Abstract
Objective
To investigate the central mechanism of moxibustion in treating chronic inflammatory visceral pain (CIVP) and its analgesic effect from the perspective of the p38 mitogen-activated protein kinase (MAPK)/Ets-like transcription factor 1 (ELK1) signaling pathway in the spinal cord.
Methods
Clean-grade male Sprague-Dawley rats were randomly divided into a normal group, a model group, a herb-partitioned moxibustion (HPM) group, a sham-HPM group, a p38 MAPK inhibitor group, and a dimethyl sulfoxide (DMSO) group. CIVP rat models were prepared using an enema mixture of 2,4,6-trinitrobenzene sulfonic acid solution and 50% ethanol. The HPM group was treated with HPM; the sham-HPM group was treated the same as the HPM group, but the moxa cones were not ignited; rats in the p38 MAPK inhibitor group received L5-L6 intrathecal injection of p38 MAPK inhibitor (SB203580); rats in the DMSO group received L5-L6 intrathecal injection of 2% DMSO. Abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were used to observe pain-related behaviors in each group. Hematoxylin-eosin staining was used to observe the morphological changes in rat colon tissue. Western blotting and real-time quantitative reverse-transcription polymerase chain reaction were used to detect the phosphorylated protein and mRNA expression of apoptosis signal-regulating kinase 1 (ASK1), MAPK kinase (MKK) 3/6, p38 MAPK, ELK1, and mitogen and stress-activated protein kinase 1 (MSK1) in the spinal cord.
Results
Compared with the normal group, CIVP rats had severe colonic inflammatory injuries, and the pathological injury scores increased significantly, along with increased AWR scores under different colorectal distension (CRD) stimulation pressures and decreased MWT and TWL; the mRNA and phosphorylated protein expression of p38 MAPK, ELK1, MSK1, ASK1, MKK3, and MKK6 all increased in the spinal cord (P<0.01). After HPM treatment, the colon injuries were repaired, and the pathological injury scores decreased; under different CRD stimulation pressures, the AWR scores decreased, and the MWT and TWL increased; the mRNA and phosphorylated protein expression of p38 MAPK, ELK1, ASK1, and MKK3 in the spinal cord also decreased, with statistically significant differences compared with the model group and the sham-HPM group (P<0.01). There were no significant differences in the above indicators between the HPM group and the p38 MAPK inhibitor group (P>0.05), and the same was true regarding the comparisons between the model group and the DMSO group.
Conclusion
HPM exerted analgesic effects via downregulating the mRNA and phosphorylated protein expression of ASK1, MKK3, p38 MAPK, and ELK1 in the spinal cord of CIVP rats. The inhibition of spinal p38 MAPK/ELK1 signaling pathway activation may be one of the mechanisms by which HPM relieves pain in CIVP.
摘要
目的
