Effects of Patient-Controlled Transcutaneous Electrical Acupoint Stimulation on Cancer Induced Bone Pain Relief in Patients with Non-Small Cell Lung Cancer: Study Protocol for a Randomized Controlled Trial.

Background: Transcutaneous Electrical Acupoint Stimulation (TEAS) therapy opens up the possibility for individuals with Cancer-induced bone pain (CIBP) to receive a home-based, patient-controlled approach to pain management. The aim of this study is designed to evaluate the efficacy of patient-controlled TEAS (PC-TEAS) for relieving CIBP in patients with non-small cell lung cancer (NSCLC). Methods/Design: This is a study protocol for a prospective, triple-blind, randomized controlled trial. We anticipate enrolling 188 participants with NSCLC bone metastases who are also using potent opioid analgesics from 4 Chinese medical centers. These participants will be randomly assigned in a 1:1 ratio to either the true PC-TEAS or the sham PC-TEAS group. All participants will receive standard adjuvant oncology therapy. The true group will undergo patient-controlled TEAS intervention as needed, while the sham group will follow the same treatment schedule but with non-conductive gel patches. Each treatment course will span 7 days, with a total of 4 courses administered. There will be 4 assessment time points: baseline, the conclusion of weeks 4, 8, and 12. The primary outcome of this investigation is the response rate of the average pain on the Brief Pain Inventory (BPI) scale at week 4 after treatment. Secondary outcomes include pain related indicators, quality of life scale, mood scales, and routine blood counts on the assessment days. Any adverse events will be promptly addressed and reported if they occur. We will manage trial data using the EDC platform, with a data monitoring committee providing regular quality oversight. Discussion: PC-TEAS interventions offer an attempt to achieve home-based acupuncture treatment and the feasibility of achieving triple blinding in acupuncture research. This study is designed to provide more rigorous trial evidence for the adjuvant treatment of cancer-related pain by acupuncture and to explore a safe and effective integrative medicine scheme for CIBP. Trial Registration: ClinicalTrials.gov NCT05730972, registered February 16, 2023.

Folic acid relieves bone cancer pain by downregulating P2X2/3 receptors in rats.

BACKGROUND: Bone cancer pain (BCP) remains a clinical challenge due to the limited and side effects of therapeutic methods. Folic acid has been known as an FDA approved dietary supplement and proved to have an analgesic effect in neuropathic pain. Here we investigate the role and mechanism of folic acid in bone cancer pain of a rat model. METHODS: Walker 256 tumor cells were inoculated into the left tibia of rats to induce bone cancer pain model. Pain reflex were assessed by paw withdrawal threshold (PWT) response to Von Frey filaments and paw withdrawal latency (PWL) response to thermal stimulation. Folic acid was injected intraperitoneally to evaluate its analgesic effect in rats with bone cancer pain. Western blotting and qPCR were used to determine P2X2/3 receptor protein and mRNA levels in ipsilateral L4-6 dorsal root ganglion (DRG) and spinal dorsal horn (SDH). RESULTS: The PWT and PWL of rats with bone cancer pain were obviously decreased compared to the naïve and sham rats. Interestingly, continuous folic acid treatment significantly increased the PWT and PWL of rats with bone cancer pain. P2X2 and P2X3 receptors were clearly upregulated at both mRNA and protein expression in L4-6 DRG and SDH of rats with bone cancer pain. P2X2 and P2X3 receptors were mainly localized with CGRP (calcitonin gene-related peptide) or IB4 (isolectin B4) positive neurons in L4-6 DRG of rats with bone cancer pain. Notably, continuous folic acid treatment significantly reduced the expression of P2X2 and P2X3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Finally, intrathecal injection of A317491 (a selective antagonist of P2X2/3 receptors) markedly elevated the PWT and PWL of rats with bone cancer pain. CONCLUSION: These results suggest that folic acid has an effective antinociceptive effect on bone cancer pain, which is mediated by downregulating P2X2/3 receptors in L4-6 DRG and SDH of rats with bone cancer pain. Folic acid may be a novel therapeutic strategy in cancer patients for pain relief.

Regulatory mechanism of Scutellaria baicalensis Georgi on bone cancer pain based on network pharmacology and experimental verification.

