Study On The Assessment Of DNA Damage By High-content Imaging In Combination With Intelligent Recognition And Its Application In Drug Screening

DNA damage,the abnormal change of DNA physical or chemical structure,plays an important role in the pathology of cancer and aging-related diseases,as well as in the damage caused by ultraviolet/ionizing radiation or chemotherapy drugs.Therefore,active compounds that can reduce DNA damage or promote DNA damage repair have aroused general interest in the field of drug research and development.However,most of the existing DNA-damage-assessing methods such as methods based on gel electrophoresis and radiolabeling,are low-throughput or lack sensitivity.With the advancement of instrumental analysis and high-content imaging technology which is characterized by microscopic imaging combined with fluorescent labeling,it has become possible to accurately analyze the degree of DNA damage in the nucleus.In order to automatically analyze the DNA damage and apply the method to high-throughput drug screening,it is necessary to solve a series of problems such as the construction of cell models,the optimization of imaging methods,the intelligent recognition of images,the confirmation of lead compounds and the investigation of Mode of Action.In this thesis,cell lines were constructed,in which the p53 binding protein 1(53BP1)was labeled with enhanced green fluorescent protein(EGFP),so that the DNA damage can be evaluated by analyzing the 53BP1 foci.High-throughput acquisition of single-cell DNA damage images was realized.And a deep-learning-based intelligent recognition method of DNA damage was established,which provides a feasible way to screen and discover anti-DNA damage compounds.Focusing on two DNA damage-related processes,the ionizing-radiation-induced DNA damage and the chemotherapy drug doxorubicin(DOX)-induced DNA damage in cardiomyocytes,three new DNA damage evaluation models were established and applied to screening from a compound library containing315 ingredients from traditional Chinese medicine.Eight active compounds were found,and the cardioprotective efficacy of one active compound isoliquiritigenin(ISL)and the Mode of Action of ISL were further studied.The main contents of the thesis are as follows:1.A deep-learning-based method(Foci Net)to intelligently identify DNA damage in cells was established,by which the DNA damage status of each single cell under ionizing radiation was accurately identified.And the Foci Net was also applied to the screening for regulators of DNA damage.First,a He La cell line stably expressing EGFP-53BP1 was constructed to visualize the formation of foci in the nucleus.By optimizing imaging methods,radiation doses,positive compound’s concentrations,etc.,a high-content imaging method for radiation-induced DNA damage was established,and was applied to a compound-library-based high-throughput screening.To solve the problem of processing more than 5,000 dual-color fluorescent images,6,000 single-cell images were manually annotated for training the classification network,and a deep learning model,Foci Net,was constructed based on U-Net and VGG-19 convolutional neural networks.The Foci Net model achieves single-cell segmentation through the U-Net-based segmentation module,and determines the DNA damage status of each cell through the VGG-19-based classification module,thereby evaluating the degree of DNA damage in the cell population.The results show that the classification accuracy of Foci Net for foci-positive cells,foci-negative cells and non-signaling cells reaches 99.02%,and it can accurately reflect the change law of the radiation dose and the positive compound’s concentration.It also outperforms traditional thresholding methods for foci images analysis in both cell segmentation and generalizability across different imaging platforms.Using this method,three active compounds(evodiamine,herbacetin and ISL)were discovered from the compound library,and comet assay showed that ISL reduced the radiation-induced double-strand breaks.2.On the basis of the above methods,in order to treat cardiotoxicity of anthracyclines such as DOX,an evaluation method for DNA damage in cardiomyocytes induced by DOX was established,and a high-throughput screening was carried out for cardioprotective compounds against DOX.First,the H9c2 cardiomyocyte cell line stably expressing EGFP-53BP1 was constructed.The increase of double-strand breaks shown in gel electrophoresis and the increase of γ-H2 AX shown in Western Blot(WB),suggesting that DOX can induce the DNA damage in cardiomyocytes.Further,the imaging parameters,the incubation times of DOX,etc.,were optimized,to establish a high-content screening method.The established method can effectively reflect the protective effects of positive compounds(dexrazoxane and resveratrol)on DOX-injured cardiomyocytes.Using this method,twelve active compounds from traditional Chinese medicine were found to potentially reduce DOX-induced DNA damage.Among them,six compounds(kaempferol,cryptochlorogenic acid,scutellarin,quercetin,kaempferide,ISL)effectively reduced the injury of H9c2 cells caused by DOX.3.In order to verify the above hit compounds,micropatterned neonatal rat cardiomyocytes(NRCMs)were constructed to evaluate the protective effect of compounds on DOX-induced myocardial injury model under the quasi-physiological conditions with biomimetic myocardial arrangement and continuous beating.The micropatterned hydrogel was optimized by 3D printing technology,and biomimetic cultured cardiomyocytes with aligned arrangement and high maturity of myofilament were obtained.Microscopic imaging analysis showed that its contraction frequency was about(32.67 ± 8.07)rpm,which was higher than(19.67 ± 4.27)rpm of traditionally cultured NRCMs.Calcium transient assay showed that it also had stable conduction of electrical impulse,which was similar to in vivo myocardium.On the basis,a DOXinduced DNA damage model in micropatterned NRCMs was constructed.Through intelligent image recognition by Foci Net,it was found that the recovery rate of dexrazoxane has increased from(61.96 ± 6.51)% in traditionally cultured NRCMs to(94.07 ± 5.56)% in micropatterned NRCMs,suggesting that the DOX-induced micropatterned NRCMs injury model could better evaluate the cardioprotective activity of compounds against DOX.Further verification of the hit compounds on the model showed that ISL could inhibit the DOX-induced 53BP1 aggregation and the DOXinduced decay of contraction frequency.4.Using in vitro cell model and animal model,the cardioprotective efficacy of the active ingredient ISL was verified.First,on H9c2 cardiomyocytes,ISL was found to dosedependently inhibit DOX-induced aggregation of 53BP1.The comet assay and WB showed that ISL reduced DOX-induced DNA double-strand breaks(P < 0.01)and DNA damage markers γ-H2AX(P < 0.01),which further suggests that ISL reduces DOXinduced DNA damage in H9c2 cardiomyocytes.Subsequently,the cell viability was detected,and the level of ATP was detected by luminescence-based method.The proportion of apoptotic cells was quantitatively detected by fluorescence imaging,and the protein expression was detected by WB to study the protective effect of ISL on cardiomyocytes.The results showed that compared with the DOX-treated group,ISL dose-dependently increased cell viability and ATP levels,dose-dependently decreased the proportion of apoptotic cells,and decreased the expressions of pro-apoptosis-related proteins BAX and Caspase 3.In a DOX-induced myocardial injury model in mice,ISL ameliorated DOX-induced decline of cardiac function,disintegration of myocardial sarcomere and swelling of myocardial mitochondria.Finally,the antioxidant activity of ISL was determined by free-radical-scavenging assay,and the levels of reactive oxygen species and peroxynitrite ions in cells were determined by fluorescence imaging.What’s more,the expression levels of HO-1 and topoisomerase II were determined by WB to explore the DNA protective mechanism of ISL.The results showed that ISL had a scavenging effect on free radicals in vitro.On H9c2 cells,compared with the DOX-treated group,the levels of reactive oxygen species and peroxynitrite ions in the ISL group were decreased,the expression of HO-1 was increased,and the expression of topoisomerase II was decreased,suggesting that the protective effect of ISL against DOX-induced DNA damage may be related to the inhibition of oxidative stress and the reduction of topoisomerase Ⅱ.