摘要
目的通过细胞和分子生物实验,观察平肝益肾方对MPP+损伤人神经母细胞瘤母细胞(SHSY5Y)细胞活性、生长情况以及自噬相关蛋白Beclin1、微管相关蛋白1轻链3 B(LC3B)表达的影响,初步探讨平肝益肾方对帕金森病(PD)神经保护作用的可能机制。方法以SH-SY5Y细胞为研究对象,分为空白组、模型组、平肝益肾方组、3-甲基腺嘌呤(3-MA)组,空白组换用完全培养基培养细胞,其余组均加入0.437 mmol/L MPP+,构建MPP+损伤SH-SY5Y细胞模型,平肝益肾方组加入193.6μg/ml平肝益肾方药液,3-MA组加入193.6μg/ml平肝益肾方药液和5 mmol/L 3-MA试剂。用MTT法检测其细胞活性;在细胞实时无标记细胞功能分析仪x CELLigence RTCA系统上检测其细胞生长时slope值平均值变化;蛋白质印迹法检测自噬相关蛋白Beclin1、LC3B表达。结果与空白组比较,模型组细胞活性显著降低(P<0.01),曲线slope值平均值降低,自噬相关蛋白Beclin1、LC3B着色明显减少且着色强度明显减少(P<0.01,P<0.01);与模型组比较,平肝益肾方组细胞活性显著升高(P<0.01),曲线slope值平均值升高,自噬相关蛋白Beclin1、LC3B着色明显增多且着色强度显著增强,表达显著升高(P<0.01,P<0.05);与平肝益肾方组比较,3-MA组细胞活性降低(P<0.05),曲线slope值平均值降低,自噬相关蛋白Beclin1、LC3B着色明显减少且着色显著减弱,表达显著降低(P<0.01,P<0.05)。结论平肝益肾方明显增强细胞活性,细胞逐渐增殖,通过上调神经细胞自噬活性,自噬相关蛋白Beclin1、LC3B表达增加,保护神经细胞,是其对PD神经保护作用的可能机制。
Objective To observe the cell viability,growth and autophagy-related protein Beclin1 and microtubule-associated protein 1 light chain of Pinggan Yishen Decoction on MPP+injured human neuroblastoma blast cells(SH-SY5 Y)through cellular and molecular biological experiments 3 B(LC3 B)expression.It was a preliminary study of the possible mechanism of the neuroprotective effect of Pinggan.Method SH-SY5 Y cells were used as the research object,and they were divided into blank group,model group,Pinggan Yishen Recipe group,3-methyladenine(3-MA)group.The blank group was replaced with complete culture medium and the other groups 0.437 mmol/L MPP+was added to construct an MPP+injury SH-SY5 Y cell model.The Pinggan Yishen Recipe group was added with 193.6μg/ml Pinggan Yishen Recipe liquid,and the 3-MA group was added 193.6μg/ml Pinggan Yishen Recipe Chemical solution and 5 mmol/L 3-MA reagent.MTT method was used to detect its cell activity;the cell real-time unlabeled cell function analyzer(x CELLigence)RTCA system was used to detect the change of slope value during cell growth;Western blotting was used to detect the expression of autophagy-related proteins Beclin1 and LC3 B.Result Compared with the blank group,the cell activity of the model group was significantly reduced(P<0.01),the slope value of the curve was reduced,the autophagy-related proteins Beclin1 and LC3 B were significantly reduced and the staining intensity was significantly reduced(P<0.01,P<0.01).Compared with model group the cell activity of Pinggan Yishen Recipe group increased significantly(P<0.01),the slope value of the curve increased,the autophagy-related proteins Beclin1 and LC3 B were stained significantly and the staining intensity was significantly enhanced,and the expression was significantly increased(P<0.01,P<0.05);Compared with the Pinggan Yishen Recipe group,the cell activity of the 3-MA group was reduced(P<0.05),the slope value of the curve was reduced,the autophagy-related proteins Beclin1 and LC3 B were significantly less colored and the coloring was significantly weakened,and the expression was significantly reduced(P<0.01,P<0.05).Conclusion Pinggan Yishen Decoction significantly enhances cell activity and proliferation ability.By up-regulating the autophagy activity of nerve cells,the expression of autophagy-related proteins Beclin1 and LC3 B increases and protects nerve cells,which is a possible mechanism of its neuroprotective effect on PD.
作者
刘京京
高鑫
卢芳
刘树民
LIU Jing-jing;GAO Xin;LU Fang;LIU Shu-min(Institute of Chinese Medicine,Heilongjiang University of Traditional Chinese Medicine,Harbin Heilongjiang 150040;School of Continuing Education,Heilongjiang University of Traditional Chinese Medicine,Harbin Heilongjiang 150040)
出处
《世界中西医结合杂志》
2020年第12期2246-2251,共6页
World Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金项目(404G405)
黑龙江中医药大学优秀创新人才支持计划资助(HLJRCL2019_01)。
作者简介
通信作者:刘树民,Email:keji-liu@163.com。
