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Pain

. 2023 Apr 7;164(9):1965–1975. doi: 10.1097/j.pain.0000000000002892

Local analgesia of electroacupuncture is mediated by the recruitment of neutrophils and released β-endorphins

Jing-tao Shi a,b, Wan-ying Cao a, Xiao-Ning Zhang a, Hong-Ye Wan a,✉, Yang-Shuai Su a, Zheng-Yang Qu a, Rui Wang c,✉, Wei He a, Xiang-Hong Jing a, Xiao-Yu Wang a,*

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PMCID: PMC10436362  PMID: 37027145

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Supplemental Digital Content is Available in the Text.

Electroacupuncture recruited β-endorphins (β-END)-containing ICAM-1+/CD11b+ immune cells and increased the β-END content at the site of inflammatory.

Keywords: Acupuncture analgesia, Inflammatory pain, Neuroimmune, Sympathetic nerves, β-endorphin

Abstract

The efficacy of acupuncture in treating pain diseases has been recognized in clinical practice, and its mechanism of action has been a hot topic in academic acupuncture research. Previous basic research on acupuncture analgesia has focused mostly on the nervous system, with few studies addressing the immune system as a potential pathway of acupuncture analgesia. In this study, we investigated the effect of electroacupuncture (EA) on the β-endorphins (β-END) content, END-containing leukocyte type and number, sympathetic neurotransmitter norepinephrine (NE), and chemokine gene expression‎ in inflamed tissues. To induce inflammatory pain, about 200 µL of complete Frester adjuvant (CFA) was injected into the unilateral medial femoral muscle of adult Wistar rats. Electroacupuncture treatment was performed for 3 days beginning on day 4 after CFA injection, with parameters of 2/100 Hz, 2 mA, and 30 minutes per treatment. The weight-bearing experiment and enzyme-linked immunosorbent assay showed that EA treatment significantly relieved spontaneous pain-like behaviors and increased the level of β-END in inflamed tissue. Injection of anti-END antibody in inflamed tissue blocked this analgesic effect. Flow cytometry and immunofluorescence staining revealed that the EA-induced increase in β-END was derived from opioid-containing ICAM-1+/CD11b+ immune cells in inflamed tissue. In addition, EA treatment increased the NE content and expression‎ of β2 adrenergic receptor (ADR-β2) in inflammatory tissues and upregulated Cxcl1 and Cxcl6 gene expression‎ levels. These findings provide new evidence for the peripheral analgesic effect of acupuncture treatment by recruiting β-END–containing ICAM-1+/CD11b+ immune cells and increasing the β-END content at the site of inflammation.

초록
통증 질환 치료에 대한 침술의 효능은 임상에서 인정되어 왔으며, 

그 작용 메커니즘은 학계 침술 연구에서 뜨거운 주제였습니다. 

침술 진통에 대한 이전의 기초 연구는 

대부분 신경계에 초점을 맞추었으며 

침술 진통의 잠재적 경로로서

 면역계를 다룬 연구는 거의 없었습니다. 

이 연구에서는 

전기침술(EA)이 

염증 조직에서 β-엔도르핀(β-END) 함량, 

END 함유 백혈구 유형 및 수, 

교감 신경전달물질 노르에피네프린(NE), 

케모카인 유전자 발현에 미치는 영향을 조사했습니다. 

염증성 통증을 유도하기 위해 성인 위스타 쥐의 일측 대퇴 내측 근육에 약 200µL의 완전한 프레스터 보조제(CFA)를 주입했습니다. 

전기침 치료는 CFA 주입 후 4일째부터 

3일간 2/100Hz, 

2mA, 

1회당 30분의 파라미터로 실시했습니다. 

체중 부하 실험과 효소 결합 면역 흡착 분석 결과, 

전침 치료는 

자발적인 통증 유사 행동을 현저히 완화하고 

염증 조직에서 β-END 수치를 증가시키는 것으로 나타났습니다. 

염증 조직에 

항-END 항체를 주입하면 

이러한 진통 효과가 차단되었습니다. 

anti-END antibody

유세포 분석 및 면역 형광 염색 결과, 

EA로 인한 β-END의 증가는 

염증 조직에서 오피오이드 함유 ICAM-1+/CD11b+ 면역 세포에서 유래한 것으로 나타났습니다. 

또한

EA 치료는 

염증 조직에서 

β2 아드레날린 수용체(ADR-β2)의 NE 함량과 발현을 증가시키고 

Cxcl1 및 Cxcl6 유전자 발현 수준을 상향 조절했습니다. 

케모카인은 화학 주성, 종양 성장, 혈관 신생 등을 포함한 많은 생물학적 행동을 매개하는 큰 계열입니다. 이 계열의 한 구성원인 CXC 하위 계열은 동일한 능력을 가지고 있습니다. CXC 케모카인은 다양한 범주의 면역 세포를 모집 및 이동시키고, 증식, 침입 및 전이와 같은 종양의 병리학적인 행동을 조절하며, 혈관 신생을 활성화하는 등의 기능을 합니다. 이러한 특성으로 인해 CXCL 서브패밀리는 종양 및 염증성 질환과 광범위하고 밀접하게 연관되어 있습니다. 연구가 점점 더 집중적으로 진행됨에 따라 CXCL의 구체적인 역할이 더 잘 설명되고 있으며 바이오마커와 표적을 포함한 CXCL의 치료 응용도 심도 있게 설명되고 있습니다. 이 리뷰에서는 다양한 질병에서 CXCL 가족 구성원의 역할을 요약합니다. 케모카인은 백혈구와 종양 세포의 생물학적 행동을 조절하고 암종과 염증의 발생에 필수적인 역할을 합니다. 케모카인의 한 하위 계열인 CXCL은 면역 세포의 모집과 종양의 병리학적 과정에 관여하는 등 관련 기능을 통해 이러한 질환에서 빼놓을 수 없는 존재입니다.2. CXCL 및 관련 질환과 관련된 연구는 주로 암과 염증성 질환에 초점을 맞추고 있습니다. 유방암, 폐암, 위암, 간암, 대장암, 난소암, 췌장암, 전립선암, 흑색종 등과 같은 다양한 암에 CXCL이 밀접하게 관여한다는 것이 입증되었습니다. 또한 CXCL은 관절염, 전신성 홍반성 루푸스, 만성 폐쇄성 폐질환, 간염 등 다양한 장기 및 시스템에 나타나는 염증성 질환에도 밀접하게 관여합니다.3. 최근에는 CXCL이 호중구, 단핵구, 대식세포 등 백혈구의 이동을 통해 염증과 면역 반응을 조절한다는 사실이 확인되었습니다. 또한 STAT3 및 NF-κB와 같은 다양한 신호 전달 경로를 활성화하여 종양 세포의 증식, 침입, 전이 및 혈관 신생을 조절하여 암 발생을 가속화합니다. 그러나 CXCL이 이러한 질병에서 어떻게 기능을 발휘하는지에 대한 구체적인 메커니즘은 아직 완전히 밝혀지지 않았습니다.4. 많은 연구에서 암, 염증성 질환 및 대사성 질환의 잠재적 바이오마커, 표적 및 지표로 CXCL을 고려했습니다. 이러한 질환의 진단, 예후 및 치료에서 CXCL의 잠재적 가치는 광범위하게 인정되고 있지만, 임상에서의 활용은 아직 완전히 밝혀지지 않았습니다.

이러한 연구 결과는 

침 치료의 말초 진통 효과에 대한 새로운 증거를 제공하며, 

β-END 함유 ICAM-1+/CD11b+ 면역 세포를 모집하고 

염증 부위의 β-END 함량을 증가시킴으로써 

침 치료의 말초 진통 효과에 대한 새로운 증거를 제공합니다.

1. Introduction

Acupuncture has been recognized in clinical practice to effectively relieve pain, and the therapeutic effect of acupuncture usually persists over a long period of time.38 Numerous physiological mechanisms have been proposed to explain the pain-relieving effect of acupuncture. For example, in inflammatory muscle pain rats, low-strength electroacupuncture (EA) has been shown to reduce the introduction of pain information through the spinal gate–control mechanism, significantly alleviating pain and inhibiting the abnormal electromyography.7 Previous studies have also demonstrated that heterotopic EA stimulation triggers the pain-inhibiting effect of diffuse noxious inhibitory controls (DNICs).15 In addition, Han9 found that acupuncture applied at specific frequencies to certain body sites can stimulate the release of endogenous opioid peptides in the central nervous system and activate opioid receptors to induce antinociception. Therefore, it has been deduced that acupuncture controlled pain transmission in the central nervous system by neuronal and endocrine pathways. However, the acupuncture-induced effect observed in this research was typically transient, as demonstrated by the fact that the inhibition of the C-fiber reflex caused by EA lasted 5 minutes after treatment.44 In addition, endogenous opioid peptides are rapidly degraded by peripheral blood proteases, giving a half-life of approximately 40 minutes for β-endorphins (β-END).42 These mechanisms could not fully explain why there is long-term pain relief after the acupuncture treatment.

