[Bone-edge electroacupuncture relieves pain by regulating expression of GRK5, ß-arrestin 2 and PKCα proteins in locus coeruleus in bone cancer pain rats with morphine tolerance].

OBJECTIVE: To observe the effect of bone-edge electroacupuncture (EA) intervention on mechanical pain threshold (PT) and expression of G protein-coupled receptor kinase (GRK5), ß-arrestin 2, total and phosphorylated PKC alpha (p-PKCα) proteins in the locus coeruleus (LC) of rats with bone cancer pain induced morphine tolerance, so as to reveal its partial central mechanisms underlying pain relief. METHODS: Forty SD rats were randomly divided into 5 groups, namely sham bone cancer, bone cancer pain, morphine tolerance, bone-edge EA, and sham EA (n＝ 8 rats in each group). The bone cancer with morphine tolerance model was established by intramedullary injection of MRMT-1 cells into the tibial cavity, and then intraperitoneal injection of morphine hydrochloride injection. After successful establishment of morphine tolerance model, the bone-edge EA (2 Hz/100 Hz，0.5－1.5 mA) was applied to bilateral "Zusanli" (ST36) and "Kunlun" (BL60) for 30 min, once a day for 7 days, after inserting the needle-tip to the tibial bone surface. The ipsilateral mechanical paw withdrawal thresholds (PWTs) were detected dynamically. The expression levels of GRK5, ß-arrestin 2, PKCα and p-PKCα in the LC area were measured by Western blot. RESULTS: The PWTs of bone cancer pain rats were decreased on day 10 after inoculation of cancer cells (P<0.01). After i．p. of morphine for 11 days, no analgesic effect and pain tolerance appeared (P>0.05). The PWTs were significantly increased in the bone-edge EA intervention group (P<0.01), not in the sham EA group (P>0.05). In comparison with the sham bone cancer group, the expression of GRK5 protein in morphine tolerance group was significantly decreased (P<0.01); compared with morphine tolerance group, the expression of GRK5 protein in bone-edge EA group was increased(P<0.01). In comparison with the sham bone cancer group, the expression of ß-arrestin 2 and p-PKCα in bone cancer group significantly increased (P<0.01). After the intervention, the increased ß-arrestin 2 and p-PKCα expressions were reversed in the bone-edge EA group (P<0.01); compared with morphine tolerance group and sham EA group, the expression of PKCα protein was decreased(P<0.01). CONCLUSION: Bone-edge EA can effectively relieve morphine tolerance in bone cancer pain rats, which may be related to its functions in up-regulating GRK5 protein and down-regulating ß-arrestin 2, PKCα and p-PKCα proteins in LC.　.

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

OBJECTIVE: To observe the effect of electroacupuncture (EA) on pain behavior and expression of µ-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 µL of human Walker 256 breast cancer cells (MRMT-1 breast cancer cells, 1 x104 cells/µL) 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 10th 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 10th to 28th 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 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.