从调节脊髓p38丝裂原活化蛋白激酶(MAPK)/Ets样转录因子1(ELK1)信号通路角度, 探讨艾灸治 疗慢性炎性内脏痛(CIVP)的中枢机制及其镇痛效应。
方法
清洁级雄性Sprague-Dawley大鼠随机分为正常 组、模型组、隔药灸组、假隔药灸组、p38 MAPK抑制剂组和二甲基亚砜(DMSO)组。采用2,4,6-三硝基苯 磺酸合50%乙醇灌肠制备CIVP大鼠模型。隔药灸组给予隔附子饼灸治疗; 假隔药灸组处理同隔药灸组, 但不 点燃艾炷; p38 MAPK抑制剂组在大鼠L5-L6间鞘内注射SB203580; DMSO组在L5-L6间鞘内注射2%DMSO。通过 腹壁撤回反射(AWR)评分、机械刺激缩足阈值(MWT)及热缩足潜伏期(TWL)观察各组大鼠痛行为变化; 应用 苏木素-伊红染色镜下观察各组大鼠结肠组织形态学变化; 采用免疫印迹法和实时荧光定量聚合酶链反应 检测各组大鼠脊髓凋亡信号调节激酶1(ASK1)、MAPK激酶(MKK)3/6、p38 MAPK、ELK1和丝裂原应激活化 蛋白激酶1(MSK1)磷酸化蛋白和mRNA的表达。
结果
与正常组比较, CIVP大鼠结肠炎性损伤严重, 病理损伤 评分明显升高; 不同压力结直肠扩张(CRD)刺激下AWR评分均增加, MWT及TWL均降低; 脊髓p38 MAPK、 ELK1、MSK1、ASK1、MKK3/6磷酸化蛋白和mRNA表达显著增加(P<0.01)。经隔药灸治疗后, 大鼠结肠损伤 有所修复, 病理损伤评分降低; 不同压力CRD刺激下, AWR评分均减少, MWT、TWL升高; 脊髓p38 MAPK、 ELK1、ASK1、MKK3磷酸化蛋白和mRNA表达降低, 与模型组、假隔药灸组比较, 差异均有统计学意义 (P<0.01)。隔药灸组与p38 MAPK抑制剂组相比、DMSO组与模型组相比, 上述指标均无显著差异(P>0.05)。
结论
隔药灸能通过下调CIVP大鼠脊髓ASK1、MKK3、p38 MAPK、ELK1磷酸化蛋白和mRNA的表达发挥镇 痛效应。抑制脊髓p38 MAPK/ELK1信号通路活化可能是隔药灸缓解疼痛,治疗CIVP的机制之一。
Similar content being viewed by others
References
ZIELIŃSKA A, SAŁAGA M, WŁODARCZYK M, FICHNA J. Focus on current and future management possibilities in inflammatory bowel disease-related chronic pain. Int J Colorectal Dis, 2019, 34(2): 217–227.
ZHANG Y, LIU J L, HAN X, JIANG H, ZHANG L M, HU J C, SHI L, LI J X. Long-term trends in the burden of inflammatory bowel disease in China over three decades: a joinpoint regression and age-period-cohort analysis based on GBD 2019. Front Public Health, 2022, 10: 994619.
MORRISON G, VAN LANGENBERG D R, GIBSON S J, GIBSON P R. Chronic pain in inflammatory bowel disease: characteristics and associations of a hospital-based cohort. Inflamm Bowel Dis, 2013, 19(6): 1210–1217.
COATES M D, SETH N, CLARKE K, ABDUL-BAKI H, MAHONEY N, WALTER V, REGUEIRO M D, RAMOSRIVERS C, KOUTROUBAKIS I E, BIELEFELDT K, BINION D G. Opioid analgesics do not improve abdominal pain or quality of life in Crohn’s disease. Dig Dis Sci, 2020, 65(8): 2379–2387.
BERRY S K, TAKAKURA W, BRESEE C, MELMED G Y. Pain in inflammatory bowel disease is not improved during hospitalization: the impact of opioids on pain and healthcare utilization. Dig Dis Sci, 2020, 65(6): 1777–1783.
XIE J, HUANG Y, WU H G, LI J. Acupuncture and moxibustion for treatment of Crohn’s disease: a brief review. World J Gastroenterol, 2022, 28(25): 3001–3003.
LI H, YE X F, SU Y S, HE W, ZHANG J B, ZHANG Q, ZHAN L B, JING X H. Mechanism of acupuncture and moxibustion on promoting mucosal healing in ulcerative colitis. Zhonghua Zhongxiyi Jiehe Zazhi, 2023, 29(9): 847–856.
STEIN D J. Massage acupuncture, moxibustion, and other forms of complementary and alternative medicine in inflammatory bowel disease. Gastroenterol Clin North Am, 2017, 46(4): 875–880.
LIN Y Y, ZHAO J M, JI Y J, MA Z, ZHENG H D, HUANG Y, CUI Y H, LU Y, WU H G. Typical ulcerative colitis treated by herbs-partitioned moxibustion: a case report. World J Clin Cases, 2020, 8(8): 1515–1524.
