Notch1抑制剂下调Ras同源基因家族激酶对糖尿病周围神经病变的影响

Effect of Notch1 inhibitor on down-regulation of Ras homolog gene family member kinase in diabetic peripheral neuropathy

目的观察Notch1抑制剂(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)对糖尿病周围神经病变(DPN)大鼠的影响及可能的作用机制。方法选取24只健康6周龄雄性SD大鼠,体质量(210±10)g,体质量范围为200~220 g,将大鼠随机分为常规组、DPN组和DAPT组,每组8只。SD大鼠适应性喂养1周后DPN组和DAPT组每只按65 mg/kg标准腹腔注射链脲佐菌素(STZ)构建糖尿病模型,常规组腹腔注射等体积的柠檬酸钠缓冲液。3 d后测尾静脉随机血糖≥16.7 mmol/L证明STZ注射成功,经进一步检测血糖≥16.7 mmol/L并持续至实验结束。糖尿病鼠模型建立后2周予DAPT组大鼠以0.1 mg/(kg·d)的DAPT腹腔注射,连续3 d。检测全部大鼠坐骨神经传导速度、痛温觉阈值,检测Notch1、Ras同源基因家族激酶(ROCK)1、ROCK2的表达。结果DPN组7 d血糖[(17.63±0.41)mmol/L]、14 d血糖[(17.95±1.22)mmol/L]、30 d血糖[(20.81±3.21)mmol/L]和60 d血糖[(26.08±3.56)mmol/L]高于常规组[(6.35±0.72)mmol/L、(6.33±0.62)mmol/L、(6.05±0.74)mmol/L、(7.54±0.65)mmol/L];DAPT组60 d血糖水平[(21.17±1.44)mmol/L]低于DPN组[(26.08±3.56)mmol/L];DPN组30 d体质量[(217.61±18.35)g]、60 d体质量[(200.75±13.19)g]小于常规组[(308.25±16.72)g、(344.13±15.34)g];DPN组60 d坐骨神经传导速度[(36.25±2.82)s]慢于常规组[(50.25±1.67)s];DAPT组60 d坐骨神经传导速度[(40.63±3.70)m/s]快于DPN组[(36.25±2.82)m/s];DPN组60 d热痛觉阈值[(25.50±1.60)s]高于常规组[(14.13±1.25)s];DAPT组60 d热痛觉阈值[(17.25±1.67)s]低于DPN组[(25.50±1.60)s],差异均有统计学意义(P<0.05)。DPN组病变髓鞘比例较常规组高,DAPT组病变髓鞘比例较DPN组低;DPN组Notch1平均免疫荧光密度较常规组显著升高,DAPT组Notch1平均免疫荧光密度较DPN组显著下降;DPN组ROCK1平均免疫荧光密度较常规组显著升高,DAPT组ROCK1平均免疫荧光密度较DPN组显著下降;DPN组ROCK2平均免疫荧光密度较常规组显著升高,DAPT组ROCK2平均免疫荧光密度较DPN组显著下降,差异均有统计学意义(P<0.05)。结论Notch1抑制剂DAPT可能通过下调ROCK对DPN产生保护作用。

Objective:To observe Notch1 inhibitor[3,5-Difluorophenacetyl-L-alanyl-S-phenyl glycine-2-butyl Ester(DAPT)]on diabetic peripheral neuropathy(DPN)rats and its possible mechanism.Methods:A total of 24 healthy 6-week-old male SD rats with a body mass of(210±10)g and a body mass range of 200 to 220 g were selected from the Endocrine Laboratory of The Affiliated Hospital of Jining Medical College.The rats were randomly divided into the conventional group,the DPN group and the DAPT group,with 8 rats in each group.One week after the SD rats were adaptively fed,the DPN group and the DAPT group of rats were each injected with streptozotocin(STZ)at a standard intraperitoneal dose of 65 mg/kg,and a diabetes model was established.The conventional group of rats were intraperitoneally injected with equal volume sodium citrate buffer.After 3 days,random blood glucose of tail vein≥16.7 mmol/L was measured,which proved that STZ injection was successful.After further detection,blood glucose≥16.7 mmol/L was measured and continued until the end of the experiment.Two weeks after the establishment of diabetic rat model,rats in the DAPT group were intraperitoneally injected with 0.1 mg/(kg·d)DAPT for 3 consecutive days.All rats’sciatic nerve conduction velocity and pain and temperature thresholds were detected,and the expressions of Notch1 and Ras homologous family kinases(ROCK)1 and ROCK2 were detected.Results:The blood glucose of DPN group at 7 days[(17.63±0.41)mmol/L],14 days[(17.95±1.22)mmol/L],30 days[(20.81±3.21)mmol/L]and 60 days[(26.08±3.56)mmol/L]in DPN group were higher than those in conventional group[(6.35±0.72)mmol/L,(6.33±0.62)mmol/L,(6.05±0.74)mmol/L,(7.54±0.65)mmol/L].The blood glucose level in the DAPT group[(21.17±1.44)mmol/L]was lower than that in the DPN group[(26.08±3.56)mmol/L].The body mass of DPN group at 30 days[(217.61±18.35)g]and 60 days[(200.75±13.19)g]was less than that of conventional group[(308.25±16.72)g,(344.13±15.34)g].The results showed that the sciatic nerve conduction velocity in the DPN group[(36.25±2.82)s]was slower than that in conventional group[(50.25±1.67)s];the sciatic nerve conduction velocity in the DAPT group[(40.63±3.70)m/s]was faster than that in the DPN group[(36.25±2.82)m/s];the thermal pain threshold of DPN group[(25.50±1.60)s]was higher than that of conventional group[(14.13±1.25)s];the thermal pain threshold of DAPT group at 60 days[(17.25±1.67)s]was lower than that of DPN group[(25.50±1.60)s],and the differences were statistically significant(P<0.05).The proportion of myelin sheath in DPN group was higher than that in conventional group,and that in DAPT group was lower than that in DPN group;the average immunofluorescence density of Notch1 in DPN group was significantly higher than that in conventional group,and that in DAPT group was significantly lower than that in DPN group;the average immunofluorescence density of ROCK1 in DPN group was significantly higher than that in conventional group,while that in DAPT group was significantly lower than that in DPN group;the average immunofluorescence density of ROCK2 in DPN group was significantly higher than that in conventional group,while that in DAPT group was significantly lower than that in DPN group(P<0.05).Conclusion:Notch1 inhibitor DAPT may have a protective effect on DPN by down-regulating ROCK.