| Purpose:Recurrence and metastasis is the main cause of death in patients with triple negative breast cancer after operation,Traditional Chinese medicine intervention to prevent recurrence and metastasis,prolong survival and improve quality of life is still an important clinical topic.For patients with triple negative breast cancer who have completed adjuvant therapy or do not need adjuvant therapy,TCM aims to prevent recurrence and metastasis,improve symptoms and quality of life,Clinically,TCM takes the principle of strengthening body resistance and eliminating evil as the treatment principle to carry out TCM consolidation treatment,The oncology department of the Affiliated Hospital of Liaoning University of TCM has been treating patients with malignant tumors with traditional Chinese medicine under the guidance of the "unity of form and spirit theory" for many years,and achieved certain results,Through a retrospective clinical study,to evaluate the effect of long-term regular Chai hu long mu decoction treatment under the guidance of the unity of form and spirit theory on the survival time and form and spirit damage of patients with triple negative breast cancer after operation,Further in vivo and in vitro experimental studies were carried out to explore the partial mechanism of Chai hu long mu decoction in inhibiting the recurrence and metastasis of triple negative breast cancer.Material and method:1.Retrospective clinical study 0n Chai hu long mu decoction inhibiting the recurrence and metastasis of triple negative breast cancer after operationa retrospective clinical study Using the method of retrospective clinical study,Objective to analyze the clinical data of Triple negative breast cancer patients with stage one to three who were followed up for 3 years.All patients were treated with conventional Western Medicine according to the clinical guidelines of the national comprehensive cancer network(NCCN).Taking regular traditional Chinese medicine treatment as the exposure factor,it is divided into high exposure traditional Chinese medicine treatment group and low exposure traditional Chinese medicine group,The high exposure traditional Chinese medicine treatment group is defined as having received routine treatment of Western medicine and oral traditional decoction of Chinese medicine with or without anti-tumor Chinese patent medicine for ≥ 6months annually or cumulative 18 months or more,The low exposure traditional Chinese medicine treatment group is defined as those who do not meet the high exposure conditions after routine treatment of Western medicine and oral traditional decoction of Chinese medicine.Observed the site of recurrence and metastasis and the time of recurrence and metastasis / death,Statistical analysis of survival prognosis and related influencing factors,changes in the shape(detailed recurrence and metastasis of tumor)and spirit(symptom score,function score,weight,KPS score)injuries,and related adverse effects were performed in the two groups after TCM intervention.2.Cell experimental study of Chai hu long mu decoction containing serum inhibiting the metastasis of triple negative breast cancercarry out in vitro experimental research,SD rats were randomly divided into blank control group,Chai hu long mu decoction low concentration group,Chai hu long mu decoction medium concentration group and Chai hu long mu decoction high concentration group,The proliferation inhibition rate of MDA-MB-231 cells was detected by CCK-8 method,the cell invasion ability was detected by Transwell chamber test,and the cell migration ability was detected by scratch test,Take Western blot to test the E-cadherin protein and vimentin protein,the markers of epithelial mesenchymal transition,And further detect TGF-β/ Smad signaling pathway key protein TGF-β1,Smad3 and Transcription factor Snail protein.3.Animal experimental study of Chai hu long mu decoction containing serum inhibiting metastasis of triple negative breast cancerUsing in vivo experimental research,BALB/c nude mice were randomly divided into blank control group,model control group,Chai hu long mu decoction low concentration group,Chai hu long mu decoction medium concentration group and Chai hu long mu decoction high concentration group,In addition to the blank control group,the Triple negative breast cancer bearing nude mice models were prepared in the other groups.Nude mice were observed for general status,number of voluntary activities in 5 min,body weight,and tumor weight,By immunohistochemistry and Western blot to test E-cadherin,vimentin,TGF-β1,Snail protein expression level.Results:1 Retrospective clinical study 0n Chai hu long mu decoction inhibiting the recurrence and metastasis of triple negative breast cancer after operation1.1 General baseline characteristicsA total of 150 eligible cases were included,including 62 in the high exposure group and88 in the low exposure group,There were 149 females and 1 male,As of last follow-up,there were 27 recurrent metastases and 7 deaths(all due to disease progression),Comparing the general baseline characteristics of triple negative breast cancer patients,there was no significant difference between the two groups(P > 0.05),and the results were comparable.1.2 Survival prognosis(1)disease-free survival and overall survival of the two groups:The high exposure group had a longer 3-year DFS than the low exposure group,Moreover,the 3-year disease-free survival rate of the high exposure group was improved by 16.9% compared with that of the low exposure group(P < 0.05);The difference trend of OS results and overall survival rate will be followed up.