基于c-JNK/CXCL1信号通路研究杜仲腰痛丸对腰椎间盘突出症大鼠脊髓背角组织炎症的抑制作用

Inhibitory effect of Duzhong Yaotong Pill on inflammation of spinal dorsal horn in rats with lumbar discs herniation based on c-JNK/CXCL1 signal pathway

摘要

目的: 分析杜仲腰痛丸调节c-Jun氨基末端激酶(c-Jun N-terminal kinase,c-JNK)/趋化因子配体1[chemokine(C-X-C motif)ligand 1,CXCL1]信号通路对腰椎间盘突出症(lumbar intervertebral disc protrusion,LDH)大鼠脊髓背角组织炎症的影响。方法: 将75只大鼠随机分为假手术组,模型组,杜仲腰痛丸低、中、高剂量组。模型复制2 h后,杜仲腰痛丸剂量组灌胃给予杜仲腰痛丸溶液,假手术组和模型组灌胃给予生理盐水。干预后观察各组大鼠一般情况,痛觉过敏实验测定机械刺激缩足反射阈值(paw withdrawal mechanical threshold,PWMT)和热刺激缩足反射潜伏期(paw withdrawal thermal latency,PWTL),ELISA检测大鼠脊髓背角组织白细胞介素6(interleukin-6,IL-6)和IL-1β水平,HE染色法观察大鼠背根神经节组织病理学变化,TUNEL染色检测脊髓细胞凋亡情况,qRT-PCR检测离子钙结合接头分子1(ion calcium binding junction molecule 1,Iba-1)、c-JNK、p-c-JNK、CXCL1mRNA表达,Westernblot检测Iba-1、c-JNK、p-c-JNK、CXCL1通路蛋白表达。结果: 与假手术组相比,模型组大鼠神经细胞萎缩,细胞核和尼氏小体减少,细胞凋亡率明显升高,IL-6、IL-β、Iba-1、p-c-JNK、CXCL1mRNA表达及蛋白表达升高,PWMT、PWTL下降(P<0.05);与模型组相比,杜仲腰痛丸低、中、高剂量组神经细胞形态恢复,核仁清晰,尼氏小体增加,细胞凋亡率明显下降,IL-6、IL-β、Iba-1、p-c-JNK、CXCL1mRNA表达及蛋白表达下降,PWMT、PWTL升高(P<0.05)。c-JNK的mRNA表达及蛋白表达相对表达水平组间比较差异无统计学意义(P>0.05)。结论: 杜仲腰痛丸能够抑制LDH大鼠脊髓背角组织炎症反应,作用机制可能与抑制c-JNK/CXCL1信号通路有关。

Abstract

Objective: To investigate the effect of Duzhong Yaotong Pill on spinal dorsal horn inflammation in rats with lumbar disc herniation (LDH) by regulating the c-Jun N-terminal kinase (c-JNK)/chemokine (C-X-C motif) ligand 1 (CXCL1) signaling pathway. Methods: Seventy-five rats were randomly divided into a sham operation group, a model group, and low-, medium-, and high-dose Duzhong Yaotong Pill groups. Two hours after model establishment, the Duzhong Yaotong Pill groups were gavaged with Duzhong Yaotong Pill solution, while the sham operation group and the model group were gavaged with normal saline. The general condition of the rats in each group was observed. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured by hyperalgesia testing. The levels of interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the spinal dorsal horn tissue were detected by ELISA. Histopathological changes in the dorsal root ganglion were observed by HE staining. Spinal cord cell apoptosis was detected by TUNEL staining. The mRNA expression of ionized calcium-binding adapter molecule 1 (Iba-1), c-JNK, p-c-JNK and CXCL1 was detected by qRT-PCR, and the protein expression of Iba-1, c-JNk, p-d-JNK and CXCL1 was detected by Westernblot. Results: Compared with the sham operation group, the model group showed neuronal atrophy, reduced nuclei and Nissl bodies, a significantly increased apoptosis rate, increased levels of IL-6, IL-β, Iba-1, p-c-JNK, and CXCL1, and decreased PWMT and PWTL (all P<0.05). Compared with the model group, the low-, medium-, and high-dose Duzhong Yaotong Pill groups showed recovery of neuronal morphology, distinct nucleoli, increased Nissl bodies, a significantly decreased apoptosis rate, decreased levels of IL-6, IL-β, decreased mRNA expression and protein expression of Iba-1, p-c-JNK, and CXCL1, and increased PWMT and PWTL (all P<0.05). There were no significant differences in the mRNA and protein expression levels of c-JNK among groups (P>0.05). Conclusion: Duzhong Yaotong Pill can inhibit spinal dorsal horn inflammation in LDH rats, and its mechanism may be related to the inhibition of the c-JNK/CXCL1 signaling pathway.

