Study Of The Mechanisms Of XiaozhengZhitong Paste (XZP) On Bone Cancer Pain By Modulating Osteoclasts Activation And PAR2/TRPV1 Signaling Pathway

Cancer pain is one of the most common symptoms for cancer patients. Most of cancer patients suffered pain due to the cancer itself (the tumor pressed on bones, nerves, or other organs), the treatment, or the tests done to diagnose cancer. It was reported that 75%-90% cancer patients experienced pain during their illness and up to 50% of cancer pain is undertreated. It was said that one quarter of the patients had newly diagnosed malignancies, one third of the patients are undergoing treatment, and three quarters of the patients with advanced disease experienced pain. For those patients with metastasis to the other places, pain is especially prevalent. And it was reported that up to 80% of the cancer patients who have bone metastasis experienced pain. Pain management is important in oncologic care and essential for maximizing patient outcomes. Mounting evidence showed that unrelieved pain significantly comprised overall quality of life and effective pain control was associated with survival. Health care practitioners depend heavily on opioid therapies for cancer pain. Although this therapy is very effective, it is with a lot of side effects, such as constipation, urinary retention, nausea, sedation, respiratory depression, myoclonus, delirium, sexual dysfunction, and hyperalgesia. So it is urgent to seek new treatment of cancer pain and new methods to improve the quality of life of patients with cancer pain.Complementary and alternative medicine (CAM) including Chinese medicine and Chinese herbs, which are noninvasive and generally considered to be relatively free of toxicity, are used as an adjunct therapy with standard pain management techniques. There is considerable evidence that Chinese medicine especial for topical treatment of Chinese herbs are useful for cancer pain and Chinese medicine plus "three ladder therapy" are more useful for cancer pain than "three ladder therapy" alone. According to traditional Chinese medicine (TCM) theory, cancer pain is the result of tumor-caused stagnation and insufficiency of Qi and blood low in the body. Thus the main therapeutic goals are to promote Qi and activate blood by sotening hard lumps, dispelling nodes, and warming the channels. Our previous study has demonstrated that herbal analgesic paste, Xiaozheng Zhitong Paste (XZP) containing six herbs of Xuejie (Dragon’s blood), Yanhusuo (Corydalis rhizoma), Ruxiang (Olibanum), Moyao (Myrrha), Qingdai (Natural Indigo), and Bingpian (Borneolum Syn-theticum), signiicantly alleviated cancer pain including bone cancer pain in patients with middle/late stage cancer. However, the mechanisms underlying action of XZP in alleviating bone cancer pain have not been systemically explored. Illuminating the mechanisms underlying action of XZP in alleviating bone cancer pain may explore the new treatment of cancer pain and the foundation for seeking new cancer pain medicine.Objective:To explore the effects and mechanisms of Xiaozheng Zhitong Paste (XZP) on bone cancer pain.Methods:(1) Make Xiaozheng Zhitong cataplasm and study the transdermal behavior of Xiaozheng Zhitong cataplasm in rats. With tetrahydropalmatine as the index, the Franz diffusion cell method was adopted for the experiment. Sample content was determined with High Performance Liquid Chromatography (HPLC).(2) Female Wistar rats weighing 150-180 g were randomly and equally divided into sham control group and bone cancer pain group. The bone cancer pain group was then randomly and equally divided into high dose group, medium dose group, low dose group, placebo group and positive control group. Mechanical behavior test was performed before tumor inoculation and on days 3,6,9,12,15,18, and 21 after tumor inoculation. Thermal behavior test was performed before tumor inoculation and on days 2,5,8,11,14,17, and 20 after tumor inoculation. The cancer-related osteolytic lesions in the tibia of individual rats were examined by X-ray radiology at 21 days after the inoculation. The rats were anaesthetized and exposed to X-ray (Emerald 125) at 40 KVP for 1/20 s, followed by the development of X-ray film (Henry Schein blue sensitive film) using a film developer (Konica SRX-101). Bone mineral content (BMC) and bone mineral density (BMD) were analyzed by radiology. The tibias were scanned by micro-CT and reconstructed with 8μm isotropic voxel size on a micro-CT system (eXplore Locus SP, GE Medical Systems). The left tibia from each animal was dissected, fixed in 10% formalin overnight, decalcified in 15% EDTA-PBS for 7 days, and paraffin-embedded. The tissue sections (6 μm) were stained with hematoxylin and eosin (HE).