Context: Scutellaria baicalensis Georgi (SBG) may relieve bone cancer pain (BCP) by regulating cell proliferation, angiogenesis, and apoptosis. Objective: The mechanism of SBG in the treatment of BCP remains to be further explored. Methods: The active compounds and targets of SBG were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and SwissTargetPrediction databases. BCP-related targets were screened from NCBI and GeneCards databases. Additionally, Cytoscape software was applied to construct network diagrams, and OmicShare platform was used to enrich Gene Ontology (GO) and pathways. Finally, the verification of active compounds and core targets was performed based on quantitative real-time PCR (qRT-PCR). Results: Interestingly, we identified baicalein and wogonin as the main active components of SBG. A total of 41 SBG targets, including VEGFA, IL6, MAPK3, JUN and TNF, were obtained in the treatment of BCP. In addition, pathways in cancer may be an essential way of SBG in the treatment of BCP. Experimental verification had shown that baicalein and wogonin were significantly related to BCP core targets. Conclusions: The active components of SBG have been clarified, and the mechanism of the active components in treating BCP has been predicted and verified, which provides an experimental and theoretical basis for the in-depth elucidation of the pharmacodynamics material basis and mechanism of SBG.

Phosphoproteomic Profiling of Rat's Dorsal Root Ganglia Reveals mTOR as a Potential Target in Bone Cancer Pain and Electro-Acupuncture's Analgesia.

Bone cancer pain (BCP) is a clinical refractory mixed pain involving neuropathic and inflammatory pain, with the underlying mechanisms remaining largely unknown. Electro-acupuncture (EA) can partly alleviate BCP according to previous research. We aim to explore the proteins and major pathways involved in BCP and EA treatment through phosphoproteomic profiling. BCP rat model was built by tibial inoculation of MRMT-1 mammary gland carcinoma cells. Mechanical hyperalgesia determined by paw withdrawal thresholds (PWTs) and bone destruction manifested on the radiographs confirmed the success of modeling, which were attenuated by EA treatment. The differentially expressed phosphorylated proteins (DEPs) co-regulated by BCP modeling and EA treatment in rat dorsal root ganglions (DRGs) were analyzed through PEX100 Protein microarray. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were significantly enriched in mammalian target of rapamycin (mTOR) signaling pathway. The phosphorylations of mTOR at Ser2448 and Thr2446 were increased in BCP and downregulated by EA. In addition, the phosphorylation of S6K and Akt, markers of the mTOR complex, were also increased in BCP and downregulated by EA. Inhibition of mTOR signaling alleviated the PWTs of BCP rats, while the mTOR agonist impaired the analgesic effect of EA. Thus, our study provided a landscape of protein phosphorylation changes in DRGs of EA-treated BCP rats and revealed that mTOR signaling can be potentially targeted to alleviate BCP by EA treatment.

Electroacupuncture Attenuates Morphine Tolerance in Rats with Bone Cancer Pain by Inhibiting PI3K/Akt/JNK1/2 Signaling Pathway in the Spinal Dorsal Horn.

PURPOSE: Morphine is often used for the treatment of moderate and severe cancer pain, but long-term use can lead to morphine tolerance. Methods for effectively inhibiting morphine tolerance and the related mechanism of action are of great significance for the treatment of cancer pain. Previous studies have shown that electroacupuncture (EA) can inhibit the occurrence of morphine tolerance, but the mechanism is not yet clear. The aim of the present study was to explore the signaling pathway by which EA attenuates the development of bone cancer pain (BCP)-morphine tolerance (MT). MATERIALS AND METHODS: Changes in the paw withdrawal threshold (PWT) of rats with bone cancer pain-morphine tolerance were observed in a study of EA combined with intrathecal injection of a PI3K inhibitor (LY294002) or agonist (insulin-like growth factor-1 [IGF-1]). We also tested the protein expression of phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-Akt), phosphorylated c-Jun NH2-terminal kinase 1/2 (p-JNK1/2), and ß-arrestin2 in the L4-6 spinal dorsal horn of rats. RESULTS: The protein expression of p-PI3K, p-Akt, p-JNK1/2, and ß-arrestin2 was upregulated in the L4-6 spinal dorsal horn of rats with bone cancer pain and bone cancer pain-morphine tolerance. EA delayed the occurrence of morphine tolerance in rats with bone cancer pain and downregulated the protein expression of p-PI3K, p-Akt, p-JNK1/2, and ß-arrestin2 in the L4-6 spinal dorsal horn of rats with bone cancer pain-morphine tolerance. Intrathecal injection of LY294002 attenuated the development of morphine tolerance and downregulated the protein expression of p-Akt, p-JNK1/2, and ß-arrestin2 in the spinal dorsal horn of rats with bone cancer pain-morphine tolerance. In addition, the inhibitory effect of EA on morphine tolerance was reversed by IGF-1. CONCLUSION: The mechanism underlying the ability of EA to attenuate morphine tolerance may be associated with inhibition of the PI3K/Akt/JNK1/2 signaling pathway.

