温络通洗剂调节CX3CL1/CX3CR1信号轴防治化疗致周围神经疼痛的作用机制研究

Chemotherapy induced peripheral neuropathic pain (CIPNP) is the most common dose-limiting adverse effect of chemotherapeutic agents. It severely impairs the patients’ quality of life, which can lead to dose reductions, discontinuation of treatment, and ultimately affect overall survival. Unfortunately, there is no satisfying therapeutics in clinical practice. TCM treatment has shown promising effect, however, the underlying mechanisms are urgently to be explained. Wen-Luo-Tong (WLT) lotion is a well proved clinical recipes, which has been applied clinically to relieving CIPN for almost 20 years. A multicenter randomized controlled trial has shown that WLT treatment significantly alleviated pain with good tolerance. Animal experiment showed WLT treatment had neuroprotective effect without impairing the tumor inhibiting rate of chemotherapeutic agent. The updated researches have shown that CX3CL1/CX3CR1 axis played important role in the course of the occurrence and development of CIPNP. And metabolites of linoleic acid (LA) might induce CIPNP via activating CX3CL1, in which process CYP450 was the key enzyme to regulate LA metabolism. Additionally, researches indicated that NF-κB and JAK/STAT3 signal pathways were also involved in regulation of CX3CL1/CX3CR1 axis. Our Pilot study found that WLT down-regulated the metabolism pathway of LA and the expression of CX3CL1 in spinal cord of CIPNP model rats. Therefore, we put a question: Are the mechanisms of WLT alleviating CIPNP related to regulating CYP450 and/or signal pathways of NF-κB and JAK/STAT3? To answer it, we design this study, which takes CX3CL1/CX3CR1 axis as breakthrough point and applies techniques of molecular biology and bioinformatics, to explore the mechanisms of WLT treating CIPNP from macroscopic and molecular perspectives. We are hoping to explain scientific connotation of Warming and Activating Meridian, and to provide a new theoretical basis for TCM management of CIPNP.

CIPNP是常见化疗不良反应，严重影响生活质量，且临床尚无有效防治方法。中医药对改善CIPNP疗效显著，但其科学内涵亟待阐明。温络通是临床验方，前期临床研究证明其可缓解CIPNP，安全性好；基础研究发现其神经保护作用，且不影响化疗效果。最新研究表明，CX3CL1/CX3CR1轴在CIPNP发病过程中发挥重要作用。而LA代谢产物可激活CX3CL1诱发CIPNP，CYP450是关键酶。同时，NF-κB、JAK/STAT3信号通路对CX3CL1/CX3CR1轴有调节作用。预实验发现，温络通抑制LA代谢及CX3CL1表达。那么，温络通防治CIPNP机制是否与其调节CYP450及NF-κB、JAK/STAT3有关？由此我们以CX3CL1/CX3CR1轴为切入点，利用分子生物学、生物信息学等技术，探讨温络通的作用机理，以期阐释“温经通络”法科学内涵，为中医药防治CIPNP提供新的理论依据。

CIPNP是常见化疗不良反应，研究表明：CX3CL1/CX3CR1轴在CIPNP发病过程中发挥重要作用。而亚油酸（LA）代谢产物可激活CX3CL1诱发CIPNP，CYP450是关键酶。同时，NF-κB、JAK/STAT3信号通路对CX3CL1/CX3CR1轴有调节作用。为此本课题提出假说：温络通洗剂缓解CIPNP的机制可能包括：（1）通过影响CYP450抑制LA代谢通路，（2）通过抑制NF-κB、JAK/STAT3信号通路，从而调节CX3CL1/CX3CR1信号轴。基于此，本项目开展一下研究：通过紫杉醇致CIPNP大鼠模型，观察温络通洗剂对CIPNP模型大鼠机械性痛觉过敏、热痛觉过敏等痛行为学的影响；检测不同组织中TNF-α、INF-β、IL-1β、CX3CL1等表达；检测温络通洗剂对LA下游代谢产物的影响；检测温络通对CYP450表达的影响，重点关注CYP2J；检测温络通洗剂对JNK/STAT、NF-κB信号通路的影响。结果发现：温络通洗剂外用有效缓解了模型动物机械刺激、热刺激缩足阈值的下降；温络通可有效降低血浆、脊髓、背根神经节等组织中IL-1β、TNF-α、IL-6等炎性因子及炎性趋化因子CX3CL1的表达。WB检测脊髓中p-JAK、p-STAT3、p-NF-kB的表达，结果表明温络通可抑制NF-κB、JAK/STAT3信号通路，从而调节CX3CL1/CX3CR1信号轴。ELISA检测脊髓中CYP2J3、CYP2J4含量，温络通可下调CYP2J3、CYP2J4。但LA下游脂质代谢物9,10-EpOME的靶向代谢组学检测，温络通组（ZY）与紫杉醇组（HL）无统计学差异。综上，本研究明确了温络通洗剂可以通过调节通过抑制NF-κB、JAK/STAT3信号通路，从而调节CX3CL1/CX3CR1信号轴。但其对LA代谢通路的影响机制有待进一步研究。