Clinical Research And The Effect Of Signal Transduction Mechanism On Treatment Of Metastasis Tumor By Yishengukangfang

Osseous metastasis tumor is a common complication of late malignant tumors. The osseous metastasis tumor can affect the bones all through the body, and most frequently in the thoracolumbar spine. Most osseous metastasis tumors are multiple lesions but just a few are single lesions. The cardinal symptom of osseous metastasis tumor is pain; if the tumor or the pathological fracture affects the spinal nerve, cauda equine or spine, the specific radiating pain, zonesthesia and paraplegia will occur. If the pain thus incurred is not treated and controlled, such symptom can develop into major complications such as the pathological fracture, functional impairment, hypercalcinemia, bone marrow function repression and the paraplegia arising from the spinal nerve compression except that the pain may affect sleep and diet. The objective to treat the osseous metastasis tumor is to reduce the symptom, and prevent relevant complications such as bone fracture, raise the quality of life and increase the lifetime. The treatment of osseous metastasis tumor is one of the difficult and hot spots in clinic treatment. Since all the treatment by western medicine may have some adverse effects and addiction, disadvantageous to the rational use of drugs, it is an important orientation to seek for the medicines with less adverse effect and without addiction for the treatment of osseous metastasis tumor and osseous metastasis pain. Chinese medical science and traditional Chinese drugs can play an important role in the treatment of the pain of osseous metastasis tumor with insignificant toxicity and side effects and better effectiveness in treatment. The strictly designed clinical randomized control study and the in-depth research on the mechanism of drug action are very important. Based on the clinical differential treatment at early stage, the clinical research on the approach of Bushenhuayu to treat the pain caused by osseous metastasis tumor was developed, and the research on the signal transmission mechanism of the osteolytic osseous metastasis tumor and the molecular biological mechanism of abirritation were extensively conducted in this study. This study consists of five parts:(1) the clinical research on the traditional Chinese drug of Bushenhuayu to treat the pain caused by osseous metastasis tumor;(2) The establishment of an osteolytic osseous metastasis model by Injection of the cells of adenocarcinoma of lung into the left cardiac ventricle in nude mice to evaluate the effect of the Prescription for Yishengukang on inhibiting the occurrence of osteolytic osseous metastasis;(3) the regulating effect of the Prescription for Yishengukang on FAP signal transduction mechanism of osteolytic osseous metastasis tumor;(4) the regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model of osseous metastasis tumor pain;(5) Probe into the mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung from the prospect of OPG/RANKL/RANK system.Part Ⅰ:the clinical research on the traditional Chinese drug of Bushenhuayu to treat the pain caused by osseous metastasis tumor[Objective] Taking the patients suffering from severe pain caused by osseous metastasis tumor as the subjects investigated, evaluation on the definite therapeutic effect of the traditional Chinese drug of Bushenhuayu at the earlier stage was conducted.[Methods] The patients defined by the criteria for diagnosis and suffering from severe pain caused by osseous metastasis tumor were randomly divided into the treatment group and the control group and totally51observed patients were included and26cases in Chinese drug group and25cases in control group according to the design code for clinical trial, adopting the principle of parallel, randomized and controlled clinical study, and based on differentiation standard of TCM. The patients in Chinese drug group who took orally the Prescription for Yishengukang, a traditional Chinese drug of Bushenhuayu, in addition to Oxycodone&Acetaminophen Tablets (Tylox) were compared with those in the group with pure oral administration Oxycodone&Acetaminophen Tablets (Tylox). The key indicators in respect to the scores of pain influence, physical strength status (Karnofsky performance score), pain score (NRS performance score) and the dosage of Oxycodone&Acetaminophen Tablets (Tylox) were compared between the groups before and after treatment; and the therapeutic action of differential treatment by Bushenhuayu on the pain caused by osseous metastasis tumor were comprehensively analyzed, scientifically evaluated and observed.[Results]1. A baseline analysis was conducted on the ages of all the cases in the groups as well as the proportion of men to women in the groups to find out a proportion of46.15%and53.85%between male patients and female patients in Chinese drug group, and a proportion of48%and52%between male patients and female patients in control group and χ2test results indicated no significant difference statistically in the proportion of male-female in the two groups (P>0.05). The ages between the Chinese drug group and the control group were respectively65.15±11.43and63.28±13.48, and the t test results indicated no significant difference statistically in the ages of the two groups (P>0.05)2. Through the comparison of the effectiveness ratio (case number and percentage) of scores of pain influence among the cases in the two groups, the results indicated the effectiveness ratio of scores of pain influence in Chinese drug group and the control group was respectively65.38%and28%and the comparison of the effectiveness ratio of scores of pain influence among the cases in the two groups had significant difference after treatment (P<0.05)3. After14days of treatment, there were9cases of significant effect,13case of effectiveness and4case of inefficacy in Chinese drug group in terms of KRS score with