During inflammation, tissue damage caused by pathogen invasion induces the release of vascular active substances and chemokines by resident tissue cells, which recruit immune cells from the blood vessels to the inflammatory site.12 Tissue-resident and recruited immune cells secrete inflammatory mediators, including cytokines, lipid mediators, and growth factors, which activate nociceptor sensory neurons to produce pain.26 Meanwhile, opioid-containing immune cells migrate preferentially to the site of pain, releasing opioid peptides that activate opioid receptors on peripheral nociceptive neurons to induce antinociception.35 Endogenous opioid peptides, including β-endorphins (β-END), enkephalins (ENK), and dynorphins (DYN), have been implicated in peripheral analgesia during the early stages of inflammation,5 whereas antinociception is mediated only by β-END in the late stages of inflammation.20 Owing to the presence of inflammation, opioid-producing leukocytes are progressively recruited into the pain site, resulting in the constant availability of endogenous opiopeptides and local long-term analgesic effects.

Nociceptive stimuli produced by acupuncture have been considered as one of the reasons for analgesia in previous studies.3,43,44 Noxious stimuli can locally activate more sensory nerve fibers by axon reflexes37; Meanwhile, it can also locally cause symptoms such as flushing and sweating by sympathetic reflexes.13,17 Calcitonin gene-related peptide (CGRP) released from sensory nerve fibers plays the role in vasodilation and immunosuppression. The capsaicin-induced CGRP release is sufficient to cause significant vasodilatation.1 Calcitonin gene-related peptide can support IL-5, constrain IL-13 expression‎, and constrain type 2 inflammation.24 There are also more connections between the neurotransmitter norepinephrine produced by postganglionic sympathetic nerves and immune cells. Norepinephrine can regulate CD4+ T-lymphocyte and B-lymphocyte function.11,32 Whether these 2 types of nerve fibers and their neurotransmitters can regulate local pain through the immune system is our concern.

1. 서론

침술은 임상에서 통증을 효과적으로 완화하는 것으로 인정되어 왔으며 침술의 치료 효과는 일반적으로 장기간에 걸쳐 지속됩니다.38 침술의 통증 완화 효과를 설명하기 위해 수많은 생리적 메커니즘이 제안되었습니다. 예를 들어, 염증성 근육통 쥐에서 저강도 전기침술(EA)은 척추 게이트 제어 메커니즘을 통한 통증 정보의 유입을 감소시켜 통증을 현저히 완화하고 비정상적인 근전도 검사를 억제하는 것으로 나타났습니다.7

이전 연구에서도 이종성 EA 자극이 확산성 독성 억제 조절(DNIC)의 통증 억제 효과를 유발한다는 사실이 입증되었습니다.15

또한 한9은

특정 신체 부위에 특정 주파수로 침을 놓으면

중추 신경계에서 내인성 오피오이드 펩타이드의 방출을 자극하고

오피오이드 수용체를 활성화하여 항통각을 유도할 수 있다는 사실을 발견했습니다.

뇌 기능은 신경전달물질과 신경펩티드를 포함한 화학적 메신저에 의해 조절됩니다. 최근 연구에 따르면 특정 신체 부위에 특정 주파수의 침술이나 전기 자극을 가하면 중추신경계에서 특정 신경 펩타이드의 방출을 촉진하여 심오한 생리적 효과를 이끌어내고 심지어 자가 치유 메커니즘을 활성화할 수 있는 것으로 나타났습니다. 이러한 신경생물학적 반응을 조절하는 조건에 대한 조사는 이론적, 임상적 의미를 가질 수 있습니다.

따라서

침술은

경 및 내분비 경로를 통해 중추 신경계에서 통증 전달을 조절한다고 추론되었습니다.

그러나

이 연구에서 관찰된 침으로 인한 효과는 일반적으로 일시적이었는데,

이는 침으로 인한 C 섬유 반사의 억제가 치료 후 5분 동안 지속되었다는 사실에서 알 수 있습니다.44

또한

내인성 오피오이드 펩타이드는

말초 혈액 프로테아제에 의해 빠르게 분해되어

β-엔돌핀(β-END)의 반감기가 약 40분입니다.42

이러한 메커니즘은

침 치료 후 장기간 통증 완화되는 이유를

완전히 설명할 수 없습니다.

염증 중에 병원체 침입으로 인한 조직 손상은

상주 조직 세포에 의해 혈관 활성 물질과 케모카인의 방출을 유도하고,

이는 혈관에서 염증 부위로 면역 세포를 모집합니다.12

조직 상주 및 모집된 면역 세포는

사이토카인, 지질 매개체 및 성장 인자를 포함한 염증 매개체를 분비하여

통각 수용체 감각 뉴런을 활성화하여 통증을 유발합니다.26

한편

오피오이드 함유 면역 세포는

통증 부위로 우선적으로 이동하여

말초 통각 신경세포의 오피오이드 수용체를 활성화하는 오피오이드 펩타이드를 방출하여

항통각을 유도합니다.35

β-엔돌핀(β-END), 엔케팔린(ENK), 다이노르핀(DYN)을 포함한 내인성 오피오이드 펩타이드는

염증 초기 단계에서 말초 진통에 관여하는 반면,5

항통각은 염증 후기 단계에서 β-END에 의해서만 매개됩니다.20

염증의 존재로 인해 오피오이드 생성 백혈구가

통증 부위에 점진적으로 모집되어

내인성 오피오펩타이드의 지속적인 가용성과 국소 장기 진통 효과를 가져옵니다.

침술에 의해 생성되는 통각 자극은

이전 연구에서 진통의 원인 중 하나로 간주되었습니다.3,43,44

noxious 자극은

축삭 반사에 의해 더 많은 감각 신경 섬유를

국소적으로 활성화시킬 수 있습니다37;

축삭 반사[1] (또는 플레어 반응)[2] 는 신체의 말초 신경에 의해 자극된 반응으로, 신경 세포 본체에서 떨어져 나와 가지를 뻗어 목표 기관을 자극합니다. 반사는 자극에 반응하는 단일 반응으로 신체 신경계의 전체 신호의 구성 요소를 구성합니다. 뉴런은 축삭, 수상 돌기 및 세포체를 통해 이러한 반사 신호를 처리하고 전달하는 흥분성 세포입니다. 축삭은 신경 세포체에서 다른 신경 세포, 국소 근육 조직, 땀샘 및 세동맥으로 돌출된 세포 간 통신을 직접적으로 촉진합니다. 축삭 반사에서 신호는 자극 부위의 축삭 중간에서 시작하여 척수 반사에 존재하는 통합 센터와 화학 시냅스를 모두 건너뛰고 효과 기관으로 직접 신호를 전달합니다. 자극은 하나의 분기된 축삭으로 제한되거나[3] 축삭이 두 개의 분열로 분기되어 주변 조직에 일반적인 반응을 일으키지 않는 뉴런으로 제한됩니다. 축삭 반사 아크는 척수 반사 아크와 구별됩니다. 척수 반사 경로에서 구심성 뉴런은 척수 인터뉴런 에 정보를 전달합니다. 이러한 뉴런은 집단적으로 작용하여 들어오는 자극을 처리하고 이해하며 통합 센터 역할을 하는 효과 뉴런을 자극합니다.[4] 통합 센터를 떠난 효과 뉴런은 반사가 발생한 원래 조직에 반응을 전달하여 반응을 일으킵니다. 축삭 반사는 신호가 발생한 단일 뉴런의 국소 신경 세포에만 국한된 반응을 일으킵니다.[5] 축삭 반사 경로에는 척수 반사에서 뉴런 간의 통신을 중계하는 통합 센터나 시냅스가 포함되어 있지 않습니다. 따라서 자극은 신경 세포체에 들어가지 않고 효과 기관으로 우회되므로 축삭 반사는 구심성 자극이 구심성 뉴런을 자극하기 전에 중추 신경계를 통과하는 진정한 반사가 아님을 나타냅니다. 축삭 반사가 발견되고 중추 신경계의 통합 센터와 시냅스를 우회하는 “새로운 유형의 말초 반사”로 설명되었습니다. 축삭 반사의 발견으로 축삭 반사가 국소 세동맥을 활성화하여 혈관 확장 및 근육 수축을 일으킨다는 사실이 밝혀졌습니다. 이러한 근육 수축은 천식 환자에게서 관찰되었는데, 방출된 신경 펩타이드가 기도의 평활근을 수축시켰습니다. 마찬가지로 체온 운동 신경 말단에서 콜린성 물질이 방출되면 땀샘을 자극하여 열에 반응하여 땀을 흘리도록 유도하는 축삭 반사가 유발됩니다. 축삭 반사는 피부의 피부 수용체에서 신호를 전달함으로써 가능합니다.

