He Yajun, Sun Lin, Feng Haoyu, Li Jisheng, Zhang Nan, Wang Zhiqiang
Department of Orthopaedics, Shanxi Bethune Hospital Affiliated to Shanxi Medical University, Taiyuan Shanxi, 030032, P.R.China.
Department of Orthopaedics, Shanxi Bethune Hospital Affiliated to Shanxi Medical University, Taiyuan Shanxi, 030032, P.R.China;School of Public Health, Shanxi Medical University, Taiyuan Shanxi, 030000, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2020 Oct 15;34(10):1298-1304. doi: 10.7507/1002-1892.202002116.
To explore the effect and potential mechanism of glycyrrhizin (GL) by inhibiting high mobility group box 1 (HMGB1) on glial scar formation after spinal cord injury (SCI) in rats.
Seventy-two female Sprague Dawley rats were randomly divided into sham group ( =12), SCI model group (SCI group, =36), GL intervention group (SCI+GL group, =12), and nuclear factor κB (NF-κB) inhibitor [pynolidine dithiocarbamate (PDTC)] intervention group (SCI+PDTC group, =12). The SCI models of SCI group, SCI+GL group, and SCI+PDTC group were made by modified Allen's method, the sham group was only exposed the spinal cord without any injury. First of all, Basso-Beattie-Bresnahan (BBB) score of hind limbs and slope test were performed in SCI group at 1, 2, and 3 weeks after operation; Western blot was used to detect the expressions of glial fibrillary acidic protein (GFAP) and HMGB1 proteins. Compared with the sham group, the most significant time point in the SCI group was selected for subsequent experiment, in which the most significant glial scar was formed. Then, behavioral tests (BBB score of hind limbs and slope test), histological observation of spinal cord tissue structure, Western blot detection of HMGB1, GFAP, and NF-κB proteins, and immunohistochemical staining observation of GFAP and chondroitin sulfate proteoglycan (CSPG) were used to explore the effect of GL on the formation of glial scar after SCI and its potential mechanism.
The BBB score and slope angle of the SCI group increased gradually with time, which were significantly lower than those of the sham group at each time point ( <0.05). Western blot detection showed that the relative expressions of HMGB1 and GFAP proteins in the SCI group at 1, 2, and 3 weeks after operation were significantly higher than those in sham group ( <0.05). The change was most obvious at 3 weeks after SCI, therefore the spinal cord tissue was selected for subsequent experiments at this time point. At 3 weeks after operation, compared with the SCI group, BBB score and slope angle of SCI+GL group significantly increased ( <0.05); the relative expressions of HMGB1, GFAP, and NF-κB proteins detected by Western blot and the expressions of GFAP and CSPG proteins detected by immunohistochemical staining significantly decreased ( <0.05); the disorder of spinal cord tissue by HE staining improved, inflammatory cell infiltration reduced, and glial scar formation decreased. At 3 weeks after operation, the expressions of NF-κB, GFAP, and CSPG proteins of the SCI+PDTC group significantly reduced when compared with the SCI group ( <0.05); and the expression of NF-κB protein significantly decreased and the expressions of GFAP and CSPG proteins significantly increased when compared with the SCI+GL group ( <0.05).
After SCI in rats, the application of GL to inhibit the expression of HMGB1 can reduce the expression of GFAP and CSPG in the injured spinal cord, then reduce the formation of glial scars and promote the recovery of motor function of the hind limbs, and GL may play a role in inhibiting glial scar through HMGB1/NF-κB pathway.
探讨甘草酸（GL）通过抑制高迁移率族蛋白B1（HMGB1）对大鼠脊髓损伤（SCI）后胶质瘢痕形成的影响及潜在机制。
将72只雌性Sprague Dawley大鼠随机分为假手术组（n = 12）、SCI模型组（SCI组，n = 36）、GL干预组（SCI + GL组，n = 12）和核因子κB（NF - κB）抑制剂[吡咯烷二硫代氨基甲酸盐（PDTC）]干预组（SCI + PDTC组，n = 12）。SCI组、SCI + GL组和SCI + PDTC组采用改良Allen法制备SCI模型，假手术组仅暴露脊髓，不造成任何损伤。首先，在术后1、2和3周对SCI组进行后肢Basso - Beattie - Bresnahan（BBB）评分和斜板试验；采用蛋白质免疫印迹法检测胶质纤维酸性蛋白（GFAP）和HMGB1蛋白的表达。与假手术组相比，选择SCI组中最显著的时间点进行后续实验，此时形成了最显著的胶质瘢痕。然后，采用行为学测试（后肢BBB评分和斜板试验）、脊髓组织结构的组织学观察、蛋白质免疫印迹法检测HMGB1、GFAP和NF - κB蛋白以及免疫组织化学染色观察GFAP和硫酸软骨素蛋白聚糖（CSPG），以探讨GL对SCI后胶质瘢痕形成的影响及其潜在机制。
SCI组的BBB评分和斜板角度随时间逐渐增加，但在每个时间点均显著低于假手术组（P < 0.05）。蛋白质免疫印迹法检测显示，SCI组术后1、2和3周HMGB1和GFAP蛋白的相对表达均显著高于假手术组（P < 0.05）。SCI后3周变化最为明显，因此选择此时的脊髓组织进行后续实验。术后3周，与SCI组相比，SCI + GL组的BBB评分和斜板角度显著增加（P < 0.05）；蛋白质免疫印迹法检测的HMGB1、GFAP和NF - κB蛋白的相对表达以及免疫组织化学染色检测的GFAP和CSPG蛋白的表达均显著降低（P < 0.05）；苏木精 - 伊红（HE）染色显示脊髓组织紊乱改善，炎性细胞浸润减少，胶质瘢痕形成减少。术后3周，与SCI组相比，SCI + PDTC组的NF - κB、GFAP和CSPG蛋白表达显著降低（P < 0.05）；与SCI + GL组相比，NF - κB蛋白表达显著降低，GFAP和CSPG蛋白表达显著增加（P < 0.05）。
大鼠SCI后，应用GL抑制HMGB1表达可降低损伤脊髓中GFAP和CSPG的表达，进而减少胶质瘢痕形成，促进后肢运动功能恢复，GL可能通过HMGB1/NF - κB途径在抑制胶质瘢痕中发挥作用。
