Effects of Curcumin on Biological Behavior and NF- κ B/TNF- α Pathway in Mice with Metastatic Bone Pain of Breast Cancer Induced by Walker 256 Cells

Background. The active ingredient curcumin of traditional Chinese medicine was selected as the research object to investigate the possible mechanism of breast cancer metastatic bone pain in mouse walker 256 cells and the effect of curcumin on the NF-κB/TNF-α pathway in order to provide a new idea for clinical treatment of breast cancer metastatic bone pain. Methods. By establishing an animal model of breast cancer bone metastasis in walker 256 cells, the biological behavior of nude mice was observed on the 8 th day after successful modeling. Meanwhile, the low dose group, middle dose group and high dose group of mice were given 15 mg/kg, 25 mg/kg, 50 mg/kg of curcumin solution intraperitoneally in 21 days, and the right cavity bone and spinal cord distended in mice (L4-L6) tissues were used to detect related factors, Immunohistochemical method was used to detect c-fos in spinal cord. Expression levels of RANK, NF-κB and TNF-α were detected by RT-PCR and Western blot. Meanwhile, serum levels of Cox2, il-6, leukotriene and PGE2 were detected. Results. Observing the biological behavior index of nude mice, we found that the mechanical pain and thermal pain threshold decreased (p < 0.05), and the cold pain and spontaneous pain scores increased significantly


Introduction
Breast cancer was one of the most common malignant tumors in women, and most patients with advanced breast cancer were often accompanied by bone invasion, which could easily cause intractable bone pain, pathological fracture and a series of bone-related events. Curcumin was the main active component extracted from the roots of turmeric, which had the effects of anticoagulant, hypolipid, antioxidation, anti-inflammatory, anti-rheumatic and anti-tumor [1].
Meanwhile, curcumin had certain effects on the physiological function, pathological process and bone metabolism of the nervous system, and could alleviate the inflammatory reaction, inhibit the peripheral or central sensitization of chronic pain [2]. In this study, the animal model of metastatic bone pain caused by breast cancer in Walker 256 cells was established. Curcumin, an active ingredient of traditional Chinese medicine was selected as the research object, and the biological behavior changes of model animals were dynamically monitored. The expression of RANK, NF-κB, TNF-α and related factors in upstream and downstream were detected by immunohistochemistry, RT-PCR and Western blot, so as to explore the possible mechanism of metastatic bone pain in breast cancer and the effect of curcumin intervention. It will provide a new idea and method Journal of Cancer Therapy for clinical treatment.

Cell Culture and Injection of Tumor Cells into Tibia
Walker 256 cells were cultured in RPMI1640 medium containing 10% calf serum and cultured at 37˚C for routine passage. When the cultured Walker 256 cells were in logarithmic growth phase, the cells were collected and injected into the right tibia of nude mice. Each animal was inoculated with 20 ul, including 2 × 10 6 cells, 5 nude mice were injected, and the heat inactivated Walker 256 cells were injected as negative control.

Biological Behavior Observation
The behavior of nude mice was observed on the 8 th day, and the PWT value was less than 6 g, and the mechanical pain threshold was stable, that is to say, the model was successful. The main observation indexes were as follows: 1) Determination of paw withdrawal threshold (PWT): Mechanical hyperalgesia was measured by foot contraction reaction stimulated by Von Frey fiber filaments.
From the 8 th day of modeling, once every other day, four kinds of vonFrey fine fibers (1 g, 2 g, 6 g and 15 g) were used to stimulate the left middle plantar of mice. The foot contraction reaction of mice was observed. Each animal was tested 5 times per fiber, at least 2 minutes between the two tests. The percentage of foot contraction response of animals to vonFrey fiber stimulation was 5 × 100%. 2) Determination of paw withdrawal latency (PWL): By using the method of hot plate experiment, the hot plate was set to (48 ± 0.1)˚C, and then the mice were put into the glass lattice on the hot plate. The legs of the mice were lifted, and the latent period of escape and licking of the feet was used as the pain threshold. The average value of three times was calculated at an interval of 10 min, and the result was hyperalgesia score. 3) Determination of cold pain sensitivity: cold plate test and acetone low temperature test were commonly used. The abnormal cold pain behavior of bone cancer pain model animals can be judged by the determination of cold stimulation hyperalgesia. 4) Observation of spontaneous pain behaviour: the spontaneous hyperalgesia behavior in the model of bone cancer was determined by spontaneous hyperalgesia behavior. Mice were placed on a smooth platform to walk freely, and the walking posture, side landing, and foot load of tumor-bearing nude mice were observed for scoring (between 0 and 3 points). 0: normal action; 1 point: mild claudication of the hind limb of the inoculation side, can be used normally but relatively insignificant; 2: the degree of claudication of the hind limb on the inoculation side was between 1 and 3; 3: severe lameness of the hind limb on the inoculation side or inability to reach the ground when the hind limb was walking.

