The Study Of The Mechanisms Underlying The Effects Of LIPUS Combined With SonoVue On Renal Glomerular Endothelial Cells

BackgroundWith the development of research on biological effects of ultrasound,the clinical application of ultrasound technology has entered the stage of disease treatment from simple disease diagnosis,which is of great significance for many specialties of clinical medicine.In the 1980s,high-pressure amplitude shock wave began to be used in the mechanical treatment of kidney stones,and "lithotripsy" quickly replaced surgery as the most common treatment option.The scope of application of ultrasound treatment is expanding.At present,the approved applications include uterine fibroid ablation,cataract extraction(phacoemulsification),surgical tissue cutting and hemostasis,percutaneous drug delivery,fracture healing,etc.Hysteromyoma ablation uses the thermal effect of high intensity focused ultrasound(HIFU).At the same time of myoma ablation,it will cause damage to surrounding tissues and blood vessels,causing inflammatory reaction.Compared with HIFU,the advantage of low-intensity pulse ultrasound(LIPUS)is that it has a limited and controllable range of action,negligible thermal effect,and can inhibit inflammatory reaction,promote fracture healing and promote soft tissue regeneration.As adverse biological effects may occur in the process of ultrasound treatment,standardization,ultrasound dosimetry,benefit assurance and risk minimization of side effects must be carefully considered in the application to ensure the safety of therapeutic ultrasound and the best benefit risk ratio of patients.On the other hand,the development of ultrasound contrast agent has gone through several stages.SonoVue is of the most widely application of ultrasound contrast agents approved for marketing in China,which has the characteristics of good stability,not easy to crack and strong harmonic signal.However,there are still many problems to be solved in the treatment of some diseases,such as the possible adverse effects on the surrounding normal cells,which need further research.Renal tumors seriously affect the quality of life,will cause the reduction of nephron and the deterioration of renal function.Many patients can not tolerate the surgical treatment,non-surgical alternative treatment is very necessary.The purpose of this study is to explore the safety and clinical value of LIPUS,LIPUS and SonoVue in the treatment of renal tumors.The existing problem is that before a new treatment method is used in animal and human experiments,it is necessary to observe its effect on normal kidney cells.If there is no damage to normal cells,then follow-up animal experiments can be carried out;if there is damage to normal cells,then follow-up can not be carried out.Therefore,we take hrgec as the research object,whether LIPUS combined with SonoVue technology has effect on hrgec,and provide necessary basic experimental data for clinical treatment.ObjectivesThe purpose of this part is to compare the effects of LIPUS or SonoVue alone or in combination on the proliferation and apoptosis of HRGEC in vitro,explore its potential mechanism and preliminarily explore the safety of this technology.MethodsHRGEC cultured in vitro were divided into four groups and treated with four different methods:(1)normal control group(2)LIPUS ultrasound treatment group(3)SonoVue treatment group(4)LIPUS and SonoVue combined treatment group.The ultrasound irradiation was low-intensity pulse ultrasound,frequency 1 MHz,duty cycle 20%,action time 5 min,SonoVue contrast agent concentration 20%.1.The toxic effects of different treatment methods on HRGECFirst of all,after the cells were treated with different methods,the proliferation ability of HRGEC was measured by MTT test,and the cell viability was evaluated by trypan blue exclusion test,so as to judge the toxic effect of the above-mentioned different treatment methods on cells.2.Effect of different treatment methods on apoptosis of HRGEC cellsHRGEC were treated with different methods and cultured for 24 hours.The cells were collected and analyzed by FITC-Annexin V/PI flow cytometry.3.Effect of different treatment methods on ERK pathwayWestern blot was used to detect the total expression and phosphorylation level of ERK protein in HRGEC,so as to evaluate the effect of LIPUS and SonoVue on ERK signaling pathway.Then HRGEC were treated with LIPUS and SonoVue,and the expression of phosphorylated ERK1/2 was detected at different