The Role And Mechanism Of Angiotensin System In Biological Behavior Of ADSCs Regulated By Low-energy Lasers

Background:Nerve and humoral factors intervene in many aspects of tissue repair and reconstruction after repair.In addition to its role as an important stress hormone system,the renin-angiotensin system(RAS)plays an important role as a multi-effect regulator in the repair of local tissue organ damage.Currently,the theory that adipose tissue is the target organ of the RAS system has been proposed,demonstrating the presence of RAS-related components in adipose tissue.Low-energy laser with laser biological effects,can promote cell proliferation,secretion of a variety of cytokines(including RAS system components),used in clinical wound repair and tissue regeneration,however,its cellular mechanism is unclear,and that lacks in-depth study on the expression of adipose-derived stem cells(ADSCs)and its influence on the biological behaviors of low-energy laser-regulated stem cells proliferation and differentiation.Objective:1.The h ADSCs system of human adipose-derived stem cells was cultured in vitro.The effect of two kinds of low-energy laser light sources,He-Ne laser and Ga Al As laser,on the growth and proliferation of ADSCs was observed.An the same time,we also observed the effects of He-Ne laser and g Ga Al As laser irradiation on the secretory function of ADSCs,especially the changes of important components in renin-angiotensin.2.The possible molecular mechanisms underlying the biological effects of low energy lasers on ADSCs.3.It was demonstrated that there is an independent renin-angiotensin system in adipose-derived stem cells.The effects of ANGII and ANG1-7,which are important components of the renin-angiotensin system,on the growth of ADSCs and the dose-effect relationship were observed,and the optimal dose was selected.Under these conditions,the effects of blockers of the ANGII and ANG1-7 downstream substrate receptors(AT1,AT2,and Mas)on the proliferation and differentiation of ADSCs were studied,and the pathways leading to changes were analyzed by CCK8.Methods:1.Human adipose tissue was isolated and cultured in ADSCs by collagenase digestion.After observing the growth state of stem cells,the phenotypic markers of cells and the ability of differentiation induction were used for identification.Next,we observed the effect of laser energy on 2 J/cm2 He-Ne laser and 4J/cm2 Ga Al As irradiated second-generation ADSCs to observe the effect on proliferation of ADSCs.At the same time,ELISA was used to detect the secretion capacity of TGF-?1,platelet-derived growth factor(PDGF),angiotensin II(Ang II)and angiotensin 1-7(Ang 1-7).2.The second generation of ADSCs was treated with 2J/cm2 He-Ne laser and4J/cm2 Ga Al As laser irradiation.Western blotting was used to detect the extracellular signal of ADSC before and after low-energy laser energy irradiation.Regulation of signal changes by kinases(ERK),phospho-ERK,(AKT),(phospho-AKT),(P38),(JNK).3.We demonstrated the presence of AT1,AT2,and MAS1 in ADSCs using RT-PCR.Next,using the enzyme-linked immunosorbent assay CCK8 to detect the proliferation of ADSCs and establish the optimal concentration of ANGII and ANG1-7,under this condition,by observing the receptor resistance of the downstream substrates of ANGII and ANG1-7 in the renin-angiotensin system.The effect of stagnant agents on the proliferation of ADSCs was used to analyze the effect of renin-angiotensin components on ADSCs.Results:1.The cells cultured in vitro were obtained by enzyme digestion.And the cells which labeled with CD59,CD29 and CD44 with a large number of positive cells were screened by cell surface markers adhered to the spindle and grew into the shape of a spindle.The ADSCs were induced by continuous culture Lipid staining showed lipid droplets of lipid droplets,osteogenic induction staining visible red calcium nodules and into cartilage can be seen visible dye blue proteoglycans.Compared with the blank control group,the absorbance results of the second-generation ADSCs irradiated by low-energy laser energy of 2J / cm2 He-Ne and 4J / cm2 Ga Al As laser irradiation were significantly promoted the proliferation of adipose-derived stem cells,which was significantly different from the control group(p <0.05).Low-energy laser irradiation can affect the secretion of ADSCs,the secretion of Ang II cytokines is inhibited,and the secretion of Ang 1-7,PDGF,TGF-? and other cytokines play a role in promoting.2.Low-energy laser irradiation inhibited the phosphorylation of AKT,P-ERK and P38 in h ADSCs,with significant difference between the two groups(P <0.05),increased P-AKT and JNK phosphorylation,with significant difference between the two groups.3.RT-PCR results showed that the expression of AT1,AT2 and MAS1 genes on adipose-derived stem cells.And we found cell proliferation experiments showed that ANGII significantly promoted the proliferation of ADSCs(P<0.05),and the concentration of 10-6 mol/l was the most obvious.Ang 1-7 significantly inhibited the proliferation of ADSCs(P<0.05),and the concentration of 10-7 mol/l was the most obvious.The ANGII,AT1 receptor blockers Losartan,AT2 receptor blockers PD123,319,Ang1-7,and Mas1 receptor blocker A779 showed significant differences in the proliferation of ADSCs by enzyme-linked immunosorbent assay CCK8(P<0.05).Conclusion:1.Low-energy laser therapy can enhance the biological behavior of ADSC through the P-AKT and JNK phosphorylation pathway,which may help to improve the Therapeutic effect of ADSCs.ANG? secreted by ADSCs and Ang 1-7 Regulate each other and play a balancing role in the biological behavior of adipose-derived stem cells.2.ADSCs adipose-derived stem cells exist independent renin-angiotensin system,the expression level of Ang II secreted by ADSCs decreased and the expression level of Ang l-7 increased after irradiation with low-energy laser.3.We found that adipose-derived stem cell activity was regulated by RAS,and the ACE-ANGII-AT1R/AT2 R pathway and ACE2-ANG(1-7)-MAS axis pathway played an important role.Angiotensin II promotes the proliferation and differentiation of ADSCs.The intrinsic receptor AT1 mediates the growth promoting effect of ANGII,while AT2 receptor mediates the growth inhibition of ANGII.The downstream substrate MAS of ANG1-7 inhibits the proliferation and differentiation of ADSCs,and antagonizes the positive regulation of ANGII on cells.