| Objective:(1)To analyze the research status of traditional Chinese medicine in the treatment of osteosarcoma by bibliometrics and visualization techniques,and to reveal the dynamic changes and development trend,so as to provide ideas and basis for the research of TCM in the treatment of osteosarcoma.(2)To develop and evaluate the TCM syndrome element diagnostic scale for osteosarcoma by using the procedure and method of international common scale development,and to explore the method of using the scale for the standardization and objectivity study of the syndrome.Apply the scale to clinical data differentiation preliminarily.(3)The study aimed to explore the active compounds,targets,pathways and mechanism of Yiqi-Sanyu-Jiedu formula in the treatment of osteosarcoma with network pharmacology and molecular docking technology.Through REGγ/RNF149/B-RAF/MEK/ERK signaling pathway,deeply analyzed the pharmacodynamic mechanism of the prescription of Yiqi-Sanyu-Jiedu formula and the effective component berberine,to provide a new idea for clinical treatment.Methods:(1)Retrieving CNKI database of Chinese medicine in the treatment of osteosarcoma of related literature.Use scientific metrology tools Cite Space V to map and do the co-occurrence,clustering and emergent analysis.(2)Literature survey and expert consultation were used to preliminarily determine the diagnostic items of osteosarcoma syndrome elements.By multi-center clinical epidemiological methods to collect patients with osteosarcoma of the diagnostic information and the general situation of traditional Chinese medicine,using the correlation analysis,stepwise regression,factor analysis to justify filtered entry syndrome elements,using regression analysis method to carry on the diagnostic entry points,curve method for the determination of syndrome diagnosis threshold factors of osteosarcoma factors of TCM.Then the diagnosis scale was established.Delphi method was used to conduct expert questionnaire survey of "TCM syndrome element diagnostic scale for osteosarcoma" and peer evaluation was conducted.To evaluate the reliability and validity of the scale,43 clinical samples were used for clinical verification.A total of177 patients with osteosarcoma were investigated.The developed scale was used for syndrome differentiation to investigate the distribution of syndrome elements of osteosarcoma.(3)All the effective components and action targets of astragalus,paris polyphylla and coptis were searched.Potential active compounds were screened.Target genes related to osteosarcoma were obtained.The drug target and disease target were intersected,and the drug-target-disease network was constructed.PPI network was obtained for the treatment of osteosarcoma with Yiqi-Sanyu-Jiedu formula,and the Network Analyzer was used for topology analysis.The core targets were enriched by GO function and KEGG pathway.The compound was molecularly docked with proteins.Adopting Realtime PCR,Western Blot,protein purification technology,such as immune coprecipitation technical analysis REGγ/RNF149/B-RAF/MEK/ERK signaling pathway in its role in the development of osteosarcoma.Results:(1)197 articles were included,and the number of articles was on the rise.Fujian,Shanghai and Guangdong have a large number of published literatures and are in the leading position of research.The cooperation among different regions,institutions of higher learning and different authors is relatively small.Among them,curcumin,pien tze huang,huadouxin and bee toxin are studied most extensively and deeply.Tripterine,cell proliferation and immunosuppression may be the focus of future development.(2)Scale development results: The TCM syndrome element diagnostic scale for osteosarcoma included 5 syndrome elements,and the final screening items,score and diagnostic threshold of each syndrome element were as follows: total score of blood stasis syndrome = complexion gloom×2+ local tingling pain×8+ pain aggravation at night×4+ body pain×3+ purple tongue darkness×3,and the diagnostic threshold was 11 points.The total score of qi deficiency syndrome =mental fatigue×3+ fatigue×5+ lazy speech with little qi×6+ weak tongue×3+ white moss×1+ fine pulse×2,and the diagnostic threshold was 9 points.The total score of qi stagnation syndrome = hypochondriac pain×8+ good breath×6+ pulse string×6,and the diagnostic threshold was 12 points.Total score of blood deficiency syndrome =colorless complexion ×6+ dizziness×5+ palpitation×1+ insomnia × amnesia×3+ weak tongue×3+ weak pulse×2,and the diagnostic threshold was 9 points.Total score of phlegm obstruction syndrome = nausea×3+ vomiting×2+ mouth mucosa×7+ joint muscle pain×4+ mucosa×4,and the diagnostic threshold was 10 points.Scale evaluation results: the overall Kappa value was 0.816,and the overall Cronbach’s αcoefficient value was 0.809.Three common factors were selected,the cumulative contribution rate of the total variance was 70.295%,and the factor load was all greater than the minimum acceptable value.There was a good correlation between the scores of various syndrome elements and the total scores of the scale.The results of syndrome differentiation by using the scale showed that the combination of two syndrome types was the most,followed by the combination of three syndrome types,and the rest of the colligation forms were few.The main syndromes are blood stasis and qi deficiency,and there are other syndromes.(3)A total of 49 active ingredients and 23 key active ingredients were screened.PPI network was used to obtain 17 key target proteins of Yiqi-Sanyu-Jiedu formula for the treatment of osteosarcoma.The key targets involved AKT1,MAPK1,PTGS2 and VEGFA.GO functional enrichment analysis resulted in 1720 GO items(P≤0.05),among which 1583 were BP items,60 were CC items,and 107 were MF items.KEGG pathway enrichment analysis resulted in 112 signaling pathways(P≤0.05),involving pi3k-akt signaling pathway,MAPK signaling pathway,and hypoxia-inducible factor-1 signaling pathway.Molecular docking results showed that kaenophenol,berberine and quercetin had strong affinity with AKT1 and MAPK1 proteins of osteosarcoma.Berberine,the active ingredient of Yiqi-Sanyu-Jiedu formula,can inhibit the proliferation of osteosarcoma cells;RNF149 expression was also up-regulated and p-MEK and p-ERK protein levels were inhibited,which was consistent with the effect of silencing REGγ in osteosarcoma cells.Conclusion: The research on osteosarcoma has made great progress.Strengthening academic cooperation between different regions and universities is an important aspect to promote the development of disciplines and academic development in the future.Currently,the basic research focus of TCM treatment of osteosarcoma is how to regulate the proliferation and apoptosis of osteosarcoma cells.The establishment of standardized TCM syndrome standards for osteosarcoma is the focus of future research.The traditional Chinese medicine research on osteosarcoma is still in its embryonic stage,which needs constant long-term development and exploration.(2)The TCM syndrome element diagnostic scale for osteosarcoma includes 5 syndrome elements and 25 items,which has good reliability and validity.Preliminary application studies show that the scale can provide standard criteria for diagnosis of clinical osteosarcoma syndrome differentiation,objectively describe the distribution of syndromes,and can provide a reference for the standardized study of TCM syndromes.(3)Yiqi-Sanyu-Jiedu formula and its effective components through regulating REGγ/RNF149/B-RAF/MEK/ERK signaling pathway signaling pathway,so as to participate in the malignant biological behavior of inhibition of osteosarcoma. |
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