烟酰胺腺嘌呤二核苷酸磷酸氧化酶4阻断剂对糖尿病神经病变大鼠坐骨神经凋亡的影响

目的探讨糖尿病周围神经病变(DPN)大鼠坐骨神经烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(Nox4)表达改变及Nox4阻断剂VAS2870对DPN大鼠坐骨神经凋亡的影响。 方法40只8周龄雄性SD大鼠，平均体重( 203.5±6.4)g，30只大鼠一次性腹腔注射链脲佐菌素(STZ)，其中20只患糖尿病，余10只大鼠淘汰；其余大鼠腹腔注射等体积柠檬酸溶液作为对照组(n＝10)。8周后糖尿病大鼠建立DPN模型，采用随机数字表法将DPN大鼠分为2组，VAS2870组(n＝10，尾静脉注射VAS2870 1 mg/kg，每日1次，共7 d)和DPN组(n＝10，尾静脉注射等体积0.9%无菌NaCl溶液，每日1次，共7 d)。用PowerLab数据记录分析系统检测坐骨神经运动神经传导速度(MNCV)和感觉神经传导速度(SNCV)，用荧光实时定量聚合酶链反应(PCR)检测坐骨神经Nox4、半胱氨酸天冬酶-3(caspase-3)、B细胞淋巴瘤因子-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA表达，用蛋白印迹检测坐骨神经Nox4、caspase-3、Bcl-2、Bax蛋白表达水平，采用单因素方差分析比较各组间的差异，用Tukey检验进行两两比较。 结果DPN大鼠坐骨神经MNCV(41.21±7.36)m/s]及SNCV(30.39±7.66)m/s]均低于VAS2870组(49.14±8.67)m/s，(38.95±6.24)m/s]，VAS2870对MNCV及SNCV的下降具有减轻作用(q＝3.58，4.53；均P<0.05)。DPN大鼠坐骨神经Nox4(4.72±0.42)、caspase-3(5.02±0.43)、Bax(5.63±0.82) mRNA(q＝7.02，6.40，7.65；均P<0.05)及蛋白水平(0.86±0.10，0.78±0.17，0.95±0.13，q＝7.83，9.15，6.87；均P<0.05)较对照组mRNA(5.79±0.53，6.90±0.79，7.39±0.67)及蛋白水平(0.59±0.13，0.45±0.14，0.64±0.14)表达上调，Bcl-2 mRNA(6.81±0.60，q＝6.31，P<0.05)及蛋白水平(0.44±0.12，q＝6.20，P<0.05)表达下调。VAS2870组Nox4、caspase-3、Bax mRNA(5.31±0.44，5.71±0.60，6.41±0.95)(q＝3.66，2.98，4.02，3.38；均P<0.05)及蛋白水平(0.72±0.13，0.60±0.18，0.79±0.15)(q＝4.24，5.78，3.83；均P<0.05)均低于DPN组，Bcl-2 mRNA(6.29±0.47)的及蛋白水平(0.56±0.12)表达均高于DPN组(q＝3.38，2.81；均P<0.05)，VAS2870部分逆转了Nox4、caspase-3、Bax、Bcl-2 mRNA及蛋白的表达水平。 结论DPN大鼠坐骨神经Nox4表达水平增高，通过调节凋亡相关蛋白caspase-3、Bax及Bcl-2表达促进坐骨神经凋亡；Nox4阻断剂通过减轻坐骨神经凋亡发挥恢复DPN大鼠坐骨神经传导速度的作用。

Effect of nicotinamide adenine dinucleotide phosphate oxidase 4 blocker on sciatic nerve apoptosis in diabetic neuropathy rats

ObjectiveTo investigate the nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression in sciatic nerve of diabetic peripheral neuropathy (DPN) rats, and the effect of VAS2870, a Nox4 blocker, on the apoptosis of the sciatic nerve of DPN rats. Methods8 weeks old male SD rats, with an average weight of (203.5±6.4)g, were developed diabetes after one time intraperitoneal injection of STZ. The other rats were intraperitoneally injected with equal volume citric acid solution as the control group (n=10). DPN model (n=20) was established in diabetic rats after 8 weeks, and DPN rats were divided into two groups by random number table method: VAS2870 group (n=10, VAS2870 1 mg/kg was injected into tail vein once a day for 7 days) and DPN group (n=10, 0.9% sterile NaCl solution was injected into tail vein once a day for 7 days). The motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) of sciatic nerve were detected by PowerLab data recording and analysis system. The expressions of Nox4, caspase-3, B-cell lymphoma factor-2(Bcl-2) and Bcl-2 associated X protein(Bax) mRNA of sciatic nerve were detected by fluorescence real time quantitative PCR reaction, and the expressions of Nox4, caspase-3, Bcl-2 and Bax protein of sciatic nerve were detected by western blot assay. ResultsThe MNCV (41.21±7.36)m/s] and SNCV (30.39±7.66)m/s] in DPN group were lower than those in VAS2870 group (49.14±8.67)m/s, (38.95±6.24)m/s], VAS2870 could alleviate the decrease of MNCV and SNCV of sciatic nerve(q=3.58, 4.53, P<0.05). Compared with the expression of mRNA (5.79±0.53, 6.90±0.79, 7.39±0.67) and protein (0.59±0.13, 0.45±0.14, 0.64±0.14) in control group, the expressions of Nox4 (4.72±0.42), caspase-3 (5.02±0.43), Bax (5.63±0.82) mRNA (q=7.02, 6.40, 7.65, all P<0.05) and protein (0.86±0.10, 0.78±0.17, 0.95±0.13, q=7.83, 9.15, 6.87, all P<0.05) in DPN rats were up-regulated, while the expressions of Bcl-2 mRNA (6.81±0.60, q=6.31, P<0.05) and protein (0.44±0.12, q=6.20, P<0.05)were down-regulated. Compared with DPN group, the expression of Nox4, caspase-3, Bax mRNA (5.31±0.44, 5.71±0.60, 6.41±0.95)(q=3.66, 2.98, 4.02, 3.38, all P<0.05) and protein (0.72±0.13, 0.60±0.18, 0.79±0.15) (q=4.24, 5.78, 3.83, all P<0.05) in VAS2870 group were down-regulated, Bcl-2 mRNA (6.29±0.47) and protein (0.56±0.12) were up-regulated (q=3.38, 2.81, all P<0.05), VAS2870 partially reversed the expression changes of Nox4, caspase-3, Bax, Bcl-2 mRNA and protein. ConclusionsThe expression of Nox4 is increased in the sciatic nerve of DPN rats, and the expression of caspase-3, Bax and Bcl-2 is regulated by Nox4 to promote the apoptosis of the sciatic nerve. Nox4 blocker can restore the conduction velocity of the sciatic nerve in DPN rats by reducing the apoptosis of sciatic nerve.