| Background and purposeUltrasound is a sound wave with a frequency higher than 20,000 Hz.Because of its good directivity and strong penetration ability,it is easy to obtain more concentrated acoustic energy and has a far transmission distance in water,so it is used for distance measurement,speed measurement,cleaning,sterilization and disinfection and then has many applications in medicine,military,agriculture and industry.Because ultrasound has the advantages of non-invasive,deep penetration,remote operation,etc.,it is widely used in medical diagnosis and treatment.The frequency of ultrasound used in diagnosis is usually 1 MHz-30 MHz.At present,the focused thermal energy effect of ultrasound has been applied in medical diagnosis and treatment,such as radiofrequency ablation,ultrasonic knife,etc;the Doppler frequency shift and echo scanning effect of ultrasound has been applied in Doppler color ultrasound and B-ultrasound;the ultrasonic cavitation effect has been applied in teeth cleaning and treatment.Ultrasound produces ultrasonic radiation force when acting on an object.Radiation force refers to the total amount of radiant energy of all wavelengths emitted by the unit surface area of the object in all directions in the hemispherical space per unit time.When the ultrasonic radiation force acts on a cell,it will produce shear stress around the cell surface,which will then affect various physiological functions of the cell.In the current research,ultrasonic radiation force was mainly sensed and transducted by mechanically sensitive protein on cell surface,which finally effect cell physiological functions.For example,when the mechanically sensitive ion channels?Mscl?was expressed in mouse neuron cells,the stimulation of low-intensity ultrasound can activate mice Neuronal cells by Mscl protein on cell surface;the ultrasonic stimulation can regulate the sensory neuron of C.elegans through the transient receptor potential channel Trp-4,and then affect C.elegans'behavior;the ultrasonic stimulation can also act on the mechanically sensitive ion channel protein Piezo1 to activate calcium ions influx,whihc increases the expression of nuclear c-Fos in the neurons of the first generation of mice and affects the learning and memory functions of mice.Piezo1 protein is widely distributed in mammals and is sensitive to ultrasound.The effect of ultrasound on the mechanically sensitive ion channel Piezo1 protein is requied much more and further studies.The protein Fam38A?Piezo1?is a non-selective calcium ion mechanically sensitive channel protein reported in Sciece.Piezo1 protein is locatded in human16q24.3,most of which are distributed on the plasma membrane and endoplasmic reticulum membrane of the trilobate spiral.It consists of 2521 amino acids with a molecular weight of 286790 d.It is expressed in human lung,bladder,kidney,skin,skeletal muscle,cardiovascular system,etc.A non-selective calcium ion mechanically sensitive channel,it can transduce mechanical forces into biological signals.In mammals,the transduction of mechanical signals affects embryonic development,touch,pain,proprioception,hearing,vascular tone,and blood flow regulation.It plays an important role in maintain the normal function of organs and tissues in human body.Piezo1 protein is a mechanically sensitive ion protein.In the current research,it has been found that the cyclic stretching force can promote the invasion and apoptosis of human osteosarcoma cells and inhibit cell proliferation;the cyclic stretching force can also induce the calcium influx and ATP release in urinary epithelial cells.The blood perfusion can induce the release of ATP and the dependent activation of the downstream signaling pathway P2Y2/Gq/G11.When the fluid shear force acts on Piezo1,it has a significant effect on the formation of blood vessels and the arrangement of endothelial cells,and the shear force produced by orbital oscillation can promote the alignment of fetal embryo endothelial cells in the direction of blood flow.It is then speculated that the action of ultrasound-induced radiation on vascular endothelial cells may affect the biological function of endothelial cells.Therefore,this project established a ultrasonic stimulation platform and fabricated the Piezo1 protein expression inhibition human umbilical vein endothelial cells?HUVECs?lines,to determine the regulation of ultrasonic stimulation on HUVECs and Piezo1 protein using the ultrasonic stimulation on Piezo1 expression inhibition group and control group cells,to determine the effects of ultrasonic stimulation and Piezo1 protein on the biological effects of HUVECs by detecting the related factors,and to explore a new treatment plan for the treatment of cardiovascular diseases.Methods1.Use an ultrasonic signal generator,an ultrasonic conversion probe and a confocal microscope to build a real-time ultrasonic-laser confocal detection platform.2.Design the si RNA sequence,use the RNA interference technology,transiently transfect the si RNA to construct Piezo1 protein expression inhibition HUVECs cell line:Piezo1 inhibition group and control group.3.Use the Fluo-4,AM,a calcium fluorescent probe,to detect intracellular calcium ion content,set the extracellular calcium ion environmental parameters of Piezo1 protein expression inhibition HUVECs cell line,the ultrasonic stimulation power and duration parameters.4.Use the Fluorescein-luciferase method to detect ATP content in HUVECs cell lines;use the ELISA method to detect the content of myosin light chain kinase?MLCK?in cell lines to explore the effects of ultrasonic stimulation and Piezo1protein on cell permeability.5.Use the Fluorescent probe DAF-FM DA method to detect the content of nitric