Study On The Interaction Between Lipid Droplets And Mitochondria Using Super-resolution Small Molecule Probes And Its Application In Drug Evaluation

The interaction between lipid droplets and mitochondria plays a key role in the normal operation and function of cell life activities.The imbalance of the homeostasis of the interaction between lipid droplets and mitochondria caused by pathological conditions will lead to the disturbance of lipid storage and metabolism of cells,resulting in a variety of energy metabolism diseases.Screening and evaluation of drugs regulating lipid droplet interaction with mitochondria are of great significance for the diagnosis and treatment of energy metabolism diseases.However,the conventional methods for analyzing and labeling the interaction between lipid droplets and mitochondria still cannot meet our requirements for the study of their fine structure changes.Therefore,there is an urgent need to develop innovative techniques and methods that can reveal the dynamics of the interaction between lipid droplet with mitochondria at the nanoscale.The Structured illumination microscopy(SIM),a kind of ultra-resolution imaging technique widely used in recent years,was used for higher imaging speed due to lower requirements for sample preparation and illumination intensity.SIM has become a powerful tool for studying the interactions between drug-regulated organelles in living cells.The organelle probes that can be used for SIM dynamic imaging should not only be targeted,but also have higher requirements for the photostability of the probes.At present,many commercial mitochondrial fluorescence probes with excellent imaging performance have been developed.However,the commercial probes that can be used to image lipid droplets with super resolution still have problems such as non-specific labeling and easy photobleaching,which limit the study on the mechanism of mitochondria-lipid droplet interaction.Therefore,it is urgent to develop lipid droplet probes with excellent SIM super-resolution imaging performance to study the fine structure,dynamics and interaction between lipid droplet and mitochondria.Therefore,this paper first designed and synthesized a lipid droplet probe with excellent fluorescence performance,and applied it to the study of lipid droplet micro-dynamics in living cells and the imaging of multi-organelle interaction.The difference of contact sites between mitochondria and lipid droplet under different physiological conditions was captured,and the method of distance analysis between contact sites was established.The effects of drugs such as Lipopolysaccharide(LPS)on the contact site of lipid droplets and mitochondria were assessed.Then,in order to further explore the application of the interaction of lipid droplet between with mitochondria in drug evaluation,a three-color automatic identification analysis technique was developed,and a new super-resolution autophagy level analysis method was established.Finally,the developed techniques and probes were used to screen and evaluate the regulatory effects of Hyaluronic acid(HA)with different molecular weights on autophagy levels in different cells.This study provides technical support and theoretical basis for evaluating organelle interactions using super-resolution imaging.The main research contents are as follows:1.The contact sites between lipid droplets and mitochondria were traced by superresolution small molecule probesC-Py,a fluorescence probe targeted lipid droplet with D-π-A structure,consisting of a coumarin group and a pyridine unit,was designed and synthesized.The results showed that C-Py not only has the lipophilic characteristics required for targeting lipid droplets,but also has high photostability,low fluorescence background and good cell permeability,which effectively overcomes the shortcomings of traditional lipid droplet fluorescence probes such as high background noise and poor optical stability.Quantitative analysis using SIM imaging technology,the lipid droplets in living cells in quantity,morphology and dynamic process,and through long-term imaging capture the lipid droplets and mitochondria formed between contact site,set up for the contact distance between mitochondria and lipid drops of quantitative method(Cd=|XR-XG|(nm),Cd is the absolute distance between the edge of the red mitochondria(XR)and the edge of the green lipid droplet(XG)on the X axis).This method was used to reveal the phenomenon of reduced contact sites during the interaction between lipid droplets and mitochondria in living cells induced by LPS,which provided an important theoretical basis for the study of the interaction between lipid droplets and mitochondria in different physiological states.2.Establishment of a super-resolved autophagy