Study On Pharmacology And Pharmacodynamic Basis Of Xiaozheng Sanjie Zhitong Zhitong Plaster

Objective:This article takes Xiaozheng Sanjie Zhitong Zhitong Plaster as the research object,explore its effective ingredients and mechanism of action.Using UPLC-Q-Exactive electrostatic field orbitrap high-resolution mass spectrometry technology,comprehensive analysis and characterization of the chemical components of Xiaozheng Sanjie Zhitong Analgesic Plaster,screen the active ingredients;copy related animal models and systematically evaluate the pharmacodynamics of Xiaozheng Sanjie Zhitong Analgesic Plaster;in vivo experiments observe the general state of Xiaozheng Sanjie Zhitong Zhitong Plaster on the animal model of breast cancer in nude mice.In vivo experiments were conducted to observe the effects of Xiaozheng Sanjie Zhitong Zhitong Plaster on the general status,volume of allogeneic tumors and tumor inhibition rate of nude mouse breast cancer animal models,detect VEGF,Bcl,Caspase-3 gene and protein expression,and culture breast cancer cells in vitro.It is MCF-7 cell line.By detecting and observing the survival rate,apoptosis rate,VEGF,Bcl,Bax,Caspase-3 gene and protein changes of the target cell line in vitro,it is clarified that Xiaozheng Sanjie Zhitong Zhitong Plaster is used to treat breast cancer mechanisms.Method:1.Using UPLC-Q-Exactive electrostatic field orbitrap high-resolution mass spectrometry technology,comprehensive analysis and characterization of the chemical components of Xiaozheng Sanjie Zhitong Analgesic Plaster,the components with high content and strong biological activity were further screened out,and their chemical components were determined by UPLC MS/MS.2.Select 4-6 weeks old female BALB/C-nu pure nude mice,MCF-7 cell suspension cells were inoculated into the second breast fat layer on the left side of the mouse to establish a nude mouse breast cancer tumor-bearing model.After modeling,they were randomly divided into model group(Group A),positive control group(fluorouracil solution)(Group B),low-dose group of Xiaozheng Sanjie Zhitong Zhitong plaster(Group C),middle-dose group of Xiaozheng Sanjie Zhitong Zhitong plaster(Group D)and the high-dose group of Xiaozheng Sanjie Zhitong Zhitong Plaster(Group E)consist of five groups,each with 12 rats.The model group was given an equal volume mixture of beeswax and sesame oil,and topical application on the skin where the mouse cells were inoculated.The positive control group was given 5-FU(intraperitoneal injection,20mg·kg-1·3d-1),and the other three groups were given Xiaozheng Sanjie Zhitong The pain-relieving plaster is applied locally at low,medium and high doses,in which the four groups except the positive control group are administered once a day for 21 consecutive days,and the positive control group is administered once a week for three consecutive weeks.Regularly observe the weight of the mice,record the changes in general conditions,and measure the length(a)and width(b)of the tumor with a vernier caliper.Calculate the approximate tumor volume(V)V=1/2ab2(unit cm3);calculate the tumor inhibition rate in each group;observe the tumor-bearing tissue and tumor tissue density under light microscope;immunohistochemical method to detect the tumor-bearing tissue and summarize CD34 positive expression,And observe the angiogenesis,Real Time PCR and Westernblot methods were used to detect VEGF,Bcl-2 and Caspase-3 gene and protein expression in tumor-bearing tissues.3.In the pharmacodynamics research,the estradiol-induced mammary gland hyperplasia model in rats and rabbits,rat agar granuloma,electrical and thermal stimulation pain model,blood stasis type model,etc.were used to evaluate Xiaozheng Sanjie Zhitong Zhitong Plaster.Inhibit mammary gland hyperplasia,influence on granuloma weight,anti-inflammatory and analgesic,and influence on blood viscosity and other pharmacodynamic changes.4.Breast cancer cell line MCF-7 cells were cultured in vitro,resuscitated,and passaged stable,MTT method was used to detect the activity of MCF-7 cells,to detect the survival rate and apoptosis rate of breast cancer cells in Xiaozheng Sanjie Zhitong Zhitong Plaster Solution,and to determine Xiaozheng San The best intervention concentration and intervention time of Jiezhitong Plaster Solution are for follow-up experimental application.5.The stable passage breast cancer cell line MCF-7 cells were