SONG Hong-li, FENG Yong-li, WANG Yao-han, FENG Li

1. Oncology Department, Beijing Hospital of Integrated Traditional Chinese and Western Medicine, Beijing, 100038,China

2. Traditional Chinese Medicine Department ,Cancer Hospital Chinese Academy of Medical Sciences, Beijing, 100021,China

Keywords:Yishen Qutong ranula Cancer induced bone pain Traditional Chinese medicine

ABSTRACT Objective: To study the effects of Yishen Qutong granula on pain sensitization and bone destruction of rats with bone cancer pain. Methods: Walker256 cells were passaged in ascites and injected into the tibia of female Wistar rats to prepare the bone cancer pain model. On the 14th day after model establishment,60 rats were randomly divided into model group,sham-operated group, Yishen Qutong granula low, middle, high dose group and tramadol hydrochloride positive control group. After continuous administration for 14 days, the mechanical pain threshold, thermal threshold and weight-bearing difference of both hind limbs were observed. Results: Compared with the model group, Yishen Qutong groups increased the mechanical pain threshold, thermal pain threshold and reduced the weight difference of both hind limbs (P＜0.05). Compared with the positive drug group, there was no significant difference in increasing the mechanical pain threshold and thermal pain threshold of rats in the medium dose group of Yishen Qutong (P＞0.05), and Yishen Qutong granula significantly reduced the weight-bearing difference of hind limbs in all groups (P＜0.05). Conclusion:Yishen Qutong granula can relieve pain sensitization and alleviate bone damage in rats with bone cancer pain.

1. Introduction

Among cancer patients receiving anti-tumor therapy, the incidence of cancer pain is as high as 60%, and even as high as 90% in patients with advanced cancer[1]. Bone cancer pain is the most common form, and bone metastasis is considered to be the most common cause of bone cancer pain[2, 3]. Bone cancer pain runs through the diagnosis and treatment process of cancer patients, which is not only related to the disease itself, but also can be caused by cancerrelated treatment. The clinical effect of opioids in the treatment of bone cancer pain is not good, not only prone to nausea, constipation and other side effects, but also opioids can slow down or even stop bone remodeling, increase bone fragility, and seriously affect the quality of life of patients[4,5]. Different from other types of pain,bone cancer pain has the characteristics of acute, inflammatory and neuropathic pain, show the hyperalgesia and pain hypersensitivity,and its molecular mechanism is very complicated, involved in tumor cells, bone cells, inflammatory microenvironment and neurons[6],good bone cancer pain animal model of bone cancer pain is of great significance and the research of the mechanism[7, 8].

Until the late 20th century, all animal models of bone cancer pain relied on systemic injection of tumor cells. As tumors metastasized in liver, lung, brain and other important organs as well as multiple parts of bone, the health of animals was poor, and the number and location of metastases were uncertain, which had a great impact on pain measurement[8,9]. Local implanted bone cancer pain model is widely used at present. In 2006, MAO-Ying et al. [10] implanted rat Walker256 breast cancer cells into the tibial bone marrow cavity of rats, which is a relatively mature bone cancer pain model in China. In this study, a rat bone cancer pain model was established by referring to MAO-Ying et al. [10-12] to observe the effects of Yishen Qutong granule on pain sensitization and bone destruction in rats with bone cancer pain based on the principle of Fuzheng,detoxifying and removing stasis.

2. Materials and methods

2. 1 Animals and cells

Adult female Wistar rats (weight 200g±20 g) and young female Wistar rats (weight 70-100 g), SPF grade, purchased from Beijing SBF Experimental Animal Technology Co., Ltd., Certificate No.syxk (Beijing) 2016-003. Rats were fed at a temperature of 24±0.5℃ and fed and drank freely. The experiment was conducted after 1 week of adaptive feeding in the laboratory. Walker256 breast cancer cell line was purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd. and stored in liquid nitrogen.