(2)relevant prognostic factors affecting DFS after TNBC:The results of K-M univariate analysis showed that the status of vascular invasion,axillary lymph node status,and exposure to TCM were associated with DFS of TNBC patients(P <0.05),According to the results of univariate analysis,we carried vascular invasion,axillary lymph node metastasis and exposure to TCM as independent variables into the Cox proportional hazards multivariate regression model,All three turned out to be independent predictors of DFS(P < 0.05),In addition,patients in the low exposure group had 3.724 times the risk of recurrent metastasis than those in the high exposure group(95% CI 1.399,9.915).(3)explore the potential beneficiaries of high exposure traditional Chinese medicine treatment:Subgroup analysis the results of the study showed that the disease-free survival rates of the high exposure group versus the low exposure group were 98.0%,77.9% in the subgroup negative for vascular invasion,In the subgroup with positive vascular invasion,the disease-free survival rates of the two groups were 69.2%,54.5%,In the subgroup with no axillary lymph node metastasis,the disease-free survival rates of the two groups were 97.9%,77.3%,In the subgroup with ≤3 axillary lymph nodes,the disease-free survival rates were 75.0%,80.0% in both groups,In the subgroup with > 3 axillary lymph nodes,the disease-free survival rates were 66.7%,42.9% in both groups,It is suggested that patients with negative vascular infiltration and no axillary lymph node metastasis may become the potential beneficiaries of high exposure traditional Chinese medicine treatment after TNBC,However,in subgroup analysis,only one patient in the high exposure group with negative vascular invasion and no axillary lymph node metastasis developed recurrent metastasis,Therefore,the results of subgroup analysis to draw potential benefit population need to be further demonstrated by large sample data.1.3 Form and spirit injury(1)form injury: The mean form injury scores were smaller after treatment with high exposure TCM than with low exposure TCM(P<0.05).(2)mental injury:(1)Mean syndrome score,intra group comparison: mean syndrome score in the low exposure group decreased after traditional Chinese medicine treatment(P < 0.05),and mean syndrome score in the high exposure group decreased significantly after traditional Chinese medicine treatment(P <0.001);Comparison between groups: after treatment with traditional Chinese medicine,the mean syndrome score in the high exposure group was significantly lower than that in the low exposure group(P < 0.001).(2)Functional domain score,Intra group comparison: the score of emotional function in the low exposure group increased after traditional Chinese medicine treatment(P < 0.05),and the overall function score in the high exposure group increased significantly after traditional Chinese medicine treatment(P < 0.001);Comparison between groups: after treatment with traditional Chinese medicine,the overall functional score of high exposure group was higher than that of low exposure group(P < 0.05).(3)Weight,There was no significant increase or decrease in body weight between the two groups(P > 0.05).(4)KPS score,Intra group comparison: the KPS score of low exposure group increased after traditional Chinese medicine treatment(P < 0.05),and that of high exposure group increased significantly after traditional Chinese medicine treatment(P < 0.001);Comparison between groups: after treatment with traditional Chinese medicine,the KPS score of high exposure group was higher than that of low exposure group(P < 0.05).1.4 Adverse reactionsDuring oral administration of traditional Chinese medicine,the incidence of adverse reactions was 8.1% in patients in the high exposure group,6.8% in patients in the low exposure group,The difference between the two groups was not significant after testing(P =0.762 > 0.05),The main adverse reactions of the two groups were gastrointestinal discomfort,which was alleviated after administration of an adjusted Chinese herbal.2.Cell experimental study of Chai hu long mu decoction containing serum inhibiting the metastasis of triple negative breast cancer2.1 Test results of CCK-8 methodCompared with the blank control group,at the same time,with the increase of drug containing serum concentration,the inhibition rate of cell proliferation increased,and the difference between different concentrations was statistically significant(P < 0.001);In addition,the inhibition rate of cell proliferation increased with the extension of intervention time in the same concentration group.There was significant difference between the same concentration group in different time periods(P < 0.05).2.2 Results of Transwell invasion experimenCompared with the blank control group,the number of invasive cells