level analysis method based on the interaction between lipid droplets and mitochondriaBased on the probe C-Py has a wide spectrum of absorption and emission,in order to avoid cross-color interference during simultaneous imaging of multiple organelles with super resolution,a commercial probe with a narrow spectrum,BODIPY,was used as the base structure.A probe,targeted lipid droplet,BDPQ,with conjugate planar structure with narrow spectrum,low interdye interference,higher fluorescence intensity,higher photobleaching resistance and lower fluorescence background was designed and synthesized to study the fine structure changes of lipid droplets and other organelles under different stimulus conditions of SIM simultaneous imaging.The changes of autophagy on the interaction between lipid droplets with mitochondria under starvation and on Carbonyl cyanide 3-chlorophenylhydrazone(CCCP)are tracked by BDPQ.The results showed that starvation for 16 h could promote the autophagy of lipid droplets,but had no effect on mitochondrial autophagy,while 10 μmol/L CCCP could significantly promote the autophagy of lipid droplets and mitochondria.In order to evaluate the regulatory effects of different stimulus conditions on autophagy levels,we used Matlab software to develop a super-resolved autophagy level analysis method based on the interaction between lipid droplets and mitochondria.The analysis method was combined with SIM imaging to effectively analyze the difference of autophagy level under starvation and CCCP stimulation.The results showed that starvation for 16 h stimulated a low level of autophagy(the ratio of lipid droplets,mitochondria to lysosomes colocalization was 6.3%± 0.5%),and 10 μmol/L of CCCP for 12 h stimulated a high level of autophagy(the ratio of lipid droplets,mitochondria to lysosomes colocalization was 13.0%±4%).In addition,the autophagy induction of CCCP was performed on the cells with autophagy related genes ATG13 and FIP200 knocked out,and the results were analyzed using the super-resolution autophagy level analysis method,so as to verify the feasibility of this quantitative analysis method for autophagy level.The results showed that autophagy levels were significantly inhibited when ATG13 and FIP200 were knocked out,indicating the feasibility of the super-resolution autophagy level analysis method established in this paper,which provides an effective strategy for the determination of autophagy levels under different stimulus conditions and the evaluation of related drugs.3.Drug evaluation based on super-resolution autophagy level analysis methodNanoscale pharmacodynamic evaluation by super-resolution micro-imaging is a promising new high-throughput screening strategy.Based on this,in combination with the super-resolution autophagy analysis method of the interaction between lipid droplets with mitochondria and the SIM imaging technique,we evaluated the regulatory effects of Hyaluronic acid(HA)with different molecular weights on the autophagy level of tumor cells(HeLa cells)and human fibroblasts(AT3 cells)at the organelle level.It was found that different molecular weights of HA had significant differences in the number of lipid droplets and the regulation of lipid autophagy level in HeLa cells and AT3 cells.o-HA and low molecular weight(30 kDa,200 kDa)HA had no significant effect on lipid droplets in HeLa and AT3 cells.High molecular weight(1080 kDa,2000 kDa)HA can significantly reduce the number of lipid droplets in HeLa cells,has no significant effect on AT3 cells,but can significantly improve the level of lipid autophagy in these two cells.We evaluated the regulatory effects of HA with different molecular weight on autophagy levels in Hela and AT3 cells by using the super-resolution autophagy level analysis method established in this study.The results showed that o-HA and low molecular weight HA had no significant effect on the autophagy levels of Hela and AT3 cells.High molecular weight HA can significantly promote the autophagy level of Hela cells and AT3 cells,and the promotion effect on the autophagy level of HeLa cells is significantly greater than AT3 cells.In addition,we found that high molecular weight HA has no significant difference in autophagy level of HeLa cells compared with the positive autophagy drug rapamycin,suggesting that high molecular weight HA may be a potential autophagy inducing drug.The above results prove that the super-resolved autophagy level analysis method proposed in this study can evaluate the regulatory effects of different drugs on the level of autophagy according to the changes in organelle interactions,providing a new analytical method and research direction for drug screening and evaluation at the nanoscale and the diagnosis and treatment of related diseases.