Effect Of Human Umbilical Cord Blood Exosomes On The Secretion Of Inflammatory Factors From Macrophages Induced By Lipopolysaccharide

Background:Dry eye is a common multi-factor chronic ocular surface disease in clinical practice.Due to aging,the widespread popularity of smart-phone and other video terminals,its prevalence has increased gradually in recent years.At present,the drugs for the treatment of dry eye are mainly to relieve symptoms,and the therapeutic effect is not satisfactory.There is an urgent need to explore and develop more effective and personalized new drugs to meet people’s demand for the treatment of the disease.Chronic inflammation is the core factor in the pathogenesis of dry eye,and macrophages,as very important antigen-presenting cells,play a key role in the process of chronic inflammation.Stimulated by pathogens or inflammatory factors,resting macrophages are easily activated as macrophages M1(pro-inflammatory)or M2(antiinflammatory),thus regulating the inflammatory response.The exosomes are microvesicles secreted by cells,which are between 30 nm and 200 nm in size.They exist as membranous structures and have specific recognition functions.They have been widely investigated as carriers,biomarkers of biological diagnosis and medicine.They play a pivotal role in intercellular communication and immunomodulatory response,and widely exist in various body fluids and cells of organism.Autologous serum and umbilical cord serum eye drops are similar to natural tears and show a good therapeutic effect,but there is no consensus on their preparation method,quality control evaluation and treatment duration.As the exosomes from human umbilical cord blood plasma(HUCB-Exos)have many advantages,such as abundant resource,variety,easy to obtain and so on,this paper mainly uses human umbilical cord blood as raw material to obtain plasma supernatant by low-speed centrifugation,and then to extract plasma HUCB-Exos by ultracentrifugation to observe the effect of HUCB-Exos on lipopolysaccharide(LPS)under the condition of serum starvation.Investigating the relative expression of pro-inflammatory cytokines TNF-α and IL-6 secreted by macrophages induced by LPS at the level of m RNA would lay a foundation for further study on the regulatory effect and therapeutic potential of HUCB-Exos on dry eye inflammation.Objective:HUCB-Exos were isolated,obtained and identified from human umbilical cord blood.On this basis,to observe the effect of HUCB-Exos on macrophage inflammation model induced by LPS,to explore the anti-inflammatory effect of HUCB-Exos,and to provide a certain reference basis for HUCB-Exos in the treatment of ocular surface inflammation in dry eye and other eye diseases.Methods:1.The umbilical vein blood of delivery women was collected and the plasma supernatant was separated by low-speed centrifugation.The exosomes were extracted by ultracentrifugation,and the extracted HUCB-Exos were identified as follows: 1)The concentration of HUCB-Exos was determined by protein quantitative bicinchonininc acid(BCA).2)The diameter of HUCBExos was detected by dynamic light scattering(DLS).3)The morphology of HUCB-Exos was observed by transmission electron microscope(TEM).4)The marker proteins CD63,TSG101 and CD9 of HUCB-Exos were identified by western blot.2.Mouse mononuclear macrophage leukemia cell line(RAW264.7)was cultured.3.To explore the effects of LPS on the induced differentiation of RAW264.7 cells at different time(12 h and 24 h).The expression of inflammatory cytokines TNF-α and IL-6 at m RNA level was detected by real-time fluorescence quantitative polynucleotide chain reaction(RT-qPCR).The CM medium was replaced with DMEM medium to observe the differentiation degree of RAW264.7 cells induced by LPS under the condition of serum starvation,and an inflammatory model was established.RT-qPCR was used to detect the expression of TNF-α and IL-6 secreted by RAW264.7 cells induced by LPS under serum starvation.4.To explore the effect of HUCB-Exos with different concentrations on the inflammatory model of RAW264.7 cells induced by LPS,and the expression of inflammatory factors TNF-αand IL-6 at the level of m RNA was detected by RT-qPCR.Results:1.The characterization and identification results of HUCB-Exos: 1)The determination results of DLS showed that the diameter of HUCB-Exos was mainly concentrated in 99.94 nm,ranging from 30 nm to 200 nm,which conformed to the normal range of exosome’s diameter.2)The observation of TEM showed that the shape of HUCB-Exos was cup-shaped or oval-shaped,which was in accord with the general morphological description of exosomes.3)The results of western blot identification showed that HUCB-Exos simultaneously expressed CD63,TSG101 and CD9,which were consistent with the expression of marker proteins on the exosome’s surface.The identification results showed that the above three items were consistent with the characteristics of the exosomes.2.RT-qPCR results showed that when RAW264.7 cells were treated with 1 μg/m L LPS for 12 h and 24 h,the m RNA level of inflammatory cytokines TNF-α and IL-6 increased significantly(P<0.001),suggesting that the cells significantly transformed to M1 macrophages at 12 h after treatment with LPS(1 μg/m L).However,because the expression of TNF-α only doubled in numerical value,which was not conducive to the observation and comparison after HUCBExos administration in the later stage.The construction scheme of inflammation model needs to be further optimized.Therefore,then we replaced the culture supernatant of CM with DMEM to create an environment of serum starvation to observe the degree of differentiation of RAW264.7 cells induced by 1 μg/m L LPS for 12 hours.RT-qPCR results showed that 1 μg/m L LPS could significantly induce RAW264.7 to differentiate into M1 macrophages at the level of serum starvation(P<0.001).The m RNA level expression of TNF-α and IL-6 was significantly increased(P<0.05).The value changed significantly and showed statistical significance.3.The results of RT-qPCR showed that 20 μg/m L and 50 μg/m L HUCB-Exos could significantly reduce the relative expression of TNF-α and IL-6 at m RNA level.Conclusion:HUCB-Exos exert a certain anti-inflammatory effect on macrophage inflammatory model,and can significantly reduce the relative expression of inflammatory factors TNF-α and IL-6.