a effective rate of84.61%while4cases of significant effect,9case of effectiveness and12case of inefficacy in control group with a effective rate of52.0%. through rank-sum test and after14days of treatment, the effective rate of life quality of the patients in treatment group was higher than that in control group with a statistic difference (P<0.05). The KPS score of the Chinese drug group after treatment was66.92±12.89,with a extremely significant difference in statistic compared to52.31±13.66of the Chinese drug group before treatment (P<0.01). the KPS score of the control group after treatment was59.20±13.52, with no significant difference in statistic compared to56.40±11.50of the control group before treatment (P>0.05) 4. In terms of comparison of NRS score between two groups before treatment, the Chinese drug group was5.08±1.62while that of the control group was5.12±1.59with no significant difference in statistic (P>0.05), indicating that the two groups were comparable; NRS score between two groups after treatment was slightly decreased, the Chinese drug group was2.73±1.51with a significant difference (P<0.05) compared with5.08±1.62before treatment, while the control group was4.16±2.13with no significant difference compared with5.12±1.59before treatment (P>0.05); the comparison between two groups after treatment had a significant difference (P<0.05).5. Single day dosage of Tylox on the first day of two groups before treatment:the Chinese drug group and the control group respectively were6.04±1.82and6.56±2.31with no significant difference (P>0.05), indicating that the two groups were comparable; the single dosage of Tylox of two groups after treatment was slightly decreased, the single dosage of Tylox of the Chinese drug group was4.35±0.63with a significant difference in statistic(P<0.05) compared with that before treatment, while the single dosage of Tylox of the control group was5.60±2.06with no significant difference compared with that before treatment (P>0.05); the comparison between two groups after treatment had a significant difference (P<0.05).6. Safety evaluation:in this experiment, all the patients were given the inspection of liver function, renal function, blood routine examination and ECG before and after treatment and the statistic results showed no significant variance, indicating that the traditional Chinese drug of Bushenhuayu was safe in use without remarkable adverse effect or toxic effect on the human systems of heart, liver, kidney and hematopoiesis. During the experiment, no adverse reaction was found in the treatment group, indicating the traditional Chinese drug of Bushenhuayu was safe in use without remarkable adverse effect or toxic effect.[Conclusion]1. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can increase the effective rate of pain influence score;2. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can increase the survival quality of the patients;3. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can decrease the NRS score and reduce pain level;4. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu can decrease the single day dosage of Lylox;5. Treatment of the pain caused by osseous metastasis tumor by method of Bushenhuayu is safe and has no remarkable adverse reaction or remarkable adverse effect or toxic effect.Part II:The establishment of an osteolytic osseous metastasis model by injection of the cells of adenocarcinoma of lung into the left cardiac ventricle in nude mice to evaluate the effect of the Prescription for Yishengukang on inhibiting the occurrence of osteolytic osseous metastasis[Objective] An osteolytic osseous metastasis model was established in this experiment by the patented adenocarcinoma of lung cell line featured by osteolytic osseous metastasis through the method of injection into the left cardiac ventricle in nude mice, based on which the intervention effect of traditional Chinese drug was observed.[Method] The patented CPA YANG IBM cell line was used in this research to establish an osteolytic osseous metastasis model in nude mice through the method of injection into the left cardiac ventricle under the guidance of Prof. Yang Shunfang, and the occurrence of100%osseous metastasis tumor in the model groups was eventually validated by Micro SPECT/CT tomography and pathological section, which confirmed that models was successfully made. The nude mice were randomly divided into six groups with six in each group and pharmacological intervention was given on the third day when model was made. The group with lower dosage of Prescription for Yishengukang:suspension of Yishengukang Extract3g/kg, once per day x five weeks, by intragastric administration; The group with moderate dosage of Prescription for Yishengukang:suspension of Yishengukang Extract12g/kg, once per day x five weeks, by intragastric administration; The group with higher dosage of Prescription for Yishengukang:suspension of Yishengukang Extract24g/kg, once per day x five weeks, by intragastric administration; the group with Pamidronate Disodium:Pamidronate Disodium4.5mg/kg, twice per week x five weeks, by subcutaneous injection administration; group with lower dosage of Prescription for Yishengukang plus Pamidronate Disodium (Chinese drug plus western drug group): suspension of Yishengukang Extract3g/kg, once per day x five weeks, by intragastric administration, and simultaneously Pamidronate Disodium4.5mg/kg, twice per week x five weeks, by subcutaneous injection administration; model control group:NS0.5ml, twice per week x five weeks, by subcutaneous injection administration, and simultaneously NS1ml, once per day x five weeks, by subcutaneous injection administration. After5weeks, the osseous metastasis rate and the location number of adenocarcinoma of lung in the nude mice in each group, the tumor cell number/total number of cells (%) on pathological tissue section in the nude mice in each group; and the body weight of the nude mice in each group was weighed in each week to observe the decrease of body weight in each group.