(3) The numbers of osteoclasts and osteoblasts in the regions were identified by the tartrate-resistant acid phosphatase (TRAP) or alkaline phosphatase (AP) staining using the TRAP or AKP staining, respectively. Peripheral blood samples were collected from individual rats at 0,7,14, or 21 days after inoculation and the concentrations of serum tartrate-resistant acid phosphatase 5b (TRACP5b), carboxy-terminal collagen crosslinks (CTX) and osteocalcin in individual rats were determined by Enzyme-Linked Immunosorbent Assay (ELISA) using the specific kits, according to the manufacturer’s instruction. The intensity of macrophage infiltrates in the bone lesions of the different groups of rats was characterized by immunohistochemistry using anti-CD68 staining. The relative mRNA and protein expression levels of RANKL/RANK/OPG signaling pathway associated regulators such as RANKL, OPG, RANK, PTHrP and IGF-1 and inflammatory regulators including IL-8、M-CSF and TNF-a in the bone tissues were determined by TR-PCR and Western blot.(4) Peripheral blood samples were collected from individual rats and the concentrations of serum PAR2/TRPV1 signaling pathway associated inflammatory regulators such as trypsin, TNF-a and IL-1β in individual rats were determined by ELISA using the specific kits, according to the manufacturer’s instruction. The relative mRNA and protein expression levels of PAR2/TRPV1 signaling pathway associated main regulators such as PAR2, TRPV1, PKC-y and PKA in the DRG neurons, and IBA1, GFAP, c-Fos, and CGRP mRNA and protein expression in the spinal cord were determined by TR-PCR and Western blot.Results:(1) The transdermal permeability and the transmission rate of tetraydropalmatine accumulated for 24 hours were 20.20% and 0.744 μg·cm-2·h-1. The transdermal behaviors of Xiaozheng Zhitong cataplasm were ideal in conformity with zero order kinetic model. Xiaozheng Zhitong cataplasm is reliable medicine for investigating the the effects and mechanisms of Xiaozheng Zhitong Paste (XZP) on bone cancer pain.(2) While consistent levels of PWT were observed in the control rats the levels of PWT were gradually reduced throughout the observation period in the placebo group of rats. In contrast, treatment with different doses of XZP significantly mitigated the mechanical allodynia in a dose and time-dependent manner (P<0.05). A similar pattern of the PWL was observed in the different groups of rats (P<0.05). These data indicated that treatment with XZP reduced the mechanical and thermal nociceptive behaviors in rats with bone cancer. The X-ray images indicated that while loss of full thickness bicortical bone was accompanied by displaced fractures in the placebo group of rats with bone cancer, less loss of medullary bone and erosion of the cortical bone were apparent in the positive control and rats with different doses of XZP at 21 days post inoculation (P<0.05). Quantitative analysis revealed that the radiological scores in the rats with different doses of XZP were significantly less than that of the placebo group, but remained higher than that of the OPG-treated positive controls (P< 0.05). Micro-CT images showed that control group had normal bone mass and structure, and the placebo group of rats displayed decreased bone mass and bone structure destruction. Among different doses of XZP treatment, the high dose group of rats ameliorated loss of bone mass more efficiently compared with other two doses. Quantitative analyses of BMC and BMD revealed that treatment with a high dose of XZP, like OPG, significantly preserved the BMC in the rats with bone cancer at 7,14 and 21 days post inoculation (p<0.05). Similarly, treatment with medium or high dose of XZP, like OPG treatment, significantly mitigated the loss of BMD in the tibias of rats with bone cancer at 7,14 and 21 days post inoculation (P <0.05). Histological examination revealed that while many cancer cells invaded the bone tissues and destroyed the bone structure in the placebo group of rats less numbers of cancer cells invaded the bone tissues in the positive controls and rats treated with different doses of XZP. Collectively, treatment with XZP mitigated the cancer invasion-mediated bone damage and structure changes in rats.