한편 교감 반사에 의해 홍조 및 발한과 같은 증상을 국소적으로 유발할 수도 있습니다.13,17

감각 신경 섬유에서 방출되는 칼시토닌 유전자 관련 펩티드 (CGRP)는

혈관 확장 및 면역 억제에 역할을합니다.

캡사이신에 의해 유도된 CGRP 방출은

상당한 혈관 확장을 일으키기에 충분합니다.1

칼시토닌 유전자 관련 펩타이드는

IL-5를 지원하고 IL-13 발현을 제한하며

제2형 염증을 억제할 수 있습니다.24

지난 20년 동안 제2형 만성 염증성 질환의 발병 기전에 대한 이해가 크게 진전되면서 20개 이상의 새로운 표적에 대한 화합물이 확인되었고, 이 화합물들은 승인되었거나 다양한 개발 단계에 있으며, 마침내 이러한 질환의 치료를 위한 보다 표적화된 접근 방식을 촉진할 수 있게 되었습니다. 새롭게 확인된 제2형 염증의 병원성 동인과 그에 따른 치료법은 대부분 비만세포, 호산구, T세포, B세포, 상피세포 및 감각 신경과 관련이 있습니다. 상피 장벽 결함과 이상 미생물 군집은 만성 제2형 염증 질환에 대한 흥미로운 미래 약물 표적이 될 수 있습니다. 여기에서는 표적 치료제의 필요성이 높은 아토피 피부염, 만성 소양증, 만성 두드러기, 천식, 코 폴립을 동반한 만성 비부비동염 등 5가지 주요 2형 만성 염증 질환의 치료를 위한 일반적인 표적, 현재 치료법 및 새로운 치료법을 검토합니다. 이 분야의 미충족 수요와 향후 방향에 대해 논의합니다.

신경절 후 교감 신경과 면역 세포에서 생성되는

신경 전달 물질 노르에피네프린과도 더 많은 연관성이 있습니다.

노르에피네프린은

CD4+ T 림프구 및 B 림프구 기능을 조절할 수 있습니다.11,32

이 두 가지 유형의 신경 섬유와 신경 전달 물질이

면역계를 통해 국소 통증을 조절할 수 있는지 여부는

우리의 관심사입니다.

2. Methods

2.1. Animals

Male and female Wistar rats weighing 180 to 220 g were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. The animals were maintained under a standard 12-hour light–dark cycle with free access to food and water. Animal experiments were reviewed and approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (Beijing, China), and the 3 R principles were strictly followed during the experiments (project approval number: D2018-04-13-1).

2.2. Inflammatory pain model

Complete Freund adjuvant (Sigma-Aldrich, St. Louis, MO) was used to model chronic inflammatory pain. Rats were randomly assigned to the control group, Complete Freund adjuvant (CFA) group, EA group, anti-β-END + EA group, and 6-hydroxydopamine (6-OHDA) + EA group. Except for the control group, all rats received a 200 µL CFA injection into the left medial femoris muscle under isoflurane anesthesia. After more than 30 seconds, the needle was slowly withdrawn to prevent leakage. Animal experiments were reviewed and approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (Beijing, China), and the 3 R principles were strictly followed during the experiments.

2.3. Electroacupuncture treatment

The EA treatment was applied to the region of the inflamed side with the lowest mechanical pain threshold, which was examined using a small animal algometer (SMALGO-Bioseb, Aix-En-Provenc, France). Acupuncture needles were inserted 5 mm deep into the region and then connected to an 8-channel general-purpose stimulus generator (STG4008, Reutlingen, Germany). On the fourth day after CFA injection, EA treatment was applied for 3 days at an intensity of 2 mA and a frequency of 2 to 100 Hz for 30 minutes. The treatment was performed under isoflurane anesthesia. The rats in the control and CFA groups were treated identically without the use of EA treatment. Sterilized acupuncture needles (size 0.18 × 13 mm) were purchased from Zhongyan Taihe (Beijing, China).

2.4. Anti–β-endorphins serum and 6-hydroxydopamine injection

Polyclonal anti-β-END antibody was obtained from rabbit antisera and purified to a final concentration of 8 mg/mL using staphylococcal protein A-Sepharose chromatography. Thirty minutes before each EA intervention, rats in the anti-β-END + EA group were injected with 110 µL anti-β-END antibody at the site with the lowest mechanical pain threshold.

6-hydroxydopamine (Sigma-Aldrich) was dissolved in saline containing 0.2% (w/v) ascorbic acid (Sigma-Aldrich) at a concentration of 100 mg/mL. We performed local chemical sympathectomy 30 hours before the first EA intervention and 6 hours before each intervention by injecting 100 µL 6-OHDA solution into the lowest mechanical pain threshold site of 6-OHDA + EA group rats.

2.5. Weight-bearing experiment

To monitor changes in nonstimulus-evoked pain behavior of rats, the weight distribution of the hind paws was measured between the left (infected side) and right (control side) using a noninvasive bipedal balancer instrument. The rats were placed in a transparent plastic test box with an inclined board and forced to stand on independent pressure transducers with their hind paws. The force on both hind limbs of normal rats was balanced, but the force on the pain side of the hind limb was significantly weakened. Five independent weight readings of the ipsilateral and contralateral plates were collected from the control panel, with at least a 1-minute gap between each reading. Results were expressed as the average of the bilateral weight-bearing difference (the value of the healthy limb minus the value of the affected limb).21

2.6. Immunofluorescence/immunohistochemistry analysis

Rats were transcardially perfused with 250 mL of 0.9% saline and 250 mL of 4% paraformaldehyde solution while under deep anesthesia with 1% sodium pentobarbital solution (10 mL/kg). After perfusion fixation, tissues from the left femoral head to the ankle joint were harvested and postfixed for 24 hours before sectioning. Tissue sample randomly selected from a model group rat was embedded in paraffin and cut into 5-µm thick sections. Subsequently, the sections were stained with β-END and HE according to standard procedures.

The remaining tissues were sectioned with a cryostat (CM1950, Leica Biosystems, Wetzlar, Germany) at a thickness of 30 µm. Cryosections were blocked for 30 minutes at 37°C with 10% BSA solution containing 3% normal donkey serum and 0.5% Triton X-100 and then incubated at 4°C overnight with primary antibodies (dilution 1:500 in 0.1 M PB containing 0.5% Triton X-100). After 3 washes in 1 × PBS, the slices were incubated for 30 minutes at room temperature with a fluorescent-conjugated secondary antibody diluted 1:500 in 0.1 M PB containing 0.5% Triton X-100. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The sections were visualized under a laser scanning confocal microscope (FV1200, Olympus, Tokyo, Japan), after washing with 1 × PBS. The antibodies used were a mouse monoclonal anti-β-END antibody (ab54205, Abcam, Cambridge, United Kingdom), a rabbit monoclonal anti-ICAM-1 antibody (ab206398, Abcam), a rabbit polyclonal anti-CD11b antibody (PA5-79533, Thermo Fisher Scientific, Waltham, MA), Alexa Fluor 488 Phalloidin (A12379, Life Technologies, Carlsbag, CA), donkey anti-rabbit Alexa Fluor 488 (A-21203, Thermo Fisher Scientific), and donkey anti-mouse Alexa Fluor 594 (A-21206, Thermo Fisher Scientific).

2.7. Enzyme-linked immunosorbent assay

Blood was collected immediately by cardiac puncture into prechilled tubes containing EDTA after EA treatment under isoflurane anaesthesia. The blood samples were allowed to clot for 2 hours at room temperature, then centrifuged at 12,000 rpm for 30 minutes at 4°C, and the supernatant serum (plasma) were collected and subsequent stored at −80°C until analysis. Rats were sacrificed with CO2 asphyxiation after blood collection. The inflammatory tissues on the left side were extracted using a 12-mm skin extractor, snap frozen in liquid nitrogen and homogenized, and then transferred to 1.5 mL EP tubes. Each sample was divided into triplicate, one for total RNA extraction and the others for protein extraction. After adding the corresponding 2.5-fold mass volume of 5% acetic acid solution to the EP tube, it was heated at 100°C for 10 minutes and centrifuged at 12,000 rpm for 20 minutes at 25°C; or add the corresponding 10-fold mass volume of RIPA buffer (R0010, Solarbio, Beijing, China) containing PMSF and a protease inhibitor in the EP tube, left stand for 20 minutes at 4°C and centrifuge at 12,000 rpm for 20 minutes at 4°C. Total protein in the tissues were extracted from the supernatant and quantified using a Pierce BCA Protein assay kit (WH333439, Thermo Scientific). Finally, β-END (FEK-022-33, Phoenix Pharmaceuticals, Mannheim, Germany), CGRP (FEK-015-09, Phoenix Pharmaceuticals), IL-6 (N05059A-1, Merck Sharp & Dohme (MSD), Kenneworth, NJ), and NE (BAE-5200R, LDN, Nordhorn, Germany) were detected in the extracted proteins according to the manufacturer's instructions.