Grouping and Administration
After screening the basic pain threshold, the mice were randomly divided into

Histology Research
On the 21 st day, the mice in each group were fixed by intraabdominal injection of 10% chloric acid hydrate, then the blood was taken from the abdominal aortic upward, then perfused with a large amount of normal saline, and the fixed tissue immediately after 65˚C water bath for 5 min, the mixture of reagents was added, the mixture was mixed by shock, then centrifuged at 37˚C water bath for 60 min,

Physicochemical Properties of Curcumin
Curcumin has a molecular formula of C21H2006, a molecular weight of 368.37 g/mol, a melting point of 183˚C and crystalline. Curcumin was a rare pigment in the botanical world with diketones, consisting of two ortho-methylated phenols and a beta-dione. The structure of beta-dione has an enol-ketone interconversion structure, but the spectral structure proves that curcumin mainly exists as enol in solid state and solution [3]. The keto-ol interconversion isomerism is shown in Figure 1. Curcumin was extremely insoluble in water but soluble in organic solvents and more stable under acidic and neutral conditions. Under al-

Observation of Biological Behavior
After detecting the mechanical pain, heat pain, cold pain and spontaneous pain in nude mice, it was found that the mechanical and thermal pain of rats were significantly decreased after the Walker 256 cells were inoculated (p < 0.05), and the cold and spontaneous pain scores were significantly increased (p < 0.05) ( Figure 2).

Expression of c-fos in the Spinal Cord
The

Expression of RANK, NF-κB, TNF-α by RT-PCR
The expression of RANK, NF-κB and TNF-α in normal control group, solvent control group, curcumin low dose group, middle dose group and high dose

Expression of RANK, NF-κB and TNF-α by Western Blot
The expression of RANK, NF-κB and TNF-α protein in normal control group, solvent control group, curcumin low dose group, middle dose group and high dose group were detected by western blot. the expression of RANK, NF-κB and TNF-α protein in high dose group were significantly lower than that in solvent control group (p = 0.03, p = 0.02, p = 0.002), and the expression of TNF-α in middle dose group was significantly lower than that in solvent control group ( Figure 5).

Serum Levels of Cox2, IL-6, Leukotriene and PGE2
The levels of Cox 2, IL-6, leukotriene and PGE2 in serum of normal control group, solvent control group, curcumin low dose group, middle dose group and high dose group were detected. The study found that the levels of Cox2, il-6, leukotriene and PGE2 decreased continuously with the increase of curcumin dose, Among them, both the medium-dose group and the high-dose group decreased significantly (p < 0.05) ( Figure 6).

Discussion
In recent years, breast cancer had become one of the most malignant tumors with the highest morbidity and mortality in women, which was a serious threat to women's health and life. In view of the biological behavior characteristics of breast cancer, its occurrence and development reflected the multi-factorial role of bio-social-psychological model [3]. Local recurrence and distant metastasis often occur. Bone was the most common metastatic site of breast cancer. The incidence of breast cancer bone metastasis was 70% to 80% [5]. 70% of patients with advanced breast cancer had bone invasion [6], and more than 50% of advanced breast cancer had bone as the first metastasis site [7]. Therefore, chronic pain caused by bone metastasis was the most common associated symptom in patients with advanced breast cancer. Allodynia was produced by non-nociceptive stimulation; this non-nociceptive stimulation couldn't produce pain in normal physiological conditions. It was one of the important characteristics of pathological  Once bone metastases occurred in breast cancer, the circulation of bone destruction and woven bone began to initiate and cause significant pain. The receptor activator of NF-κB (RANK) was a member of the TNF receptor family.
The combination of RANK with receptor activator for nuclear κB ligand (RANKL) caused multiple kinase cascades and initiated and transmits signals of osteoclast differentiation [8]. The expression and involvement of cytokines such as downstream NF-κB and c-fos were needed in the differentiation of osteoclast precursor cells. The combination of RANK with RANKL was a key step in stimulating the activity of NF-κB [9]. As a transcription factor, nuclear factor-kappab (NF-κB) was the convergence point of many signaling pathways and played a key role in regulating inflammatory response [10]. to the intensity, time and nature of nociceptive stimulation [12].
Compared with the normal control group, the mechanical hyperalgesia threshold, the thermal hyperalgesia threshold decreased (p < 0.05), and the cold pain hyperalgesia and spontaneous hyperalgesia scores increased significantly (p < 0.05) after modeling in this study, which was basically consistent with the results reported in Medhurst SJ et al. [13]. In breast cancer, pain and hyperalgesia oc- decreased with the increase of curcumin dose (p < 0.05). Among them, the middle dose group and the high dose group decreased significantly (p < 0.05). From this, it can be seen that curcumin especially 25 mg/kg, 50 mg/kg curcumin solution can inhibit the activity of cell NF-κB and spinal cord c-fos by reducing the expression of osteoclast rank, thus reducing the expression of TNF-α and the release of inflammatory factor PGE2, while further reduced the formation of important inflammatory factor PGE2 in bone pain by reducing the production of leukotriene and inhibiting the activity of Cox2. It can be speculated that this may be a possible mechanism for curcumin to exert an analgesic effect on bone metastasis pain in breast cancer.

Conclusion
In this paper, by establishing the animal model of metastatic bone cancer and observing the biological behavioral changes of the experimental animals, we explored the mechanism of metastatic bone pain and sensitization in breast cancer from histological molecular level, and used the modern medical experimental method to select the active ingredient curcumin of traditional Chinese medicine for intervention study, which provided a new way to control the bone metastatic pain effectively. However, the pathological mechanisms of bone metastases were complex and the pathways involved in this study, whether curcumin has direct effect or indirect effect, still need to be supported by other methods. Whether curcumin can be applied directly to the clinical treatment of bone metastases, whether it can be used in combination with clinical treatment, whether it can effectively reduce the toxic effect of long-term drug treatment, how it can be applied safely and so on, these series of issues need our more in-depth study.

Data Availability
Data are available regarding this study.