Effect Of Yishen Medicine On NRK Cells’ EMT And Its Biosafety Evaluation Of Zebrafish Reproduction And Development

Background: Fibrosis is the terminal stage of many diseases,and it often has positive prompt effects.Ourpreviouswork had optimized the traditional Chinese medicine "Yishen Medicine" with independent intellectual property rights,which has an effect on the treatment of kidney fibrosis.Epithelial-mesenchymal transition(EMT)is a primitive epithelial cells transforming into mesenchymal cells under biochemical conditions to obtain high energy,migration ability and extracellular functions.The EMT process is an important molecular mechanism of kidney fibrosis.Zebrafish has the advantages of short growth cycle,obvious structural characteristics,rapid modeling,etc.,and can be widely and deeply researched on drug safety.Objective: This research aims to explore the effect of Yishen medicine on renal cells’ fibrosis by inhibiting EMT pathway at a safe dose,it could provide experimental basis for the clinical application of Yishen medicine.Methods: NRK cells were selected and treated with TGF-β1 to induce EMT.Studied the protective effect of Yishen medicine on fibrotic NRK cells.Research indicators include: 1.Observation of cell morphology;2.Cytoskeleton texture;3.Mitochondria quantity and outline;4.Calcium ion imaging technology;5.Observation of zebrafish embryos,juveniles and adults with different concentrations of drugs Various safety indicators.6.Use high-resolution liquid chromatography mass spectrometry to analyze the active ingredients of drugs,and analyze the pharmacological mechanism of Yishen medicine in the treatment of CRF in combination with the network pharmacology database.Results:(1)The contents of heavy metal elements Hg,Pd,Cd,and As in the Yishen medicine are significantly lower than the national standards,and the contents of the essential trace elements Cu,Fe,and Zn are significantly higher than the national standards.Yishen Medicine is relatively beneficial to human health;(2)Treating NRK cells with 10 ng/m L TGF-β1,the tested cells became rounded,the cytoskeleton texture deepened,and the number of mitochondria increased,which was consistent with the characteristics of epithelial-mesenchymal transformed cells;(3)After treatment with 3 mg/m L Yishen medicine,the skeletal texture of fibrotic NRK cells was significantly weakened,and the number of mitochondria was significantly reduced,indicating that the EMT pathway of the tested cells was inhibited.Compared with the positive control group(Niaoduqing),Yishen medicine is more effective in protecting cell viability;(4)For the effect of zebrafish embryo development,the Yishen medicine is safe below 3 mg/m L,and it is safe for juvenile fish growth and development,and the Yishen medicine is safe below 5 mg/m L,and is long-term toxicity to adult zebrafish.Yishen medicine is safety below 2mg/m L,and it is preliminarily proved that Yishen medicine has no adverse effects on zebrafish at all stages of development;(5)PPI analysis showed that there are 440 overlaps between the target of Yishen medicine and the target involved in CRF disease,among which TNF and IL-6 have the highest correlation.KEGG and GO analysis showed that Yishen medicine has significant correlation with cancer pathway,MAPK pathway,diabetes complications AGE-RAGE signaling pathway,cell differentiation pathway,etc.Conclusion: Yishen medicine can effectively improve the cell state of epithelial cell-mesenchymal transition and inhibit the epithelial-mesenchymal transition process,and is superior to the commercially available Niaoduqing granules,and has good biosafety In summary,Yishen medicine has better application prospect for the treatment of chronic renal failure.