Investigate The Mechanism Of Low-level Semiconductor Laser On Proliferation、Migration And Tube-like Structure Formation Of Vascular Endothelial Cells

Purpose:To investigate the effects of Low-level semiconductor laser/low-level laser therapy(LLLT)on the proliferation,migration and tube-like structure of vascular endothelial cells in vitro and their related molecular mechanisms.To elucidate low-energy laser therapy at the cellular and molecular level.The mechanism that promotes vascularization of prosthesis and controls the exposure of prosthetic eye sockets provides experimental evidence and provides new ideas for clinical treatment of angiogenesis-dependent diseases.Methods:1.To determine the optimal LLLT irradiation method for proliferating cells in vitro:In vitro culture of human umbilical vein endothelial cell line HUVEC cells,HUVECs treated with LLLT with different light parameters and irradiation modes,and the activity of HUVEC cells detected by CCK8 method to determine the low-energy laser light Therapeutic methods provide the best irradiation parameters and irradiation methods to promote cell proliferation in vitro.Among them,the parameters of laser parameters include:single irradiation dose/energy density(0.1-20.0 J/cm~2)and irradiation power density(2.07,5.25,9.92,20.99 And31.35mw/cm~2);irradiation mode variables include:irradiation interval time(48h,24h,12h,8h and 6h)different cumulative irradiation times(irradiation interval time is 12 hours,cumulative irradiation times were 2,4 and 6 times;Interval 24 hours,cumulative exposure 6 times).For each experiment,a control group without laser irradiation treatment was set up,and the other treatments were the same with the irradiation group.2.LLLT was treated with a single irradiation dose(1.0,2.0,4.0 J/cm~2,respectively),with an irradiation interval of 12 hours and cumulative irradiation for 6times.HUVEC was treated with LLLT and compared with a control group without LLLT treatment;(1)The effect of LLLT on the proliferation of HUVEC was detected by CCK8 assay and EdU cell proliferation assay;(2)The effect of LLLT on the migration of HUVEC was examined by scratch test;(3)The effect of endothelial cell lumen formation assay on the tube formation ability of HUVEC was examined.;(4)Methods The levels of PI3K and Akt protein phosphorylation in PI3K/Akt pathway in HUVEC were assayed,and the expression levels of eNOS,HIF-1αand VEGFA protein in cells were detected;(5)The level of VEGFA in cell culture supernatant was detected by ELISA.The situation;(6)Pretreated with LY294002(2μM)for 1h pretreatment and treated with LLLT to detect HUVEC proliferation,migration and tube-like structure formation and intracellular eNOS,HIF-1αand VEGFA expression levels.Results:1.Determination of the best irradiation method:(1)The optimal dose for promoting HUVEC proliferation is 4.0 J/cm~2.The effect of single dose of LLLT with different doses on the proliferation of HUVEC shows that a single irradiation dose of1.0 J/cm~2 can be achieved.To promote the proliferation of cells and increase the single dose(1.0-4.0 J/cm~2)in a certain range,LLLT can promote cell proliferation and continue to increase the single irradiation dose.LLLT promotes cell proliferation to weaken,even The cell proliferation was inhibited(20.0 J/cm~2);(2)different power densities on LLLT-induced cell proliferation showed that LLLT within the power density range of 2.07-31.35mW/cm~2,can promote cell proliferation,and in a single irradiation dose Under the same conditions,increasing the power density of laser irradiation can increase the proliferative effect of LLLT;(3)the best irradiation interval is 12 hours,and the LLLT with the same single irradiation dose and the same power density condition changes the irradiation interval time.The LLLT at 24h,12h and 8h could promote the proliferation of HUVEC.Among them,LLLT with a12-hour interval promoted the best cell proliferation,and the interval was too long(48h)to promote the proliferation of HUVEC.Is not obvious,the time interval is too short(6H)even inhibit cell proliferation,induce apoptosis.(4)The optimal cumulative irradiation times were 6 times.Under the conditions of a single irradiation dose,constant power density,and the same irradiation interval,the cumulative irradiation times of 6 irradiations were more effective than other groups in promoting irradiation,and cumulative irradiation Under the same number of conditions,compared with the interval time of 24h,the LLLT with a 12h interval between irradiations significantly increased the proliferation of cells.Based on the above results,it was determined that the LLLT exerted the best irradiation to promote cell proliferation in vitro:a single irradiation dose of 4.0 J/cm~2,an irradiation interval of 12 hours,and cumulative irradiation of 6 times2.The effects of LLLT on the proliferation,migration and tube-like structure formation of HUVEC:(1)The results of CCK8 and EdU cell proliferation assays showed that the cell viability increased significantly after LLLT treatment,and LLLT promoted the cell proliferation in a dose-dependent manner;(2)Scratch test results It was shown that the cell migration ability of LLLT treatment was significantly increased,and LLLT promoted the cell migration in a dose-dependent manner.With the increase of the number of laser irradiation,LLLT promoted the cell migration.(3)Endothelial cell lumen formation experiments showed that the single-shot irradiation dose of 2.0 and 4.0 J/cm~2 of LLLT treatment significantly improved the formation of tube-like structures compared to the control group and was dose-dependent.3.Western Blot and EILSA results showed that:(1)After treatment with LLLT,the phosphorylation levels of PI3K and Akt proteins increased,and the expression levels of eNOS,HIF-1α,and VEGFA increased,and the changes of protein phosphorylation and protein expression and laser The single irradiation dose was dose dependent;(2)LY294002 preconditioning 1 h pretreated with LLLT-treated HUVEC,compared with the irradiated group,LLLT promoted cell proliferation and cell migration was weakened,but had no significant effect on the formation of tube-like structure of HUVEC;Pretreatment with LY294002 for 1 h pretreatment could reduce the upregulation of eNOS,HIF-1αand VEGFA expression induced by LLLT and promote the secretion of VEGFA.Conclusions:1.LLLT up-regulates the expression of HIF-1α,eNOS and VEGFA in the cells and promotes the proliferation and migration of vascular endothelial cells through a PI3K signaling pathway-dependent manner.2.The role of LLLT in the formation of tube-like structures in vascular endothelial cells may be through activation of other signaling pathways within the cell.