2.8. Flow cytometry

Rats were anesthetized with isoflurane, and left popliteal lymph node and surrounding inflammatory tissue were dissected and processed to prepare cell suspensions according to the manufacturer's instructions (130-098-305, Miltenyi Biotec, North Rhine-Westphalen, Germany). After digesting the tissue with gentleMACS, the cell suspensions were filtered into 15 mL centrifuge tubes through 70 μm nylon strainers (352350, FALCON, Tewksbury, MA). Strainers were gently rinsed with 5 mL 1 × PBS to obtain a single-cell suspension, followed by centrifugation at 300g for 10 minutes at 4°C, and the supernatant were discarded. Cells were washed 2 times with 5 mL 1 × PBS (containing 1% FBS) by using continuous steps of resuspension, centrifugation at 300g for 5 minutes at 4°C, and supernatants discarded. After fixation and permeabilization, respectively, at room temperature according to the kit instruction (ab185917, Abcam), these cells were incubated in the dark with the following antibodies: anti-β-END antibodies conjugated with APC (ab201807, Abcam), PE Mouse anti-Rat CD11b (562105, BD Pharmingen, New Jersey), and anti-ICAM1 antibodies conjugated with CY405 (ab201798, Abcam). After incubation, cells were washed 2 times with 1X PBS (containing 1% FBS), gently resuspended in 2 mL 1 × PBS, and transferred to flow tubes. Flow cytometry was used to analyze cells after incubation (FACS Celesta, BD Bioscience). The data were then analyzed further using the FlowJo software (Tree Star).

2.9. Western blot analysis

Total proteins were isolated from tissue samples in each group using RIPA buffer for Western blot (WB) assays. The extracted proteins (40 μg per sample as determined by the BCA protein assay) were subjected to 90 minutes of sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS-PAGE) with β-actin as an internal reference (YM3028, Immunoway). The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes and blocked for 2 hours with 5% nonfat milk in TBST. Monoclonal antibodies against ADR-β2 (dilution 1:5000 in 5% nonfat milk-TBST, Abcam) were added onto the membranes and incubated at room temperature for 10 minutes, then overnight at 4°C. After washing with TBST, the membranes were incubated for 40 minutes at room temperature with 1:2000 HRP-conjugated secondary antibody–labeled IgG antibody diluted in 5% nonfat milk-TBST. Enhanced chemiluminescence (ECL) detection reagent (Amersham Pharmacia Biotech, Amersham, United Kingdom) was added to the membranes for 3 minutes, and the images were exposed to radiographic film. Finally, film autoradiograms were analyzed and quantified using the Quantity One 4. 60 software.

2.10. RNA-sequencing

RNA-sequencing (RNA-seq) analysis was performed by Allwegene Technology Inc, Beijing, China. Total RNA was extracted from the inflamed tissue samples of the control group, CFA group, and EA group, and its concentration, purity, and integrity were determined. The length of the RNA fragment was detected using the Agilent 2100 bioanalyzer instrument (Agilent). Then, a cDNA library is constructed by PCR amplification. After quality control, clean read pairs were obtained using the Illumina second-generation high-throughput sequencing platform with the PE150 sequencing strategy. Complete comparison and transcript splicing analysis was performed using star and Cufflinks software and then quantitative analysis on all genes. Gene expression‎ levels were measured using the HTSeq software and quantified as the ratio of reads mapped to a gene to the gene length in kbp and expressed as the fragments per kb of transcript per million fragments mapped (FPKM).

2.11. Quantitative real-time polymerase chain reaction

To validate the transcriptome sequencing data, we used real-time PCR to determine the mRNA levels of 3 individual genes implicated in chemotaxis. The first-strand cDNA was synthesized using 2 μg total RNA and PrimeScript RT Master Mix (Takara, Japan). cDNA of Cxcl1, Ccl5, and Cxcl6 were amplified. Quantitative real-time PCR amplification was performed using the ABI 7500 Real-Time PCR Systems (Applied Biosystems). The amplification procedure was as follows: one cycle of 30 seconds at 95°C for predegeneration followed by 40 PCR cycles of 5 seconds at 95°C and 40 seconds at 60°C. The primers used were as follows: Ccl5 forward 5′- CCTCACCGTCATCCTCGTT-3′ and reverse 5′- GACTGCAAGGTTGGAGCACT-3′, Cxcl1 forward 5′- GCACCCAAACCGAAGTCAT-3′ and reverse 5′- GGGGACACCCTTTAGCATCT-3′, and Cxcl6 forward 5′- CCCCAAGGTGGAAGTCATAG-3′ and reverse 5′- GTGCATTCCGCTTTGTTTTC-3′. The Ct values of the target genes and the reference gene were obtained from the amplification curve. Three replicates were performed for each sample, and the relative quantification of gene expression‎ was calculated using the 2−ΔΔCt method.

2.12. Statistical analysis

All data were presented as means ± SEM. To compare variables between groups, 1-way analysis of variance was used followed by the Tukey HSD test where appropriate. *#&P < 0.05 or **P < 0.01 was considered statistically significant. Statistical analysis was performed using SPSS software 21.0. Pearson correlation analysis was used to determine correlations.

3. Results

3.1. Electroacupuncture treatment alleviate inflammatory muscle pain by increasing the content of β-endorphins in inflammatory tissue

We collected the 15 days data of 8 groups that received CFA injection given by intramuscular injection in medial femoris muscle (Fig. 1A). We used weight-bearing as an index of nonstimulus-evoked nociception (Fig. 1B). Intramuscular injection of CFA resulted in persistent pain and inflammation. After injection of CFA, the difference in weight-bearing and the content of IL-6 in inflammatory tissue were significantly increased. Day 4 to 6 after CFA injection was chosen for EA treatment because of the relative stationary pain and inflammatory response (blue box showed in Figs. 1C and D). Considering sexual dimorphism in pain reaction, we compared the weight-bearing difference and IL-6 levels in inflammatory tissues between genders in these groups. Yet, we did not observe obvious gender differences (Fig. S1A and S1B, available as supplemental digital content at http://links.lww.com/PAIN/B798). On day 6 after the injection, weight-bearing differences between 4 groups of rats were determined, as shown in Figure 1F. After EA treatment, the difference in weight-bearing in rats was significantly reduced when compared with the CFA group. In addition , a local injection of the anti-End antibody neutralized the analgesic effect, as indicated by an increase in the weight-bearing difference in the anti-End + EA group. As shown in Figure 1G, the β-END content in inflammatory tissue was significantly increased in the CFA group as compared with the control group. After EA treatment, the levels of β-END in inflammatory tissue were significantly increased compared with the CFA group. However, there were no significant differences in the β-END content of peripheral blood between the control, CFA, and EA groups (Fig. 1H). We compared the analgesic effects of EA between genders in these 4 groups and did not observe obvious gender differences in the analgesic effects (Fig. S2A, S2B and S2C, available as supplemental digital content at http://links.lww.com/PAIN/B798).

3. 결과

3.1. 전기침 치료는

염증 조직의 β-엔돌핀 함량을 증가시켜

염증성 근육통을 완화합니다.

대퇴 내측 근육에 근육 주사로 CFA 주사를 투여한 8개 그룹의 15일 데이터를 수집했습니다(그림 1A). 비자극 유발 통각 반응의 지표로 체중 부하를 사용했습니다(그림 1B). CFA의 근육 내 주사는 지속적인 통증과 염증을 초래했습니다. CFA 주사 후, 체중 부하와 염증 조직의 IL-6 함량 차이가 유의하게 증가했습니다. CFA 주사 후 4~6일째는 상대적으로 통증과 염증 반응이 고정되어 있어 EA 치료를 위해 선택되었습니다(그림 1C와 D에 파란색 상자 표시). 통증 반응의 성적 이형성을 고려하여 이들 그룹의 성별에 따른 염증 조직의 체중 부담 차이와 IL-6 수치를 비교했습니다. 그러나 뚜렷한 성별 차이는 관찰되지 않았습니다(그림 S1A 및 S1B, http://links.lww.com/PAIN/B798 에서 보충 디지털 콘텐츠로 제공). 주사 후 6일째 되는 날, 그림 1F와 같이 4개 그룹 쥐의 체중 차이를 측정했습니다. EA 치료 후, 쥐의 체중 부하 차이는 CFA 그룹과 비교했을 때 현저하게 감소했습니다. 또한 , 항-End 항체의 국소 주사는 진통 효과를 중화시켰으며, 이는 항-End + EA 그룹에서 체중 부담 차이가 증가한 것으로 나타났습니다. 그림 1G에서 볼 수 있듯이, 염증 조직의 β-END 함량은 대조군에 비해 CFA 그룹에서 유의하게 증가했습니다. EA 치료 후 염증 조직의 β-END 수치는 CFA 그룹에 비해 유의하게 증가했습니다. 그러나 말초 혈액의 β-END 함량은 대조군, CFA군, EA군 간에 유의미한 차이가 없었습니다(그림 1H). 이 4개 그룹에서 성별에 따른 EA의 진통 효과를 비교한 결과, 진통 효과에서 뚜렷한 성별 차이는 관찰되지 않았습니다(그림 S2A, S2B 및 S2C, http://links.lww.com/PAIN/B798 에서 보충 디지털 콘텐츠로 제공).

