HSPB8对糖尿病神经元的保护性作用及机制研究

Study of protective effect and mechanism of HSPB8 on diabetic neurons

目的观察HSPB8在自发性2型糖尿病db/db小鼠脊髓组织中的表达,探讨HSPBB发挥神经保护性作用的机制。方法选择8周龄,雄性,db/m小鼠(db/m组),糖尿病db/db小鼠(db/db组),各10只。动态观察体重、空腹血糖;HE染色观察小鼠脊髓组织损伤情况;qRT-PCR检测HSPB8、Caspase-1、IL-18、P62和LC3基因mR-NA表达;Westerm blotting检测HSPB8、Caspase-1、IL-18 P62和LC3蛋白表达。分别用11、25.30和35 mmol/L葡萄糖浓度刺激RGC-5细胞48 h为糖尿病体外模型,Western blotting检测HSPB8、Caspase-1、IL-18、LC3和P62蛋白表达。RGC-5细胞沉默HSPB8蛋白,高糖(35 mmo/L)刺激48 h,W estem blotting检测HSPB8、Caspase-1、IL-18、LC3和P62蛋白表达。组间比较采用成组1检验和单因素方差分析。结果①HE染色结果表明db/db小鼠脊髓组织出现损伤;②qRT-PCR结果表明HSPB8、Caspase-1、IL-18、P62和LC3基因均在糖尿病脊髓组织中表达;③Western blotting结果表明,与db/m组小鼠相比,db/db小鼠受损脊髓组织中HSPB8、LC3-IL表达下降,Caspase-1、IL-18和P62表达均升高(均P<0.05);不同浓度高糖刺激后,随着血糖浓度升高,HSPB8、LC3-IL表达下降,Caspase-1、IL-18和P62表达升高(均P<0.05);④RGC-5细胞HSPB8沉默后高糖刺激48 h,Caspase-1、IL-18和P62蛋白表达均升高而LC3-IL表达下降(均P<0.05)。结论HSPB8在糖尿病神经病变中发挥重要保护性作用,其机制可能与HSPB8影响神经细胞中的炎性因子及自噬过程相关。

Objective To observe the expression of HSPB8 in spinal cord tssues of spontaneous type 2 diabetes db/db mice,and explore the neuroprotective mechanism of HSPB8.Methods Eight weeks old,male,db/m mouse(db/m group,10 mice),db/db mouse(db/db group,10 mice)were selecd.Body weight and fasting blood glucose were dy-namical observation.HE staining was used to observe spinal cord in mice.mRNA expressions of HSPB8,Caspase-1,IL-18,P62 and LC3 were detected by qRT-PCR.For the in vitro model of diabetes,RGC-5 cells were stimulated with glucose concentrations of 11,25,30 and 35 mmol/L for 48 hours,respectively.RGC-5 cells were silenced by HSPB8 protein and stimulated by high glucose(35 mmo/L)for 48 h.HSPB8,Caspase-1,IL-18,P62 and LC3 were detected by western blot.Group t-test and one-way ANOVA were used for comparison between groups.Results①HE staining results showed that spinal cord tissue of db>/db mice was damaged;②qRT PCR results showed that HSPB8,Caspase-,IL-18,P62 and LC3 were expressed in diabetic spinal cord tssues;③W estern bloting results showed that compared with the db/m group of mice,the db/db mice were damaged spinal cord tssues HSPB8,LC3-Ⅱexpression were decreased,and the Caspase 1,IL-18,P62 expression were increased(all P<0.05).With the increase of blood glucose concentra-tion,the expression of HspB8,LC3-I were decreased,and the caspase-1,IL-18 and p62 were inereased(all P<0.05);④Forty-eight hours afer RGC-5 cells HSPB8 silence sugar stimulation,the Caspase 1,IL-18 and P62 protein expression were increased and LC3-Ⅱexpression were declined(all P<0.05).Conclusion HSPB8 plays an important protective role in diabetic neuropathy,and its mechanism may be related to the efect of HSPB8 on inflammatory factors and autophagy in neurons.