2.2 Main experimental instruments and reagents

Tramadol hydrochloride injection (Grunenthal GmbH); Bme-404 electronic mechanical pain meter; Yls-21A hot and cold plate instrument; Hind limb disability measuring instrument, Italian 2Biological company (Note: yls-21A hot and cold plate instrument and hind limb disability measuring instrument are provided by jialiqun, director of China Japan Friendship Hospital)

2.3 Experimental methods

2.3.1 Animal modeling

2.3.1.1 Preparation of ascites tumor cells

Take walker256 breast cancer cells out of liquid nitrogen,place them in a 37 ℃ water bath for rapid resuscitation, put the resuscitated cells in a centrifuge at 1500 r/min, centrifuge for 5 min,discard the supernatant, add 0.5 mL sterile normal saline, the ratio of cells to normal saline is 1:1, and mix well for use. Take a female Wistar rat weighing 70-100 g, disinfect the abdomen with Iodophor,extract 0.3mL cell suspension, inject it into the abdominal cavity on the left side of the abdominal white line, soften the abdomen of the rat,and put it back into the cage for feeding. After about 3-5 days, the abdomen of the rats swelled. About 1-2 mL of yellow white turbid ascites was extracted with a syringe and injected into a centrifuge tube. It was centrifuged at 1500 r/min for 5 min. The supernatant was discarded and washed once with 0.5 mL of sterile normal saline.Then add a small amount of normal saline to mix the cells, count them under the microscope with a cell counting plate, and adjust the cell concentration to 2×107/mL, put the cell suspension on ice for use. The above operations are sterile conditions, and the extracted ascites should avoid bloody ascites as far as possible.

2.3.1.2 Modeling

Adult female Wistar rats weighing (200±20 g) were taken. After successful intraperitoneal injection of 0.3% pentobarbital sodium(1mL /100 g), the rats were fixed on the rat plate. The left hind limb was shaved and disinfected with Iodophor. Cut the medial skin of the upper tibia about 1 cm long, carefully separate the muscle with tweezers, and expose the tibial bone surface. Drill holes with a 5 mL(22G) syringe needle. The empty position is 0.5 cm below the knee joint on the medial surface of the tibia. Drill holes perpendicular to the bone surface first, and then drill holes at an angle of 45° with the bone surface. After the needle breaks through the bone surface, use a 10 μL micro syringe to extract 4 μL cell suspension and inject it into the bone marrow cavity. The micro injector needle stays for about 10 s, pull it out, and quickly seal the needle hole with sterile bone wax. Wipe the overflowing ascites tumor cells with an alcohol cotton ball, suture the incision, and wipe the wound with Iodophor, Apply a small amount of erythromycin ointment on 4 sides to prevent local infection. In sham operation group, rats were injected with the same amount of sterile normal saline into tibial bone marrow, and the rest were operated in the same model.

2.3.2 Grouping and administration

After the success of the model, the rats were randomly divided into sham operation group (A group), model group (B group), positive drug group (tramadol hydrochloride, C group), low-dose group (D group), medium dose group (E group), high-dose group (F group),with 10 rats in each group. Drug intervention began on the 14th day after the model was established. Traditional Chinese medicine group:Yishen Qutong granule was administered by gavage, and the gavage volume of rats was 1 mL/100 g. Once a day for 14 consecutive days;Dissolve the drug in clean drinking water and convert it into the required concentration according to the body surface area of rats:D group: the drug concentration is 0.175 g/mL; E group: the drug concentration was 0.35 g/mL; F group: the drug concentration was 0.7 g/mL; C group: tramadol hydrochloride injection, 10 mg/kg intraperitoneal injection, once a day for 14 d. Sham operation group and model group: gavage with equal volume of clean drinking water once a day for 14 d.

2.3.3 Behavioral observation of rat bone cancer pain model

After modeling, the rats' diet, drinking water, mental state, wound healing and hind limb movement were observed every day. The modeling day was counted as 0 d. The mechanical pain threshold,thermal pain threshold and hind limb weight difference were measured before modeling and on the 4th, 7th, 10th, 14th, 21st and 28th days after modeling. The measurement period was from 9:00 to 16:00.

