| ObjectiveDry eye is a common ocular surface disease,and its incidence is increasing in recent years.This study aims to explore the role and molecular mechanism of extracellular vesicles of human adipose stem cells in regulating ocular surface inflammation and inhibiting dry eye response,and to study the possibility of its clinical transformation,so as to provide a new method and idea for the prevention and treatment of dry eye in clinical practice.MethodsAnimal experiment: Female C57BL/6 mice of 6-8 weeks were selected and randomly divided into four groups(CON group,CON+ h ADSC-Evs group,DS+PBS group,DS+h ADSC-Evs group),with 6 mice in each group.Mice in DS+PBS group and DS+ h ADSCEvs group were injected with scopolamine subcutaneous on their hindlimbs 4 times a day(9:00,12:00,15:00,18:00).PBS and human adipose-derived stem cell extracellular vesicles were used for spot eye treatment,4 times a day,the time was consistent with the time of scopolamine injection.Mice were placed at a constant temperature(24±2℃),constant humidity(30±5%)and constant wind speed(2.5±0.5km/s)for 5 consecutive days.Mice in the CON group and the CON+ h ADSC-Evs group were reared in a normal environment and treated with PBS or human adipose derived stem cell extracellular vesicular point-eye therapy 4 times a day,as described above.After 5 days,tear secretion and fluorescein sodium staining were detected in each group.Complete eye tissues(including eyeball,complete upper and lower eyelid and palpebral conjunctiva)of mice in each group were collected,fixed with 4% paraformaldehyde,embedded with paraffin,sected with periodate acid-Schiff(PAS)staining,and the number of positive cells was statistically analyzed.γ-H2 Ax,Muc-5AC,NLRP3,Caspase-1,IL-1β immunofluorescence staining,combined with q RT-PCR assay,were used to detect the difference of NLRP3 pathway activation in the eye surface of mice in each group.The conjunctiva and cornea of mice in each group were taken for q RT-PCR test to detect the indexes of inflammatory factors in the conjunctiva of mice in each group.Cell experiment: Human corneal epithelial cell line HCECs were divided into CON group,450 m Os M group and 450 m Os M + h ADSC-Evs group with 6-well,12-well or 24-well plates.After 24 hours of cell starvation,the 450 m Os M group and the 450 m Os M +h ADSC-Evs group were replaced with 450 m Os M hyperosmotic medium,and the h ADSCEvs group was added with human adipose-derived stem cell extracellular vesicles.CCK-8kit was used to detect cell viability after treatment for different time according to experimental requirements.Cell apoptosis was detected by TUNEL staining kit.The expression of inflammatory cytokines and activation of NLRP3-IL-1β pathway were detected by q RT-PCR,Western Blot and immunofluorescence.ResultsCompared with the DS+PBS group,the lacrimal secretion was significantly increased,the corneal clinical score was significantly improved,and the number of conjunctival goblet cells and Muc-5AC expression were significantly restored;compared with the hypertonic control group,the activity of HCEC cells was significantly restored and the cells withered The expression of inflammatory factors(TNF-α,IL-1 β,IL-17,IL-6)in the ocular surface(cornea and conjunctiva)of the mice treated with adipose derived stem cells extracellular vesicles was significantly decreased,which was verified by cell experiment;QRT-PCR and Western blot results showed that adipose derived stem cells extracellular vesicles could significantly inhibit the expression of NLRP3,ASC,TNF-α,IL-1 β,IL-17,IL-6 in the ocular surface(cornea and conjunctiva)of the mice treated with adipose derived stem cells extracellular vesicles,Caspase-1 and IL-1β were up-regulated,which was verified in animal experiments.ConclusionThe extracellular vesicles of adipose derived stem cells have the potential to treat dry eye,and its therapeutic effect may be closely related to inhibiting the activation of NLRP3 inflammasome. |
PDF Full Text Request