oxide?NO?in HUVECs cell lines;use the ELISA method to detect the content of the cyclic guanosine phosphate?c GMP?and angiotensin??Ang-??in cell lines to determine the effects of ultrasonic stimulation and Piezo1 protein on vasoconstriction and relaxation;use the Fluorescent probe method to detect the content of reactive oxygen species?ROS?in HUVECs cell lines to determine the effect of ultrasonic stimulation and Piezo1 protein on intracellular ROS content.6.the RNAsequence information of HUVECs sample simulated was collected and analysed;use the ELISA method to detect human epithelial neutrophil activation peptide 78?ENA-78/CXCL5?and human neutrophil gelatinase-related lipocalin?NGAL/LCN2?to determine the effects of ultrasonic stimulation and Piezo1 protein on the immune-related inflammatory factors.Results1.Construction of cell model:The Piezo1 protein expression inhibition HUVECs cell line was contructed by the transient transfection of si RNA.It was successfully constructed with an inhibition efficiency about 80%.2.Ultrasonic stimulation environment and stimulation power:The extracellular calcium environment is conducive to calcium ion influx.During the ultrasonic stimulation process,it requires a certain concentration of calcium ions in the extracellular fluid for calcium ion transport.The intracellular calcium ion concentration was stable after 40 minutes incubation;the ultrasonic stimulation duration and power affected the influx of calcium ions.When the ultrasonic stimulation duration was 15 s,the stimulation of 1 w power caused most of the cells to break up and die.At 0.2 w power,only a few cells would break up.In the subsequent experiment,0.2 was the appropriate power;When the power was 0.2 w and the stimulation duration was 1 s,5 s,and 10 s,it was found that the cell morphology had a slightly change when the stimulation time was 10 s.In the subsequent experiment,the ultrasonic stimulation used less than 10 s was considered as the appropriate time.3.Ultrasound stimulation of Piezo1 protein regulates intracellular calcium concentration,ATP and MLCK:Under the calcium environment,when the different ultrasonic parameters acted on Piezo1 inhibition group and control group cells;at 0.2 w,after 1 s,5 s,8 s,and 10 s ultrasonic stimulation duration,it was found that as the stimulation time increased,the intracellular calcium ion flux continued to increase,and the intracellular calcium flux of the Piezo1 inhibition group was lower than that of the control group;by detecting the ATP content in the Piezo1 inhibition group and the control group,it was found that with the increase of stimulation time,the amount of ATP consumed in the cells gradually increased,and the ATP consumed in the Piezo1 inhibition group was lower than that in the control group;by detecting the MLCK in the Piezo1 inhibition group and the control group,It was found that with the increase of stimulation duration,the content of MLCK in the cells gradually increased,and the content of MLCK in the Piezo1 inhibition group was lower than that in the control group.4.Ultrasound stimulation of Piezo1 protein regulates intracellular NO,c GMP and Ang-?:When ultrasonic stimulation was applied to Piezo1 inhibition group and control group cells,the intracellular NO content was detected.With the increase of stimulation time,the intracellular NO content gradually decreased,and the NO production in the Piezo1 inhibition group was less than that in the control group;the c GMP content in the Piezo1 inhibition group and the control group was detected,and it was found that the intracellular c GMP content gradually increased as the stimulation time increased,and the c GMP in the Piezo1 inhibition group content was lower than that of the control group.The content of Ang-?in the Piezo1 inhibition group and the control group was detected,and it was found that as the stimulation time increased,the content of Ang-?in the cells had a slight difference after 3 s stimulation,but the difference was not obvious.There is not much difference in Ang-?content between the Piezo1 inhibition group and the control group.5.Regulation of intracellular ROS by ultrasound stimulation of Piezo1protein:After ultrasonic stimulation was applied to HUVECs,the intracellular ROS content was detected,and it was found that the intracellular ROS content gradually decreased with stimulation duration;after Piezo1 inhibition group and control group cells were exposed to 3s stimulation time,it was found that the ROS content in the Piezo1 inhibition group was much lower than that in the control group.6.Ultrasound stimulation of Piezo1 protein regulates intracellular immune-related factors CXCL5 and LCN2:When HUVECs were exposed to ultrasonic stimulation for 3s,RNAsequence data analysis showed that the CXCL5content in the ultrasonic group decreased and the LCN2 content increased;when the ultrasonic stimulation acted on the Piezo1 inhibition group and control group cells for1s,3s,5s,the content of CXCL5 was detected.It was found that with the increase of the stimulation time,the content of CXCL5 gradually decreased,and the Piezo1inhibition group reduced the content of CXCL5 in the cells;As the time of ultrasonic stimulation increased,the content of LCN2 in the cells gradually decreased,however the Piezo1 inhibition group increased the content of LCN2 in the cells.Conclusion1.Ultrasound stimulation of Piezo1 protein can regulate the permeability of cell membrane.2.Ultrasound stimulation of Piezo1 protein has the ability to cause vasodilation and reduce the content of ROS in cells.3.Ultrasound stimulation of Piezo1 protein can regulate cellular immune-related inflammatory factors. |
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