枸杞多糖通过调节热休克蛋白B8介导的自噬改善糖尿病神经病理性疼痛的研究

Lycium barbarum polysaccharides relieve diabetic neuropathy pain through regulating autophagy mediated by HSPB8

目的探讨枸杞多糖(LBP)通过调节热休克蛋白B8(HSPB8)介导的自噬改善糖尿病神经病理性疼痛(DNP)及其作用机制。方法 62只SD大鼠随机选取10只为正常对照组(NC),其余腹腔注射STZ建立糖尿病大鼠模型,随机分为糖尿病组(DM)、LBP组、α-硫辛酸组(LA),每组各15只。每4周检测各组大鼠的机械缩足反射阈值(PWT)和热缩足反射潜伏期(PWL)。治疗12周后,采用Western blot、免疫组织化学法检测腰段脊髓神经中HSPB8、Bcl-2相关永生化基因3(BAG3)、微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、P62蛋白表达量。结果与NC组比较,DM组在4、8、12周时PWT、PWL降低(P<0. 05或P<0. 01)。与DM组比较,LBP组在4、8、12周时PWT、PWL升高(P<0. 05或P<0. 01)。Western blot法检测显示,与NC组比较,DM组HSPB8、BAG3、LC3-Ⅱ蛋白表达量降低[(0. 17±0. 04)vs(0. 13±0. 04)、(0. 35±0. 11)vs(0. 14±0. 04)、(0. 31±0. 03)vs(0. 17±0. 05),P<0. 05或P<0. 01],P62蛋白表达量升高[(0. 37±0. 03)vs(0. 63±0. 08),(P<0. 01)]。与DM组比较,LBP组HSPB8、BAG3、LC3-Ⅱ蛋白表达量升高[(0. 13±0. 04)vs(0. 21±0. 03)、(0. 14±0. 04)vs(0. 32±0. 09)、(0. 17±0. 05)vs(0. 34±0. 08),P<0. 01],P62蛋白表达量降低[(0. 63±0. 08)vs(0. 39±0. 14),(P<0. 01)]。免疫组织化学法检测显示,与NC组比较,DM组HSPB8、BAG3、LC3-Ⅱ蛋白表达量降低[(168. 24±10. 21)vs(144. 83±14. 53)、(146. 50±13. 48)vs(124. 83±9. 33)、(144. 33±7. 77)vs(127. 83±5. 92),P<0. 01],P62蛋白表达量升高[(146. 67±14. 10)vs(175. 83±9. 22),(P<0. 01)]。与DM组比较,LBP组HSPB8、BAG3、LC3-Ⅱ蛋白表达量升高[(144. 83±14. 53)vs(180. 0±7. 95)、(124. 83±9. 33)vs(155. 0±5. 76)、(127. 83±5. 92)vs(156. 32±5. 56),P<0. 01],P62蛋白表达量降低[(175. 83±9. 22)vs(139. 67±12. 68),(P<0. 01)]。结论 LBP可能通过提高糖尿病大鼠脊髓神经中HSPB8促进自噬缓解DNP。

ObjectiveTo explore the effect of lycium barbarum polysaccharides(LBP)in relieving diabetic neuropathy pain through regulating autophagy mediated by HSPB8 and its mechanism.Methods A total of 62 SD rats were used in this study. 10 rats were selected as the normal control group(NC),and the rest were used to establish the DM rat model with STZ. Among them,45 DM rat model were randomly divided into DM group(n=15),LBP group(n=15)and Lipoic acid group(n=15). The paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)of rats were detected every 4 weeks. The expression of HSPB8,BAG3,LC3-Ⅱ and P62 protein in spinal cord were detected by western blot and immunohistochemisty.Results weeks in DM group(P<0. 05 or P<0. 01). Compared with DM group,PWT and PWL were increased at4,8 and 12 weeks in LBP group(P<0. 05 or P<0. 01). Western blot results showed that the HSPB8,BAG3,LC3-Ⅱ protein concentration in the spinal cord were lower in DM group than in NC group[(0. 17±0. 04)vs(0. 13±0. 04),(0. 35±0. 11)vs(0. 14±0. 04),(0. 31±0. 03)vs(0. 17±0. 05),P<0. 05 or P<0. 01],while P62 protein was higher in DM group than NC group[(0. 37±0. 03)vs(0. 63±0. 08),P<0. 01]. The HSPB8,BAG3,LC3-Ⅱ protein were obviously higher in LBP group than in DM group[(0. 13±0. 04)vs(0. 21±0. 03),(0. 14±0. 04)vs(0. 32±0. 09),(0. 17±0. 05)vs(0. 34±0. 08),P<0. 01],while P62 protein was significantly lower in LBP group than in DM group[(0. 63±0. 08)vs(0. 39±0. 14),P<0. 01]. Immunohistochemical results showed that the HSPB8,BAG3,LC3-Ⅱ protein concentration in the spinal cord were decreased[(168. 24 ± 10. 21)vs(144. 83 ± 14. 53),(146. 50 ± 13. 48)vs(124. 83±9. 33),(144. 33±7. 77)vs(127. 83±5. 92),P<0. 01],while P62 protein was increased in DM group than in NC group[(146. 67±14. 10)vs(175. 83±9. 22),P<0. 01]. The HSPB8,BAG3,LC3-Ⅱprotein were increased[(144. 83 ± 14. 53)vs(180. 0 ± 7. 95),(124. 83 ± 9. 33)vs(155. 0 ± 5. 76),(127. 83±5. 92)vs(156. 32±5. 56),P<0. 01]. while P62 protein was decreased in LBP group than in DM group.autophagy sequentially in spinal cord of the diabetic rat.