divided into two groups,the control group and the experimental group,the control group did not intervene,the experimental group was given Xiaozheng Sanjie Zhitong Zhitong plaster solution for intervention,and the Real Time PCR method was used to detect MCF-7 cells The expression of VEGF,Bcl-2 and Caspase-3 m RNA.6.The stable MCF-7 cells were divided into two groups,a control group and an experimental group.The control group did not intervene.The experimental group was treated with Xiaozheng Sanjie Zhitong Zhitong Plaster Solution for intervention.Westernblot method was used to detect VEGF,Bcl,Bax,and Caspase-3,the expression of cleaved caspase-3 protein.Result:1.Xiaozheng Sanjie Zhitong Pain Relief Plaster has targeted properties,Plaster content,adhesion,weight difference,and content determination.The research results show that the self-made Plaster is a yellow-brown semi-solid preparation;the Plaster content is tentatively determined to be no less than each patch 24.994g/100 cm~2.The initial adhesion is no less than 17 steel balls;the RSD of the weight of 10 Xiaozheng Sanjie Zhitong Analgesic Plasters is1.41%.2.The thin-layer identification method of turmeric,garcinia cambogia,turmeric,angelica,and Tiankuzi in Xiaozheng Sanjie Zhitong Pain Relief Plaster has shown that the identification methods of turmeric and other medicinal materials are reproducible,durable,and scientific and reliable.3.In animal experiments,the tumors in the blank control group grew rapidly,and the rest of the treatment groups had inhibitory effects on tumors.Among them,the positive control group and Xiaozheng Sanjie Zhitong Zhitong Plaster showed the strongest inhibition in the middle and high doses,and there was no obvious among the three groups The difference(P>0.05),while the low-dose group is inferior to the other three treatment groups(P<0.05);compared with the blank group,the mice in each treatment group showed no abnormalities in behavior,eating,response to external stimuli,gloss of fur and mental state.After HE staining and observation under light microscope,the cancer cells in the model group were arranged very tightly,with complete nuclei,uniform size,and no connective tissue.The tumor cells in each treatment group showed different degrees of cell shrinkage,accompanied by different degrees of connective tissue.Among them,the connective tissue in the high-dose and positive control drug groups has more expression than other treatment groups;in immunohistochemistry,CD34 was positively expressed in each group,indicating that neovascularization in tumor-bearing tissues can reduce tumor neovascularization after treatment.Among them,the angiogenesis of the medium,high-dose and positive control drug group is better than that of the model group.Reduced,better than the low-dose group(P<0.05).4.The results of anti-inflammatory and analgesic experiments show that Xiaozheng Sanjie Zhitong Zhitong Plaster can significantly reduce the weight of subcutaneous cotton ball granuloma and subcutaneous agar granuloma in rats.Compared with the matrix control group,it has statistical significance(P<0.05).At the same time,Xiaozheng Sanjie Zhitong Pain Relief Plaster can effectively reduce the pain response of mice caused by electrical and thermal stimulation,which is better than the matrix control group(P<0.05).5.In the pharmacodynamic experiment of promoting blood circulation and removing blood stasis,Xiaozheng Sanjie Zhitong Zhitong Plaster can effectively reduce blood viscosity(including high shear rate and low shear rate)and plasma viscosity of rats with blood stasis(subcutaneous injection of epinephrine+ice water immersion),reduced viscosity,erythrocyte sedimentation rate,fibrinogen content,external thrombus index,external thrombus length and wet weight;increased the blood flow rate,diameter of the capillaries of the ears of normal mice,and the number of capillaries with blood cells flowing in the fixed area.The above results were better than the blood stasis model matrix group(P<0.05).6.In the pharmacodynamic experiment on mammary gland hyperplasia,compared with the matrix control group of mammary gland hyperplasia model,Xiaozheng Sanjie Zhitong Zhitong Plaster can effectively reduce the mammary gland hyperplasia caused by estradiol in rats and rabbits(P<0.05),manifested as reducing the weight of the breast,the height of the nipple,the degree of breast lesions and the pathological changes of the breast tissue in the breast hyperplasia caused by estradiol,the reduction of the serum estradiol level in rabbits and the pathological changes of the breast tissue(P<0.05).7.In the part of animal experimental gene detection,the results showed that VEGF and Bcl-2 in tumor-bearing tissues in the model group were significantly increased,which was significantly higher than the other four treatment groups(P<0.05),and Caspase-3 was significantly lower than the other four groups.