| Cancer induced Bone pain(CIBP)is the most common form of pain in patients with advanced cancer pain.It is generally believed that the occurrence of bone cancer pain may be related to the pain-generating substances produced by tumor cells and inflammatory cells,the sustained activation and activation of osteoclasts,and the nerve compression and injury caused by tumor expansion.While,the activation of p38MAPK signaling pathway in spinal cord is becoming more and more important in bone cancer.At present,the main clinical treatment is opioids,adjuvant radiotherapy,chemotherapy,hyperthermia and other combined treatment.Due to the side effects of western medical treatment,many patients with cancer pain cannot be effectively controlled in clinical practice,which seriously affects their quality of life.Traditional Chinese medicine(TCM)has abundant means to treat cancer pain,with few side effects,safe and effective,and can alleviate the side effects brought by western medicine treatment.It has certain advantages in the treatment of cancer pain.Yishen Gukang decoction is a patent prescription of Professor Feng Li,which has proven its effectiveness and safety in treating cancer pain in several clinical studies.However,the molecular mechanism of its treatment for bone cancer pain is still unclear.The previous study has shown that Yishen Gukang Decoction can inhibit the reduction of metastasis and promote the osteogenesis in vitro.Based on the previous research,this study developed Yishen Gukang into a Chinese patent medicine granule preparation,which is convenient for patients to take and carry,and has better quality control.At the same time,by observing the effect of Yishen Gukang Decoction on the pain behavior of rats with bone cancer pain,revealing its molecular mechanism of inhibiting bone cancer pain,it can provide a theoretical basis for the development of new therapeutic drugs for cancer pain.Objective1.Carry out the research on the process of Yishengu Kangfang Granule Preparation,and obtain a more scientific and stable dosage form that is convenient for patients to take,laying a foundation for the future research of clinical preparations.2.Explore the molecular mechanism of Yishen Gukang decoction in inhibiting CIBP,which provided a new method for anti-cancer pain of traditional Chinese medicine.MethodsPart one:According to the analysis of active ingredients of Yishen Gukang decoction,to determine its extraction process and moulding process,and to study the quality control by TLC identification.Part two:Study on the Mechanism of Yishen Gukang Decoction in Inhibiting Bone Cancer Pain:1.Establish a rat model of CIBP and observe the effect of Yishen Gukang decoction on its behavior.Female Wistar rats were randomly divided into sham operation group and model group,Ascites passage of Walker 256 breast cancer cells was used.Ascites tumor cells were implanted into the bone marrow cavity of the tibia of the left hind limb of rats,and the sham operation group was injected with physiological saline.The rats were randomly divided into sham-operated group,model group,Yishen Gukang granule low dose group,middle dose group,high dose group and tramadol hydrochloride positive control group on the 14th day after model establishment.There were 10 rats in each group for 14 days after drug intervention.The body weight,mechanical pain threshold,thermal pain threshold and weight difference of both hind limbs were observed before and on the 4th,7th,10th,14th,21st and 28th day after modeling.The X-ray changes of tibia in the model group were observed on the 7th,14th,21st and 28th day.On the 28th day,the tibia tissues of rats in each group were stained with HE to observe the effects of Yishen Gukang on the bone tissues of rats.2.Observing the effect of Yishen Gukang decoction on key molecules of p38MAPK signaling pathway and related inflammatory molecules in spinal dorsal horn of rats with CIBP.The relatived mRNA levels of p38MAPK,TNF-?,IL-1?and COX-2 in spinal dorsal horn of rats were determined by RT-PCR;The expression of p38MAPK and p-p38MAPK protein in the dorsal horn of spinal cord in rats was determined by the Western blot;The expression of p38MAPK in the spinal cord of rats in each group was observed by immunohistochemistry;The levels of TNF-a,IL-1?p,cox-2,IL-6 and PGE2 in peripheral blood of rats in each group were determined by ELISA.3.Observing the effect of Yishen Gukang decoction on OPG/RANK/RANKL signaling pathway in rats of CIBP.The expressions of OPG,RANK