(3) Bone cancer is associated with aberrant activation of osteoclasts, leading to bone reabsorption and damage, which also induce the compensated activation of osteoblasts. To understand the potential mechanisms underlying the action of XZP, the activity of osteoclasts and osteoblasts in the tibia lesions of rats was characterized by BAP and TRAP staining, respectively. Obviously, high intensity of osteoclast staining was observed in the rats with bone cancer. Quantitative analyses indicated that the mean intensity of TRAP staining in the rats receiving XZP was significantly reduced, as compared with that in the placebo controls (p<0.05). Similarly, high levels of BAP staining were observed in the rats with bone cancer, but the relative levels of BAP staining in the rats with XZP treatment were significantly lower than that in the placebo group at 14 or 21 days post inoculation (p<0.05). These suggest that bone cancer promoted the activation of osteoclasts, which compensated to activate osteoblasts in the bone cancer lesions. Furthermore, ELISA analyses indicated that in comparison with that in the placebo group, the levels of serum TRAP5b, a biomarker of bone resorption, in rats with XZP were significantly reduced at 14 or 21 days post inoculation and the effect of treatment with different doses of XZP appeared to be dose-dependent (p<0.05). A similar pattern of the levels of serum CTX, a biomarker of bone turnover, was detected in the different groups of rats. In addition, the levels of serum osteocalcin, a hormone secreted by osteoblasts, were also significantly reduced in the rats with XZP at 14 or 21 days post inoculation (p<0.05). Together, these data indicated that treatment with XZP inhibited the cancer-related bone resorption and turnover as well as compensated bone formation in rats. Accordingly, the relative levels of RANKL, OPG, RANK, PTHrP and IGF-1, and inflammatory regulators including IL-8、M-CSF and TNF-α mRNA transcripts and protein expression in the bone lesions were determined by quantitative RT-PCR and western blot. While significantly increased levels of RANKL, RANK, PTHrP and IGF-1, and inflammatory regulators including IL-8、M-CSF and TNF-α mRNA transcripts and protein expression were detected in the rats with bone cancer, as compared with that in the healthy controls. The relative levels of these gene mRNA transcripts and protein expression were significantly reduced in the rats receiving XZP treatment, similar to that in the rats with OPG treatment, as compared with that in the placebo group (p<0.05). In contrast, treatment with XZP significantly enhanced the OPG gene transcription and protein expression in the rats with bone cancer (p<0.05). Thus, treatment with XZP modulated the expression of bone metabolism-related regulators in the bone lesions, contributing to its therapeutic effects in rats with bone cancer.(4) ELISA analysis indicated that upstream levels of the PAR2 signaling pathway including trypsin, TNF-α and IL-1β serum in rats treated with XZP were significantly reduced compared with placebo controls (p<0.05); and these effects appeared to be dose-dependent. Furthermore, relative downstream levels of PAR2 signaling pathway mediators including PAR2, PKC-γ, PKA and TRPV1 mRNA transcripts in bone lesions were significantly increased in the rats with bone cancer (p<0.05), compared with the sham controls; while relative levels of these gene mRNA transcripts were significantly reduced in the rats that received XZP treatment, similar to rats with OPG treatment (p<0.05). A similar pattern was detected when protein expressions were analyzed in bone lesions in rats in the different groups (p<0.05). Together, these data indicate that XZP treatment inhibited the PAR2 signaling pathway in rats with bone cancer, which may contribute to the observed antinociceptive effects. Significantly increased levels of c-Fos, GFAP, IBA1 and CGRP mRNA transcripts were detected in rats with bone cancer, compared with the sham controls; while relative levels of their gene mRNA transcripts were significantly reduced in rats treated with XZP (p<0.05), similar to rats that received OPG treatment. A similar pattern was detected when protein expressions in the spinal cord were analyzed (p<0.05). Thus, XZP treatment inhibited the activation of astrocytes and microglial cells, contributing to its therapeutic effects in rats with bone cancer.Conclusion:1. XZP can alleviate bone cancer pain in rats.2. XZP can significantly alleviate bone cancer-related nociception by mitigating the bone damage and modulating the RANKL/RANK/OPG pathway and inflammation.3. XZP can significantly alleviate cancer-induced bone pain by inhibiting peripheral and central sensitization through modulating the PAR2/TRPV1 signaling pathway.