-3;compared with the treatment group,the expression of VEGF,Bcl-2 and Caspase-3 genes in the positive control group was not different from that in the high-dose group(P>0.05),while the middle and low-dose groups showed VEGF and Bcl-2 Higher than the high-dose and positive control group(P<0.05),the expression of Caspase-3 was lower than the second two groups(P<0.05).8.In the part of animal experiment basic protein detection,the results were consistent with gene expression,which showed that VEGF and Bcl-2protein in the model group were significantly increased,which was significantly higher than the other four treatment groups(P<0.05),and Caspase-3 protein was significantly lower In the other four groups;compared with the positive control group,the high-dose showed the same effect(P>0.05),while the middle-low-dose was inferior to the positive control group(P<0.05).9.Take human breast cancer MCF-7 for routine culture,after passage and stable recovery,prepare 7 concentrations of Xiaozheng Sanjie Zhitong Zhitong Plaster extract solution,and intervene MCF-7 cells at 24h and 48h respectively,and detect by MTT method.The best concentration is0.32mg/m L,and the best intervention time is 24h.10.Cell growth was observed under light microscope.Control group:cells died with increasing time.At 48h,a large number of cells died.At the initial 6h of culture,the cancer cells are arranged in a typical glandular shape.As time grows,the cells are tapered,with the nucleus located on the side of the cell,showing deformities such as distortions,folds,and depressions,coarse granular,uneven distribution;experimental group:in the initial 6hours of culture,compared with the control group,there was a small amount of cell death.As time increases,cell death accelerates,the arrangement is sparse,the nucleolus is more and the shape is irregular,the cytoplasm is reduced,and a large number of diffuse small voids are seen in the cytoplasm and nucleus,at the same time,deaths increased compared with the control group.11.Cytogenetics detection part:Xiaozheng Sanjie Zhitong Zhitong Plaster extract can effectively reduce the expression of VEGF and Bcl-2m RNA in MCF-7 cells,which is better than that of the control group(P<0.05),while the expression of Caspease-3m RNA between the two groups There was no difference(P>0.05).12.Cell protein detection part:Xiaozheng Sanjie Zhitong Zhitong Plaster extract can effectively reduce the expression of VEGF and Bcl-2m RNA in MCF-7 cells,which is better than the control group(P<0.05),and can significantly increase Caspase-3.The expression of cleaved caspase-3protein was better than that of the control group(P<0.05).Conclusion:1.Curcumin,the main active component of turmeric in Xiaozheng Sanjie Zhitong Pain Relief Plaster,has a content of 2.85 mg/g.Gambogic acid is not more than 15.15mg/g.2.Xiaozheng Sanjie Zhitong Pain Relief Plaster can significantly reduce the general condition of tumor-bearing nude mice,suppress tumor rate,destroy the morphology of cancer cells in tumor-bearing tissue,increase connective tissue in tumor-bearing tissue,and reduce microangiogenesis in tumor-bearing tissue.3.Xiaozheng Sanjie Zhitong Pain Relief Plaster has the effects of inhibiting breast hyperplasia,promoting blood circulation and removing blood stasis,anti-chronic inflammation,analgesia and anti-breast cancer.4.Xiaozheng Sanjie Zhitong Zhitong Plaster can significantly reduce the expression of VEGF,Bcl-2 m RNA and protein in tumor-bearing nude mice,increase the expression of Caspase-3 m RNA and protein,block the conduction of angiogenesis signals,and achieve the treatment of breast cancer.5.Xiaozheng Sanjie Zhitong Zhitong Plaster extract can significantly inhibit the expression of VEGF and Bcl-2 in MCF-7 cells,promote the expression of Caspase-3 and cleaved caspase-3,and may induce MCF-7 by activating Caspase-3 Apoptosis,prevention and treatment of breast cancer.