[Early intervention of bone-nearby acupuncture combined with electroacupuncture on morphine tolerance in bone cancer pain rats and its effect on the expression of HDAC and MOR in dorsal root ganglia].

OBJECTIVE: To observe the effect of early intervention of bone-nearby acupuncture (BNA) combined with electroacupuncture (EA) on the expression of histone deacetylase1(HDAC1), histone deacetylase 2 (HDAC2) andµ-opioid recepter (MOR) in dorsal root ganglia (DRG) of bone cancer pain-morphine tolerance (BCP-MT) rats, and to explore its possible mechanism. METHODS: A total of 35 SD rats were randomized into a sham BCP group (n=6), a BCP group (n=7), a MT group (n=7), a BNA+EA group (n=8) and a shame BNA group (n=7). Except of the sham BCP group, cancer cell inoculation operation at left tibia was given in the other 4 groups to establish the bone cancer pain model. In the MT group, the BNA+EA group and the shame BNA group, intraperitoneal injection of morphine hydrochloride was given to establish the morphine tolerance model. After the operation, bone-nearby acupuncture combined with electroacupuncture was applied at "Zusanli" (ST 36) and "Kunlun" (BL 60) in the BNA+EA group, with dilatational wave, 2 Hz/100 Hz in frequency, 0.5 to 1.5 mA in intensity. Intervention in the shame BNA group was applied at the same time and acupoints as those in the BNA+EA group, the needles were pierced the skin without any electrical stimulation. The needles were retained for 30 min, once a day for continuous 7 days in both BNA+EA and shame BNA groups. Before and 10, 11, 15, 22 days after the operation, the left paw withdrawal threshold (PWT) was measured in the 5 groups. The levels of HDAC1, HDAC2 and MOR in DRG were detected by Western blot. RESULTS: Ten days after the cancer cell inoculation operation, the PWT of the BCP, MT, BNA+EA and sham BNA groups was decreased compared with the sham BCP group (P<0.01). Eleven days after the operation, the PWT of the MT, BNA+EA and sham BNA groups was increased compared with the BCP group (P<0.01). Twenty-two days after the operation, the difference was no significant between the BCP group and MT group (P>0.05); the PWT of the BNA+EA group was increased compared with the MT and sham BNA group (P<0.01). In the BCP group, the DRG levels of HDAC1 and HDCA2 were increased, while the level of MOR was decreased compared with the sham BCP group (P<0.05, P<0.01). In the MT group, the DRG level of HDAC1 was increased compared with the BCP group (P<0.05). In the BNA+EA group, the DRG level of HDAC1 was decreased compared with the MT group and the sham BNA group (P<0.01, P<0.05), while the level of MOR was increased (P<0.01). CONCLUSION: Early intervention of bone-nearby acupuncture combined with electroacupuncture can relieve the morphine tolerance in bone cancer pain rats, it may relate to down-regulating the expression of HDAC1 and up-regulating the expression of MOR in the dorsal root ganglia.

Oxytocin ameliorates bone cancer pain by suppressing toll-like receptor 4 and proinflammatory cytokines in rat spinal cord.

Bone cancer pain is considered to be mechanistically unique compared with inflammatory or neuropathic pain states. Toll-like receptor 4 (TLR4) is a transmembrane receptor protein which has been reported to be involved in neuropathic pain. However, the role of TLR4 in bone cancer pain is still unclear. Therefore, the aim of this study is to investigate the hypothesis that oxytocin may ameliorate bone cancer pain by suppressing TLR4 in spinal cord. Behavioral analysis and molecular biological experiments were carried out. Our data demonstrated that intrathecally delivery of oxytocin significantly ameliorated the mechanical allodynia and thermal hyperalgesia in bone cancer pain rats. Moreover, oxytocin suppressed the up-regulation of TLR4 and proinflammatory cytokines TNFα and IL-1ß in spinal cord of bone cancer pain rats. Therefore, we concluded that intrathecal administration of oxytocin relieves bone cancer pain by suppressing the up-regulation of TLR4, TNFα and IL-1ß in spinal cord. Oxytocin possesses analgesic efficacy against bone cancer pain and deserves further to confirm its effectiveness in clinically relevant of cancer pain.