Figure 1.

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EA treatment increased the content of β-END in inflammatory tissues and relieved pain. (A) Experimental procedure timeline of observation of pain and local inflammatory reaction after intramuscular injection of CFA. N = 6 male and 6 female Wistar rats per group, total of 9 groups: control, day 1, day 3, day5, day 7, day 9, day 11, day 13, and day15. Injections were given intramuscularly. (B) A schematic illustration of behavioral testing. Changes in weight-bearing difference (C) and Interleukin- 6 (IL-6) levels in inflammatory tissues (D) measured at 15 days after CFA injection, *P < 0.05 vs control group, #P < 0.05 vs day 1. (E) Experimental procedure timeline of EA treatment. N = 6 male and 6 female Wistar rats per group, total of 4 groups: control, CFA, EA, and Anti-End + EA (antibody concentration: 8 mg/mL, 110 µL). (F) Weight-bearing difference was tested on day 6 after CFA injection in the indicated groups, *P < 0.05 vs control group, #P < 0.05 vs CFA group, &P < 0.05 vs EA group. β-END levels in the tissues (G) and plasma (H) were measured on day 6 after CFA injection and EA treatment, *P < 0.05 vs control group, #P < 0.05 vs CFA group. All data are shown as mean ± SEM; statistical analyses were performed using one-way ANOVA followed by the Tukey honestly significant difference (Tukey HSD) test. ANOVA, analysis of variance; β-END, β-endorphins; CFA, complete Frester adjuvant; EA, electroacupuncture.

3.2. Increased numbers of β-endorphins containing ICAM-1+/CD11b+ immune cells in inflammatory tissue were associated with electroacupuncture treatment

After CFA injection, obvious inflammatory reaction occurred in muscle tissue of the injection site (Fig. 2A). Inflammatory cell infiltration was observed in paraffin section with immunohistochemical staining of β-END. The presence of β-END positive cells was observed both in localized inflammation and draining lymph node. Based on the morphology of the nuclei, we speculated that β-END–positive cells were neutrophils, macrophages, and monocytes (Fig. 2B). In frozen section with immunofluorescence staining, confocal microscopy was performed to assess colocalization of β-END and ICAM-1 or CD11b, which were common markers of neutrophils, monocytes, and macrophages. The results showed that EA treatment led to increase the number of opioid-containing ICAM-1+/CD11b+ cells in the inflammatory tissues (Figs. 2C and D).

3.2.

염증 조직에서 ICAM-1+/CD11b+ 면역 세포를 포함하는 β-엔도르핀의 증가는

전기침 치료와 관련이 있었습니다.

CFA 주사 후, 주사 부위의 근육 조직에서 명백한 염증 반응이 발생했습니다(그림 2A). 파라핀 섹션에서 β-END의 면역 조직 화학 염색으로 염증 세포 침윤이 관찰되었습니다. β-END 양성 세포의 존재는 국소 염증과 배액 림프절 모두에서 관찰되었습니다. 핵의 형태에 근거하여 β-END 양성 세포는 호중구, 대식세포, 단핵구일 것으로 추측했습니다(그림 2B). 면역 형광 염색을 한 동결 절편에서 공초점 현미경을 사용하여 호중구, 단핵구 및 대식세포의 공통 마커인 β-END와 ICAM-1 또는 CD11b의 공동화를 평가했습니다.

그 결과,

EA 치료는

염증 조직에서 오피오이드 함유 ICAM-1+/CD11b+ 세포의 수를 증가시키는 것으로 나타났습니다(그림 2C 및 D).

Figure 2.

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EA treatment increased the number of β-END containing ICAM-1+/CD11b+ immune cells in inflammatory tissue. (A) Representative examples of muscles from rats after CFA injection and control treatment. (B) Immunohistochemistry images of sections stained with β-END showing inflammatory cell infiltration. Dark brown staining represents β-END immunoreactivity. Neutrophils exhibited a typical polymorphic nucleus, macrophages exhibited an elongated or indented oval nucleus, and monocytes exhibited an oval notched or horseshoe-shaped nucleus. (C and D) Immunofluorescence staining of inflammatory tissue using a mouse anti-β-END antibody (red), rabbit anti-ICAM-1 antibody or anti-CD11b antibody (all were marked green), and DAPI. Arrows point at double-positive cells. β-END, β-endorphins; CFA, complete Frester adjuvant; EA, electroacupuncture.

3.3. Electroacupuncture induces the release of β-endorphins from immune cells of ICAM-1+/CD11b+ in inflammatory tissues

In the early phase after CFA injection, innate immune cells in the blood circulation and local connective tissue were mobilized, resulting in the recruitment of granulocytes and monocytes in inflammatory tissue. Flow cytometry was used to measure the percentage of granulocytes (GRAs) and monocytes (MONs) in draining lymph nodes and inflammatory tissue on day 6 after CFA injection (Fig. 3A). The proportions of GRAs and MONs in the EA group were significantly higher than those in the CFA group and much higher than those in the control group (Fig. 3B). Simultaneously, the proportions of β-END+ GRAs and MONs were significantly lower in the EA group than in the CFA group, and much lower than the control group (Figs. 3C and D). Compared with the CFA group, EA treatment significantly reduced the proportions of β-END and CD11b coexpression‎ in GRAs and MONs (Figs. 3F and G). Meanwhile, the proportions of β-END and ICAM-1 coexpression‎ in GRAs and MONs were also reduced in the EA group (Figs. 3H and I). The findings suggested that the EA induced the release of β- END from immune cells of ICAM-1+/CD11b+ in inflammatory tissues.

3.3. 전기침술은 염증 조직의 ICAM-1+/CD11b+ 면역 세포에서 β-엔돌핀의 방출을 유도합니다.

CFA 주입 후 초기 단계에서 혈액 순환과 국소 결합 조직의 선천성 면역 세포가 동원되어 염증 조직에서 과립구와 단핵구가 모집되었습니다. 유세포 분석법을 사용하여 CFA 주입 후 6일째에 림프절과 염증 조직에서 과립구(GRA)와 단핵구(MON)의 비율을 측정했습니다(그림 3A). EA 그룹에서 GRA와 MON의 비율은 CFA 그룹보다 유의하게 높았으며 대조군보다 훨씬 높았습니다(그림 3B). 동시에, β-END+ GRA와 MON의 비율은 EA 그룹에서 CFA 그룹보다 유의하게 낮았으며 대조군보다 훨씬 낮았습니다(그림 3C 및 D).

CFA 그룹에 비해 EA 치료는

GRA와 MON에서 β-END와 CD11b의 동시 발현 비율을 유의하게 감소시켰습니다(그림 3F 및 G).

한편, EA 그룹에서도 GRA와 MON에서 β-END와 ICAM-1의 공동 발현 비율이 감소했습니다(그림 3H 및 I).

이 연구 결과는

EA가 염증 조직에서 ICAM-1+/CD11b+ 면역 세포로부터

β- END의 방출을 유도한다는 것을 시사합니다.

Figure 3.

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EA treatment increased proportions of GRAs and MONs and decreased the proportions of β-END+ CD11b+ and β-END+ ICAM-1+ cells on day 6 after CFA injection. (A) Flow cytometry plots showed the gating strategy for β-END+ cells in GRAs and MONs. (B) Bar graph showed the change of GRAs and MONs (% of total) on day 6 after CFA injection in 3 groups (n = 9). (C and D) Flow cytometry plots and bar graph show the expression‎ of β-END in GRAs and MONs in 3 groups (n = 9). (E) Gating strategy of β-END+ ICAM-1+ cells and β-END+ CD11b+ cells in GRAs and MONs. Flow cytometry plots and bar graphs show the proportions of β-END+ CD11b+ (F and G) and β-END+ ICAM-1+ (H and I) cells in GRAs and MONs in 3 groups. **P < 0.01 vs control group, #P < 0.05 vs CFA group. All data are shown as mean ± SEM; statistical analyses were performed using 1-way ANOVA followed by the Tukey HSD test. ANOVA, analysis of variance; β-END, β-endorphins; CFA, complete Frester adjuvant; EA, electroacupuncture; FSC, forward scatter; FSC-A, forward scatter area; GRAs, granulocytes; MONs, monocytes; SSC, side scatter; SSC-A, side scatter area; SSC-H, side scatter height.

