种活洼激活消失，回归到起始水平。此外，LPS刺激巨噬细胞激活ERK、JNK在15rain被磷酸化而激活，p38蛋白在1h左右磷酸化而被激活。SB216763预处理一定程度上抑制了LPS刺激巨噬细胞的ERK、JNK和p38蛋白磷酸化的激活。无论是小鼠肝脏炎症模型还是炎症细胞模型，GsK-3p活性抑制均促进了抗炎细胞因子白细胞介素10（IL-10）的表达，而显著抑制了促炎细胞因子IL-12、肿瘤坏死因子（TNF）α，IL-6和IL-1D的基因表达。在对照组、炎症组及SB216763干预组小鼠炎症模型中炎症因子基因表达结果显示，IL-10相对表达量分别为0．21±0．08，0．83±0．21，1．76±0．67，F=3．16，P=0．027；IL-12相对表达量分别为0．11±0．05，0．85±0．11，0．43±0．10，F=2．67，P=0．0381TNFα相对表达量分别为0．052±0．012，8．11±0．98，3．9±0．82，F=4．13，P=0．016；IL-1β相对表达量分别为0．12±0．07，2．51±0．62，1．28±0．33，F=2．22，P=0．030；IL-6相对表达量分别为0．22±0．08，6．37±0．81，2．11±0．63，F=3．21，P=0．024。结论抑制GSK-3p活性选择性地调节肝脏中枯否细胞抗炎和促炎因子的表达从而引起肝脏缺血再灌注损伤的改善，随着促炎细胞因子被抑制，使得炎症反应所诱导的肝细胞凋亡也间接地受到有效控制。

Objective To determine the mechanism underlying the therapeutic activities of glycogen synthase kinase β （GSK3β） against hepatic ischemia-reperfusion （H-IR） injury by investigating the inhibitive effects of GSKβ3 on inflammation mediated by Toll-like receptor 4 （TLR4）. Methods C57BL/6 male mice were subjected to 90 min of warm liver cephalad lobe ischemia, followed by reperfusion for various lengths of time. The mice were divided into three groups： the H-IR untreated model （control group）, and the H-IR inflammation-induced models that received an intraperitoneal injection of purified lipopolysaccharide （LPS） endotoxin alone （inflammation group） or with pretreatment of the SB216763 GSKβ-specific inhibitor （intervention group）. To create a parallel isolated cell system for detailed investigations of macrophages, marrow-derived stem cells were isolated from femurs of the H-IR control group of mice and used to derive primary macrophages. The ceils were then divided into the same three groups as the whole mouse system： control, LPS-induced inflammation model, and inflammation model with SB216763 intervention. Differential expressions of inflammation-related proteins and genes were detected by Western blotting and real-time quantitative PCR, respectively. Results The phosphorylation levels of ERK, JNK and p38 MAPK were induced in liver at 1 h after reperfusion, but then steadily decreased and returned to baseline levels by 4 h after reperfusion. In addition, the phosphorylation levels of ERK and JNK were induced in macrophages at 15 min after LPS stimulation, while the phosphorylation level of p38 MAPK was induced at 1 h; SB216763 pretreatment suppressed the LPS-slimulated ERK, JNK and p38 phosphorylation in rnacrophages. In the mouse model, GSK3β activity was found to promote the gene expression of anti-inflammatory cytokine IL-10 （control： 0.21 ± 0.08, inflammation： 0.83 ± 0.21, intervention： 1.76 ± 0.67; F = 3.16, P = 0.027） but to significantly inhibit the gene expression of pro-inflammatory cytokines IL-12 （control： 0.11 ± 0.05, inflammation： 0.85 ± 0.11, intervention： 0.43 ± 0.10;F= 2.67,P = 0.038）, TNF-ct, （control： 0.052 ± 0.012, inflammation： 8.11 ± 0.98, intervention： 3.9 ± 0.82; F= 4.13,P = 0.016）, IL-6 （control： 0.22 ± 0.08, inflammation： 6.37 ± 0.81, intervention： 2.11 ± 0.63; F = 3.21, P = 0.024）, and IL-113 （control： 0.12 ± 0.07, inflammation： 2.51 ± 0.62, and intervention： 1.28 ± 0.33; F = 2.22, P = 0.030）. Conclusion Inhibition of GSK3β selectively regulates the expression of anti-inflammatory and pro-inflammatory cytokines in liver Kupffer cells （liver macrophages）. This process may be one of the mechanisms underlying the ability of GSK3β to ameliorate hepatic ischemia-reperfusion injury, possibly because inhibition of pro-inflammatory cytokines may indirectly mediate liver cell apoptosis.