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

4

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

11

21

22

24

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

24

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

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

11

21

22

24

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

24

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.