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Xiaozheng Zhitong Paste Relieves Bone Cancer Pain in Mice by Alleviating Activation of Microglia in Spinal Cord and Damage to Neurons via Blocking PAR2/NF-κB/NLRP3 Pathway

  • role： First author第一作者
  • Affiliation：

    Guang'anmen Hospital，China Academy of Chinese Medical Sciences， Beijing 100053，China

  • Email：zheng19990718@163.com
  • Introduction：郑广达，硕士，从事经方治疗癌性疼痛的临床与基础研究，E-mail：zheng19990718@163.com
ZHENG Guangda1,  
  • Affiliation：

    Guang'anmen Hospital，China Academy of Chinese Medical Sciences， Beijing 100053，China

MENG Linghan1,  
  • Affiliation：

    Liaoning University of Traditional Chinese Medicine， Shenyang 110847，China

SHANG Lu2,  
  • Affiliation：

    Liaoning University of Traditional Chinese Medicine， Shenyang 110847，China

REN Juanxia2,  
  • Affiliation：

    Guang'anmen Hospital，China Academy of Chinese Medical Sciences， Beijing 100053，China

LI Dongtao1,  
  • Affiliation：

    Guang'anmen Hospital，China Academy of Chinese Medical Sciences， Beijing 100053，China

LIU Haixiao1,  
  • Affiliation：

    Liaoning University of Traditional Chinese Medicine， Shenyang 110847，China

WANG Lingyun2,  
  • Affiliation：

    Liaoning University of Traditional Chinese Medicine， Shenyang 110847，China

LI Changlin2,  
  • Affiliation：

    Xining Traditional Chinese Medicine Hospital，Xining 810001，China

CHEN Yaohua3,  
  • Affiliation：

    Xining Traditional Chinese Medicine Hospital，Xining 810001，China

YANG Guiping3,  
  • role： Corresponding author通信作者
  • Affiliation：

    Guang'anmen Hospital，China Academy of Chinese Medical Sciences， Beijing 100053，China

  • Email：byjtxyx@163.com
  • Introduction：鲍艳举，博士，主任医师，从事经方治疗癌性疼痛的临床与基础研究，E-mail：byjtxyx@163.com
BAO Yanju1*

résumé

ObjectiveTo investigate the effects and underlying mechanisms of Xiaozheng Zhitong Paste （XZP） on bone cancer pain （BCP）.MethodsThirty female BALB/c mice were randomly divided into five groups： a Sham group， a BCP group， a BCP+low-dose XZP group， a BCP+high-dose XZP group， and a BCP+high-dose XZP + protease-activated receptor 2 （PAR2） agonist GB-110 group. BCP mice model was constructed by injecting Lewis lung carcinoma cells into the femoral cavity of the right leg， which was followed by being treated with XZP for 21 d. After 21 d， the mice were sacrificed. Nissl staining was used to evaluate the survival of spinal cord neurons. Immunofluorescence staining was conducted to localize ionized calcium-binding adapter molecule 1 （Iba1） and neuronal nuclear antigen （NeuN） in spinal cord tissue， thereby assessing microglial activation and neuronal survival. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， transforming growth factor-β （TGF-β）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） in spinal cord tissue. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect mRNA expression levels associated with M1/M2 polarization of microglia. Western blot analysis was performed to examine the expression of proteins related to microglial polarization as well as those involved in the PAR2/nuclear factor kappa B （NF-κB）/NOD-like receptor protein 3 （NLRP3） signaling pathway in the spinal cord.ResultsCompared with the Sham group， the spinal cord neurons were damaged， the number of Nissl-positive spinal cord neurons in the spinal cord tissue was significantly reduced （P<0.01）， and the rate of NeuN-positive cells was significantly decreased （P<0.01）. The spinal cord microglia were activated， the inflammatory level of the spinal cord tissue was enhanced， and Iba1 staining was significantly enhanced （P<0.01）. The levels of IL-1β， TNF-α， IL-6， TGF-β， IL-4 and IL-10 were significantly increased （P<0.01）. The mRNA expressions of IL-1β， TNF-α and inducible nitric oxide synthase （iNOS） were significantly increased （P<0.01）， and the expression of PAR2， NLRP3， ASC and NF-κB p65 proteins in the spinal cord tissue of the BCP mice was significantly enhanced （P<0.01）. Compared with the BCP group， high-dose XZP treatment significantly increased the number of Nissl-positive spinal cord neurons in the BCP mice （P<0.01）， significantly enhanced the rate of NeuN-positive cells in the spinal cord tissue， and significantly weakened Iba1 staining （P<0.01）. In addition， the levels of IL-1β， TNF-α， and IL-6 were significantly decreased， while the levels of TGF-β， IL-4， and IL-10 were significantly increased （P<0.05， P<0.01）. The mRNA expression levels of IL-1β， TNF-α， and iNOS were decreased， whereas those of cluster of differentiation 206 （CD206）， arginase-1 （Arg-1）， and YM1/2 were significantly increased （P<0.05， P<0.01）. Low-dose and high-dose XZP treatment significantly decreased the expression of PAR2， NLRP3， ASC， and NF-κB p65 proteins in the spinal cord tissue （P<0.05， P<0.01）. These effects could all be significantly eliminated by the PAR2 agonist GB-110.ConclusionXZP can mitigate BCP in mice， which may be achieved through blocking the activated PAR2/NF-κB/NLRP3 pathway.

mots-clés

bone cancer pain （BCP）;Xiaozheng Zhitong Paste （XZP）;bone damage;neuronal damage;protease-activated receptor 2 （PAR2）/nuclear factor kappa B （NF-κB）/NOD- like receptor protein 3 （NLRP3）

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