Early intervention of bone-nearby acupuncture combined with electroacupuncture on morphine tolerance in bone cancer pain rats and its effect on the expression of HDAC and MOR in dorsal root ganglia / 中国针灸

OBJECTIVE@#To observe the effect of early intervention of bone-nearby acupuncture (BNA) combined with electroacupuncture (EA) on the expression of histone deacetylase1(HDAC1), histone deacetylase 2 (HDAC2) andμ-opioid recepter (MOR) in dorsal root ganglia (DRG) of bone cancer pain-morphine tolerance (BCP-MT) rats, and to explore its possible mechanism.@*METHODS@#A total of 35 SD rats were randomized into a sham BCP group (=6), a BCP group (=7), a MT group (=7), a BNA+EA group (=8) and a shame BNA group (=7). Except of the sham BCP group, cancer cell inoculation operation at left tibia was given in the other 4 groups to establish the bone cancer pain model. In the MT group, the BNA+EA group and the shame BNA group, intraperitoneal injection of morphine hydrochloride was given to establish the morphine tolerance model. After the operation, bone-nearby acupuncture combined with electroacupuncture was applied at "Zusanli" (ST 36) and "Kunlun" (BL 60) in the BNA+EA group, with dilatational wave, 2 Hz/100 Hz in frequency, 0.5 to 1.5 mA in intensity. Intervention in the shame BNA group was applied at the same time and acupoints as those in the BNA+EA group, the needles were pierced the skin without any electrical stimulation. The needles were retained for 30 min, once a day for continuous 7 days in both BNA+EA and shame BNA groups. Before and 10, 11, 15, 22 days after the operation, the left paw withdrawal threshold (PWT) was measured in the 5 groups. The levels of HDAC1, HDAC2 and MOR in DRG were detected by Western blot.@*RESULTS@#Ten days after the cancer cell inoculation operation, the PWT of the BCP, MT, BNA+EA and sham BNA groups was decreased compared with the sham BCP group (0.05); the PWT of the BNA+EA group was increased compared with the MT and sham BNA group (<0.01). In the BCP group, the DRG levels of HDAC1 and HDCA2 were increased, while the level of MOR was decreased compared with the sham BCP group (<0.05, <0.01). In the MT group, the DRG level of HDAC1 was increased compared with the BCP group (<0.05). In the BNA+EA group, the DRG level of HDAC1 was decreased compared with the MT group and the sham BNA group (<0.01, <0.05), while the level of MOR was increased (<0.01).@*CONCLUSION@#Early intervention of bone-nearby acupuncture combined with electroacupuncture can relieve the morphine tolerance in bone cancer pain rats, it may relate to down-regulating the expression of HDAC1 and up-regulating the expression of MOR in the dorsal root ganglia.

[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.

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

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 (P0.05) but significantly lower than that of the sham BCP group (P0.05). 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.

Alleviating Mechanical Allodynia and Modulating Cellular Immunity Contribute to Electroacupuncture's Dual Effect on Bone Cancer Pain.

HYPOTHESIS: Electroacupuncture (EA) has been used as an alternative analgesic therapy for hundreds of years, yet its analgesic potency and therapeutic advantage against bone cancer pain (BCP) in comparison with morphine remains unclear. This study aimed to investigate the effects of EA on mechanical allodynia and cellular immunity of BCP rats, and to further explore the potential mechanism. METHODS: The BCP model was established by implanting Walker 256 mammary gland carcinoma cells into the left tibia of adult female Sprague-Dawley rats. EA (dilatational wave, 2/100 Hz, 0.5 mA-1mA-1.5 mA for 10 minutes each intensity) was applied bilaterally to Zusanli (ST 36) and Kunlun (BL 60) for 30 minutes. Both EA stimulation and morphine (10 mg/kg, intraperitoneally) was given once every other day. Naloxone (0.3 mg/kg, intraperitoneally) was injected at 30 minutes prior to EA. Mechanical allodynia were demonstrated by paw withdrawal thresholds (PWTs) which measured by dynamic plantar aesthesiometer. T cell proliferation, percentage of CD3+, CD4+ and CD8+ T lymphocytes in spleen as well as expression of interleukin-2 (IL-2) in plasma were detected by WST-8, flow cytometry, and enzyme-linked immunosorbent assay technique, respectively. RESULTS: An intratibial inoculation of Walker 256 mammary gland carcinoma cells significantly decreased PWTs to mechanical stimuli. EA stimulation alleviated mechanical allodynia in BCP rats, and the analgesic potency of EA was weaker than that of morphine. In contrast to morphine, EA stimulation of BCP rats increased splenic concanavalin A (Con A)-induced T cell proliferation and plasma IL-2 content, as well as increased the percentages of splenic CD3+CD4+ and CD3+CD8+ T cell subsets. Moreover, both the analgesic effect and the partial immunomodulation of EA were suppressed by an intraperitoneal injection of naloxone. CONCLUSION: EA could significantly alleviate BCP-induced mechanical allodynia. Although the analgesic effect of EA was weaker than that of morphine, EA had an immunomodulation effect on cellular immunity. Both analgesic and immunomodulatory effect of EA might share the same mechanism via the opioid-mediated pathway, which needs further investigation.