关键词

腰椎间盘突出症 / 杜仲腰痛丸 / 脊髓炎症 / c-JNK/CXCL1信号通路

Key words

lumbar disc herniation / Duzhong Yaotong Pill / spinal cord inflammation / c-Jun N-terminal kinase/CXCL1 signal pathway

[1] LIU YM, FENG Y, LIU YQ, et al. Chinese association for the study of pain: expert consensus on chronic postsurgical pain[J]. World J Clin Cases, 2021, 9(9): 2090-2099.
[2] 崔学军, 梁倩倩. 腰椎间盘突出症中西医结合诊疗专家共识[J]. 世界中医药, 2023, 18(7): 945-952.
[3] SHENOY K, STEKAS N, DONNALLY CJ 3rd, et al. Retrolisthesis and lumbar disc herniation: a postoperative assessment of outcomes at 8-year follow-up[J]. Spine J, 2019, 19(6): 995-1000.
[4] 张晓琳, 赵丽娜, 孔令磊. 姜黄素调节CXCL12/CXCR4信号通路对腰椎间盘突出症大鼠脊髓背角组织炎症及小胶质细胞活化的影响[J]. 免疫学杂志, 2025, 41(5): 312-317.
[5] TAŞKAYA B, TAŞKENT I., ÇAK?LL? M, et al. The effect of manual therapy on psychological factors and quality of life in lumbar disc herniation patients: a single blinded randomized clinical trial[J]. Int J Environ Res Public Health, 2024, 21(9): 1234.
[6] CIEZA A, CAUSEY K, KAMENOV K, et al. Global estimates of the need for rehabilitation based on the global burden of disease study 2019: a systematic analysis for the global burden of disease study 2019[J]. Lancet, 2021, 396(10267): 2006-2017.
[7] ZHU LG, YU CS, ZHANG XF, et al. The treatment of intervertebral disc degeneration using Traditional Chinese Medicine[J]. J Ethnopharmacol, 2020, 263: 113117.
[8] KIM CH, CHOI Y, CHUNG CK, et al. Nonsurgical treatment outcomes for surgical candidates with lumbar disc herniation: a comprehensive cohort study[J]. Sci Rep, 2021, 11(1): 3931.
[9] KÖGL N, PETR O, LÖSCHER W, et al. Lumbar disc herniation: the significance of symptom duration for the indication for surgery[J]. Dtsch Arztebl Int, 2024, 121(13): 440-448.
[10] HEIDER FC, SIEPE CJ.[Lumbar disc herniation] [J]. Orthopadie(Heidelberg, Germany), 2025, 54(1): 3-17.
[11] 秦晓宽, 孙凯, 徐卫国, 等. 腰椎间盘突出症中医循证实践指南[J]. 西部中医药, 2024, 37(5): 1-15.
[12] 葛龙, 李镜, 尚文茹, 等. 非手术疗法治疗腰椎间盘突出症的循证实践指南[J]. 中国循证医学杂志, 2024, 24(2): 125-148.
[13] 薛旭, 赵继荣, 李婧婧, 等. 杜仲腰痛丸干预腰椎间盘突出症临床疗效与药理作用的研究进展[J]. 中成药, 2025, 47(7): 2272-2278.
[14] 陈祁青, 马东, 赵继荣, 等. 基于网络药理学及动物实验的杜仲腰痛丸治疗腰椎间盘突出症慢性下肢痛的作用研究[J]. 世界科学技术-中医药现代化, 2023, 25(5): 1702-1713.
[15] 尹浩, 吉美奇, 胡志祥, 等. 三种腰椎间盘突出症大鼠模型制备方法的比较与评价[J]. 中国组织工程研究, 2025, 29(14): 2930-2936.
[16] TANG Q, HUANG YL, ZHU LR, et al. The effect of melatonin on radicular pain in a rat model of lumbar disc herniation[J]. Spine (Phila Pa 1976), 2022, 47(10): 754-763.
