| The incidence of cancer presents a rising trend. The initial symptom is pain in most cancer patients, while75%-95%sufferers with advanced cancer bear intolerant pain. An estimated one third of advanced cancer patients are plagued by bone metastasis. Meanwhile, with improved diagnostic techniques and treatment options, cancer mortality is obviously lower in malignant tumor patients and the survival time is significantly longer. Therefore, how to improve the life quality of cancer patients becomes an urgent task.The experimental data gained suggest that there is a close relationship between neurogliocyte and chronic pain. gliocytes (astrocytes, microglia) play an important role in main pain transmitters. Meanwhile, the inflammatory cytokine released from gliocytes, including NF-a, IL-1β, IL-6, are key factors which trigger pain neurons sensitization and the formation of central sensitization, which indicates that gliocytes play an important role in the primary afferent, development of algesia, pain control and the formation and maintenance of central sensitization. As the crystallization of Chinese wisdom, traditional Chinese medicine is widely accepted by patients owing to its advantages of broad-spectrum functions, little side effects and acceptable affordability. A large number of domestic and international studies have showed that traditional Chinese medicines have certain analgesic effects for both acute and chronic pain to some degree. Clinical practice also indicates that Chinese medicine can not only relieve pain, but also improve the life quality of cancer pain patients by strengthening the immune function. However, for a long time, the study of pain mechanism is hindered due to the lack of suitable experimental animal models of cancer pain, which also blocks the exploration of new and effective treatment method, including some cancer pain drugs and non-drug therapies such as acupuncture and so on. It is not until1998when Wacnik first reported mouse model of cancer pain, and then a number of experimental animal models followed in succession.Zhixin Formula, a empirical formula created by Zhang Haicen, a famous researcher from Henan Institute of Traditional Chinese Medicine, is a scientific summary of his50years’clinical experience. It contains radix angelicae, asarum, Chuanxiong, possessing the functions of warming and activating meridians, invigorating blood circulation, dispelling wind and relieving pain. Clinical studies have confirmed that Zhixin Formula has a significant effect in treating a variety of pain syndrome such as bone cancer pain. To sum up, this paper discusses the following aspects:1. Establishing animal models of cancer pain to find effective ways to relieve pain by referring to previous reports, thus exploring the analgesic effect of Zhixin Formula. A comprehensive assessment of the model is achieved by evaluating the body weight, tibia pathology, tibia X-ray, hind thermal hyperalgesia and mechanical allodynia (allodynia) and spontaneous pain of model rats.2. Observing the changes of gliocytes in spinal cord of rats and the related mechanism.1) Detecting the spinal cord radial glial cell marker protein GFAP (Glial Fibrillary Acidic Protein) in bone cancer pain rats by immunohistochemistry;2) Detecting the spinal cord NF-K B (Nuclear factor-kappa B) and SIGIRR by Western Blot in tibial cancer pain rats;3) Detecting the spinal microglia marker protein of CD11b, Toll receptor2(TLR2) and4(TLR4) by RT-PCR;4) Detecting the inflammatory agents such as TNF-α, IL-1β (Interleukin-1beta), IL-6(Interleukin-6) in the tibial bone cancer pain rats by ELISA;RESULTS:1. The establishment of bone cancer pain model Modeling the bone cancer group by injecting3×103ascites tumor into the rat marrow cavity, which is the ascites tumor came from grafting the W256tumor cells. The experimental results show that weight gain in bone cancer group was significantly slower than the blank group weight even decline(P<0.01). Autonomic movement of rats in bone cancer group is on the rise than those in the blank group(P<0.01). Compared with the blank group, mechanical pain hypersensitivity in bone cancer group decreased significantly(P<0.01). The bone structure of rats in model group shows great changes and even destruction compared with the blank group according to X-ray photograph and HE staining.2. The changes of activities and functions of gliocytes in the spinal dorsal horn in rats with bone cancer painThe experimental rats were randomly divided into the control group, bone cancer group, Western medicine group (tramadol hydrochloride,10μg·kg-1), Chinese medicine group (Wuling Analgesic Capsule,60μg·kg-1), ZF high dose treatment group, ZF normal dose group, ZF low-dose treatment group(the concentration of high-, medium-and low-dose were9g·kg-1,4.5g· kg-1,2.25g·kg-1respectively). On D14, rats were administrated by gavage for seven days, once a day. Morphological and biological technology testing experiments, including immunohistochemistry, Western Bloã€RT-PCR, were carried on in the serum, spinal surgery lateral tibia and lumbar enlargement segment (L4-L6) of rats in the21st day. 1) Immunohistochemical detection shows that intramedullary astrocytes marker protein GFAP in bone cancer pain model has increased significantly compared with the blank group, ZF normal dose group and ZF high dose group. Hypertrophy of astrocyte proliferation appears when bone cancer pain happens. Zhixin Formula can inhibit the analgesic effect on the rats.2) PCR detection of microglial marker protein CD11b shows that CD11b mRNA in bone cancer pain model group is significantly higher than the normal dose and high dose group(P<0.01), which suggests that when bone cancer pain occurs, microgliocytes manifest proliferation and activation, and Zhixin Formula has significant analgesic effect on it.3) The key immunocompetence factors of central nervous system immune in Zhixin Formula intervention group, including TLR2, TLR4mRNA, are lower than those of bone cancer group(P<0.05). Protein level of NF-κB/GAPDH in bone cancer pain model group is significantly higher than that in normal group and ZF high dose group (P<0.05). The results suggest that the central pain pathway of TLR-NF-κB is activated. By inhibiting the downstream pathway of TLR, Zhixin Formula achieves its analgesic effect.4) On the contrary, the result of NF-κB is just the opposite. The intramedullary SIGIRR protein expression in bone cancer group is also lower than ZF high dose group, ZF normal group (P<0.01). Interestingly, the expression of ZF high dose group are higher than that of the blank group, but ZF normal dose group Is the same as western medicine group, which indicates that through activating the SIGIRR so as to inhibit NF-κB, Zhixin Formula may be one of the mechanisms of reducing and treating cancer pain.5) Expression changes of inflammatory agents NF-α, IL-1β, IL-6induced by tibia bone cancer pain.The expression of IL-1βin ZF high dose group and blank group is lower than that of bone cancer group, which indicates that there is significant difference (P<0.01). The expression of IL-1βin ZF normal dosage group is also lower than the bone cancer group (P<0.05). Moreover, the expression of IL-6, TNF-αin ZF high dose treatment group and blank group is lower than the bone cancer group, which indicates that there is significant difference (P<0.01). The same condition also occurrs in ZF normal dosage treatment group (P<0.05).Conclusion:Proliferation of gliocytes combined with activation of TLR-NF-κB are mechanisms of bone cancer pain; Meanwhile, low expression changes of SIGIRR may be associated with the pathogenesis of bone cancer pain. Through activating hyperplasia of SIGIRR, inhibiting the activation and proliferation of gliocytes in spinal cord, and activating the pathway of TLR-NF-κB, Zhixin Formula has significant analgesic effects. |
