糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响

高变娥; 姚伟洁; 杨鑫伟; 史浩田; 朱笳悦; 刘仁慧; 许利平

doi:10.13422/j.cnki.syfjx.2018040115

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糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响

药理 | 更新时间：2024-01-04

- 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
- 中国实验方剂学杂志 2018年24卷第4期页码：115-123
- 作者机构：
- 作者简介：
- 基金信息：
  
  国家自然科学基金面上项目（81473642）;首都中医药研究专项面上项目（17ZY19）
- DOI：10.13422/j.cnki.syfjx.2018040115
  中图分类号： R285.5
- 纸质出版日期：2018
- 稿件说明：
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高变娥, 姚伟洁, 杨鑫伟, 等. 糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响[J]. 中国实验方剂学杂志, 2018,24(4):115-123.

GAO Bian-e, YAO Wei-jie, YANG Xin-wei, et al. Effect of Tangluoning on Endoplasmic Reticulum Stress PERK Pathway in Diabetic Peripheral Neuropathy Rats and Schwann Cells[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(4): 115-123.
高变娥, 姚伟洁, 杨鑫伟, 等. 糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响[J]. 中国实验方剂学杂志, 2018,24(4):115-123. DOI： 10.13422/j.cnki.syfjx.2018040115.

GAO Bian-e, YAO Wei-jie, YANG Xin-wei, et al. Effect of Tangluoning on Endoplasmic Reticulum Stress PERK Pathway in Diabetic Peripheral Neuropathy Rats and Schwann Cells[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(4): 115-123. DOI： 10.13422/j.cnki.syfjx.2018040115.

摘要

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。

Abstract

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

hematoxylin-eosin 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-sugar-induced 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%

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 Caspase 3 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 p-PERK

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.

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