[17] HUANG ZY, TAN HB, FU YF, et al. Neurotransmitter imbalance and amygdala synaptic plasticity in lumbar disc herniation-induced chronic pain and related emotional Disturbances: a multi-omics analysis[J]. Neuropharmacology, 2025, 271: 110405.
[18] MA LL, DENG DL, ZHANG TH, et al. STING-IFN-I pathway relieves incision induced acute postoperative pain via inhibiting the neuroinflammation in dorsal root ganglion of rats[J]. Inflamm Res, 2023, 72(8): 1551-1565.
[19] CHANG L, YE F, LUO QH, et al. Increased hyperalgesia and proinflammatory cytokines in the spinal cord and dorsal root ganglion after surgery and/or fentanyl administration in rats[J]. Anesth Analg, 2018, 126(1): 289-297.
[20] HORNUNG AL, BAKER JD, MALLOW GM, et al. Resorption of lumbar disk herniation: mechanisms, clinical predictors, and future directions[J]. JBJS Rev, 2023, 11(1): e22.
[21] LINDAHL O, REXED B. Histologic changes in spinal nerve roots of operated cases of sciatica[J]. Acta Orthop Scand, 1951, 20(3): 215-225.
[22] LU X, CHEN LH, JIANG C, et al. Microglia and macrophages contribute to the development and maintenance of sciatica in lumbar disc herniation[J]. Pain, 2023, 164(2): 362-374.
[23] SLUSARCZYK J, TROJAN E, GŁOMBIK K, et al. Targeting the NLRP3 inflammasome-related pathways via tianeptine treatment-suppressed microglia polarization to the M1 phenotype in lipopolysaccharide-stimulated cultures[J]. Int J Mol Sci, 2018, 19(7): 1965.
[24] RIFAT A, OSSOLA B, BÜRLI RW, et al. Differential contribution of THIK-1 K⁺ channels and P2X7 receptors to ATP-mediated neuroinflammation by human microglia[J]. J Neuroinflammation, 2024, 21(1): 58.
[25] ATTA AA, IBRAHIM WW, MOHAMED AF, et al. Microglia polarization in nociplastic pain: mechanisms and perspectives[J]. Inflammopharmacology, 2023, 31(3): 1053-1067.
[26] GAO L, ZHANG AP, FU L, et al. Huangqin decoction attenuates spared nerve injury (SNI)-induced neuropathic pain by modulating microglial M1/M2 polarization partially mediated by intestinal nicotinamide metabolism[J]. Phytomedicine, 2024, 129: 155594.
[27] CHEN HL, YANG L, ZHANG XLN, et al. Scutellarin acts via MAPKs pathway to promote M2 polarization of microglial cells[J]. Mol Neurobiol, 2023, 60(8): 4304-4323.
[28] 徐昕, 唐丽玮, 金丹, 等. 氯胺酮对腰椎间盘突出根性痛大鼠脊髓炎症因子的影响及机制[J]. 中国临床药理学杂志, 2019, 35(18): 2119-2121.
[29] 牛辉, 鲍朝辉, 张文明, 等. 基于c-JNK/CXCL1信号通路研究椎间盘丸对腰椎间盘突出症大鼠脊髓炎症的抑制作用[J]. 中药新药与临床药理, 2021, 32(5): 655-660.

基金资助

国家自然科学基金资助项目(82460940);兰州市科技计划资助项目(2023-2-60);甘肃省自然科学基金资助项目(23JRRA1248)