[Effects of electroacupuncture on expression of µ-opioid receptor in nucleus ceruleus in rats with bone cancer pain-morphine tolerance].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on pain behavior in rats with bone cancer pain and morphine tolerance, and to explore partial action mechanism. METHODS: Forty-two SD healthy female rats were randomly divided into a sham operation group (7 rats), a bone cancer pain group (8 rats), a morphine tolerance group (9 rats), an EA group (9 rats) and a sham EA group (9 rats). The rats in the sham operation group were treated with injection of phosphate buffer saline at medullary cavity of left-side tibia, and the rats in the remaining groups were injected with MRMT-1 breast cancer cells. After operation, no treatment was given to rats in the sham operation group and bone cancer pain group. 11 days after operation, rats in the morphine tole-rance group, EA group and sham EA group were treated with intraperitoneal injection of morphine hydrochloride, once every 12 hours, for 11 days to establish the model of bone cancer pain and morphine tolerance. One day after the establishment of this bone cancer pain model, the rats in the morphine tolerance group were injected with morphine, once every 12 hours (9:00 a.m. and 9:00 p.m.) for 7 days; the rats in the EA group and sham EA group were injected with morphine at 9:00 a.m., and treated with EA (2 Hz/100 Hz) and sham EA (only injected into the subcutaneous tissue) at bilateral "Zusanli" (ST 36) and "Kunlun" (BL 60), 30 min per treatment, once a day for 7 days. One day before cancer cell injection, 6 days, 8 days, 10 days after operation, after 30 min on 1 days, 5 days, 9 days, 11 days of morphine injection, and after 30 min on 1 days, 3 days, 5 days, 7 days of EA treatment, the paw withdrawal threshold (PWT) was measured in each group. On 11 day of morphine injection, HE staining was applied to observe the morphology and structure change of tibia in the sham operation group, bone cancer pain group and morphine tolerance group, random 2 rats in each group. On 7 days of EA treatment, fluorescent immunohistochemical method was applied to observe the expression of µ-opioid receptor positive cells in nucleus ceruleus in each group, random 4 rats in each one. RESULTS: After 10 days of the cancer cells injection, the PWT of 28 rats of bone cancer pain model (8 rats in the bone cancer pain group, 8 rats in the morphine tolerance group, 6 rats in the EA group and 6 rats in the sham EA group) was significantly lower than that of 7 rats in the sham operation group (P<0.01). After one day of morphine injection, the PWT of the morphine tolerance group, EA group and sham EA group was higher than that of the bone cancer pain group (all P<0.01); on 11 d of morphine injection, the PWT of the morphine tolerance group, EA group and sham EA group was not significantly different from that of the bone cancer pain group (all P>0.05). On 11 d of morphine injection, the tumor induced by cancer cells was observed in upper 1/3 tibia in the bone cancer pain group and morphine tolerance group, and the marrow cavity was filled with MRMT-1 cancer cells; no abnormal change was observed in the sham operation group. On 1 d, 3 d, 5 d and 7 d of EA treatment, the PWT of the cancer pain group, morphine tolerance group and sham EA group was lower than that of the EA group (all P<0.01). On 7 d of EA treatment, the positive expression of MOR in nucleus ceruleus in the cancer pain group, morphine tolerance group, EA group and sham EA group was lower than that in the sham operation group (P<0.01, P<0.05), and that in the cancer pain group, morphine tolerance group and sham EA group was lower than that in the EA group (all P<0.01). CONCLUSIONS: EA can improve mechanical pain threshold in rats with bone cancer pain-morphine tolerance, and improve the abnormal pain, which is likely to be involved with improvement of the MOR positive cells expression in nucleus ceruleus by EA.

