| ObjectiveCancer introduced Bone pain is one of the common complications in patients with advanced cancer,which severely reduces the patient’s ability of daily living.Among them,the activation of nerve growth factor(NGF)can be widely seen in the model of cancerous bone metastasis,and it can often exert an effect by activating the PI3K-AKT pathway.Scorpion is a traditional Chinese medicine monomer commonly used for analgesia,which can significantly relieve pain.Shentong Zhuyu Decoction is one of the classic prescriptions for promoting Qi and removing blood stasis,and is often used in the treatment of arthralgia.Clinical application of Quanxie or Shentong Zhuyu Decoction can relieve the pain symptoms of patients with bone metastasis pain to a certain extent.In this study,we searched the database and the traditional Chinese medicine inheritance auxiliary system to find the best compatibility of the whole scorpion for the treatment of cancerous bone metastasis;A model of cancer introduced bone pain was constructed by direct injection of Walker256 breast cancer cells into the tibia,to observe and analysis the dose-dependent analgesic effect of Xingqi Huayu Recipe containing scorpion(Xiewu Shentong Zhuyu Decoction Decoction)on rat,the effect on the expression of NGF and its high affinity receptor protein tyrosine kinase A(Trk A),PI3K-AKT pathway related proteins PI3 K,p-AKT,AKT and astrocyte marker protein GFAP.To explore whether the Xingqi Huayu Recipe containing scorpion can play an analgesic effect by reducing the production of NGF,inhibiting the activation of the PI3K-AKT pathway and the activation of astrocytes.MethodBy searching the databases of Wan Fang,CNKI,Cqvip that contain whole scorpion oral prescriptions for the treatment of cancer pain,build a database based on the traditional Chinese medicine inheritance auxiliary system(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,V2.5).Use the software data analysis module and data mining methods to analyze the frequency of single drugs,drug combinations,and find its optimal combination.Twenty-four healthy,SPF and female SD rats,weighing 220±20g,were randomly divided into four groups(n=6/group),including model group(CIBP+Vehicle),model+Decoction Low,Middle and high-dose groups(CIBP+L,M,H);a rat model of breast cancer tibial metastasis pain was established.After the pain occurred,normal saline and different concentrations of Decoction(Concentration of 1.6 g/m L,3.2 g/m L,6.4 g/m L,2m L/d),the gavage volume is 1m L/100g/d,then measure the 50% paw withdrawal threshold.27 healthy,SPF and female SD rats,weighing 220±20g,were randomly divided into three groups(n=9/group),including sham operation group(Sham+ Vehicle),model group(CIBP+Vehicle),and model administration Group(CIBP+XWST),a rat model of breast cancer bone metastasis pain was established.The administration group was given Xiewu Shentong Zhuyu Decoction,2 m L/d,for 7 days after the model was established.On 3,5,7,9,11,and 13 days after the model,50% paw withdrawal threshold and hindlimb application score were used to evaluate the behavioral performance of pain in rats;on the13 th day,X-rays of the tibia were taken to determine the degree of bone destruction;Anatomy and observe the appearance of the tibia on the sick side of the rat;Extract spinal cord lumbar enlargement tissue.The protein expression of NGF,Trk A,PI3 K,p-AKT,GFAP was detected by Western blot;the immunoreactivity of GFAP was detected by immunofluorescence to reflect its activation;the expression of NGF in the serum and spinal cord tissue of each group of rats was detected by Elisa.Results1.Cluster analysis by TCM inheritance assistant system showed that scorpion and centipede were commonly used analgesic drug pairs for the treatment of cancerous bone metastasis pain.2.The analgesic effect of Xiewu Shentong Zhuyu Decoction produced by intragastric administration at low doses.With the increase of dose,the analgesic effect of middle and high doses increases,but the mortality rate of high doses is higher.Therefore,the medium-dose Xiewu Shentong Zhuyu Decoction was finally selected;3.The results of X-ray photography and anatomical observation showed that compared with the healthy side and the sham operation group,the tibia on the affected side had obvious bone destruction.The quantitative score of bone destruction shows that Xiewu Shentong Zhuyu Decoction can reduce the degree of bone destruction;4.After the first administration of Xiewu Shentong Zhuyu Decoction,it can be seen that compared with the CIBP+Vehicle group,the 50% paw withdrawal threshold of rats in the CIBP+XWST group was significantly increased,and the analgesic effect was best two hours after the administration.After continuous administration for 7 days,compared with the Sham+Vehicle group,50% paw withdrawal threshold of the rats in the CIBP+Vehicle group and the CIBP+XWST group was significantly lower,and the hindlimb application score was significantly increased;compared with the CIBP+Vehicle group,the CIBP+XWST group was larger The 50% paw withdrawal threshold was significantly increased,and the score of hindlimb application was significantly reduced.Compared with the CIBP+Vehicle group,the weight of rats in the CIBP+Vehicle group and the CIBP+XWST group was significantly reduced,while the weight of the CIBP+Vehicle group and the CIBP+XWST group had no significant difference.5.Western blot was used to detect the protein expressions of NGF,Trk A,PI3 K,pAKT and GFAP in spinal lumbar enlargement segment of CIBP model rats.The result is the grayscale ratio of target protein expression to internal reference protein GAPDH.Results Compared with SHAM + Vehicle group,The expression of NGF,TRKA,PI3 K,p-AKT,and GFAP protein were up-regulated,and the expression of these proteins could be down-regulated after the administration of the decoction.and the results were statistically significant.The content of NGF in the spinal cord was measured by ELISA.Compared with the Sham+ Vehicle group,the content of NGF in CIBP model rats was increased,while Xiewushentongzhuyu decoction could reduce the level of NGF.The immunofluorescence staining method was used to detect the immunoreactivity of spinal astrocyte-labeled protein GFAP.Results Compared with Sham+Vehicle group,the immune response signal of spinal cord GFAP in CIBP model rats was enhanced,and the degree of enhancement was decreased by Xiewu Shentong Zhuyu Tang.ConclusionXiewu Shentong Zhuyu Decoction can significantly improve the pain behavior of rats with CIBP,including inhibiting 50% paw withdrawal threshold in rats and improving the motor function of the affected hind limbs.Its mechanism is related to down-regulation of NGF-Trk A expression in the spinal cord,inhibition of PI3K-AKT signaling pathway and activation of astrocytes. |
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