3.4. The sympathetic nerve is associated with the analgesic effect of electroacupuncture treatment

On day 6 after CFA injection, CGRP released by sensory nerve fibers and Crcp (CGRP receptor component) gene expression‎ in inflammatory tissue was significantly increased in the CFA group. However, no significant difference in CGRP contents or Crcp gene expression‎ was observed between the CFA and EA groups (Figs. 4A and B). Meanwhile, CFA injection led to a decrease in NE release from sympathetic nerves, whereas EA treatment increased the NE content and expression‎ of β2 adrenergic receptor (ADR-β2) in inflammatory tissues (Figs. 4C and F). To elucidate the role of the sympathetic nerve in this process, we used chemical sympathectomy by local intramuscular injection of 6-OHDA. After sympathetic ablation, there was a drastic reduction of NE content in the 6-OHDA + EA group (Fig. 4C). The local injection of 6-OHDA neutralized the analgesic effect of EA treatment, as indicated by the increase in the weight-bearing difference in the 6-OHDA + EA group (Fig. 4D).

3.4. 교감신경은 전기침 치료의 진통 효과와 관련이 있습니다.

CFA 주사 후 6일째, 감각 신경 섬유에서 방출되는 CGRP와 염증 조직에서 Crcp (CGRP 수용체 성분) 유전자 발현이 CFA 그룹에서 유의하게 증가했습니다. 그러나 CFA 그룹과 EA 그룹 간에는 CGRP 함량이나 Crcp 유전자 발현에 유의미한 차이가 관찰되지 않았습니다(그림 4A 및 B).

한편, CFA 주입은 교감신경에서 NE 방출을 감소시킨 반면,

EA 치료는

염증 조직에서 β2 아드레날린 수용체(ADR-β2)의 NE 함량과 발현을 증가시켰습니다(그림 4C 및 F).

이 과정에서 교감신경의 역할을 규명하기 위해 6-OHDA의 국소 근육 주사를 통한 화학적 교감신경 절제술을 사용했습니다. 교감신경 절제술 후, 6-OHDA + EA 그룹에서 NE 함량이 급격히 감소했습니다(그림 4C). 6-OHDA의 국소 주사는 6-OHDA + EA 그룹의 체중 부담 차이의 증가에서 알 수 있듯이 EA 치료의 진통 효과를 중화시켰습니다(그림 4D).

Figure 4.

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Sympathetic nerve mediating the analgesic effect of EA treatment. (A) Compared with the control group, content of CGRP in inflammatory tissue was higher in the CFA group, but it was not significantly different between the CFA group and EA group (n = 9). (B) Expression‎ of gene Crcp in FPKM was generated from whole-transcriptome sequencing in result 5. Compared with the control group, expression‎ of gene Crcp in inflammatory tissue was higher in the CFA group, but it was not significantly different between the CFA group and EA group (n = 5). (C) Contents of NE was measured on day 6 after CFA injection in the control, CFA, EA, and 6-OHDA + EA groups (n = 8). *P < 0.05 vs control group, #P < 0.05 vs CFA group, &P < 0.05 vs EA group. (D) Weight-bearing difference was tested on day 6 after CFA injection in the control, CFA, EA, and 6-OHDA + EA groups (n = 8). *P < 0.05 vs control group, #P < 0.05 vs CFA group, &P < 0.05 vs EA group. (E) Expression‎ of β2 adrenergic receptor as determined by the Western blotting test. (F) Compared with the control group, ADR-β2 expression‎ in inflammatory tissue was lower in the CFA group. ADR-β2 expression‎ in the EA group was significantly higher compared with the CFA group (n = 8). *P < 0.05 vs control group, #P < 0.05 vs CFA group. All data are shown as mean ± SEM; statistical analyses were performed using 1-way ANOVA followed by the Tukey HSD test. ADR-β2, β2 adrenergic receptor; ANOVA, analysis of variance; CFA, complete Frester adjuvant; CGRP, calcitonin gene-related peptide; EA, electroacupuncture; FPKM, fragments per kb of transcript per million fragments mapped; NE, norepinephrine; OHDA, hydroxydopamine.

3.5. RNA-sequencing analysis and validation of quantitative real-time PCR

To further investigate the effect of EA on the migration of opioid-containing cells, we used RNA-seq to perform transcriptomic analysis of inflammatory tissues. As shown in Figure 5A, the mRNA levels of cell migration–associated genes obtained from RNA-seq were plotted as a heat map. Differentially expressed genes between the CFA and EA groups were selected and drawn as Violin plots. As shown in Figure 5B, EA treatment significantly upregulated Cxcl1 and Cxcl6 gene expression‎ levels and downregulated Ccl5 gene expression‎ levels when compared with the CFA group. Although there was no statistically significant difference in Icam1 gene expression‎ between the CFA and EA groups, there was a tendency toward significance. The expression‎ levels of the chemokine genes were then validated using quantitative real-time PCR. The variations in genes expression‎ were consistent with the RNA-seq analysis results (Fig. 5C). To demonstrate the correlation between sympathetic nerves and chemokine genes, we analyzed the correlation between the NE content and Cxcl1 gene expression‎ in inflammatory tissues. Pearson correlation coefficient analysis revealed a positive correlation, r = 0.5080, P = 0.0445(Fig. 5D).

3.5. RNA 시퀀싱 분석 및 정량적 실시간 PCR의 검증

오피오이드 함유 세포의 이동에 대한 EA의 영향을 추가로 조사하기 위해 RNA-seq을 사용하여 염증 조직의 전사체 분석을 수행했습니다. 그림 5A에서 볼 수 있듯이, RNA-seq에서 얻은 세포 이동 관련 유전자의 mRNA 수준을 히트 맵으로 표시했습니다. CFA 그룹과 EA 그룹 간에 다르게 발현되는 유전자를 선별하여 바이올린 플롯으로 그렸습니다. 그림 5B에서 볼 수 있듯이, EA 처리는 CFA 그룹과 비교했을 때 Cxcl1 및 Cxcl6 유전자 발현 수준을 유의미하게 상향 조절하고 Ccl5 유전자 발현 수준을 하향 조절했습니다. CFA 그룹과 EA 그룹 간에 Icam1 유전자 발현에 통계적으로 유의미한 차이는 없었지만 유의미한 경향을 보였습니다. 그런 다음 정량적 실시간 PCR을 사용하여 케모카인 유전자의 발현 수준을 검증했습니다. 유전자 발현의 변화는 RNA-seq 분석 결과와 일치했습니다(그림 5C). 교감신경과 케모카인 유전자 간의 상관관계를 입증하기 위해 염증 조직에서 NE 함량과 Cxcl1 유전자 발현의 상관관계를 분석했습니다. 피어슨 상관관계 계수 분석 결과 r = 0.5080, P = 0.0445로 양의 상관관계가 있는 것으로 나타났습니다(그림 5D).

Figure 5.

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RNA-sequencing analysis and qRT-PCR verification of genes related to cell migration. (A) Heat map of cell migration–associated genes. Different heat map colors represent the relative mRNA expression‎ levels of genes calculated by Log2 (FPKM + 1). (B) Violin plots of expression‎ values for differentially expressed genes between CFA and EA groups. Results are expressed as FPKM. Data shown are the mean FPKM  ±  SEM (n = 5). *P < 0.05 vs CFA group. (C) Significant differentially expressed genes as determined by real-time PCR (n = 8). (D) Correlation between NE and the gene expression‎ of Cxcl1 in inflammatory tissues based on the Pearson correlation analysis. All data are expressed as the mean ± SEM. *P < 0.05 vs CFA group. CFA, complete Frester adjuvant; EA, electroacupuncture; FPKM, fragments per kb of transcript per million fragments mapped; NE, norepinephrine.

4. Discussion

The major finding of this study is that EA exerts a local analgesic effect during peripheral inflammation by activating sympathetic nerve fibers and promoting the migration of β-END containing ICAM-1+/CD11b+ immune cells to the pain site. This is demonstrated by the following findings: (1) EA treatment significantly alleviated spontaneous pain behavior in rats by increasing the amount of β-END in local inflammatory tissue, whereas local subcutaneous administration of an anti-End antibody blocked this analgesia; (2) EA treatment significantly increased the number of β-END containing ICAM-1+/CD11b+ immune cells in inflammatory tissues; (3) EA treatment significantly decreased the proportion of cells with β-END co-expressed with ICAM-1 or CD11B in granulocytes and monocytes; (4) finally, EA upregulated the expression‎ levels of NE and β2-ADR in inflamed tissue, and 6-OHDA treatment completely offset the analgesic effect of EA.