and RANKL in the tibia of rats in each group were detected by immunohistochemistry,and the MOD values of the positive proteins were calculated.Results Part one:According to the characteristics of the active ingredients of Yishen Gukang decoction,The water absorption rate of the medicinal materials was 200%,and orthogonal test,comparative experiment,enlargement and validation test were carried out.The verification test confirmed that the extraction process was performed by boiling water for 3 times,and the three times of water addition were 10 times,8 times,and 8 times,respectively,and boiling for 1.5 hours each time.After granulation,a mixture of the base dextrin and mannitol(8:1)was determined,and the ratio of the base to the dry paste was 1:2.It is obtained by a one-step granulation method.Conduct thin-layer identification analysis of prescription drugs and establish quality control standards.The microbial degree,heavy metal,arsenic and extract of the obtained granules were all in compliance with the relevant provisions of the 2015 edition of the Pharmacopoeia.Part two:Study on the Mechanism of Yishen Gukang Decoction in Inhibiting Bone Cancer Pain:1.Behavioral aspects:The weight,PWL and TWL of model group rats were lower than those of sham-operated group(p<0.05),the difference of weight-bearing of both hind limbs was greater than that of sham-operated group(p<0.05).The weight of rats in the intervention group was higher than that in the model group(p<0.05);PWL and TWL were increased and the difference of weight-bearing of both hind limbs was reduced in the treatment groups,what's more,Yishen Gukang medium dose group and tramadol hydrochloride group could significantly increase PWL and TWL(p<0.05),and there was no significant difference between the two groups(p>0.05),Yishen Gukang decoction significantly reduced the weight-bearing difference of hind limbs in all groups(p<0.05),and the effect was better than that of the positive group(p<0.05).X-ray examination of the tibia of the operated limbs in the model group showed that the bone destruction was progressively aggravated with time.HE staining of tibia showed that the bone tissue of model group and positive drug group was damaged seriously.Yishen Gukang decoction group was lighter than model group and positive drug group,and the medium dose group had the lightest bone destruction.3.From results of RT-PCR,we can know that Yishen Gukang middle and high dose group could significantly reduce the levels of p38MAPK,TNF-alpha and COX-2 in spinal cord of rats(p<0.05),while it could significantly reduce the levels of IL-lbeta in each groups(p<0.05).Western blot results showed that compared with model group,Yishen Gukang decoction could significantly reduce the expression of p38MAPK protein in spinal cord of rats(p<0.05),and each group of Yishen Gukang decoction could significantly reduce the content of p-p38MAPK protein(p<0.05).The results of immunohistochemistry showed that the expression of p38MAPK in spinal dorsal horn of rats was significantly down-regulated in the low,middle and high dose groups of Yishen Gukang decoction compared with the model group(p<0.05),and the medium dose group had the best effect.ELISA results showed that compared with the model group,each group of Yishen Gukang decoction could significantly reduce the levels of TNF-a,IL-6 and PGE2 in serum of bone cancer pain model rats(p<0.05),while the medium dose group and high dose group could significantly reduce the levels of IL-1? and COX-2(p<0.05).4.Immunohistochemical results showed that compared with the model group,each group of Yishen Gukang decoction could significantly increase the expression of OPG protein and down-regulate the expression of RANKL,while the medium and high dose groups could significantly down-regulate the expression of RANK.Conclusion1.The process of Yishen Gukang Granules is clear,and the quality control conforms to the relevant provisions of Pharmacopoeia 2015 edition.2.Yishen Gukang decoction can effectively relieve symptoms of cancer induced bone pain and reduce bone destruction,what's more,the medium dose group having the best effect.3.Yishen Gukang Decoction may inhibit bone cancer pain by regulating p38MAPK signaling pathway and inhibiting related inflammatory molecules.4.Yishen Gukang decoction may inhibit osteoclast activation and reduce bone destruction by regulating OPG/RANK/RANKL signaling pathway,thereby reducing bone cancer pain. |
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