Electroacupuncture intervention relieves pain possibly by promoting MOR endocytosis in locus coeruleus in bone cancer pain rats with morphine tolerance

SHI Ren-yi; FU Tao-fang; CAI Yang-qian; DU Jun-ying; FANG Jun-fan; FANG Jian-qiao; LIANG Yi

doi:10.13702/j.1000-0607.180635

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Electroacupuncture intervention relieves pain possibly by promoting MOR endocytosis in locus coeruleus in bone cancer pain rats with morphine tolerance

更新时间：2023-08-11

- Electroacupuncture intervention relieves pain possibly by promoting MOR endocytosis in locus coeruleus in bone cancer pain rats with morphine tolerance
- Acupuncture Research Vol. 44, Issue 3, Pages: 161-169(2019)
- 作者机构：
  
  浙江中医药大学第三临床医学院针灸神经生物学实验室
- 作者简介：
- 基金信息：
- DOI：10.13702/j.1000-0607.180635
  CLC：
- Published：2019
- 稿件说明：
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SHI Ren-yi, FU Tao-fang, CAI Yang-qian, et al. Electroacupuncture intervention relieves pain possibly by promoting MOR endocytosis in locus coeruleus in bone cancer pain rats with morphine tolerance[J]. Acupuncture research, 2019, 44(3): 161-169.
DOI：

SHI Ren-yi, FU Tao-fang, CAI Yang-qian, et al. Electroacupuncture intervention relieves pain possibly by promoting MOR endocytosis in locus coeruleus in bone cancer pain rats with morphine tolerance[J]. Acupuncture research, 2019, 44(3): 161-169. DOI： 10.13702/j.1000-0607.180635.

摘要

目的:观察电针对骨癌痛-吗啡耐受大鼠痛行为的影响

探讨电针抗骨癌痛大鼠吗啡耐受效应的作用机制。方法:通过胫骨内接种MRMT-1乳腺癌细胞(3×10

个)诱发骨癌痛

联合腹腔注射盐酸吗啡注射液(10mg·kg4个)诱发骨癌痛

联合腹腔注射盐酸吗啡注射液(10mg·kg

(-1)

·d(-1)·d

(-1)

分两次注射

连续注射11d)建立骨癌痛-吗啡耐受大鼠模型。第1部分:23只健康雌性SD大鼠随机分为假手术组(n=6)、骨癌痛组(n=9)、吗啡耐受组(n=8)。第2部分:61只健康雌性SD大鼠随机分为假手术组(n=11)、骨癌痛组(n=11)、吗啡耐受组(n=13)、电针组(n=13)和假电针组(n=13)。成功诱导吗啡耐受后1d(接种癌细胞后第22天)电针

于每日第1次吗啡腹腔注射后即刻电针

穴位选用双侧"足三里"和"昆仑"

每次30min

每日1次

连续治疗7d。于癌细胞接种前和接种后10、11、21、22、24、26和28d时检测大鼠患侧机械缩足阈(PWTs);胫骨X线及HE染色观察胫骨组织形态;免疫荧光组织化学法(IHC)检测大鼠蓝斑核(LC)内μ阿片受体(MOR)、Rab5阳性细胞表达及其共表达情况。结果:癌细胞接种后10d

骨癌痛组、吗啡耐受组、电针组和假电针组大鼠PWTs均显著低于同期假手术组(P

0.01);接种后21d(即吗啡持续注射后11d)

4组大鼠PWTs均显著低于同期假手术组(P

0.01);接种后22～28d(即电针1～7d)

电针组大鼠患侧PWTs显著高于同期骨癌痛组、吗啡耐受组和假电针组(P

0.01)。X线结果显示骨癌痛大鼠左侧胫骨近端上1/3处骨皮质破坏

呈不连续状

且局部软组织可见明显肿胀。HE染色结果显示

假手术组大鼠胫骨结构完整

骨髓腔内未见MRMT-1癌细胞;骨癌痛组和吗啡耐受组骨髓腔内见大量MRMT-1癌细胞。IHC结果显示:骨癌痛组、吗啡耐受组大鼠LC内MOR、Rab5阳性细胞率及MOR(-1)

