基于H4K16ac介导的细胞自噬探讨电针血清对脑缺血后神经元的保护机制  

Protective mechanism of electroacupuncture serum on neurons after cerebral ischemia based on H4K16AC-mediated autophagy

作　　者：徐疏影 沈燕[2] 彭拥军[1] 杨沙[2] 李文倩 Xu Shuying;Shen Yan;Peng Yongjun;Yang Sha;Li Wenqian(Hospital affiliated to Nanjing University of Chinese Medicine,Jiangsu 210029,China;First Teaching Hospital of Tianjin University of Traditional Chinese Medicine,China National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion,Tianjin 300192,China)

机构地区：[1]南京中医药大学附属医院,江苏210029 [2]天津中医药大学第一附属医院国家中医针灸临床医学研究中心

出　　处：《北京中医药大学学报》2021年第4期366-373,共8页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.81973932,No.81574060,No.81001556);天津市自然科学基金重点项目(No.18JCZDJC99200);长江学者和创新团队发展计划项目(No.IRT1167)。

摘　　要：目的从组蛋白H4第16位赖氨酸的乙酰化(H4K16ac)对细胞自噬调控的角度,研究电针血清对脑缺血后神经元的保护机制。方法电针血清的制备:建立改良的大鼠急性局灶性脑缺血再灌注模型,在造模成功后5 min和16 h进行电针干预,针刺人中、百会穴,并接电针治疗仪治疗30 min。第2次电针治疗后7 h取大鼠血清备用。细胞实验:将大鼠大脑皮质神经元细胞分为对照组、模型组、电针组、组蛋白乙酰转移酶MOF(hMOF)抑制剂组与细胞沉默调节蛋白1(Sirt1)抑制剂组,除对照组外,其余各组剥夺氧、糖3 h后再复氧、复糖24 h,并于复氧、复糖的同时给予2%电针血清、15 g/L的hMOF siRNA及8 g/L的Sirt1 siRNA处理。CCK-8检测细胞处理48 h后细胞增殖情况;流式细胞术检测细胞处理48 h后细胞凋亡情况;Western blot检测细胞中hMOF、Sirt1、组蛋白H4第16位赖氨酸的乙酰化(H4K16ac)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、自噬相关蛋白Beclin1表达;qPCR检测hMOF、Sirt1、Beclin1 mRNA表达;染色质免疫沉淀技术(ChIP)检测各组神经元细胞的H4K16ac在自噬靶基因Beclin1启动子区的结合程度。结果与模型组相比,电针干预可使细胞增殖活性提高(P<0.05),细胞凋亡率下降(P<0.05)。与模型组比较,电针组和hMOF抑制组剂均可降低hMOF和H4K16ac蛋白的表达(P<0.05),升高Sirt1、LC3-II和Beclin1的表达(P<0.05);而与Sirt1抑制剂组差异无统计学意义(P>0.05)。qPCR结果显示,与模型组比较,电针组和hMOF抑制组剂均可抑制hMOF mRNA表达(P<0.05),而Sirt1和Beclin1 mRNA表达上调(P<0.05)。且与模型组相比,电针组在自噬靶基因Beclin1启动子区域中H4K16ac的富集量增加(P<0.05)。结论电针血清对糖-氧剥夺再灌注损伤神经元细胞具有保护作用,其抗脑缺血再灌注损伤的作用可能是电针通过调节组蛋白H4K16ac的表达进而调控细胞自噬,从而减轻脑缺血再灌注损伤,发挥对神经细胞的保护作用。Objective To study the protective mechanism of electroacupuncture serum on neurons after cerebral ischemia from the perspective of autophagy regulation by acetylation of histone H4 16 lysine(H4 K16 ac). Methods 1) Preparation of electroacupuncture serum: The improved rat model of acute focal cerebral ischemia reperfusion was established. Electroacupuncture intervention was performed 5 at min and 16 h after the model was successfully established. Acupuncture was performed at the points of Renzhong(GV 26) and Baihui(GV 20), and electroacupuncture treatment was applied for 30 mins. The samples were collected 7 h after the second electroacupuncture treatment, and the serum of the rats was used for reserve. 2) Cell experiment: The rat cerebral cortex neuron cells were divided into control group, model group, electroacupuncture group, hMOF inhibitor group and Sirt1 inhibitor group. All cells were deprived of oxygen and sugar for 3 h and then re-oxygenated and re-glycated for 24 h except the control group. Meanwhile, all these cells were treated with 2% electroacupuncture serum, 15 g/L hMOF siRNA and 8 g/L Sirt1 siRNA. Cell proliferation was measured by using CCK-8 after 48 h of cell treatment. Cell apoptosis was measured by using flow cytometry after 48 h treatment. hMOF, Sirt1, H4 K16 ac, LC3-Ⅱ, and Beclin1 protein expression in rat cerebral cortex neurons were measured with Western blot assay. The expression of hMOF, Sirt1 and Beclin1 mRNA was detected by using qPCR. ChIP was used to detect the binding degree of H4 K16 ac of neurons in each group in the Beclin1 promoter region of autophagy target gene. Results Compared with the model group, the cell proliferation activity was significantly increased(P<0.05), and the cell apoptosis rate was significantly decreased(P<0.05). Compared with model group, both electroacupuncture and hMOF inhibitor decreased the expression of hMOF and H4 K16 ac proteins(P<0.05), and increased the expression of Sirt1, LC3-II and Beclin1(P<0.05). There was no significant difference between S

关 键 词：电针血清 大鼠大脑皮质神经元细胞 大鼠神经元组蛋白H4第16位赖氨酸的乙酰化 组蛋白乙酰转移酶MOF 组蛋白去乙酰化酶Sirtl 1 细胞自噬 

分 类 号：R245[医药卫生—针灸推拿学]