2.3.3.1 Measurement method of mechanical pain threshold of rats

The rats were placed in a plexiglass cage with a volume of 9×12×18 cm. The bottom of the cage was a metal orifice plate with an aperture of about 0.6 cm. Before measurement, the rats were placed in the cage for 20-30 min. After they were quiet, the soles of both hind legs of the rats were stimulated vertically with a measuring probe (avoiding the foot pad). After the foot lifting measurement, the rats were stimulated five times within 1 min, with an interval of more than 10 s. The computer displayed the arithmetic mean value of the measured peak value was the mechanical pain threshold of rats.

2.3.3.2 Measurement method of thermal pain threshold

Set the temperature of the hot plate to 55±0.2 ℃, put the rats into the hot plate instrument, press the timing switch to start timing, and then press the time button when the rats lick their hind feet. The measurement time is the thermal pain threshold of rats. Before the experiment, rats with a pain threshold value of less than 30 s for 4 s were excluded. In order to prevent scalding, the upper limit of time was 30 s. The pain threshold value of those who did not lick their hind feet for more than 30 s was recorded as 30 s. Each rat shall be measured for 3 times to obtain the average value, and the interval between each measurement shall be at least 5 min。

2.3.3.3 Measurement method of weight difference of hind limbs of rats

Put the rats in the organic box of the hind limb disability measuring instrument, put the body in the middle of the organic box as far as possible, put the forelimbs on the slope of the organic box, and step on two independent sensors at the bottom of the box respectively.After the rats adapt to silence for 2 min, start the measurement. The measurement time is 5 s. The instrument displays the weight value of both hind limbs. Each rat is measured for 3 times continuously,take the arithmetic mean value, and calculate the difference. Weight difference of hind limb = weight bearing value of right hind limb -weight bearing value of left hind limb.

2.3.3.4 Imaging evaluation

The model group rats were treated with 0.3% pentobarbital on the 7th, 14th, 21st and 28th days after modeling. The rats were anesthetized by intraperitoneal injection of sodium toLate. The left hind limb was irradiated by X-ray in the radiology department of the Institute of orthopedics and traumatology, Chinese Academy of traditional Chinese medicine, and the bone condition was observed.

2.3.3.5 Pathological evaluation

Three rats in each group were randomly selected on the 28th day after modeling to take materials from the left hind limb.Before taking materials, they fasted for more than 8 h. After X-ray examination, blood was taken from the abdominal aorta, and then the left hind limb was quickly cut off, the skin and muscle were stripped, and the tibia was fixed with 4% polymethylaldehyde fixation solution. After decalcification with 10% EDTA for 4 weeks,they were embedded in paraffin and sectioned. He staining was used to observe the tumor destruction of bone tissue in each group under microscope.

2.4 Statistical processing

SPSS25.0 statistical software is used for analysis. The measurement data are expressed as mean ± standard deviation (±s). The normal distribution data are subject to t-test, and the non normal distribution data are subject to nonparametric test. P＜0.05 means that the difference is statistically significant.

3. Results

3.1 Model evaluation

Behavioral and imaging methods were used to evaluate whether the model was successful. The mechanical pain threshold and thermal pain threshold of the model rats decreased continuously after modeling, and reached a relatively stable threshold on the 14th day after modeling, indicating that the formation of mechanical hyperalgesia and thermal hyperalgesia is consistent with the characteristics of bone cancer pain (see the following for specific results).

Imaging changes: X-ray examination showed that there was no obvious bone abnormality in tibia in each group on day 7, and a small number of defect foci were found in tibial cortex on day 14,indicating that the modeling was successful. In the model group,bone tissue was further damaged on 21 and 28 days, and severe damage of tibial cortex, incomplete trabecular bone, and severe swelling of tibial tissue were observed on 28 days after modeling,indicating lytic bone destruction.