Morin Suppresses Astrocyte Activation and Regulates Cytokine Release in Bone Cancer Pain Rat Models.

As inflammatory and immune responses are involved in pathophysiology of debilitating neuropathic pain, reagents that can modulate these two responses may have therapeutic potential. Morin, derived from the moraceae family of plants, benefits inflammation-related diseases, but its antinociceptive effects on cancer pain remain elusive. In the present study, we investigated antinociceptive effects of morin on bone cancer pain using a rat model, where rats were subject to implantation of Walker 256 mammary gland carcinoma cells into the tibia. Morin (5-20 mg/kg) dose-dependently attenuated behavioral hypersensitivities, including mechanical allodynia and free movement pain, which was accompanied by downregulation of astrocyte marker glial fibrillary acidic protein in the spinal cord in cancer-bearing rats. Treatment with morin also induced reduction of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 and upregulation of an antiinflammatory cytokine IL-10. Furthermore, intrathecal injection of AM630 (an antagonist of cannabinoid receptor 2, CB2 ), but not naloxone (an antagonist of opioid receptors), significantly blocked morin attenuation of behavioral hypersensitivities. Taken together, these results suggest that morin suppresses astrocyte activation and neuro-inflammation induced by bone cancer pain and its antinociceptive effects on bone cancer pain may be associated with activation of CB2 receptors in the spinal cord. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous inhibition of NMDA and mGlu1/5 receptors by levo-corydalmine in rat spinal cord attenuates bone cancer pain.

Bone cancer pain is a challenge for its not completely clarified mechanism and broad clinical morbidity. Therefore, novel and more effective drugs are urgent needed for improvement of patients' quality of life. Glutamate receptors have been associated with the development of the central sensitization of chronic pain. Inhibition of N-methyl-d-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors can effectively attenuate bone cancer pain, respectively. Herein, our results indicated that levo-Corydalmine (l-CDL), a compound from Corydalis yanhusuo W.T. Wang, which has been used in traditional Chinese medicine for pain relief could effectively attenuate bone cancer pain induced by tibia bone cavity tumor cell implantation (TCI) through simultaneously inhibiting the NMDA and mGlu1/5 receptors in rat spinal cord without notable side effects. Both intragastric and intrathecal administration of l-CDL significantly alleviated the mechanical hypersensitivity induced by TCI in rats, and the analgesic effect of l-CDL could be reversed by intrathecal administration of NMDA receptor agonist NMDA and mGlu1/5 receptor agonist DHPG but not AMPA receptor agonist AMPA. l-CDL could also selectively suppress NMDA and DHPG induced rapid rise in Ca2+ oscillations in primary cultures neurons of spinal cord in vitro. The antinociception of l-CDL were partially mediated by the reduced phosphorylation of PKC γ and ERK1/2 in spinal cord of TCI rats in a NMDA and mGlu1/5 dependent manner. In conclusion, these results suggested that l-CDL attenuates TCI induced bone cancer pain through simultaneously inhibiting the NMDA and mGlu1/5 receptors and the downstream PKC γ, ERK1/2 signaling pathways in the spinal cord.

Effects of electroacupuncture on expression of μ-opioid receptor in nucleus ceruleus in rats with bone cancer pain-morphine tolerance / 中国针灸

