Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (10): 1560-1566.doi: 10.12307/2023.302
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Zhang Min1, 2, Bai Shulin3, Li Shenghong1, 2, Fan Zhibo1, 2, Xie Yijia1, 2, Xu Xiaomei1, 2
Received:
2022-04-16
Accepted:
2022-06-02
Online:
2023-04-08
Published:
2022-09-08
Contact:
Xu Xiaomei, MD, Associate professor, Master’s supervisor, Department of Orthodontics, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China
About author:
Zhang Min, Master candidate, Physician, Department of Orthodontics, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China
Supported by:
CLC Number:
Zhang Min, Bai Shulin, Li Shenghong, Fan Zhibo, Xie Yijia, Xu Xiaomei. Effects of high mobility group box 1 and ERK1/2 pathway on autophagy of human periodontal ligament cells under tensile stress[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(10): 1560-1566.
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2.1 适宜强度的张应力可诱导人牙周膜细胞内自噬的发生和上调HMGB1的表达 第3-5代人牙周膜细胞施加不同时间的周期性牵张力后,采用RT-qPCR检测LC3-Ⅱ、Beclin-1、HMGB1的表达,结果可见随着张应力加载时间的延长,LC3-Ⅱ和Beclin-1及HMGB1 mRNA表达均呈现先增加后降低的趋势。LC3-Ⅱ、Beclin-1表达量在3 h时达到高峰(P < 0.001),随后开始下降,加力24 h其表达量下降至与对照组相比无明显差异(P > 0.05)。HMGB1 mRNA表达在张应力加载1 h后开始增加(P < 0.05),于3 h后达到高峰(P < 0.001),随后开始下降,同样加力24 h其表达量与对照组相比无差异,见图2。"
2.2 周期性张应力下人牙周膜细胞中HMGB1发生核质转位 张应力作用不同时间进行HMGB1免疫荧光染色,在高倍镜(×200)下观察可见未加载张应力时HMGB1主要位于细胞核内,胞质未见分布;张应力加载3 h后,胞核、胞质中均可见红染的HMGB1;张应力加载6 h后,胞质中HMGB1分布进一步增加,见图3A。 为进一步确认人牙周膜细胞中HMGB1定位随张应力加载时间的关系,在0,1,3,6,12,24 h张应力加载完成后分别提取胞核胞浆蛋白,采用Western blot分别定量检测胞核胞浆中HMGB1的表达,结果可见:在胞核中,加力3 h时HMGB1表达随张应力加载逐渐减少(P < 0.01),加力6 h时胞核中HMGB1表达进一步减少(P < 0.01),但12 h与6 h时相比未有明显减少(P > 0.05),而后加力24 h HMGB1表达进一步减少(P < 0.001);在胞浆中,HMGB1表达呈现逐渐增加的趋势,加力3 h时HMGB1表达开始增加(P < 0.01),12 h和24 h胞浆中HMGB1表达虽与对照组相比有明显增加(P < 0.001),但与6 h时相比未有明显增加,见图3B。从蛋白层面上证实了随张应力加载HMGB1出现了由胞核到胞质的转位。 "
2.3 HMGB1通过核质转位参与周期性张应力作用下人牙周膜细胞的自噬诱导 为验证HMGB1通过核质转位参与调控自噬,使用7.5 mmol/L丙酮酸乙酯(HMGB1胞质转位抑制剂)预处理细胞24 h后加力3 h,通过RT-qPCR,Western blot观察加力状态下自噬变化情况。 RT-qPCR和Western blot结果显示:丙酮酸乙酯组LC3-Ⅱ、Beclin-1和HMGB1表达与对照组相比均无明显差异,LC3-Ⅱ和Beclin-1在其他各组表达趋势类似。结果显示:与对照组相比,加力3 h时LC3-Ⅱ、 Beclin-1表达增加(P < 0.05)。丙酮酸乙酯+加力3 h组LC3-Ⅱ和Beclin-1表达较加力3 h组明显减少(P < 0.05),可见抑制HMGB1转位可下调张应力引起的自噬。同时,作者还发现,HMGB1在加力3 h组表达有增加(P < 0.05),抑制转位后,丙酮酸乙酯+加力3 h组HMGB1 mRNA表达与加力3 h组相比均无明显差异(P > 0.05),但其蛋白的表达减少(P < 0.05),见图4。 "
2.4 HMGB1通过ERK1/2通路参与调控张应力作用下人牙周膜细胞的自噬 采用10 μmol/L PD98059抑制ERK1/2信号通路,通过RT-qPCR,Western blot观察张应力下自噬变化情况。结果显示:与张应力加载3 h相比,PD98059+3 h组LC3-Ⅱ、Beclin-1 mRNA及蛋白的表达均减少;而单纯抑制ERK1/2通路,HMGB1的表达未见明显变化(P > 0.05),这表明抑制ERK1/2通路并不会影响加力状态下HMGB1的增加。此外,与PD98059组相比,加力3 h可逆转PD98059导致的自噬下调。结果证实了抑制ERK1/2信号通路后,加载张应力条件下HMGB1导致的自噬受到抑制,见图5。"
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