Fig 1 X-ray of tibia of rats with bone cancer pain in each group

3.2 Effect of Yishen Qutong granula on the mechanical pain threshold of bone cancer pain model rats

Mechanical nociceptive sensitization can be measured by measuring mechanical pain thresholds. As can be seen from Table 1, the pain threshold of rats in the model group decreased to below 9 and leveled off after 14 days of modeling. From 7 d to 28 d after modeling, the pain threshold of model group and Yishen Qutong granule group was lower than that of sham operation group(P＜0.05); At 21 d and 28 d of modeling, the pain threshold of rats in yishen Qutong Granule group and positive control group was higher than that in model group (P＜0.05), while there was no statistically significant difference between the medium-dose group and the positive control group (P＞0.05).

3.3 Effect of Yishen Qutong granula on the thermal pain threshold of bone cancer pain model rats: Effect of Yishen Qutong Granule on heat pain threshold in rats with bone cancer pain

Hypersensitivity to heat pain can be measured by measuring the reaction time of rats to heat stimulation. Table 2 shows that the threshold of heat pain in the modeling group continued to decrease 4 d after modeling, while the threshold of heat pain in the sham group rose to the preoperative level. On day 14 after modeling, the threshold of heat pain in the model group and the Yishen Qutong Granule group was significantly lower than that in the sham group(P＜0.05); At 21 d and 28 d after modeling, the heat pain threshold of Yishen Qutong granule medium-dose group and positive control group was significantly higher than that of model group (P＜0.05),and there was no significant difference between the medium-dose group and the positive control group (P＞0.05).

Tab 1 Effect of Yishen Qutong granula on mechanical pain threshold in rats with bone cancer(g,n=10, ±s )

Tab 1 Effect of Yishen Qutong granula on mechanical pain threshold in rats with bone cancer(g,n=10, ±s )

Note: Compared with A group: *P＜0.05; compared with B group: #P＜0.05; compared with C group: △P＜0.05.

Group 0 d 4 d 7 d 14 d 21 d 28 d A group 26.43±3.66 22.87±2.65 23.77±2.43 26.43±2.80 26.20±3.34 26.44±3.20 B group 25.49±2.74 21.33±3.31 18.04±3.26* 8.91±2.64* 8.24±3.27* 7.86±1.58*C group 24.75±2.56 21.03±4.23 17.51±2.38* 10.15±3.79* 20.83±3.85*# 21.87±4.45*#D group 25.64±4.10 20.94±5.43 18.55±3.49* 9.49±2.72* 16.13±3.55*#△ 12.98±3.02*#△E group 25.99±3.07 22.47±3.90 18.51±4.13* 10.19±3.15* 17.64±3.09*# 18.29±3.69*#F group 25.24±3.41 21.63±4.29 18.26±3.40* 10.61±2.15* 13.83±4.80*#△ 13.98±6.85*#△

Tab 2 Effect of Yishen Qutong granula on thermal pain threshold in rats with bone cancer(s,n=10, ±s)

Tab 2 Effect of Yishen Qutong granula on thermal pain threshold in rats with bone cancer(s,n=10, ±s)

Note: Compared with A group: *P＜0.05; compared with B group: #P＜0.05; compared with C group: △P＜0.05.

Group 0 d 4 d 7 d 14 d 21 d 28 d A group 8.23±2.43 7.14±2.13 7.18±1.52 6.94±1.46 7.21±1.43 7.52 ±1.18 B group 7.58±1.21 7.00±1.42 5.96±0.87 4.67±0.64* 4.54±0.63* 4.10±0.53*C group 8.27±1.04 6.97±1.76 6.58±1.55 5.02±0.96* 5.35±1.04*# 6.20±1.41*#D group 7.91±1.30 7.85±2.22 6.33±1.17 4.81±1.18* 5.09±0.75* 4.57±0.98*△E group 7.43±1.25 7.50±1.63 7.13±1.83 5.07±1.09* 5.42±0.69*# 5.74±0.89*#F group 7.34±1.52 6.51±1.17 6.37±1.01 5.01±0.86* 4.56±0.90* 4.61±0.68*△

3.4 Effect of Yishen

Qutong granula on the weight difference of both hind limbs of bone cancer pain model rats: From the 7th day after modeling, the weight difference of hind limbs in the modeling group was higher than that in the sham operation group (P＜0.05); On the 21st and 28th days after modeling, the weight difference of hind limbs in Yishen Qutong formula group and positive drug group was smaller than that in model group (P＜0.05); On the 21st day after modeling, the weightbearing difference of the middle dose and high dose groups was significantly lower than that of the positive drug group (P＜0.05). On the 28th day after modeling, the weight-bearing difference of the hind limbs of the rats in the low dose, middle dose and high dose groups was lower than that of the positive drug group (P＜0.05).

