胰岛素对急性重症胆管炎脓毒症大鼠骨骼肌代谢的影响

目的 探讨胰岛素对大鼠急性重症胆管炎（ACST）脓毒症状态下泛素系统介导骨骼肌蛋白质分解的调理作用。方法 20只雄性SD大鼠按月龄、体重同窝饲养，随机分为假手术组（SO）、ACST脓毒症模型组（AS）、低剂量胰岛素ACST脓毒症模型组（LIAS，胰岛素给予量为2.4 mU·kg-1·min-1）、高剂量胰岛素ACST脓毒症模型组（HIAS，胰岛素给予量4.8 mU·kg-1·min-1），每组5只。所有大鼠均行正常血糖钳夹模型制备，并通过调节25%葡萄糖的输注率（GIR）将血糖维持在5～6 mmol/L的稳态；只有当血糖超过11.9 mmol/L时才需加用胰岛素。采用高效液相-色谱分析法检测伸趾长肌内3-甲基组氨酸（3-MH）；RT-PCR法检测伸趾长肌内编码泛素、蛋白酶体C2亚基的mRNA表达变化。结果 AS组大鼠伸趾长肌内3-MH含量为（3.69±0.20）nmol/g，较SO组（2.07±0.13）nmol/g显著升高（P＜0.001）；且显著高于HIAS组（2.39±0.21）nmol/g（P＜0.001）和LIAS组（2.87±0.19）nmol/g（P＜0.001）。HIAS组大鼠伸趾长肌内3-MH含量较LIAS组显著降低（P＜0.001）。AS组大鼠伸趾长肌内泛素mRNA表达较SO组明显升高，其相对表达量增加了19.70倍。LIAS组泛素mRNA相对表达量为6.96（范围为4.17～11.67）、HIAS组相对表达量为1.89（范围为1.43～2.48），与AS组比较均明显下降（P=0.028，0.009）。HIAS组泛素mRNA表达水平低于LIAS组（P=0.009）。AS组大鼠伸趾长肌内蛋白酶体C2亚基mRNA表达较SO组明显升高，其相对表达量增加了191.34倍。LIAS组蛋白酶体C2亚基mRNA相对表达量为31.12（15.74～61.39）、HIAS组蛋白酶体C2亚基mRNA相对表达量为7.67（4.08～14.50），与AS组比较表达均明显下降（P值均为0.009）。HIAS组C2亚基mRNA表达水平低于LIAS组（P=0.009）。结论 急性重症胆管炎脓毒症状态下，胰岛素可抑制骨骼肌蛋白降解，该作用可能是在基因水平通过抑制泛素-蛋白酶体通路表达和活化而实现的。

Effect of insulin on metabolism of the skeletal muscles in acute cholangitis of severe type rats with sepsis

objective To study the function of insulin on ubiquitin-proteasome induced skeletal muscles proteolysis during acute cholangitis of severe type (ACST) in rats. Methods Twenty male Sprague-Dawley rats were randomly divided into sham group (SO group), ACST sepsis group (AS group), low-dose insulin ACST sep-sis group (LIAS group, 2.4 mU·kg-1·min-1), and high-dose insulin ACST sepsis group (HIAS group, 4.8 mU· kg-1·min-1), with 5 rats in every group. All rats were performed as euglycemic insulin clamp and kept the plasma glucose level in physiologic range (5~6 mmol/L) through adjusting glucose infusion rate (GIR) of 25% glucose. When the plasma glucose was higher than 11.9 mmol/L, more insulin was used. The concentration of 3 meth-ylhistidine (3-MH) in extensor digitorium longus (EDL) muscles were determined by high performance liquid chromatography. The mRNA levels of ubiquitin and C2 subunit of proteasome in the EDL muscle was evaluated by RT-PCR. Results The concentration of 3-MH in the EDL muscles of AS group was higher (3.69±0.20 nmol/g), compared with SO group (2.07±0.13 nmol/g, P<0.001), HIAS group (2.39±0.21 nmol/g, P<0.001), and LIAS group (2.87±0.19 nmol/g, P<0.001). The concentration of 3-MH in HIAS group were decreased significantly, compared with LIAS group (P<0.001). Ubiquitin mRNA level in EDL muscles of the AS group increased signifi-cantly, compared with that in the SO group, with an increase of 19.70 times. The relative expression of ubiquitin mRNA in the LIAS group and HIAS group were 6.96 (4.17~11.67) and 1.89 (1.43~2.48), which decreased significantly (P=0.028, 0.009), compared with AS group. The ubiquitin mRNA expression level of HIAS group was lower than that of LIAS group (P=0.009). The C2 subunits mRNA level in EDL muscles of the AS group in-creased significantly, compared with that of SO group, with an increase of 191.34 times. The relative expression of C2 subunits mRNA in the LIAS group and HIAS group were 31.12 (15.74~61.39) and 7.67 (4.081~14.50), which decreased significantly (both P=0.009), compared with AS group. The C2 subunits mRNA expression level of HIAS group was lower than that of LIAS group (P=0.009). Conclusion Insulin can effectively alleviate skeletal muscles proteolysis during ACST induced sepsis in rats. This phenomenon may depend on inhibiting the activity of ubiquitin proteasome pathway at gene level.