Effect and mechanism of glycyrrhizin on glial scar formation after spinal cord injury in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HE Yajun ¹ ,  SUN Lin ¹ , FENG Haoyu ¹ , LI Jisheng ¹ , ZHANG Nan ^1,2 , WANG Zhiqiang ¹

1. Department of Orthopaedics, Shanxi Bethune Hospital Affiliated to Shanxi Medical University, Taiyuan Shanxi, 030032, P.R.China;
2. School of Public Health, Shanxi Medical University, Taiyuan Shanxi, 030000, P.R.China;

Corresponding author：

SUN Lin, Email: sunlin_9999@163.com

Keywords：

Spinal cord injury; glial scar; glycyrrhizin; high mobility group box 1; nuclear factor κB; rat

DOI：

10.7507/1002-1892.202002116

Video：

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Objective 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.Methods Seventy-two female Sprague Dawley rats were randomly divided into sham group (n=12), SCI model group (SCI group, n=36), GL intervention group (SCI+GL group, n=12), and nuclear factor κB (NF-κB) inhibitor [pynolidine dithiocarbamate (PDTC)] intervention group (SCI+PDTC group, n=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.Results 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 (P<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 (P<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 (P<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 (P<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 (P<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 (P<0.05).Conclusion 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.

Citation： HE Yajun, SUN Lin, FENG Haoyu, LI Jisheng, ZHANG Nan, WANG Zhiqiang. Effect and mechanism of glycyrrhizin on glial scar formation after spinal cord injury in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(10): 1298-1304. doi: 10.7507/1002-1892.202002116 Copy

1.	Okada S, Hara M, Kobayakawa K, et al. Astrocyte reactivity and astrogliosis after spinal cord injury. Neurosci Res, 2018, 126: 39-43.
2.	Tran AP, Warren PM, Silver J. Regulation of autophagy by inhibitory CSPG interactions with receptor PTPσ and its impact on plasticity and regeneration after spinal cord injury. Exp Neurol, 2020, 328: 113276.
3.	李星, 李佳音, 肖志峰, 等. 脊髓损伤后胶质瘢痕在神经再生过程中作用的探讨. 中国修复重建外科杂志, 2018, 32(8): 973-978.
4.	Orr MB, Gensel JC. Spinal cord injury scarring and inflammation: therapies targeting glial and inflammatory responses. Neurotherapeutics, 2018, 15(3): 541-553.
5.	孙麟, 马迅, 刘强, 等. 丙酮酸乙酯通过抑制高迁移率族蛋白 1 减轻大鼠脊髓损伤后水肿及体外氧糖剥夺/复氧后星形胶质细胞肿胀的研究. 中华创伤骨科杂志, 2018, 20(12): 1079-1086.
6.	Mu SW, Dang Y, Fan YC, et al. Effect of HMGB1 and RAGE on brain injury and the protective mechanism of glycyrrhizin in intracranial-sinus occlusion followed by mechanical thrombectomy recanalization. Int J Mol Med, 2019, 44(3): 813-822.
7.	Sun L, Li M, Ma X, et al. Inhibiting high mobility group Box-1 reduces early spinal cord edema and attenuates astrocyte activation and aquaporin-4 expression after spinal cord injury in rats. J Neurotrauma, 2019, 36(3): 421-435.
8.	吕聪, 孙麟, 冯皓宇, 等. 减少氧糖剥夺/复氧后脊髓星形胶质细胞凋亡: 抑制高迁移率族蛋白 B1/核转录因子 κB 通路的作用. 中国组织工程研究, 2019, 23(33): 5353-5359.
9.	Pinho-Ribeiro FA, Fattori V, Zarpelon AC, et al. Pyrrolidine dithiocarbamate inhibits superoxide anion-induced pain and inflammation in the paw skin and spinal cord by targeting NF-κB and oxidative stress. Inflammopharmacology, 2016, 24(2-3): 97-107.