Tab 3 Effect of Yishen Qutong granula on weight-bearing difference of both hind limbs in rats with bone cancer(g,n=10, ±s )

Tab 3 Effect of Yishen Qutong granula on weight-bearing difference of both hind limbs in rats with bone cancer(g,n=10, ±s )

Note: Compared with A group: *P＜0.05; compared with B group: #P＜0.05; compared with C group: △P＜0.05.

Group 0 d 4 d 7 d 14 d 21 d 28 d A group 0.10±1.03 5.58±2.77 2.28±1.35 1.09±1.79 0.63±1.61 -0.39±2.00 B group 0.35±1.64 5.92±2.53 9.11±2.40* 15.48±5.00* 23.02±4.72* 36.21±10.79*C group 0.81±3.18 6.42±2.41 8.08±1.77* 14.08±3.78* 16.66±2.79*# 26.58±8.74*#D group -0.31±1.83 6.59±2.83 8.30±1.78* 14.93±3.71* 13.11±4.83*# 19.38±3.58*#△E group -0.36±1.75 6.29±2.17 8.62±1.79* 13.39±4.22* 9.03±3.06*#△ 8.54±4.96*#△F group 1.01±4.36 6.74±2.43 7.59±2.42* 14.30±3.44* 10.81±3.49*#△ 15.68±3.77*#△

3.5 Pathological evaluation

On the 28th day after modeling, we stained the tibia of rats in each group with he to observe the effect of Yishen Qutong granula on bone tissue of rats with bone cancer pain. It can be seen from the HE staining pictures of tibia of rats in each group in Figure 2 that the bone cortex in the sham operation group is complete and the bone trabeculae are closely arranged. However, the trabeculae of tibia in each group were damaged, and tumor cells were found in the medullary cavity. In the model group and the positive drug group, the bone cortex was severely damaged, the bone trabecula almost disappeared, and a large number of tumor cells infiltrated in the medullary cavity and invaded outside the bone. The bone destruction in each group was between the sham operation group and the positive drug group, and the bone destruction in the middle dose group was the least.

Fig 2 Pathological pictures of tibia of rats in each group with HE staining

4. Discussion

Bone cancer pain has both inflammatory pain and neuropathic pain pathological mechanisms, and its clinical manifestations are persistent background pain, burst pain and pain sensitization[13].Such pain is often unable to be alleviated and may develop into chronic pain with poor clinical control, severely damaging patients'functional status and shortening their survival[14]. The advanced treatment of bone cancer pain still is given priority to with opioid therapy, but opioids bone cancer pain patients, with large dose of common nausea and lethargy, delirium, constipation or intestinal obstruction serious adverse reactions, such as lower the quality of survival, and opioids does not stop tumor bone destruction,sustainable alternatives to drugs and in the clinical treatments are urgently needed. Traditional Chinese medicine has flexible and diverse treatment methods for bone cancer pain, including internal administration of Traditional Chinese medicine, acupuncture,drug application, acupoint injection, ear point, etc., which are safe and effective without obvious side effects. The combination of Traditional Chinese and Western medicine can reduce toxicity and increase efficiency, which is highly recognized by patients and has obvious clinical advantages[15].

