Low-intensity pulsed ultrasound activates adipose-derived stem cells to enhance intracavernosal injection treatment for neurogenic erectile dysfunction
Objective To explore the efficacy and molecular mechanisms of low-intensity pulsed ultrasound(LI PUS)in enhancing adipose-derived stem cell(ADSC)i ntracavernosal injection(ICI)for the treatment of cavernous nerve injury-induced erectile dysfunction(CNI-ED).Methods A rat model of CNI-ED was established surgically and divided into five groups(25 rats in total),5 rats in each group:Sham group,Cavernous Nerve Injury(CNI)group,ADSC-ICI treatment group,LIPUS treatment group,and LIPUS combined with ADSC-ICI treatment(LIPUS+ADSC-ICI)group.Erectile function was evalua-ted by measuring intracavemosal pressure(ICP)/mean arterial pressure(MAP).Using 5-ethynyl-2'-de-oxyuridine(EdU)labeling,Western blotting,hematoxylin-eosin(HE)staining,Masson staining,and cell viability analysis,we explored cellular phenotypes and molecular mechanisms.comparisons between two groups were conducted using Student's t-test,and comparisons among multiple groups were performed u-sing one-way analysis of variance.Results ICP/MAP levels in the LIPUS+ADSC-ICI group exceeded CNI,ADSC-ICI,or LIPUS groups(0.79±0.06 vs.0.51±0.02,0.59±0.02,0.36±0.05,F=106.30,P＜0.05).Inflammation and fibrosis in penile tissue were lower in the LIPUS+ADSC-ICI group than in CNI,ADSC-ICI,or LIPUS groups(31.13±2.05 vs.80.70±4.30,70.36±3.83,56.35±3.37,F=472.10,P＜0.05).At 1st,3rd and 5th day after injection,ADSC retention in LIPUS+ADSC-ICI group was higher than that in ADSC group(6.49±0.78 vs.3.43±0.61;5.04±0.69 vs.2.04±0.46;4.09±0.54vs.1.36±0.41,t=6.93,8.04,9.10,P＜0.05);The proliferation capacity of AD-SC after LIPUS treatment and injection was higher than that of ADSC-ICI,LIPUS alone treatment group or CNI group(6.50±0.76 vs.3.78±0.88,3.23±0.63,1.17±0.38,F=85.23,P＜0.05).Platelet and endothelial cell adhesion molecule 1(CD31),endothelial nitric oxide synthases(eNOS),alpha smooth muscle actin(a-SMA),and calponin1 expression were higher in the LIPUS+ADSC-ICI group than CNI,ADSC-ICI,or LIPUS groups(3.34±0.16 vs.1.12±0.15,1.87±0.24,2.51±0.14,F=76.46,P＜0.05;2.24±0.20 vs.0.98±0.08,1.30±0.09,1.93±0.13,F=144.40,P＜0.05;2.71±0.14 vs.1.07±0.24,1.64±0.32,2.01±0.20,F=68.23,P＜0.05;2.42±0.12 vs.1.20±0.10,1.48±0.09,2.10±0.10,F=235.50,P＜0.05).Integrin levels were higher in LIPUS+ADSC-ICI group than in ADSC-ICI or LIPUS groups(2.99±0.14 vs.2.05±0.28,2.61±0.10,F=89.96,P＜0.05).Piezo1 knockdown reduced Integrin expression(1.83±0.42 vs.4.77±0.19,t=18.18,P＜0.05)and proliferation(111.60±2.81 vs.190.10±4.59,t=25.25,P＜0.05).Mechanis-tically,yes1 associated transcriptional regulator(YAP),ww domain containing transcription regulator 1(TAZ),F-actin,and protein tyrosine kinase 2(FAK)were higher,while phosphorylation yes1 associated transcriptional regulator(p-YAP)was lower in the LIPUS+ADSC-ICI group than CNI,ADSC-ICI,or LI-PUS groups(2.61±0.21vs.1.49±0.14,1.92±0.20,1.26±0.06,F=108.30,P＜0.05;2.08±0.41vs.1.44±0.17,1.78±0.24,1.24±0.20,F=236.70,P＜0.05;2.80±0.08vs.1.42±0.17,2.55±0.14,1.14±0.16,F=61.00,P＜0.05;2.71±0.14vs.1.52±0.14,2.37±0.18,1.24±0.18,F=184.90,P＜0.05;1.26±0.12vs.2.23±0.16,1.80±0.12,3.58±0.10,F=128.00,P＜0.05).Conclusion LIPUS activates the YAP/TAZ pathway by activating the Piezol-Integran axis on the ADSC membrane,regulating the proliferation and differentiation of autologous ADSCs and enhancing ADSC-ICI therapy.
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