In the present and previous studies, the nervous system is often the target of acupuncture analgesia mechanisms. Acupuncture has been shown to suppress or block the introduction of damaging sensory impulses in different levels of the central nervous system, including the spinal dorsal angle and PAG, thus exerting an analgesic effect.43 However, few studies have examined the immune system as a potential pathway for pain control with acupuncture treatment. Numerous studies in the field of pain have found that immune cells containing opioids preferentially migrate to the injured tissues, releasing opiopeptides, and activating the opioid receptors on peripheral sensory nerve terminals, thereby inhibiting inflammatory pain.10 Researchers discovered that both central and peripheral opioid peptides were implicated in the antinociceptive effect of early inflammation by activating the corresponding receptors. Antinociception was primarily induced at a later stage of inflammation by the interaction between leukocyte-derived β-END and peripheral opioid receptors.20 In this study, local injection of β-END–neutralizing antibodies counteracted the analgesic effect of acupuncture analgesia, indicating that acupuncture may mediate analgesia by increasing β-END levels in peripheral tissues.

Selectors mediate opioid-containing immune cells in normal tissues by rolling close to vessel walls, trafficking between the blood and lymphatic systems, and very rarely into the tissue. When inflammation occurs, the cells activated by inflammatory media and chemokines adhere to the vascular wall of the inflammatory site, exiting the circulation and extravasating to inflammatory foci.14 The binding of ICAM-1 to leukocyte β2 integrin is essential for leukocyte transendothelial migration.41 Blockade of β2 integrins or ICAM-1 by monoclonal antibodies significantly decreased the infiltration of leukocytes expressed opioid peptides in inflamed tissue and abolished stress-induced peripheral antinociception.18,19 Mac-1 (CD11b/CD18) plays a role in the adhesion of stimulated neutrophils to the vascular endothelium during CFA-induced local inflammation.6,30 According to previous studies, the peripheral endogenous opioid peptides that mediate the local antinociceptive effect are mainly derived from monocytes or macrophages during the late stages of inflammation.4,28 Acupuncture was shown to achieve endogenous pain control by increasing the expression‎ of chemokine CXCL10 and the number of infiltrating CXCR3+ macrophages expressing opiopeptides.40 We now extend these findings by demonstrating that EA treatment selectively targeted opioid-containing ICAM-1+/CD11b+ cells to the pain site and increased local tissue content of β-END. However, the nonspecific nature of ICAM-1 and CD11B makes it difficult to distinguish between leukocyte types that produce β-END.

In this study, we found that the activation of the sympathetic nerve was involved in the local analgesic effect mediated by EA, and chemical sympathectomy completely abolished intrinsic opioid analgesia. The sympathetic nervous system stimulated adrenergic receptors on inflammatory cells by producing NE and releasing β-END into peripheral inflamed tissue.2 In addition, the NE secreted by sympathetic nerves regulates immunological response primarily by β2-adrenergic receptors (β2-AR) expressed on lymphocytes.34 Although most studies suggest that β2-AR agonists have immunosuppressive effects, numbers studies suggest that β2-AR activation on immune cells can enhance inflammation by MAPK signaling pathways.16 The enhanced expression‎ of endothelial cell ICAM-1 and the subcutaneous recruitment of opioid peptide–containing neutrophils and monocytes during painful paw inflammation have been demonstrated to be caused by sympathetic fibers.22 The use of β-blocker decreases the expression‎ of chemokine receptor 2 (CCR2) and peripheral blood leukocytes infiltration into the site of damage.8 By acting on β-ARs expressed on nonhematopoietic cells, the sympathetic nerve has been demonstrated to generate circadian changes in the expression‎ of endothelial cell adhesion molecules and chemokines and to regulate rhythmic recruitment of leukocytes to the inflammatory site.33 Some researchers have observed that the activation of β2-AR inhibited antigen-primed T cells egress from LNs by interactions with CCR7 and CXCR4.25 The effect of SNS on cell migration may provide insight into the endogenous opioid analgesic mechanism induced by EA.

After that, we eval‎uated the effect of EA on the expression‎ of chemokine genes, which play an important role in the regulation of leukocytes migration and the perception of pain.36 Cxcl1 and Cxcl6 have been shown to act as specific chemoattractants for polymorphonuclear neutrophils.27 In addition, some investigators discovered that Cxcl1 activates the Mac-1/ICAM-1 pathway promoting adhesion between leukocytes and endothelial cells.39 Apart from promoting cell recruitment, CXCL1 induces Ca2+-regulated opioid release by activating the chemokine receptor CXCR2 on neutrophils and thereby inhibiting inflammatory pain.29 Ccl5 exhibits a strong chemotactic activity toward T lymphocytes, monocytes, and macrophages expressing CCR5 receptor.23,31 Because the results reported here demonstrate that EA treatment upregulated the transcription of Cxcl1 and Cxcl6, we hypothesized that neutrophils are the primary type of opioid-containing leukocytes recruited to inflammatory sites (Fig. 6). Although the downregulation of Ccl5 gene expression‎ was observed, its role in EA-induced opioid-obtaining immune cells emigration is unknown.

4. 토론

이 연구의 주요 결과는

교감 신경 섬유를 활성화하고

ICAM-1+/CD11b+ 면역 세포를 포함하는 β-END의 통증 부위로의 이동을 촉진함으로써

말초 염증 동안

EA가 국소 진통 효과를 발휘한다는 것입니다.

이는 다음 연구 결과를 통해 입증되었습니다:

(1) EA 치료는 국소 염증 조직에서 β-END의 양을 증가시켜 쥐의 자발적 통증 행동을 유의하게 완화시킨 반면, 항-End 항체의 국소 피하 투여는 이러한 진통을 차단했습니다.

(2) EA 치료는 염증 조직에서 ICAM-1+/CD11b+ 면역 세포를 포함하는 β-END의 수를 유의하게 증가시켰습니다;

(3) EA 치료는 과립구 및 단핵구에서 ICAM-1 또는 CD11B와 공동 발현되는 β-END를 가진 세포의 비율을 유의하게 감소시켰습니다.

(4) 마지막으로, EA는 염증 조직에서 NE 및 β2-ADR의 발현 수준을 상향 조절했으며 6-OHDA 치료는 EA의 진통 효과를 완전히 상쇄했습니다.

현재와 이전의 연구에서 신경계는 종종 침술 진통 메커니즘의 표적이 됩니다. 침술은 척추 등각과 PAG를 포함한 중추신경계의 여러 수준에서 손상된 감각 자극의 유입을 억제하거나 차단하여 진통 효과를 발휘하는 것으로 나타났습니다.43

그러나

침술 치료를 통한 통증 조절의 잠재적 경로로서

면역 체계를 조사한 연구는 거의 없습니다.

통증 분야의 수많은 연구에서

오피오이드를 함유한 면역 세포가

손상된 조직으로 우선적으로 이동하여

오피오펩타이드를 방출하고

말초 감각 신경 말단의 오피오이드 수용체를 활성화하여

염증성 통증을 억제한다는 사실이 밝혀졌습니다.10

연구자들은

중추 및 말초 오피오이드 펩타이드가 모두 해당 수용체를 활성화하여

초기 염증의 항통각 효과에 관여한다는 사실을 발견했습니다.

항통각은

주로 백혈구 유래 β-END와 말초 오피오이드 수용체 간의 상호작용에 의해

염증의 후기 단계에서 유도되었습니다.20

이 연구에서

β-END 중화 항체의 국소 주사는

침술 진통의 진통 효과를 상쇄하여

침술이 말초 조직의 β-END 수준을 높여 진통을 매개할 수 있음을 시사합니다.

선택체는 혈관벽 가까이에서 굴러다니며 혈액과 림프계 사이를 이동하거나 매우 드물게 조직 내로 이동하여 정상 조직에서 오피오이드 함유 면역 세포를 매개합니다. 염증이 발생하면 염증 매개체와 케모카인에 의해 활성화된 세포가 염증 부위의 혈관벽에 부착되어 순환계를 빠져나와 염증 병소로 혈관 외로 이동합니다.14 백혈구 β2 인테그린에 대한 ICAM-1의 결합은 백혈구 내피 세포 이동에 필수적입니다.41 단일 클론 항체에 의한 β2 인테그린 또는 ICAM-1의 차단은 염증 조직에서 오피오이드 펩타이드를 발현하는 백혈구의 침윤을 유의하게 감소시키고 스트레스 유발 말초 항통증을 폐지했습니다.18,19 Mac-1(CD11b/CD18)은 CFA 유발 국소 염증 동안 자극된 호중구가 혈관 내피에 부착하는 역할을 합니다.6,30 이전 연구에 따르면, 국소 항통각 효과를 매개하는 말초 내인성 오피오이드 펩타이드는 주로 염증 후기 단계에서 단핵구 또는 대식세포에서 유래합니다.4,28

침술은

케모카인 CXCL10의 발현과 오피오펩타이드를 발현하는

침윤성 CXCR3+ 대식세포의 수를 증가시킴으로써

내인성 통증 조절을 달성하는 것으로 나타났습니다.40

우리는 이제 이러한 결과를 확장하여

침 치료가 오피오이드 함유 ICAM-1+/CD11b+ 세포를 통증 부위로 선택적으로 표적화하고

β-END의 국소 조직 함량을 증가시킴을 입증함으로써

이러한 발견을 확대했습니다.