BAO C H, WANG D, LIU P, SHI Y, JIN X M, WU L Y, ZENG X Q, ZHANG J Y, LIU H R, WU H G. Effect of electro-acupuncture and moxibustion on brain connectivity in patients with Crohn’s disease: a resting-state fMRI study. Front Hum Neurosci, 2017, 11: 559.
BAO C H, LIU P, LIU H R, JIN X M, CALHOUN V D, WU L Y, SHI Y, ZHANG J Y, ZENG X Q, MA L L, QIN W, ZHANG J Z, LIU X M, TIAN J, WU H G. Different brain responses to electro-acupuncture and moxibustion treatment in patients with Crohn’s disease. Sci Rep, 2016, 6: 36636.
YUAN T, MANOHAR K, LATORRE R, OROCK A, GREENWOOD-VAN MEERVELD B. Inhibition of microglial activation in the amygdala reverses stress-induced abdominal pain in the male rat. Cell Mol Gastroenterol Hepatol, 2020, 10(3): 527–543.
FENG Y J, LI Y Y. The role of p38 mitogen-activated protein kinase in the pathogenesis of inflammatory bowel disease. J Dig Dis, 2011, 12(5): 327–332.
MAI L J, ZHU X, HUANG F, HE H W, FAN W G. p38 mitogen-activated protein kinase and pain. Life Sci, 2020, 256: 117885.
YANG K Y, BAE W S, KIM M J, BAE Y C, KIM Y J, KIM H J, NAM S H, AHN D K. Participation of the central p38 and ERK1/2 pathways in IL-1β-induced sensitization of nociception in rats. Prog Neuropsychopharmacol Biol Psychiatry, 2013, 46: 98–104.
HUANG Y, ZHANG D, LI Z Y, YANG Y T, WU L J, ZHANG J, ZHI F Y, LI X Y, SHI Z, HONG J, MA X P. Moxibustion eases chronic inflammatory visceral pain in rats via MAPK signaling pathway in the spinal cord. J Pain Res, 2019, 12: 2999–3012.
LI Z Y, HUANG Y, YANG Y T, ZHANG D, ZHAO Y, HONG J, LIU J, WU L J, ZHANG C H, WU H G, ZHANG J, MA X P. Moxibustion eases chronic inflammatory visceral pain through regulating MEK, ERK and CREB in rats. World J Gastroenterol, 2017, 23(34): 6220–6230.
ZHOU Q Q, PRICE D D, CAUDLE R M, VERNE G N. Visceral and somatic hypersensitivity in TNBS-induced colitis in rats. Dig Dis Sci, 2008, 53(2): 429–435.
CHEN Z, XIE F, BAO M, LI X, CHAO Y, LIN C, GUO R, ZHANG C, WU A, YUE Y, GUAN Y, WANG Y. Activation of p38 MAPK in the rostral ventromedial medulla by visceral noxious inputs transmitted via the dorsal columns may contribute to pelvic organ cross-sensitization in rats with endometriosis. Neuroscience, 2015, 291: 272–278.
AL-CHAER E D, KAWASAKI M, PASRICHA P J. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterology, 2000, 119(5): 1276–1285.
CHAPLAN S R, BACH F W, POGREL J W, CHUNG J M, YAKSH T L. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods, 1994, 53(1): 55–63.
HARGREAVES K, DUBNER R, BROWN F, FLORES C, JORIS J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain, 1988, 32(1): 77–88.
ARAKI Y, ANDOH A, FUJIYAMA Y, BAMBA T. Development of dextran sulphate sodium-induced experimental colitis is suppressed in genetically mast cell-deficient Ws/Ws rats. Clin Exp Immunol, 2000, 119(2): 264–269.
GRUNDY L, ERICKSON A, BRIERLEY S M. Visceral pain. Annu Rev Physiol, 2019, 81: 261–284.
DOCHERTY M J, JONES R C3RD, WALLACE M S. Managing pain in inflammatory bowel disease. Gastroenterol Hepatol (N Y), 2011, 7(9): 592–601.