time points(12 hours,24 hours),4.Function recovery experiment to verify the function of ERK signal pathwayHRGEC were pretreated with 10 ?mol/l PD98059(ERK signaling pathway inhibitor)for 1 hour,and then treated with LIPUS,SonoVue alone or LIPUS combined with SonoVue.Western blot was used to detect the level of phosphorylated ERK1/2 protein in each group.In addition,the proliferation rate,viability and apoptosis of PD98059 cells were compared.Results1.LIPUS combined with SonoVue inhibits HRGEC cell viabilityHRGEC treated with LIPUS or SonoVue had no significant difference in cell viability and proliferation(P>0.05),while the combination of LIPUS and SonoVue could significantly reduce cell viability and proliferation compared with other groups(average P<0.01).2.The effect of LIPUS combined with SonoVue on apoptosis of HRGEC cellsAlthough HRGEC treated with LIPUS and/or SonoVue did not cause serious damage to cells,LIPUS combined with SonoVue led to a significant increase in the apoptosis of HRGEC compared to the control group(average P<0.05).3.The combination of LIPUS and SonoVue affects HRGEC by inhibiting ERK pathway activationThe expression of ERK protein was similar in different treatment groups.The phosphorylation level of ERK1/2 did not change in HRGEC treated with LIPUS or SonoVue alone,but decreased significantly in HRGEC treated with LIPUS and SonoVue.The expression of phosphorylated ERK1/2 in HRGEC treated with LIPUS and SonoVue decreased at 12h,and decreased more significantly after 24h,i.e.the inhibition was more obvious with the increase of treatment time.In addition,after PD98059 pretreatment,there was no significant difference in the proliferation rate,viability and apoptosis of HRGEC in four different treatment groups(P>0.05).Conclusion:Compared with LIPUS or SonoVue alone,the joint action of both can induce apoptosis and inhibit proliferation effect of HRGEC via inhabiting ERK signaling pathway.Background Endothelium forms the interface between circulating blood and supplying tissue,which is very important for the stability of vascular biology.There are four kinds of intercellular connections: tight connection,adhesion connection,gap connection and desmosomes.The endothelial cell connection mainly includes tight connection(TJs)and adhesion connection(AJs).AJs contributes to the stability of cell-cell adhesion and the regulation of actin cytoskeleton and intracellular signal.AJs is mainlycom posed of Ca2 + ve and belongs to Ca2 + dependent adhesion molecule family.Similar to classical cadherin,ve cadherin binds to pi20 catenin and 3-Catenin,which connects cadherin catenin complex to actin cytoskeleton through a-Catenin.Ve cadherin is widely distributed and expressedin endothelial cells.Importantly,VE-cadherin is very important for the formation of stable and mature blood vessels.In addition,VE-cadherin blocking antibody can increase the microvascular permeability.In addition,ve cadherin regulates the signal pathway of TJ structure and tissue.Therefore,VE-cadherin is the target of many molecular pathways involving vascular leakage.Vascular permeability is finely regulated in normal blood vessels and can adapt to many environmental changes in the surrounding micro environment,including angiogenesis,shear stress,flow and inflammation.Intercellular connections,especially VE-cadherin,are actively involved in these changes of vascular barrier.In fact,the integrity of VE-cadherin can be changed in many ways,including phosphorylation,destabilization of catenin interaction,vascularization,abscission and contraction of myosin,all of which ultimately increase the permeability of endothelial cells.Glomerular endothelial cells are typical vascular endothelial cells.Some studies have shown that VE cadherin is widely distributed in this cell and participates in the development of most kidney diseases.The results show that ultrasound contrast agent can reduce the cavitation threshold of ultrasound,increase the cavitation effect of ultrasound and "perforate"(acoustic pore effect)on the cell membrane,so as to increase the permeability of cell membrane.At present,there are many studies on the effects of ultrasound and / or ultrasound contrast agents on cell membrane permeability,but few studies on the effects of ultrasound and ultrasound contrast agents on cell-cell permeability and molecular mechanism.Previous experiments showed that LIPUS combined with SonoVue could inhibit the proliferation of HRGEC,promote cell apoptosis