<p><b>OBJECTIVE</b>To observe the effects of electroacupuncture (EA) on pain behavior in rats with bone cancer pain and morphine tolerance, and to explore partial action mechanism.</p><p><b>METHODS</b>Forty-two SD healthy female rats were randomly divided into a sham operation group (7 rats), a bone cancer pain group (8 rats), a morphine tolerance group (9 rats), an EA group (9 rats) and a sham EA group (9 rats). The rats in the sham operation group were treated with injection of phosphate buffer saline at medullary cavity of left-side tibia, and the rats in the remaining groups were injected with MRMT-1 breast cancer cells. After operation, no treatment was given to rats in the sham operation group and bone cancer pain group. 11 days after operation, rats in the morphine tole-rance group, EA group and sham EA group were treated with intraperitoneal injection of morphine hydrochloride, once every 12 hours, for 11 days to establish the model of bone cancer pain and morphine tolerance. One day after the establishment of this bone cancer pain model, the rats in the morphine tolerance group were injected with morphine, once every 12 hours (9:00 a.m. and 9:00 p.m.) for 7 days; the rats in the EA group and sham EA group were injected with morphine at 9:00 a.m., and treated with EA (2 Hz/100 Hz) and sham EA (only injected into the subcutaneous tissue) at bilateral "Zusanli" (ST 36) and "Kunlun" (BL 60), 30 min per treatment, once a day for 7 days. One day before cancer cell injection, 6 days, 8 days, 10 days after operation, after 30 min on 1 days, 5 days, 9 days, 11 days of morphine injection, and after 30 min on 1 days, 3 days, 5 days, 7 days of EA treatment, the paw withdrawal threshold (PWT) was measured in each group. On 11 day of morphine injection, HE staining was applied to observe the morphology and structure change of tibia in the sham operation group, bone cancer pain group and morphine tolerance group, random 2 rats in each group. On 7 days of EA treatment, fluorescent immunohistochemical method was applied to observe the expression of μ-opioid receptor positive cells in nucleus ceruleus in each group, random 4 rats in each one.</p><p><b>RESULTS</b>After 10 days of the cancer cells injection, the PWT of 28 rats of bone cancer pain model (8 rats in the bone cancer pain group, 8 rats in the morphine tolerance group, 6 rats in the EA group and 6 rats in the sham EA group) was significantly lower than that of 7 rats in the sham operation group (<0.01). After one day of morphine injection, the PWT of the morphine tolerance group, EA group and sham EA group was higher than that of the bone cancer pain group (all<0.01); on 11 d of morphine injection, the PWT of the morphine tolerance group, EA group and sham EA group was not significantly different from that of the bone cancer pain group (all>0.05). On 11 d of morphine injection, the tumor induced by cancer cells was observed in upper 1/3 tibia in the bone cancer pain group and morphine tolerance group, and the marrow cavity was filled with MRMT-1 cancer cells; no abnormal change was observed in the sham operation group. On 1 d, 3 d, 5 d and 7 d of EA treatment, the PWT of the cancer pain group, morphine tolerance group and sham EA group was lower than that of the EA group (all<0.01). On 7 d of EA treatment, the positive expression of MOR in nucleus ceruleus in the cancer pain group, morphine tolerance group, EA group and sham EA group was lower than that in the sham operation group (<0.01,<0.05), and that in the cancer pain group, morphine tolerance group and sham EA group was lower than that in the EA group (all<0.01).</p><p><b>CONCLUSIONS</b>EA can improve mechanical pain threshold in rats with bone cancer pain-morphine tolerance, and improve the abnormal pain, which is likely to be involved with improvement of the MOR positive cells expression in nucleus ceruleus by EA.</p>

External application of traditional Chinese medicine in the treatment of bone cancer pain: a meta-analysis.

BACKGROUND: Bone cancer pain presents a clinical challenge with limitations of current treatments. Many patients seek additional therapies that may relieve pain. Many external applications of traditional Chinese medicines (EAs-TCMs) have been evaluated in clinical trials, but fewer are known about them outside of China. The objective of this study is to assess the efficacy for bone cancer pain. METHODS: A systematic literature search was conducted in seven databases until December 2014 to identify randomized controlled trials (RCTs) about EAs-TCMs in the treatment of bone cancer pain. The primary outcome was total pain relief rate. The secondary outcomes were adverse events at the end of treatment course. The methodological quality of RCTs was assessed independently using six-item criteria according to the Cochrane Collaboration. All data were analyzed using Review Manager 5.2.0. We included any RCTs evaluating an EA-TCM for the treatment of bone cancer pain. We conducted a meta-analysis. RESULTS: We included six RCTs with 534 patients. In general, the reporting of methodological issues was poor. Compared with morphine sulfate sustained release tablets (MSSRTs) or radiotherapy or bisphosphonates, we analyzed data from five trials reporting on complete response effect score (relative risk (RR) = 5.38, 95% confidence interval (CI) = 2.80-10.31, P < 0.00001) and partial response (RR = 1.18, 95% CI = 1.02-1.37, P = 0.02) and six trials reporting on total pain relief rate (RR = 1.49, 95% CI = 1.43-1.67, P < 0.00001). Six RCTs showed significant effects of EA-TCM for improving pain relief in patients with bone cancer pain. In addition, no severe adverse events were found. CONCLUSION: This systematic review showed positive but weak evidence of EA-TCM for bone cancer pain because of the poor methodological quality and the small quantity of the included trials. Future rigorously designed RCTs are required.