FANG J Q, SHAO X M. New trains of thoughts about acupuncture analgesia: acupuncture analgesia may involve multi-dimensional regulation of pain. Zhen Ci Yan Jiu, 2017, 42(1): 85–89.
YANG M X, CHEN X Z, BO L N, LAO L X, CHEN J, YU S Y, YU Z, TANG H Z, YI L, WU X, YANG J, LIANG F R. Moxibustion for pain relief in patients with primary dysmenorrhea: a randomized controlled trial. PLoS One, 2017, 12(2): e0170952.
HUANG R J, ZHAO J M, WU L Y, DOU C Z, LIU H R, WENG Z J, LU Y, SHI Y, WANG X M, ZHOU C L, WU H G. Mechanisms underlying the analgesic effect of moxibustion on visceral pain in irritable bowel syndrome: a review. Evid Based Complement Alternat Med, 2014, 2014: 895914.
DUBOIS M Y, CHEN L. Of low back pain and moxibustion. Pain Med, 2014, 15(8): 1243–1244.
KIM T H, KIM K H, KANG J W, LEE M, KANG K W, KIM J E, KIM J H, LEE S, SHIN M S, JUNG S Y, KIM A R, PARK H J, JUNG H J, SONG H S, KIM H J, CHOI J B, HONG K E, CHOI S M. Moxibustion treatment for knee osteoarthritis: a multi-centre, non-blinded, randomised controlled trial on the effectiveness and safety of the moxibustion treatment versus usual care in knee osteoarthritis patients. PLoS One, 2014, 9(7): e101973.
ZHAO J M, LU J H, YIN X J, CHEN X K, CHEN Y H, TANG W J, JIN X M, WU L Y, BAO C H, WU H G, SHI Y. Comparison of electroacupuncture and moxibustion on brain-gut function in patients with diarrhea-predominant irritable bowel syndrome: a randomized controlled trial. Zhonghua Zhongxiyi Jiehe Zazhi, 2015, 21(11): 855–865.
ZHAO J M, LU J H, YIN X J, WU L Y, BAO C H, CHEN X K, CHEN Y H, TANG W J, JIN X M, WU H G, SHI Y. Comparison of electroacupuncture and mild-warm moxibustion on brain-gut function in patients with constipation-predominant irritable bowel syndrome: a randomized controlled trial. Zhonghua Zhongxiyi Jiehe Zazhi, 2018, 24(5): 328–335.
ZHU Y, WU Z Y, MA X P, LIU H R, BAO C H, YANG L, CUI Y H, ZHOU C L, WANG X M, WANG Y M, ZHANG Z W, ZHANG H, JIA H P, WU H G. Brain regions involved in moxibustion-induced analgesia in irritable bowel syndrome with diarrhea: a functional magnetic resonance imaging study. BMC Complement Altern Med, 2014, 14: 500.
LIU S M, SHI Q M, ZHU Q C, ZOU T, LI G, HUANG A, WU B, PENG L C, SONG M M, WU Q, XIE Q Y, LIN W J, XIE W, WEN S Y, ZHANG Z D, LÜ Q L, ZOU L F, ZHANG X, YING M F, LI G D, LIANG S D. P2X7 receptor of rat dorsal root ganglia is involved in the effect of moxibustion on visceral hyperalgesia. Purinergic Signal, 2015, 11(2): 161–169.
HUANG Y, YANG Y T, LIU X X, ZHAO Y, FENG X M, ZHANG D, WU H G, ZHU Y, HUANG W Y, MA X P. Effect of herbal-partitioned moxibustion at Tianshu (ST25) and Qihai (CV6) on pain-related behavior and emotion in rats with chronic inflammatory visceral pain. J Acupunct Tuina Sci, 2015, 13(1): 1–8.
LI Z Y, YANG Y T, HONG J, ZHANG D, HUANG X F, WU L J, WU H G, SHI Z, LIU J, ZHU Y, MA X P. Extracellular signal-regulated kinase, substance P and neurokinin-1 are involved in the analgesic mechanism of herb-partitioned moxibustion. Neural Regen Res, 2017, 12(9): 1472–1478.
