Chaenomelis Fructus Extracts Protects Spinal Cord Injury in Rats by Regulating HMGB/TLR4/NF-B Signaling Pathway

YANG Bin; HUANG Jun-qing; ZHANG Ji-wei

doi:10.13422/j.cnki.syfjx.20181330

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Chaenomelis Fructus Extracts Protects Spinal Cord Injury in Rats by Regulating HMGB/TLR4/NF-B Signaling Pathway

更新时间：2024-01-04

- Chaenomelis Fructus Extracts Protects Spinal Cord Injury in Rats by Regulating HMGB/TLR4/NF-B Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 24, Issue 15, Pages: 167-175(2018)
- 作者机构：
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- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20181330
  CLC： R285.5
- Published：2018
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YANG Bin, HUANG Jun-qing, ZHANG Ji-wei. Chaenomelis Fructus Extracts Protects Spinal Cord Injury in Rats by Regulating HMGB/TLR4/NF-B Signaling Pathway[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(15): 167-175.
DOI：

YANG Bin, HUANG Jun-qing, ZHANG Ji-wei. Chaenomelis Fructus Extracts Protects Spinal Cord Injury in Rats by Regulating HMGB/TLR4/NF-B Signaling Pathway[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(15): 167-175. DOI： 10.13422/j.cnki.syfjx.20181330.

摘要

目的：探讨木瓜醇提取物对脊髓损伤（spinal cord injury，SCI）大鼠神经功能的保护作用，并初步探讨其可能机制。方法：制备SCI大鼠模型，根据处理方式分为假手术组（Sham组），模型组（SCI组），木瓜提取物治疗组（Pap组）；重组高迁移率族蛋白B1（high mobility group histone B1，HMGB1）处理组（rHMGB1组）；重组HMGB1+木瓜提取物处理组（rHMGB1+Pap组）。应用basso beattie bresnahan（BBB）评分评估大鼠后肢运动功能；检测脊髓组织含水量评估脊髓水肿情况；应用苏木素-伊红（HE）染色进行组织学评估；运用酶联免疫吸附检测白细胞介素-1β（interleuki-1β，IL-1β），肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α），白细胞介素-6（interleuki-6，IL-6）水平；原位末端转移酶标记法（TUNEL）检测脊髓组织细胞凋亡情况；应用免疫组织化学和蛋白免疫印迹法（Western blot）检测组织中HMGB1，Toll样受体4（Toll-like receptors 4，TLR4），核转录因子κB（nuclear factor-κB，NF-κB）p65蛋白表达情况；Western blot检测半胱氨酸蛋白酶（Caspase）-3蛋白；应用实时荧光定量PCR（Real-time PCR）检测HMGB1，TLR4，NF-κB p65 mRNA表达情况。结果：与Sham组比较，SCI组大鼠BBB评分明显降低（P<0.05）。SCI组和rHMGB1组脊髓组织松散，呈现嗜中性粒细胞浸润的组织学损伤；而Pap组和rHMGB1+Pap组中组织学损伤情况得到明显改善。与Sham组比较，SCI组大鼠脊髓组织中含水量，HMGB1，TLR4，NF-κB p65 mRNA和蛋白表达水平，IL-1β，IL-6，TNF-α水平，凋亡阳性细胞数，Caspase-3蛋白表达水平均明显增加（P<0.05）。Pap组和rHMGB1+Pap组含水量，HMGB1，TLR4，NF-κB p65 mRNA和蛋白表达水平，IL-1β，IL-6，TNF-α水平，凋亡阳性细胞数，Caspase-3蛋白表达水平均明显低于SCI组（P<0.05）。rHMGB1+Pap组含水量，HMGB1，TLR4，NF-κB p65 mRNA和蛋白表达水平，IL-1β，IL-6，TNF-α水平，凋亡阳性细胞数，Caspase-3蛋白表达水平明显低于rHMGB1组，差异均有统计学意义（P<0.05）。结论：木瓜提取物能够通过抑制HMGB1/TLR4/NF-κB p65信号通路活化，减轻SCI后的炎症反应、减少脊髓组织细胞凋亡，从而诱导脊髓组织修复和运动功能恢复。

Abstract

Objective: To investigate the protective effect of Chaenomelis Fructus extracts on neurological function in rats with spinal cord injury (SCI)

and explore its possible mechanism. Method: SCI rat model was prepared and was divided into sham operation group (Sham group)

model group (SCI group)

Chaenomelis Fructus alcohol extracts treatment group (Pap group)

recombinant high mobility group histone B1(HMGB1)treatment group (rHMGB1 group)

and recombinant HMGB1+ Chaenomelis Fructus extracts treatment group (rHMGB1+Pap group) according to the treatment methods. Rat hindlimb motor function was evaluated by Basso Beattie Bresnahan(BBB)score; water content in spinal cord tissue was detected to assess the spinal cord edema; Histological evaluation was performed by hematoxylin-eosin (HE) staining. The levels of interleuki-1β(IL-1β)

tumor necrosis factor-α(TNF-α)

and interleuki-6(IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). Terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptosis of spinal cord cells. The protein expression levels of HMGB1

Toll-like receptors 4 (TLR4) and nuclear factor-κB (NF-κB) p65 were detected by immunohistochemistry and Western blot

and their mRNA expression levels were detected by real time fluorescence quantitative polymerase chain reaction (Real-time PCR). Cysteine aspartate-specific protease-3(Caspase-3) was detected by Western blot. Result: As compared with the Sham group

the BBB score of the SCI group was significantly lower (P <0.05); spinal cord tissue was loose in SCI group and rHMGB1 group

showing histological damage of neutrophil infiltration; and histological damage was significantly improved in Pap group and rHMGB1+Pap group. As compared with Sham group

the water content in spinal cord tissue

mRNA and protein expression levels of HMGB1

TLR4

NF-κB p65 mRNA and protein

and IL-1β

IL-6

TNF-α levels

count of apoptosis-positive cells

and Caspase-3 protein expression level were significantly increased in SCI group (P<0.05). The water content in spinal cord tissue

mRNA and protein expression levels of HMGB1

TLR4

NF-κB p65 mRNA and protein

and IL-1β

IL-6

TNF-α levels

count of apoptosis-positive cells

and Caspase-3 protein expression level in Pap and rHMGB1+Pap group were significantly lower than the SCI group (P<0.05). The water content in spinal cord tissue

mRNA and protein expression levels of HMGB1

TLR4

NF-κB p65 mRNA and protein

and IL-1β

IL-6

TNF-α levels

count of apoptotic-positive cells and Caspase-3 protein expression level in rHMGB1+Pap group were significantly lower than those in rHMGB1 group

with statistically significant differences (P<0.05). Conclusion: Chaenomelis Fructus extracts can induce spinal cord tissue repair and motor function recovery by inhibiting the activation of HMGB1

TLR4

NF-κB p65 signaling pathway

reducing the inflammatory response after SCI and reducing the apoptosis of spinal cord tissue.

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