The pain of bone cancer can be classified as "arthralgia", "bone gangrene" and "stone gangrene" in Chinese medicine. At present,most doctors believe that qi stagnation, blood stasis, cold coagulation,phlegm knot and heat toxin are the main causes, and "pain caused by ungeneral conditions" and "pain caused by unglorious conditions"are the main pathogenesis[16,17]. Group according to the etiology and pathogenesis, therefore, in order to "centralizer, detoxification,blood" for the treatment of "kidney expelling pain granule" in the early several clinical trials have confirmed that the treatment of advanced cancer patients with moderately severe pain, the pain of bone metastases, renal expelling particles can not only relieve pain pain in patients with continuous pain symptoms and the outbreak of pain, improve the patient's quality of life, It can also effectively reduce the dosage of opioids and significantly reduce their adverse reactions[18-20]. The yishen Qutong granules used in this experiment are processed by one-step plasmid method through water extraction process by clinical Pharmacy Institute of Beijing Health Bureau. The granules not only retain the active ingredients of drugs, but also are convenient for patients to carry and take.

In this study, ascites tumor cells of Walk256 breast cancer cells were obtained from ascites of young female mice and then implanted into the tibia of adult female mice. The modeling rate of this method was as high as 85%. The success of modeling was evaluated comprehensively by imaging and behavior to avoid the error caused by modeling failure. In this experiment, yellow-white turbid ascites was used for ascites with high concentration of tumor cells. In the later stage of the experiment, the rats were generally in a better state, and the mortality rate was significantly lower than that of rats with ascites tumor cells extracted from bloody ascites.Pain sensitization in rats with bone cancer pain was evaluated by measuring mechanical pain threshold, thermal pain threshold and hindlimb weight bearing difference. We observed that four days after modeling, mechanical pain threshold and thermal pain threshold were decreased in all rats, and weight difference of hind limbs increased, which was considered as neuropathic pain caused by surgical trauma. But after 4 d, along with the bone wound healing,poor control in the pain threshold, weight index gradually returned to normal, and module of rats, over time cancer cells grow gradually,and the erosion of bone tissue, the tumor microenvironment inflammatory cell activation, release a large number of inflammatory factor, causing pain sensitization[21], and therefore visible pain rat mechanical pain threshold, thermal pain threshold gradually reduce,The weight difference gradually increases. In this study, tramadol hydrochloride was used as a positive control drug, and tramadol is a second-step analgesic commonly used in clinical practice. Studies have shown that tramadol hydrochloride can significantly relieve hyperalgesia in neuropathic pain rats[22]. Through experiments, we observed that mechanical pain threshold of bone cancer pain rats decreased significantly from 7 d after modeling, and weight bearing difference of both hind limbs increased significantly, while heat pain threshold decreased significantly from 14 d, and heat hyperalgesia occurred later than mechanical hyperalgesia. In a number of neuropathic and inflammatory pain animal experiments, Mechanical hyperalgesia and heat hyperalgesia in rats can appear within 1-3 d after modeling, or even within 4 h at the earliest[23], which also indicates that cancer pain is different from inflammatory pain and neuropathic pain. After 1 week of drug intervention, both yishen Qutong granule group and positive drug group could significantly improve mechanical hyperalgesia and hyperalgesia in rats with bone cancer pain. Drug intervention after 2 weeks, pain kidney expelling particles dose group and positive medicine group rats mechanical pain threshold, thermal pain threshold is 1 week before, and dosage group and positive medicine group effect, low, high dose group and improve thermal hyperalgesia, but pain kidney expelling particles is low, medium and high dose group in the aspect of improving bone cancer pain pain hypersensitivity in rats effect is better than that of tramadol hydrochloride. This may be related to the fact that the tumor progression and tramadol hydrochloride could not control the damage of the tumor to the bone tissue, and HE staining showed that the bone damage in the tramadol hydrochloride group was more serious than that in the Yishen Qutong granule group.

The above shows that although tramadol hydrochloride has a good analgesic effect, it can not alleviate the progress of osteolytic bone destruction in bone cancer pain model rats, while the medium dose group of Yishen Qutong granula can not only effectively alleviate mechanical and thermal hyperalgesia in bone cancer pain model rats,but also delay tumor progression and improve the quality of life of bone cancer pain model rats.

Author’s contribution:

Song Hong-li, Feng Yong-li and Wang Yao-han are responsible for the experimental operation of the subject, Song Hong-li is responsible for the writing and data analysis of the article, and Professor Feng Li is responsible for the subject and article guidance.All authors declare no conflict of interest.