Study of the analgesic activities, chronic toxicity and addictive potential of Jia-Yuan-Qing pill in rats.

Jia-Yuan-Qing pill (JYQP) composed of Porcellio laevis Latreille, Corydalis Rhizoma and Radix Cynanchi Paniculati at a ratio of 9:7:7 has been found to be an effective analgesic agent. The present study aimed to evaluate the safety, addictive potential and anti-cancer pain activity of JYQP in a rat model. During the 6-month chronic toxicity test, no significant changes in general behavior, defecation, postural abnormalities, dietary or water intake or blood biochemical parameters were observed in male and female rats. Although a high dose of JYQP (5 g/kg) caused swelling of the liver, spleen and kidney in male and female rats, no pathological changes were observed in all organs examined via hematoxylin and eosin staining. The analgesic effect of JYQP on bone cancer pain was successfully confirmed in a rat model of Walker 256 cell-induced bone cancer. In contrast to morphine, in a physical dependence test, JYQP produced no withdrawal symptoms following chronic administration. The data from this study provide experimental evidence supporting the clinical use of JYQP as an effective, safe and non-addictive agent for the treatment of bone cancer pain.

Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.

Accumulating evidence suggests that activation of spinal microglia contributes to the development of inflammatory and neuropathic pain. However, the role of spinal microglia in the maintenance of chronic pain remains controversial. Bone cancer pain shares features of inflammatory and neuropathic pain, but the temporal activation of microglia and astrocytes in this model is not well defined. Here, we report an unconventional role of spinal microglia in the maintenance of advanced-phase bone cancer pain in a female rat model. Bone cancer elicited delayed and persistent microglial activation in the spinal dorsal horn on days 14 and 21, but not on day 7. In contrast, bone cancer induced rapid and persistent astrocytic activation on days 7-21. Spinal inhibition of microglia by minocycline at 14 d effectively reduced bone cancer-induced allodynia and hyperalgesia. However, pretreatment of minocycline in the first week did not affect the development of cancer pain. Bone cancer increased ATP levels in CSF, and upregulated P2X7 receptor, phosphorylated p38, and IL-18 in spinal microglia. Spinal inhibition of P2X7/p-38/IL-18 pathway reduced advanced-phase bone cancer pain and suppressed hyperactivity of spinal wide dynamic range (WDR) neurons. IL-18 induced allodynia and hyperalgesia after intrathecal injection, elicited mechanical hyperactivity of WDR neurons in vivo, and increased the frequency of mEPSCs in spinal lamina IIo nociceptive synapses in spinal cord slices. Together, our findings demonstrate a novel role of microglia in maintaining advanced phase cancer pain in females via producing the proinflammatory cytokine IL-18 to enhance synaptic transmission of spinal cord nociceptive neurons.

Analgesic effect of intrathecal ginsenosides in a murine bone cancer pain.

BACKGROUND: Bone cancer pain has a disruptive effect on the cancer patient's quality of life. Although ginsenosides have been used as traditional medicine in Eastern Medicine, the effect on bone cancer pain has not been thoroughly studied. The aim of this study was to determine whether ginsenosides may alter the bone cancer pain at the spinal level. METHODS: NCTC 2472 tumor cells (2.5 × 10(5)) were injected into the femur of adult male C3H/HeJ mice to evoke bone tumor and bone cancer pain. To develop bone tumor, radiologic pictures were obtained. To assess pain, the withdrawal threshold was measured by applying a von Frey filament to the tumor cells inoculation site. The effect of intrathecal ginsenosides was investigated. Effect of ginsenosides (150, 500, 1,000 µg) was examined at 15, 30, 60, 90, 120 min after intrathecal delivery. RESULTS: The intrafemoral injection of NCTC 2472 tumor cells induced a radiological bone tumor. The withdrawal threshold with tumor development was significantly decreased compared to the sham animals. Intrathecal ginsenosides effectively increased the withdrawal threshold in the bone cancer site. CONCLUSIONS: NCTC 2472 tumor cells injection into the mice femur caused bone tumor and bone cancer pain. Intrathecal ginsenosides attenuated the bone cancer-related pain behavior. Therefore, spinal ginsenosides may be an alternative analgesic for treating bone cancer pain.