分两次注射

于每日第1次吗啡腹腔注射后即刻电针

穴位选用双侧"足三里"和"昆仑"

每次30min

每日1次

骨癌痛组、吗啡耐受组、电针组和假电针组大鼠PWTs均显著低于同期假手术组(P

0.01);接种后21d(即吗啡持续注射后11d)

4组大鼠PWTs均显著低于同期假手术组(P

0.01);接种后22～28d(即电针1～7d)

电针组大鼠患侧PWTs显著高于同期骨癌痛组、吗啡耐受组和假电针组(P

0.01)。X线结果显示骨癌痛大鼠左侧胫骨近端上1/3处骨皮质破坏

呈不连续状

且局部软组织可见明显肿胀。HE染色结果显示

假手术组大鼠胫骨结构完整

骨髓腔内未见MRMT-1癌细胞;骨癌痛组和吗啡耐受组骨髓腔内见大量MRMT-1癌细胞。IHC结果显示:骨癌痛组、吗啡耐受组大鼠LC内MOR、Rab5阳性细胞率及MOR

/Rab5+/Rab5

阳性率均低于假手术组(P

0.01);吗啡耐受组大鼠LC内Rab5阳性细胞率及MOR+阳性率均低于假手术组(P

0.01);吗啡耐受组大鼠LC内Rab5阳性细胞率及MOR

/Rab5+/Rab5

阳性率均低于同期骨癌痛组(P

0.05);电针组大鼠LC内MOR、Rab5阳性细胞率及MOR+阳性率均低于同期骨癌痛组(P

0.05);电针组大鼠LC内MOR、Rab5阳性细胞率及MOR

/Rab5+/Rab5

阳性率均显著高于同期骨癌痛组、吗啡耐受组和假电针组(P

0.01)。结论:电针可缓解骨癌痛-吗啡耐受大鼠的机械痛阈

部分翻转吗啡耐受现象;该效应可能与其提高大鼠LC内MOR表达、促进MOR内吞有关。Objective To observe the effect of electroacupuncture(EA)on pain behavior and expression of!-opioid receptor(MOR)and Rab5(an important protein molecule for internalization of MOR)in the locus coeruleus(LC)region in bone cancer pain(BCP)rats with morphine tolerance(MT)

so as to explore its mechanisms underlying improvement of BCP and MT.Methods The present study included two parts.In the first part

23 female SD rats were randomized into sham BCP(n=6)

BCP(n=9)and BCP+MT(n=8)groups

and in the second part

61 female SD rats were randomized into 5 groups:sham BCP(n=11)

BCP(n=11)

BCP+MT(n=13)

BCP+MT+EA(n=13)and BCP+MT+sham EA(n=13).The BCP morphine tolerance(BCP+MT)model was established by injection of 10 !L of human Walker 256 breast cancer cells(MRMT-1 breast cancer cells

1 x10+阳性率均显著高于同期骨癌痛组、吗啡耐受组和假电针组(P

0.01)。结论:电针可缓解骨癌痛-吗啡耐受大鼠的机械痛阈

部分翻转吗啡耐受现象;该效应可能与其提高大鼠LC内MOR表达、促进MOR内吞有关。

Abstract

Objective To observe the effect of electroacupuncture(EA)on pain behavior and expression of!-opioid receptor(MOR)and Rab5(an important protein molecule for internalization of MOR)in the locus coeruleus(LC)region in bone cancer pain(BCP)rats with morphine tolerance(MT)

so as to explore its mechanisms underlying improvement of BCP and MT.Methods The present study included two parts.In the first part

23 female SD rats were randomized into sham BCP(n=6)

BCP(n=9)and BCP+MT(n=8)groups

and in the second part

61 female SD rats were randomized into 5 groups:sham BCP(n=11)

BCP(n=11)

BCP+MT(n=13)

BCP+MT+EA(n=13)and BCP+MT+sham EA(n=13).The BCP morphine tolerance(BCP+MT)model was established by injection of 10 !L of human Walker 256 breast cancer cells(MRMT-1 breast cancer cells

