糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响

Effect of Tangluoning on Endoplasmic Reticulum Stress PERK Pathway in Diabetic Peripheral Neuropathy Rats and Schwann Cells

目的：观察糖络宁对糖尿病周围神经病变（DPN）大鼠和雪旺细胞内质网应激RNA依赖的蛋白激酶样内质网激酶（PERK）通路相关蛋白和mRNA表达的影响。方法：60只SD雄性大鼠,除空白组外,其余各组大鼠采用高脂饲料喂养联合腹腔注射链脲佐菌素（STZ,35 mg·kg^-1）复制DPN大鼠模型,随机分为模型组,氧化三甲胺组,糖络宁低、高剂量组,连续给药12周。采用苏木素-伊红（HE）染色观察大鼠坐骨神经病理学改变,免疫荧光染色法检测大鼠坐骨神经中p-PERK,磷酸化的真核翻译起始因子2a（p-eIF2a）和转录活化因子4（ATF4）蛋白的表达;建立高糖诱导的雪旺细胞模型,分为25 mmol·L^-1葡萄糖组（control）,150 mmol·L^-1葡萄糖组（model）,150 mmol·L^-1葡萄糖＋0.1%,1%和10%糖络宁组,分别干预24,48 h。采用实时荧光定量PCR（Real-time PCR）法检测细胞中PERK,核转录因子E2相关因子2（Nrf2）,血红素加氧酶-1（HO-1）,B淋巴细胞瘤-2相关的X蛋白（Bax）和天冬氨酸特异性半胱氨酸蛋白酶-3（Caspase-3）mRNA表达水平。结果：坐骨神经病理学改变：空白组大鼠坐骨神经有髓神经纤维的髓鞘结构完整致密,形态规则,排列整齐。模型组出现严重脱髓鞘现象,结构松散,形态不规则,排列紊乱。糖络宁组有轻微脱髓鞘现象,结构近似完整,形态近似规则。经积分吸光度统计,与空白组相比,模型组明显降低（P〈0.01）;雪旺细胞PERK,Nrf2,HO-1,Caspase-3和Bax mRNA表达水平,与空白组比较,同时相的模型组中PERK,Bax和Caspase-3 mRNA的表达明显升高（P〈0.05,P〈0.01）,与模型组比较,同时相的糖络宁3个含药血清剂量组中以上3个指标表达明显降低（P〈0.05,P〈0.01）,而Nrf2和HO-1 mRNA的表达明显升高（P〈0.05,P〈0.01）;坐骨神经中p-PERK,p-eIF2a和ATF4蛋白表达,与空白组比较,模型组蛋白表达量显著增加（P〈0.01）;与模型组比较,糖络宁组蛋白表达量显著减少（P〈0.01）。结论：糖络宁能够减轻坐骨神经组织的病理损伤,其防治DPN的作用机制可能与调节内质网应激时PERK相关途径包括抑制PERK/eIF2a途径同时促进PERK/Nrf2途径有关。

Objective： To observe the effect of Tangluoning on RNA-dependent protein kinase（PKR）-like ER kinase（PERK） pathway related protein and mRNA expressions of diabetic peripheral neuropathy（DPN）rats and Schwann cells.Method： A total of sixty SD male rats were used.Except for the blank control group,the rest SD male rats were treated with high-fat diet combined with streptozotocin（STZ,35 mg·kg^-1） to replicate the DPN rats model.The rats were randomly divided into model control group,trimethylamine oxide group,low-dose Tangluoning group and high-dose Tangluoning group.After 12 weeks of continuous administration,hematoxylineosin staining（HE） was adopted to observe the pathological changes in sciatic nerve under the microscope.Immunofluorescence staining was used to detect expressions of p-PERK,phosphorylated eukaryotic translation initiation factor 2α（p-eIF2a） and activating transcription factor 4（ATF4） proteins in sciatic nerve; high-sugarinduced schwann cell model was established.The experiment was divided into 25 mmol·L^-1 glucose group（control）,150 mmol·L^-1 glucose group（model）,150 mmol·L^-1 glucose ＋ 0.1%,1%,10% Tangluoning groups; they were respectively treated for 24,48 h.Real-time PCR was used to detect PERK,nuclear factor-E2-related factor 2（Nrf2）,heme oxygenase-1（HO-1）,B-cell lymphoma 2 associated X protein（Bax） and Caspase-3 mRNA expressions.Result： Pathological changes in sciatic nerve： myelinated nerve fiber had a complete dense structure and regular shape in blank control group; severe demyelination phenomenon,irregular shape and disordered arrangement were observed in model group; whereas mild demyelination phenomenon,approximately complete structure and approximately regular shape were found in Tangluoning groups.As for integral optical density,compared with the blank control group,model group exhibited a higher level（P〈0.01）; compared with model group,herb group exhibited a lower level（P〈0.01）.As for mRNA expressions of PERK,Nrf2,HO-1,Caspase 3 and Bax in Schwann cells,compared with control group,mRNA expression of PERK,Bax and Caspase3 in model group were significantly increased（P〈0.05,P〈0.01）; compared with model group,mRNA expressions of PERK,Caspase-3 and Bax in Tangluoning groups were significantly decreased（P〈0.05,P〈0.01）,and mRNA expressions of Nrf2 and HO-1 were significantly increased（P〈0.05,P〈0.01）.As for protein expressions of p-PERK,p-eIF2a and ATF4,compared with blank control group,protein expressions of pPERK,p-eIF2a and ATF4 in model group displayed an obvious increase（P〈0.01）; compared with model control group,Tangluoning groups displayed an obvious decrease（P〈0.01）.Conclusion： Tangluoning can alleviate the pathological damage of sciatic nerve tissues and regulate the PERK pathway,including down-regulating PERK/eIF2a/ATF4 pathway and up-regulating PERK/Nrf2/HO-1 pathway when endoplasmic reticulum stress（ERS）improves DPN.