decreased with the increase of drug containing serum concentration,and there was significant difference between different concentrations(P < 0.05).2.3 Scratch test resultsCompared with the blank control group,the cell migration rate gradually decreased with the increase of drug containing serum concentration at the same time,However,there was no significant difference between different concentrations at 12 h and 24h(P > 0.05),and there was significant difference between different concentrations at 36 h and 48h(P < 0.05);In the same concentration group,with the extension of intervention time,the cell migration rate increased and the effect of inhibiting migration and healing weakened.The difference in different periods of the same concentration group was statistically significant(P < 0.05).2.4 Western blot results of E-cadherin,vimentin,TGF-β1,Smad3 and snail proteinsCompared with the blank control group,with the increase of serum concentration of Chai hu long mu decoction,the expression of E-cadherin increased gradually(P < 0.05)and the expression of vimentin protein decreased gradually(P < 0.001).Compared with the blank control group,the expression of TGF-β1,Smad3 and snail protein decreased gradually with the increase of serum concentration of Chai hu long mu decoction(P < 0.001).3 Animal experimental study of Chai hu long mu decoction containing serum inhibiting metastasis of triple negative breast cancer3.1 General conditions and tumor inhibition rate of nude micegeneral status,body weight and number of voluntary activities of the nude mice:The triple negative breast cancer tumor bearing nude mouse model was prepared successfully,Chai hu long mu decoction improved the general status of tumor bearing nude mice,improved the motility of nude mice(P < 0.05),and attenuated the loss of body weight in nude mice(P <0.05).the tumor weights of the Chai hu long mu decoction group were all lower than those of the model control group(P < 0.001),and with increasing concentrations of Chai hu long mu decoction,the tumor weight gradually decreased(P < 0.001),and the tumor inhibition rate gradually increased(P < 0.001).3.2 Expression of E-cadherin and vimentin in tumor tissueIHC test results showed that compared with the model control group,with the increase of the concentration of Chai hu long mu decoction,the average integrated optical density of E-cadherin gradually increased(P < 0.05),and the average integrated optical density of vimntin gradually decreased(P < 0.001),There was no positive expression of E-cadherin and vimentin in breast tissue of blank control group.The results of Western blot showed that compared with the model control group,with the increase of the concentration of Chai hu long mu decoction,the expression of E-cadherin protein increased gradually(P < 0.001),and the expression of vimentin protein decreased gradually(P < 0.001).There was no positive expression of E-cadherin and vimentin in the breast tissue of the blank control group.3.3 Expression of TGF-β1,Smad3 and snail in tumor tissueIHC test results showed that compared with the model control group,with the increase of the concentration of Chai hu long mu decoction,the average integrated optical density of TGF-β1,Smad3 and snail in each group gradually decreased(P < 0.05),and there was no positive cell expression of TGF-β1,Smad3 and snail in the breast tissue of the blank control group.The results of Western blot showed that compared with the model control group,with the increase of the concentration of Chai hu long mu decoction,the protein expressions of TGF-β1,Smad3 and snail in each group gradually decreased(P < 0.001),and there was no positive expression of TGF-β1,Smad3 and snail in the breast tissue of the blank control group.Conclusion:1.Under the guidance of the theory of unity of form and spirit,long-term regular oral administration of Chai hu long mu decoction can improve the 3-year DFS and disease-free survival rate of patients with triple negative breast cancer after operation and after radiotherapy and chemotherapy,In this study,vascular invasion,axillary lymph node metastasis and traditional Chinese medicine exposure were independent factors affecting the disease-free survival of triple negative breast cancer patients.2.Taking Chai hu long mu decoction orally for a long time can reduce the body and spirit damage of triple negative breast cancer patients to some extent.In addition,Chai hu long mu decoction is safe.3.Chai hu long mu decoction may act by downregulating TGF-β1,Smad3,key proteins of the TGF-β/ Smad signaling pathway,and snail protein expression,a downstream transcription factor,inhibit the growth,invasion,migration,and epithelial mesenchymal transition of triple negative breast cancer.This may be one of the important mechanisms of Chai hu long mu decoction to inhibit metastasis of triple negative breast cancer.4.Chai hu long mu decoction can inhibit the growth of triple negative breast cancer tissue,maintain the weight of tumor bearing nude mice,improve the autonomous activity and general state of tumor bearing nude mice,and reduce the form and spirit damage of tumor bearing nude mice to a certain extent. |
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