西罗莫司通过磷脂酰肌醇3激酶/蛋白激酶B/雷帕霉素靶蛋白通路对淋巴管畸形的治疗作用

Therapeutic effect of sirolimus on lymphatic malformations through the phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin pathway

目的探讨西罗莫司通过磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)通路对淋巴管畸形的治疗机制研究。方法30只雌性Wistar大鼠(河南实验动物中心)在颈部皮下注射弗氏不完全佐剂构建大鼠淋巴结畸形模型,按照随机数字表法分为模型组、西罗莫司低剂量组和高剂量组,每组10只,西罗莫司低剂量组和高剂量组经尾静脉注射西罗莫司25 mg/kg和100 mg/kg,模型组注射等体积生理盐水。3组大鼠治疗6周后,计算病变组织质量和体积变化;蛋白质免疫印迹(Western blot)分析PI3K/Akt/mTOR通路及其底物p70S6K活性。免疫组织化学分析病变部位细胞核增殖抗原(Ki-67)和血管内皮生长因子(VEGF)表达水平。测量体重分析药物的安全性。组间比较采用单因素方差分析。结果模型组大鼠病变部位质量[(0.51±0.06)g]明显高于西罗莫司低剂量组[(0.41±0.04)g],差异有统计学意义(t=4.342,P<0.05)。西罗莫司低剂量组大鼠病变部位质量[(0.41±0.04)g]明显高于西罗莫司低剂量组[(0.24±0.05)g],差异有统计学意义(t=7.726,P<0.05)。模型组大鼠病变部位体积[(297.60±22.10)mm^(3)]明显高于西罗莫司低剂量组[(245.20±23.33)mm^(3)],差异有统计学意义(t=5.155,P<0.05)。西罗莫司低剂量组大鼠病变部位体积[(245.20±23.33)mm^(3)]明显高于西罗莫司高剂量组[(189.60±22.84)mm^(3)],差异有统计学意义(t=5.385,P<0.05)。模型组大鼠淋巴管畸形病灶Ki-67和VEGF表达水平(171.30±17.55、208.70±23.57)明显高于西罗莫司低剂量组(136.70±13.06、156.80±16.31),差异有统计学意义(t=5.002,5.727,P<0.05)。西罗莫司低剂量组大鼠淋巴管畸形病灶(136.70±13.06、156.80±16.31)明显高于西罗莫司高剂量组(99.30±9.18、113.20±12.65),差异有统计学意义(t=7.411、6.681,P<0.05)。模型组大鼠淋巴管畸形病灶磷酸化mTOR和磷酸化p70S6K表达水平(0.98±0.07、0.88±0.10)明显高于西罗莫司低剂量组(0.79±0.07、0.60±0.09),差异有统计学意义(t=7.704、6.453P<0.05)。西罗莫司低剂量组大鼠淋巴管畸形病灶磷酸化mTOR和磷酸化p70S6K表达水平(0.79±0.07、0.60±0.09)明显高于西罗莫司高剂量组(0.50±0.13、0.36±0.10),差异有统计学意义(t=6.231、5.691,P<0.05)。结论西罗莫司治疗淋巴管畸形具有较好的效果,且安全性较高,可能与其抑制mTOR激酶活性有关。

Objective To investigate the therapeutic mechanism of sirolimus on lymphatic malformations through the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway.Methods Totally,30 female Wistar rats from Henan Experimental Animal Center were randomly divided into model group,sirolimus low dose group,and sirolimus high dose group by a random number table.Rats in Sirolimus low dose group and sirolimus high dose group were injected with 25 mg/kg and 100 mg/kg sirolimus through the tail vein respectively,and the model group was injected with equivalent volume of saline.After 6 weeks of treatment,the mass and volume changes of the lesion tissue were calculated in the three groups.The activity of PI3K/Akt/mTOR pathway and its substrate p70S6K was analyzed by Western blotting.The expression levels of proliferation cell nuclear antigen(Ki-67)and vascular endothelial growth factor(VEGF)in the lesion site was detected by immunohistochemistry.The safety of drugs was measured.The measurement data between groups were compared using one-way analysis of variance.Results The lesion site mass of the model group[(0.51±0.06)g] was significantly greater than that of the low dose sirolimus group[(0.41±0.04)g,t=4.342,P<0.05].The lesion site mass in the low dose sirolimus group[(0.41±0.04)g] was significantly greater than that in the low dose sirolimus group[(0.24±0.05)g,t=7.726,P<0.05].The volume of the lesion site in the model group[(297.60±22.10)mm^(3)]was significantly greater than that in the low dose sirolimus group[(245.20±23.33)mm^(3),t=5.155,P<0.05].The volume of lesion sites in the low dose sirolimus group[(245.20±23.33)mm]was significantly greater than that in the high dose sirolimus group[(189.60±22.84)mm^(3),t=5.385,P<0.05].The expression levels of Ki-67 and VECF in the lymphatic malformation lesions of rats in the model group(171.30±17.55,208.70±23.57)were significantly higher than those in the low dose sirolimus group(136.70±13.06,156.80±16.31,t=5.002,5.727,P<0.05).The lesion of lymphatic malformation in the low dose sirolimus group(136.70±13.06,156.80±16.31)was significantly increased as compared with in the high dose sirolimus group(99.30±9.18,113.20±12.65,t=7.411,6.681,P<0.05).The expression levels of phosphorylated mTOR and phosphorylated p70S6K in the lymphatic malformation lesions in the model group(0.98±0.07,0.88±0.10)were significantly higher than those in the low dose sirolimus group(0.79±0.07,0.60±0.09,t=7.704,6.453 P<0.05).The expression levels of phosphorylated mTOR and phosphorylated p70S6K in the lymphatic malformation lesions in the low dose sirolimus group(0.79±0.07,0.60±0.09)were significantly higher than those in the high dose sirolimus group(0.50±0.13,0.36±0.10,t=6.231,5.691,P<0.05).Conclusion Sirolimus is effective and safe in the treatment of lymphatic malformations,which may be related to the inhibition of mTOR kinase activity.