[Results]1. The osseous metastasis rate of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group was zero and the location number of osseous metastasis was zero; while the osseous metastasis rate of the model group was100%and the location number of osseous metastasis was12; the osseous metastasis rate of the group with moderate and higher dosage of Prescription for Yishengukang was respectively50%and33%and the location number of osseous metastasis was respectively4and2; the osseous metastasis rate of the group with Pamidronate Disodium was17%and the location number of osseous metastasis was1.2. It was observed by histopathology that the osseous tissue in the nude mice in the model group was severely destroyed by tumor tissue and the marrow cavity was almost filled out by tumor cells and several OLCs (Multinuclear Giant Cells) were observed, the bone trabecula was severely destroyed, the solid tumor cell clusters was seen in some areas and the structure of bone trabecula was disappeared; at the edge of marrow cavity, most of the tumor cells were in active status and the tumor grew out resulting in severe destroy of bone trabecula and extensive invasion, attenuation and defection of cortical bone; as for Prescription for Yishengukang, though a certain proportion of osseous metastasis occurred in the group with higher dosage, the extent for the bone mass to be destroyed by tumor cells was minor; a certain tumor cells were seen in marrow cavity but the destroy to bone trabecula and the damage to cortical bone were obviously minor compared with that of the model group and the cortical bone was relatively intact. The ratio between tumor cells and total cells of the model group was obviously higher than that of the group with Prescription for Yishengukang, namely, the quantity of tumor cell inside the osseous metastasis focus of the model group was obviously higher than that of the group with Prescription for Yishengukang (P<0.05).3. The body weight decrease rate of the nude mice model group was obviously higher than that of the group with lower dosage of Prescription for Yishengukang (P<0.05).The above results indicated with respect to a whole and at the cellular level that Prescription for Yishengukang had a certain inhibition on the osseous metastasis of adenocarcinoma of lung in nude mice.[Conclusion]1. An osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice as successfully established;2. Prescription for Yishengukang could decrease the osseous metastasis rate and the number of osseous metastasis location of adenocarcinoma of lung in nude mice;3. Prescription for Yishengukang could decrease the ratio between the tumor cells and the total cells inside the osseous metastasis focus of adenocarcinoma of lung in nude mice and could mitigate the destroy extent of the osseous metastasis focus to the bone mass;4. Prescription for Yishengukang could decrease e concentration of alkaline phosphatase in the serum of nude mice with osseous metastasis of adenocarcinoma of lung;5. Prescription for Yishengukang could improve the whole body condition of the nude mice with osseous metastasis of adenocarcinoma of lung and could inhibit their body weight decrease. Part III:The regulating effect of the Prescription for Yishengukang on FAP signal transduction mechanism of osteolytic osseous metastasis tumor[Objective] Further probe into the regulating effect of the Prescription for Yishengukang on the molecular biology mechanism for FAP signal transduction was conducted in this research based on the work finished at earlier stages.[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of CaNAβ,a target associating with the signal transduction of FAK, CD49and CaN/NFAT at the junction between osseous metastasis tumor and the bones as well as the osseous metastasis tumor in nude mice in each group and the expression of chemokine receptor CXCR4. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.[Results]1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that the FAK and CD49protein in the model group was over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of FAK and CD49could be inhibited (P<0.01), therefore, Prescription for Yishengukang could inhibit the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the over expression of FAK and CD49, namely, Prescription for Yishengukang could inhibit the detachment and metastasis of tumor cells by stopping the FAP signal transduction pathway mediated by FAK to actualize the effect on inhibition of tumor metastasis;2. High expression of CXCR4in the model group vs. low expression of CXCR4(P <0.01) in the group with lower dosage of Prescription for Yishengukang, the Chinese drug plus western drug group and the group with Pamidronate Disodium indicated that Prescription for Yishengukang could inhibit the chemotaxis effect of bony tissue on the tumor cells by inhibiting the expression of CXCR4and could inhibit the occurrence of osseous metastasis of tumor;3. High expression of CaNAβ in the model group vs. low expression of CaNAβ (P<0.01) in the group with lower dosage of Prescription for Yishengukang, the Chinese drug plus western drug group and the group with Pamidronate Disodium indicated that Prescription for Yishengukang could inhibit the destroy of tumor tissue to the bone mass by inhibiting the expression of CaNAβ, namely inhibiting the osteolytic bone resorption caused by osteoclast activation and could inhibit the osteolytic damage to bone mass in local areas and eliminate the foundation for tumor cell to "settle down" and expand.