MOLONEY R D, JOHNSON A C, O’MAHONY S M, DINAN T G, GREENWOOD-VAN MEERVELD B, CRYAN J F. Stress and the microbiota-gut-brain axis in visceral pain: relevance to irritable bowel syndrome. CNS Neurosci Ther, 2016, 22(2): 102–117.
ZHANG Y K, HUANG Z J, LIU S, LIU Y P, SONG A A, SONG X J. WNT signaling underlies the pathogenesis of neuropathic pain in rodents. J Clin Invest, 2013, 123(5): 2268–2286.
CHEN W J, GUO S D, WANG S G. MicroRNA-16 alleviates inflammatory pain by targeting Ras-related protein 23 (RAB23) and inhibiting p38 MAPK activation. Med Sci Monit, 2016, 22: 3894–3901.
ANAND P, AZIZ Q, WILLERT R, VAN OUDENHOVE L. Peripheral and central mechanisms of visceral sensitization in man. Neurogastroenterol Motil, 2007, 19 (1 Suppl): 29–46.
ZHANG T, ZHANG N, ZHANG R, ZHAO W D, CHEN Y, WANG Z L, XU B, ZHANG M N, SHI X R, ZHANG Q Q, GUO Y Y, XIAO J, CHEN D, FANG Q. Preemptive intrathecal administration of endomorphins relieves inflammatory pain in male mice via inhibition of p38 MAPK signaling and regulation of inflammatory cytokines. J Neuroinflammation, 2018, 15(1): 320.
QU Y J, JIA L, ZHANG X, WEI H, YUE S W. MAPK pathways are involved in neuropathic pain in rats with chronic compression of the dorsal root ganglion. Evid Based Complement Alternat Med, 2016, 2016: 6153215.
ONO K, HAN J. The p38 signal transduction pathway: activation and function. Cell Signal, 2000, 12(1): 1–13.
SHARROCKS A D. Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture. Biochem Soc Trans, 2002, 30(2): 1–9.
ARTHUR J S. MSK activation and physiological roles. Front Biosci, 2008, 13: 5866–5879.
Acknowledgments
This work was supported by the Projects of National Natural Science Foundation of China (国家自然科学基金 项目, No. 82104985, No. 81273843, No. 81674073, No. 81202754); Projects of Natural Science Foundation of Shanghai (上海市自然科学基金项目, No. 23ZR1460100, No. 23ZR1460000); Outstanding Leader Plan of Shanghai (上海市领军人才项目, No. 060).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of Interest
Author WU Huangan is an editor-in-chief of the Journal of Acupuncture and Tuina Science; author MA Xiaopeng is a member of the Editorial Board of Journal of Acupuncture and Tuina Science, and authors YANG Guang and HONG Jue are the editors serving for Journal of Acupuncture and Tuina Science. The paper was handled by other editors and has undergone a rigorous peer review process. Authors WU Huangan, MA Xiaopeng, YANG Guang, and HONG Jue were not involved in the journal’s review or decisions related to this manuscript.
Statement of Human and Animal Rights
The treatment of animals in this experiment conformed to the ethical criteria.
Additional information
Co-first Authors: ZHANG Dan, M.D., associate researcher; LI Zhiyuan, M.D., attending physician
Rights and permissions
About this article
Cite this article
Zhang, D., Li, Z., Yu, H. et al. Mechanism of moxibustion in treating chronic inflammatory visceral pain: regulation of the p38 MAPK/ELK1 signaling pathway in the spinal cord. J. Acupunct. Tuina. Sci. (2024). https://doi.org/10.1007/s11726-024-1425-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11726-024-1425-5
Keywords
- Moxibustion Therapy
- Medicinal Cake-partitioned Moxibustion
- Inflammatory Bowel Diseases
- Visceral Pain
- Visceral Hypersensitivity
- p38 Mitogen-activated Protein Kinase Signaling Pathway
- Rats