不同类型GSK-3β抑制剂对重症急性胰腺炎大鼠肾损伤的作用及量效关系

The effects of different GSK-3β inhibitors and dose-response relationship in severe acute pancreatitis associated kidney injury in rats

目的 观察TDZD-8 干预大鼠重症急性胰腺炎（SAP）肾损伤的量效关系，并将三种常用GSK-3β 抑制剂TDZD-8、氯化锂（LiCL）、SB216763 对该模型的作用效果进行对比，以探讨针对SAP 并发肾损伤大鼠模型最有效的GSK-3β 抑制剂类别及其有效、安全的最佳剂量。方法 96 只SPF 级雄性Wistar 大鼠，随机（随机数字法）分为8 组（n=12） ：假手术组（SO 组）、重症急性胰腺炎组（SAP 组）、TDZD-8 预处理组0.25、0.5、1.0、2.0 mg/kg（TD 组，分别标记为TD1、TD2、TD3、TD4 组），LiCL 预处理组 （L 组） 和SB216763 预处理组（SB 组），胰胆管逆行注射5% 牛磺胆酸钠制作SAP模型。术后12 h 剖杀各组大鼠，测定各组大鼠腹水量、血清AMY、Cr、BUN 和ALT 水平，并观察胰腺、肾脏组织病理学变化。结果 SAP 组腹水量、AMY、Cr、BUN、ALT 水平以及胰腺、肾脏病理评分均较SO 组显著升高（P〈0.05） ；TD1 组几乎对SAP 无缓解作用，TD2、TD3、TD4、L 组和SB 组均能不同程度地减少SAP 大鼠的腹水量，降低血清AMY、Cr、BUN 水平，并显著降低胰腺组织病理评分，差异有统计学意义（P〈0.05） ；TD2、TD3 均能不同程度地减少ALT 水平（P〈0.05），而TD4 组ALT 水平较高，与SAP 组相似；TD2 对各个指标的效果不如TD3 组作用显著，两者比较各项指标差异均有统计学意义（P〈0.05）。 对SAP 大鼠腹水量和ALT 水平的改善作用，TD 组中最佳剂量组TD3组与L 组和SB 组比较，差异无统计学意义（P〉0.05） ；而TD3 组的AMY、Cr、BUN 水平以及胰腺、肾脏病理评分均较L 组和SB 组降低更显著，差异有统计学意义（P〈0.05） ；L 组Cr、BUN 水平和胰腺、肾脏病理学评分与SB 组比较均降低更显著，差异有统计学意义（P〈0.05） ；Western blot 检测发现各组中GSK-3β 蛋白表达均处于较一致的水平，差异无统计学意义（P〉0.05） ；而p-GSK-3β ser9在 SAP 组表达低于SO 组，差异有统计学意义（P〈0.05） ；TD3、L、SB 三组中的p-GSK-3β ser9 均较SAP 组明显增强，其中以TD3 组表达最强，同时 L 组表达强于SB 组，组间比较差异均有统计学意义（P〈0.05）。结论 通过对TDZD-8、LiCL 和SB216763 对大鼠SAP 肾损伤模型的作用比较可知TDZD-8 是针对该模型最有效的GSK-3β 抑制剂。对于牛磺胆酸钠诱导的SAP 并发肾损伤大鼠模型，静脉给予TDZD-8 1 mg/kg 预处理对该模型是安全有效的最佳剂量。

Objective To observe the dose-response relationship of the GSK-3β inhibitor TDZD- 8 in severe acute pancreatitis （SAP） associated kidney injury in rats. In order to identify the most effective class of GSK-3β inhibitor and its effective and reasonable safe dose in SAP associated kidney injury model in rats by comparing three kinds of frequently-used GSK-3β inhibitor TDZD-8, lithium chloride （LiCL）, SB216763 in this model. Methods Totally 96 SPF male Wistar rats were randomly（random number） divided into 8 groups （n=12）： sham operation group （SO group）, severe acute pancreatitis group （SAP group）, TDZD-8 pretreatment groups （TD group, marked TD1, TD2, TD3 and TD4 group, respectively） at different dosage （0.25, 0.5, 1.0 and 2.0 mg/kg）, LiCL pretreatment groups （L group, 40 mg/kg）, and SB216763 pretreatment group （SB group, 1 mg/kg）. SAP model was induced by retrograde infusion of 5% sodium taurocholate into the biliopancreatic duct. Rats in each group were sacrificed at 12 h after operation. Then the mortality, quantity of ascites, serum AMY, Cr, BUN and ALT were recorded, and the pathological changes of pancreatic tissues and kidney tissues were observed. Results Compared with the SO group, the levels of ascites, serum AMY, Cr, BUN, ALT and pancreatic and renal pathologic score in the SAP group were all significantly increased （P〈0.05）. Compared with the TD~ group, quantity of ascites, serum AMY, Cr, BUN,ALT and pancreatic tissue pathological grading were reduced in different degrees in the TD2, TD3 and TD4 groups with statistically significant difference （P〈0.05）; ALT values were reduce in different degrees in the TD2 and TD3 groups as compared with the SAP group （P〈0.05）, while ALT value in the TD4 group was similar to that in the SAP group; compared with the TD2 group, all the indexes in the TD3 group were significant better （P〈0.05）; Compared with TD3 group （the best group in TD group）, the levels of ascites and serum ALT in the L group and SB group had no significant difference （P〉0.05）, but the levels of AMY, Cr, BUN, ALT, pancreatic and renal pathologic score were significantly reduced in the TD3 group than those in the L and SB groups （P〈0.05）; compared with the SB group, the values of Cr, BUN, pancreatic and renal pathologic score in the L group were lower （P〈0.05）. GSK-3B protein expression in all groups showed no obvious difference （P〉0.05）, while p-GSK-3β set9 protein expression in the SAP group was lower than that in the SO group （P〈0.05）, and p-GSK-3β ser9 protein expression in the TD3, L and SB groups were stronger than that in the SAP group. Among them, p-GSK-3β ser9 protein expression was highest in the TD3 group, followed by the L group, finally the SB group, and the differences were statistically significant （P〈0.05）. Conclusions Among the three different GSK-3β inhibitors, TDZD-8 is the most effective GSK-3β inhibitor for SAP associated with kidney injury in rats. The GSK-3β inhibitor TDZD-8 1 mg/kg administered intravenously is safe, effective and optimal dosage for attenuating the severity of severe acute pancreatitis associated with kidney injury.