and possibly affect renal glomerular filtration by inhibiting the activation of ERK1/2signaling pathway.However,whether LIPUS and / or SonoVue alone or in combination affect the intercellular permeability of glomerular vascular endothelial cells,whether VE-cadherin is involved in it,and whether its upstream regulatory mechanism is unclear.Objectives The point of this part is to explore the effects of LIPUS and SonoVue on the adhesion of HRGEC,to seek for relevant influence factors and explore the mechanism of glomerular filtration barrier damage.Methods HRGEC cultured in vitro were divided into four groups and treated with four different methods:(1)normal control group(2)LIPUS ultrasound treatment group(3)SonoVue treatment group(4)LIPUS and SonoVue combined treatment group.The ultrasound irradiation was low-intensity pulse ultrasound,frequency 1 MHz,duty cycle 20%,action time 5 min,SonoVue contrast agent concentration 20%.1.The effect of different treatment methods on the paracellulerpermeability ofHRGECcells Transwell cell permeability test was used to detect the intensity of FITC-dextran fluorescence in the inferior chamber and compare the intercellular permeability of hrgec cells under different treatment conditions.2.Effect of different treatment methods on the expression of VE-cadherin The expression of VE-cadherin at mRNA and protein levels was detected by real-time PCR and Western blot.At the same time,the expression of VE-cadherin in hrgec cells was observed by immunofluorescence.3.The effect of different treatment methods onthe expression of ETS-related gene(ERG)and its role in the altered paracelluler permeability Western blot was used to detect the expression of ERG protein in HRGEC.The expression of VE-cadherin protein was detected by Western blot.FITC dextran Transwell test was used to detect the changes of intercellular permeability after the interference of ERG expression.On the other hand,after overexpression of ERG gene in HRGEC,the cells were treated with LIPUS,SonoVue and LIPUS combined with SonoVue.Transwell experiment was used to evaluate the permeability difference between the cells in each group after overexpression of ERG gene,and to determine whether the above phenotype changes induced by different treatment methods were caused by the down regulation of ERG gene.Results1.The combination of LIPUS and SonoVue increases the paracelluler permeability of monolayer HRGEC The permeability of HRGECcells treated with LIPUS and SonoVue alone was higher than that of the control group,but the combination of LIPUS and SonoVue could significantly increase the permeability of HRGECcells(P < 0.01),and the difference was statistically significant(P < 0.05).2.The combination of LIPUS and SonoVue reduces the expression of VE-cadherin After treatment with LIPUS or SonoVue,the expression of VE-cadherin did not significantly change compared with the control group;when the cells were treated with LIPUS and SonoVue,the expression of VE-cadherin mRNA and protein decreased significantly compared with other groups.At the same time,immunofluorescence results showed that the expression of VE cadherin protein in the junction of the cells was significantly lower than that of other groups.These results showed that LIPUS combined with SonoVue treatment could increase the expression of VE-cadherin protein,reduce the intercellular adhesion,and thus affect the paracellulerpermeability of cells.3.The combination of LIPUS and SonoVue affects paracelluler permeability by inhibiting the expression of ERG In addition,the expression level of ERG protein in HRGEC decreased significantly after the treatment of LIPUS and SonoVue.When the expression of ERG was disturbed,the expression level of VE cadherin also decreased significantly,which further indicated that ERG was the upstream regulator of VE cadherin.After the interference of ERG gene,the cell permeability increased significantly,which was similar to the result of the combination of LIPUS and SonoVue.After overexpression of ERG gene,hrgec cells were treated with LIPUS,SonoVue and LIPUS combined with SonoVue.There was no significant difference in permeability between the cells in each group(P > 0.05).Conclusion:LIPUS combined with SonoVue can inhibit the expression of VE-cadherinby down regulating the ERG gene,thus reducing the adhesion between HRGECcells and increasing the paracelluler permeability.This is the potential mechanism of mechanism of glomerular filtration banier damage.