[Conclusion]1. The Prescription for Yishengukang could inhibit the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the over expression of FAK and CD49, namely, Prescription for Yishengukang could inhibit the detachment and metastasis of tumor cells by stopping the FAP signal transduction pathway mediated by FAK to actualize the effect on inhibition of tumor metastasis;2. The Prescription for Yishengukang could inhibit the chemotaxis effect of bony tissue on the tumor cells by inhibiting the expression of CXCR4and could inhibit the occurrence of osseous metastasis of tumor;3. Prescription for Yishengukang could inhibit the osteolytic damage to bone mass by inhibiting the expression of CaNAβ.Part Ⅳ:The regulating effect of the Prescription for Yishengukang on the protein expression in dorsal horn of spinal cord in a rat model of osseous metastasis tumor pain[Objective] The regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model was researched. [Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model in each group. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.[Results]1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that GFAP, c-fos and P substances in dorsal horn of spinal cord in the model group were over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of the protein of GFAP, c-fos and P substances was at a lower level (P<0.01); osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the proteins in relation to pain such as GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice in these two groups was obviously lower than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the pain caused by the invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice, namely, Prescription for Yishengukang had certain abirritation by inhibiting the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in nude mice.[Conclusion]1. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of GFAP in dorsal horn of spinal cord in nude mice; 2. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of c-fos in dorsal horn of spinal cord in nude mice;3. The Prescription for Yishengukang could inhibit the pain caused by the osseous invasion and metastasis of adenocarcinoma of lung cells through the inhibition on the protein expression of P substance in dorsal horn of spinal cord in nude mice.Part V:Probe into the mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung from the prospect of OPG/RANKL/RANK system(Objective] The mechanism of the traditional Chinese drug, the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung was planned to be expatiated from the prospect of OPG/RANKL/RANK system.[Method] On the basis of osteolytic osseous metastasis model of adenocarcinoma of lung in nude mice, the method of Immunohistochemical staining was adopted to respectively observe the expression of OPG (protein expressions of osteopro tegerin), RANKL and IL-6in osseous metastasis tumor and at the junction between osseous metastasis tumor and the bones in nude mice in each group. Image analysis was conducted by image analysis software MIA4.0, and five visual fields were chosen from each section to count the optical density value of the positive substances expressed in each high magnification visual field under400Microscope Magnification.[Results]1.100%osseous metastasis occurred in the nude mice in model group and the immunohistochemistry data indicated that the OPG protein in the model group were down-regulated expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, the immunohistochemistry data indicated that the expression of the OPG protein could be increased (P<0.01); osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the OPG protein in nude mice in these two groups was obviously higher than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the osseous invasion and metastasis of adenocarcinoma of lung cells through increasing the expression of OPG protein.2. The proteins of RANKL and IL-6in nude mice in the model group were over expressed; no osseous metastasis occurred in the nude mice of the group with lower dosage of Prescription for Yishengukang and the Chinese drug plus western drug group, where, compared with the model group, the expression of proteins of RANKL and IL-6could be down-regulated (P<0.01), osseous metastasis occurred in the nude mice of the groups with moderate and higher dosage of Prescription for Yishengukang, but the expression of the proteins of RANKL and IL-6in nude mice in these two groups was lower than that of the model group (P<0.05); therefore, Prescription for Yishengukang could inhibit the osseous invasion and metastasis of adenocarcinoma of lung cells through decreasing the expression of proteins of RANKL and IL-6.[Conclusion]1. The Prescription for Yishengukang could inhibit the osseous metastasis of adenocarcinoma of lung cells through increasing OPG protein expression;2. The Prescription for Yishengukang could inhibit the osteolytic damage of tumor tissue to the bone mass through inhibiting the expression of proteins of RANKL and IL-6.[Innovation points]1. The therapeutic action by differential Treatment on the pain suffered from osseous metastasis tumor was systematically observed and the curative effect was evaluated according to the design code for clinical trial, adopting the principle of parallel, randomized and controlled clinical study.2. The patented CPA YANG1BM cell line was used in this research to establish an osteolytic osseous metastasis model in nude mice successfully through the method of injection into the left cardiac ventricle under the guidance of Prof. Yang Shunfang.3. Further probe into the regulating effect of the Prescription for Yishengukang on the molecular biology mechanism for FAP signal transduction was conducted in this research based on the work finished at earlier stages.4. Further probe into the regulating effect of the Prescription for Yishengukang on the protein expression of GFAP, c-fos and P substances in dorsal horn of spinal cord in a rat model for osseous metastasis tumor pain was conducted based on the work finished at earlier stages.5. The mechanism of the Prescription for Yishengukang to inhibit the osteolytic osseous metastasis of adenocarcinoma of lung was expatiated from the prospect of OPG/RANKL/RANK system.