10.	莫翠萍, 任力杰, 赵振富, 等. BMSCs 移植治疗大鼠脊髓损伤效果及局部细胞因子表达变化. 中国修复重建外科杂志, 2016, 30(3): 265-271.
11.	Mollica L, De Marchis F, Spitaleri A, et al. Glycyrrhizin binds to high-mobility group Box 1 protein and inhibits its cytokine activities. Chem Biol, 2007, 14(4): 431-441.
12.	de Mos M, Laferrière A, Millecamps M, et al. Role of NFkappaB in an animal model of complex regional pain syndrome-typeⅠ (CRPS-Ⅰ). J Pain, 2009, 10(11): 1161-1169.
13.	Li G, Wu X, Yang L, et al. TLR4-mediated NF-κB signaling pathway mediates HMGB1-induced pancreatic injury in mice with severe acute pancreatitis. Int J Mol Med, 2016, 37(1): 99-107.
14.	朱双龙, 段会全, 刘英富, 等. 柴胡皂苷 a 对大鼠急性脊髓损伤的神经保护作用与机制研究. 中国修复重建外科杂志, 2017, 31(7): 825-829.
15.	Wang KC, Tsai CP, Lee CL, et al. Elevated plasma high-mobility group Box 1 protein is a potential marker for neuromyelitis optica. Neuroscience, 2012, 226(18): 510-516.
16.	Yang J, Liu X, Zhou Y, et al. Hyperbaric oxygen alleviates experimental (spinal cord) injury by downregulating HMGB1/NF-κB expression. Spine (Phila Pa 1976), 2013, 38(26): E1641-1648.
17.	Li L, Ni L, Eugenin EA, et al. Toll-like receptor 9 antagonism modulates astrocyte function and preserves proximal axons following spinal cord injury. Brain Behav Immun, 2019, 80: 328-343.
18.	Papatheodorou A, Stein AB, Bank M, et al. High-mobility group box 1 (HMGB1) is elevated systemically in persons with acute or chronic traumatic spinal cord injury. J Neurotrauma, 2016, 34(3): 746-754.
19.	Sun L, Li M, Ma X, et al. Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner. J Neuroinflamm, 2017, 14(1): 231.
20.	Tang X, Davies JE, Davies SJ. Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue. J Neurosci Res, 2003, 71(3): 427-444.
21.	Schwab JM, Zhang Y, Kopp MA, et al. The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury. Exp Neurol, 2014, 258: 121-129.
22.	Li XQ, Chen FS, Tan WF, et al. Elevated microRNA-129-5p level ameliorates neuroinflammation and blood-spinal cord barrier damage after ischemia-reperfusion by inhibiting HMGB1 and the TLR3-cytokine pathway. J Neuroinflamm, 2017, 14(1): 205.
23.	Wang Q, Ding Q, Zhou Y, et al. Ethyl pyruvate attenuates spinal cord ischemic injury with a wide therapeutic window through inhibiting high-mobility group box 1 release in rabbits. Anesthesiology, 2009, 110(6): 1279-1286.
24.	Chen H, Guan B, Wang B, et al. Glycyrrhizin prevents hemorrhagic transformation and improves neurological outcome in ischemic stroke with delayed thrombolysis through targeting peroxynitrite-mediated HMGB1 signaling. Transl Stroke Res, 2019. https://doi.org/10.1007/s12975-019-00772-1.
25.	Ieong C, Sun H, Wang Q, et al. Glycyrrhizin suppresses the expressions of HMGB1 and ameliorates inflammative effect after acute subarachnoid hemorrhage in rat model. J Clin Neurosci, 2018, 47: 278-284.
26.	Su X, Wang H, Zhao J, et al. Beneficial effects of ethyl pyruvate through inhibiting high-mobility group box 1 expression and TLR4/NF-kappaB pathway after traumatic brain injury in the rat. Mediat Inflamm, 2011, 2011: 807142.
27.	Deng G, Gao Y, Cen Z, et al. miR-136-5p Regulates the inflammatory response by targeting the IKKβ/NF-κB/A20 pathway after spinal cord injury. Cell Physiol Biochem, 2018, 50(2): 512-524.