그러나 ICAM-1과 CD11B의 비특이적 특성으로 인해 β-END를 생성하는 백혈구 유형을 구별하기 어렵습니다.

이 연구에서 우리는 교감 신경의 활성화가 EA에 의해 매개되는 국소 진통 효과에 관여하고 화학적 교감 신경 절제술이 내재적 오피오이드 진통을 완전히 폐지한다는 것을 발견했습니다. 교감 신경계는 NE를 생성하고 말초 염증 조직으로 β-END를 방출하여 염증 세포의 아드레날린 수용체를 자극합니다.2 또한 교감 신경에서 분비되는 NE는 주로 림프구에 발현되는 β2- 아드레날린 수용체(β2-AR)에 의해 면역학적 반응을 조절합니다.34 대부분의 연구에 따르면 β2-AR 작용제는 면역 억제 효과가 있지만, 수치 연구에 따르면 면역 세포에서 β2-AR 활성화는 MAPK 신호 경로에 의해 염증을 강화할 수 있습니다.16 내피 세포 ICAM-1의 발현 증가와 통증성 발 염증 동안 오피오이드 펩티드 함유 호중구 및 단핵구의 피하 모집은 교감 섬유에 의한 것으로 입증되었습니다.22 β 차단제의 사용은 케모카인 수용체 2 (CCR2)의 발현과 말초 혈액 백혈구의 손상 부위로의 침윤을 감소시킵니다 .8 교감 신경은 비 조혈 세포에서 발현되는 β-AR에 작용함으로써 내피 세포 접착 분자와 케모카인의 발현에 일주기적인 변화를 일으키고 염증 부위로 백혈구의 리듬 모집을 조절하는 것으로 입증되었습니다 .33 일부 연구자들은 β2-AR의 활성화가 CCR7 및 CXCR4와의 상호 작용에 의해 항원 프라이밍 된 T 세포가 LN에서 빠져 나가는 것을 관찰했습니다 .25 세포 이동에 대한 SNS의 효과는 EA에 의해 유도되는 내인성 오피오이드 진통제 메커니즘에 대한 통찰력을 제공 할 수 있습니다.

그 후, 백혈구 이동 조절과 통증 인식에 중요한 역할을 하는 케모카인 유전자의 발현에 대한 EA의 영향을 평가했습니다.36 Cxcl1과 Cxcl6는 다형 핵 호중구의 특정 화학 유인 물질로 작용하는 것으로 나타났습니다.27 또한 일부 연구자들은 Cxcl1이 백혈구와 내피 세포 사이의 접착을 촉진하는 Mac-1/ICAM-1 경로를 활성화한다는 사실을 발견했습니다.39 세포 모집을 촉진하는 것 외에도 CXCL1은 호중구의 케모카인 수용체 CXCR2를 활성화하여 Ca2+ 조절 오피오이드 방출을 유도함으로써 염증성 통증을 억제합니다.29 Ccl5는 CCR5 수용체를 발현하는 T 림프구, 단핵구 및 대 식세포에 대해 강력한 화학 주성 활성을 나타냅니다.23,31 여기에 보고된 결과는 EA 치료가 Cxcl1 및 Cxcl6의 전사를 상향 조절한다는 것을 보여주기 때문에, 우리는 호중구가 염증 부위에 모집되는 오피오이드 함유 백혈구의 주요 유형이라는 가설을 세웠습니다(그림 6). Ccl5 유전자 발현의 하향 조절이 관찰되었지만, EA에 의한 오피오이드 획득 면역 세포 이동에서 이 유전자의 역할은 알려져 있지 않습니다.

Figure 6.

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EA exerts a local analgesic effect during peripheral inflammation by activating sympathetic nerve fibers and promoting the migration of β-END containing immune cells to the pain site. β-END, β-endorphins; CFA, complete Frester adjuvant; EA, electroacupuncture.

Apart from acupuncture, other forms of traditional Chinese medicine techniques, including cupping and scraping act on the local site to treat pain diseases. These techniques are used clinically and have a comparable analgesic effect. Cupping and scraping therapy has the potential to cause subcutaneous bleeding points and produce local inflammatory reactions (blood cells leaving the circulation system can also chemotaxis and recruit immune cells). Based on the findings of this study and the conclusions, it is reasonable to infer that similar conventional medical therapies may exert a therapeutic effect on local pain by the peripheral analgesic effect of the immune system. Nonetheless, this mechanism remains to be demonstrated experimentally.

침술 외에도 부항과 긁기 등 다른 형태의 전통 한의학 기법이 국소 부위에 작용하여 통증 질환을 치료합니다. 이러한 기법은 임상적으로 사용되며 진통 효과가 비슷합니다. 부항 요법과 긁기 요법은 피하 출혈점을 유발하고 국소 염증 반응을 일으킬 가능성이 있습니다(순환계를 떠나는 혈액 세포는 화학 작용을 일으켜 면역 세포를 모집할 수도 있습니다). 이 연구 결과와 결론을 바탕으로 유사한 기존 의료 요법이 면역 체계의 말초 진통 효과에 의해 국소 통증에 치료 효과를 발휘할 수 있다고 추론하는 것이 합리적입니다. 그럼에도 불구하고 이 메커니즘은 실험적으로 입증되어야 합니다.

5. Conclusion

Taken together, our findings demonstrate that EA exerts peripheral analgesic effects at the site of inflammation by increasing the number of endogenous opiopeptides produced by peripheral immune cells in the local tissue. In addition, EA activates the sympathetic nerves, which recruits β-END–containing ICAM-1+/CD11b+ immune cells to the site of inflammatory pain and releases opiopeptides. The modulation of chemokines by local sympathetic activation is required for peripheral opioid peptide–mediated antinociception.

5. 결론
지금까지의 연구 결과를 종합하면, 

전침(EA)는 

국소 조직에서 말초 면역 세포가 생성하는 내인성 오피오펩타이드의 수를 증가시켜 

염증 부위에 말초 진통 효과를 발휘한다는 사실이 입증되었습니다. 

또한, 

EA는 

교감 신경을 활성화하여 

β-END를 함유하는 ICAM-1+/CD11b+ 면역 세포를 염증성 통증 부위로 모집하고 

오피오펩타이드를 방출합니다. 

국소 교감신경 활성화에 의한 케모카인의 조절은 

말초 오피오이드 펩타이드 매개 항통각 작용에 필요합니다.

Conflict of interest statement

The authors have no conflict of interest to declare.

Appendix A. Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/B798.

Supplementary Material

jop-164-1965-s001.pdf (842.9KB, pdf)

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jop-164-1965-s002.pdf (207.2KB, pdf)

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Acknowledgements

This work was supported by a grant from China Academy of Chinese Medical Sciences Innovation Fund (ID: CI2021A03404) and National Natural Science Foundation of China (ID: 82130122, ID: 81973964 and ID: 81674083). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article. Data availability: All data included in this study are available on request by contact with the corresponding author. Author contributions: X.-Y. Wang and X.-H. Jing conceived and designed the study. J.-t. Shi performed the experiment of flow cytometry, Western blot, and the quantitative real-time PCR. W.-y. Cao performed the animal experiments, acquired the blood samples, tissue dissection and processing, immunohistochemistry, and confocal imaging. H.-Y. Wan analyzed the data of flow cytometry. X.-N. Zhang analyzed the data of RNA sequencing. Z.-Y. Qu offered insights and assisted in interpreting the results. Y.-S. Su and W. He wrote the article. R. Wang supervised the project and revised the article. All the authors provided feedback and approved the article. Special thanks are extended to Gong Zhou and Zhi-Yuan Hui, who engaged in the experiment of behavioral experiment.

Footnotes

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

J.-t. Shi and W.-y. Cao contributed equally.

Contributor Information

Jing-tao Shi, Email: 909208703@qq.com.

Wan-ying Cao, Email: 1040959561@qq.com.

Xiao-Ning Zhang, Email: zxnruobing@126.com.

Hong-Ye Wan, Email: redleaf2011@163.com.

Yang-Shuai Su, Email: suyangshuai@163.com.

Zheng-Yang Qu, Email: quzhengyang3@163.com.

Rui Wang, Email: wangruibit@bit.edu.cn.

Wei He, Email: hazel7811@hotmail.com.

Xiang-Hong Jing, Email: jxhtjb@263.net.

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