1 x10

cells/!L)into the bone marrow cavity at the upper part of the left tibia and intraperitoneal injection of morphine hydrochloride(10 mg/kg

once per 12 h

for 11 successive days).On day 21 after inoculation

EA(2 Hz/100 Hz

0.5-1.5 mA

increasing 0.5 mA every 10 min)was began to applied to bilateral"Zusanli"(ST30)and"Kunlun"(BL60)immediately after the first intraperitoneal injection of morphine.The treatment was performed for 30 min every time

once daily for 7 successive days.The paw withdrawal threshold(PWT)was detected before and 10

26 and 28 days after inoculation.The immunoactivity of MOR and Rab5 proteins in the LC region was detected by immunofluorescence histochemistry.Results In the first part of the study

at the 10 th day after inoculation of cancer cells

the PWT of the BCP and BCP+MT groups was significantly lower than that of the sham BCP group(P

0.05)

suggesting a success of BCP model.From day 11 to 19 after inoculation(during injection of morphine)

the PWTs of the BCP+MT group were significantly higher than those of the BCP group(P

0.01)

and on day 21

the PWT of the BCP+MT group was similar to that of the BCP group(P

0.05)but significantly lower than that of the sham BCP group(P

0.01)

suggesting a success of MT.H.E.staining showed a large quantity of MRMT-1 cancer cells in the bone marrow cavity in both BCP and BCP+MT groups.In the second part of the study

the decreased PWTs from 10 th to 28 th day after inoculation were significantly increased on day 22

26 and 28 in the BCP+MT+EA group relevant to the BCP

BCP+MT and BCP+MT+sham EA groups(P

0.01).The ratios of MOR and Rab5 positive(+)cells and MOR+/Rab5+of the left LC region were significantly lower in the BCP and BCP+MT groups than those of the sham BCP group(P

0.01)

but were considerably higher in the BCP+MT+EA group than those in the BCP

BCP+MT and BCP+MT+sham EA groups(P

0.01).The ratios of Rab5+and MOR+/Rab54 cells/!L)into the bone marrow cavity at the upper part of the left tibia and intraperitoneal injection of morphine hydrochloride(10 mg/kg

once per 12 h

for 11 successive days).On day 21 after inoculation

EA(2 Hz/100 Hz

0.5-1.5 mA

once daily for 7 successive days.The paw withdrawal threshold(PWT)was detected before and 10

26 and 28 days after inoculation.The immunoactivity of MOR and Rab5 proteins in the LC region was detected by immunofluorescence histochemistry.Results In the first part of the study

at the 10 th day after inoculation of cancer cells

the PWT of the BCP and BCP+MT groups was significantly lower than that of the sham BCP group(P

0.05)

suggesting a success of BCP model.From day 11 to 19 after inoculation(during injection of morphine)

the PWTs of the BCP+MT group were significantly higher than those of the BCP group(P

0.01)

and on day 21

the PWT of the BCP+MT group was similar to that of the BCP group(P

0.05)but significantly lower than that of the sham BCP group(P

0.01)

suggesting a success of MT.H.E.staining showed a large quantity of MRMT-1 cancer cells in the bone marrow cavity in both BCP and BCP+MT groups.In the second part of the study

the decreased PWTs from 10 th to 28 th day after inoculation were significantly increased on day 22

26 and 28 in the BCP+MT+EA group relevant to the BCP

BCP+MT and BCP+MT+sham EA groups(P

0.01).The ratios of MOR and Rab5 positive(+)cells and MOR+/Rab5+of the left LC region were significantly lower in the BCP and BCP+MT groups than those of the sham BCP group(P

0.01)

but were considerably higher in the BCP+MT+EA group than those in the BCP

BCP+MT and BCP+MT+sham EA groups(P

0.01).The ratios of Rab5+and MOR+/Rab5

cells of the BCP+MT group were significantly lower than those of the BCP group(P

0.05).No significant changes were found in the ratios of MOR+and Rab5+cells of the BCP+MT group were significantly lower than those of the BCP group(P

0.05).No significant changes were found in the ratios of MOR+and Rab5

cells and MOR+/Rab5+cells and MOR+/Rab5

cells after BCP+MT+sham EA in comparison with the BCP+MT group(P

0.05).Conclusion EA intervention can relieve pain and MT in bone cancer pain rats with MT

which may be related to its effects in increasing MOR expression and promoting endocytosis of MOR in LC region.

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