1. Okada S, Hara M, Kobayakawa K, et al. Astrocyte reactivity and astrogliosis after spinal cord injury. Neurosci Res, 2018, 126: 39-43.
2. Tran AP, Warren PM, Silver J. Regulation of autophagy by inhibitory CSPG interactions with receptor PTPσ and its impact on plasticity and regeneration after spinal cord injury. Exp Neurol, 2020, 328: 113276.
3. 李星, 李佳音, 肖志峰, 等. 脊髓损伤后胶质瘢痕在神经再生过程中作用的探讨. 中国修复重建外科杂志, 2018, 32(8): 973-978.
4. Orr MB, Gensel JC. Spinal cord injury scarring and inflammation: therapies targeting glial and inflammatory responses. Neurotherapeutics, 2018, 15(3): 541-553.
5. 孙麟, 马迅, 刘强, 等. 丙酮酸乙酯通过抑制高迁移率族蛋白 1 减轻大鼠脊髓损伤后水肿及体外氧糖剥夺/复氧后星形胶质细胞肿胀的研究. 中华创伤骨科杂志, 2018, 20(12): 1079-1086.
6. Mu SW, Dang Y, Fan YC, et al. Effect of HMGB1 and RAGE on brain injury and the protective mechanism of glycyrrhizin in intracranial-sinus occlusion followed by mechanical thrombectomy recanalization. Int J Mol Med, 2019, 44(3): 813-822.
7. Sun L, Li M, Ma X, et al. Inhibiting high mobility group Box-1 reduces early spinal cord edema and attenuates astrocyte activation and aquaporin-4 expression after spinal cord injury in rats. J Neurotrauma, 2019, 36(3): 421-435.
8. 吕聪, 孙麟, 冯皓宇, 等. 减少氧糖剥夺/复氧后脊髓星形胶质细胞凋亡: 抑制高迁移率族蛋白 B1/核转录因子 κB 通路的作用. 中国组织工程研究, 2019, 23(33): 5353-5359.
9. Pinho-Ribeiro FA, Fattori V, Zarpelon AC, et al. Pyrrolidine dithiocarbamate inhibits superoxide anion-induced pain and inflammation in the paw skin and spinal cord by targeting NF-κB and oxidative stress. Inflammopharmacology, 2016, 24(2-3): 97-107.
10. 莫翠萍, 任力杰, 赵振富, 等. BMSCs 移植治疗大鼠脊髓损伤效果及局部细胞因子表达变化. 中国修复重建外科杂志, 2016, 30(3): 265-271.
11. Mollica L, De Marchis F, Spitaleri A, et al. Glycyrrhizin binds to high-mobility group Box 1 protein and inhibits its cytokine activities. Chem Biol, 2007, 14(4): 431-441.
12. de Mos M, Laferrière A, Millecamps M, et al. Role of NFkappaB in an animal model of complex regional pain syndrome-typeⅠ (CRPS-Ⅰ). J Pain, 2009, 10(11): 1161-1169.
13. Li G, Wu X, Yang L, et al. TLR4-mediated NF-κB signaling pathway mediates HMGB1-induced pancreatic injury in mice with severe acute pancreatitis. Int J Mol Med, 2016, 37(1): 99-107.
14. 朱双龙, 段会全, 刘英富, 等. 柴胡皂苷 a 对大鼠急性脊髓损伤的神经保护作用与机制研究. 中国修复重建外科杂志, 2017, 31(7): 825-829.
15. Wang KC, Tsai CP, Lee CL, et al. Elevated plasma high-mobility group Box 1 protein is a potential marker for neuromyelitis optica. Neuroscience, 2012, 226(18): 510-516.
16. Yang J, Liu X, Zhou Y, et al. Hyperbaric oxygen alleviates experimental (spinal cord) injury by downregulating HMGB1/NF-κB expression. Spine (Phila Pa 1976), 2013, 38(26): E1641-1648.
17. Li L, Ni L, Eugenin EA, et al. Toll-like receptor 9 antagonism modulates astrocyte function and preserves proximal axons following spinal cord injury. Brain Behav Immun, 2019, 80: 328-343.
18. Papatheodorou A, Stein AB, Bank M, et al. High-mobility group box 1 (HMGB1) is elevated systemically in persons with acute or chronic traumatic spinal cord injury. J Neurotrauma, 2016, 34(3): 746-754.
19. Sun L, Li M, Ma X, et al. Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner. J Neuroinflamm, 2017, 14(1): 231.
20. Tang X, Davies JE, Davies SJ. Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue. J Neurosci Res, 2003, 71(3): 427-444.
21. Schwab JM, Zhang Y, Kopp MA, et al. The paradox of chronic neuroinflammation, systemic immune suppression, autoimmunity after traumatic chronic spinal cord injury. Exp Neurol, 2014, 258: 121-129.
22. Li XQ, Chen FS, Tan WF, et al. Elevated microRNA-129-5p level ameliorates neuroinflammation and blood-spinal cord barrier damage after ischemia-reperfusion by inhibiting HMGB1 and the TLR3-cytokine pathway. J Neuroinflamm, 2017, 14(1): 205.
23. Wang Q, Ding Q, Zhou Y, et al. Ethyl pyruvate attenuates spinal cord ischemic injury with a wide therapeutic window through inhibiting high-mobility group box 1 release in rabbits. Anesthesiology, 2009, 110(6): 1279-1286.
24. Chen H, Guan B, Wang B, et al. Glycyrrhizin prevents hemorrhagic transformation and improves neurological outcome in ischemic stroke with delayed thrombolysis through targeting peroxynitrite-mediated HMGB1 signaling. Transl Stroke Res, 2019. https://doi.org/10.1007/s12975-019-00772-1.
25. Ieong C, Sun H, Wang Q, et al. Glycyrrhizin suppresses the expressions of HMGB1 and ameliorates inflammative effect after acute subarachnoid hemorrhage in rat model. J Clin Neurosci, 2018, 47: 278-284.
26. Su X, Wang H, Zhao J, et al. Beneficial effects of ethyl pyruvate through inhibiting high-mobility group box 1 expression and TLR4/NF-kappaB pathway after traumatic brain injury in the rat. Mediat Inflamm, 2011, 2011: 807142.
27. Deng G, Gao Y, Cen Z, et al. miR-136-5p Regulates the inflammatory response by targeting the IKKβ/NF-κB/A20 pathway after spinal cord injury. Cell Physiol Biochem, 2018, 50(2): 512-524.

Chinese Journal of Reparative and Reconstructive Surgery

Effect and mechanism of glycyrrhizin on glial scar formation after spinal cord injury in rats

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