The Mechanism And Role Of Exosomes Derived From HucMSC For Allevivating Diabetic Retinopathy Via Regulating PGC-1α

Objective: Diabetic retinopathy(DR)is the most common microvascular complication of diabetes and the main cause of blindness in adults.The current treatments mainly include laser treatment,intravitreal injection of anti-angiogenic factor drugs and surgery,but there is a lack of ideal prevention and treatment programs.The purpose of this study is to explore the role and mechanism of human umbilical cord mesenchymal stem cells derived exosomes(hucMSC-Ex)in the DR model,so as to provide new strategy for the management and treatment of DR.Methods: A high-fat diet combined with streptozotocin(streptozotocin,STZ,35mg/kg)DR rat model in vivo was built.Based on the exosomes isolation and extraction technology established in the early stage of our research group,intravitreal injection of hucMSC-Ex was performed for intervention,and exosomes derived from human fetal lung fibroblasts-1 cells(HFL1-Ex),hucMSC-conditioned medium(hucMSC-CM),the exosomes-free conditioned medium(hucMSC-Ex free CM)and PBS were used as controls.16 weeks after modeling,retinal tissue HE staining was used to observe the retinal thickness and structural disorder of each group,Evans blue staining was used to detect the vascular leakage of each group of retinal tissues,and Western blot,q RT-PCR,immunohistochemistry,etc.were used to detect the level of proliferation,apoptosis,oxidative stress and DNA damage in order to comprehensive evaluate the repair effect of hucMSC-Ex on DR.In vitro,human retinal pigment epithelial cell(ARPE-19)was used as a cell model,treated with 30 m M high glucose stimulation,and hucMSC-Ex was added for intervention.Growth curve,CCK8,Western blot were applied to observe cell proliferation,viability and apoptosis,and immunofluorescence probe was used to detect the level of reactive oxygen species(ROS).Based on high-throughput sequencing,miRNAs profile analysis on hucMSC-Ex and HFL1-Ex were performed to screen for differentially expressed miRNAs that may inhibit oxidative stress,and in ARPE-19 cells,miRNAs mimics and inhibitor were transfected to clarify the molecular mechanism of miRNAs.Finally,we applied electroporation technology to prepare miRNAs engineered hucMSC-Ex,and applied engineered hucMSC-Ex to models in vivo and in vitro to explore its enhanced therapeutic efficacy.Results: In the DR model,HE staining revealed that the overall thickness of the retinal tissue was thinner,and the structure of each layer was disordered.Western blot,q RT-PCR and TUNEL staining showed that retinal cell apoptosis,oxidative stress and DNA damage were obvious.Evans blue staining results presented that the blood vessel leakage was severe,and the blood-retinal barrier was destroyed in the DR group.Compared with other control groups,the group after hucMSC-Ex treatment the overall thickness of the retina increased,the tructure of each layer was clear,the retinal vascular leakage was restored,and reduced oxidative stress and DNA damage in vivo.And in vitro,after hucMSC-Ex treatment,it could reverse the weakened proliferation and ROS production of ARPE-19 cells caused by high glucose.Peroxisome proliferator-activated receptor γ coactiva-tor-1 alpha(PGC-1α)was involved in the regulation of oxidative stress,and hucMSC-Ex could activate PGC-1α expression both in retinal tissues and retinal cells based on experiments results in vivo and vitro.According to the miRNAs sequencing results of hucMSC-Ex and HFL1-Ex,the enrichment of miRNA molecules in hucMSC-Ex,such as NC＿000016.10＿10228(referred to as miR-10228)were screened that could regulate PGC-1α.The electroporation technology was applied to electroporate miR-10228 mimics into hucMSC-Ex to prepare engineered exosomes(miR-10228 engineered hucMSC-Ex).The results showed that compared with the group injected with hucMSC-Ex alone,the miR-10228 engineered hucMSC-Ex showed stronger anti-apoptosis,anti-oxidative stress DNA damage,pro-proliferation and protection of the blood-retinal barrier,and it could alliviate the DR process more effective.Conclusions: Our findings indicate that hucMSC-Ex can activate PGC-1α and delay the progression of DR by transferring miR-10228.The miR-10228 engineered hucMSC-Ex is expected to become a new measure for DR treatment.