The Observation Of Autophagy-related Proteins In Human Trabecular Meshwork Cells And Effect Of P62 And NF-kappa B Silencing On Human Trabecular Meshwork Cell Viability

Background and aims:Autophagy is an evolutionarily highly conserved,complex cell degradation and recovery mechanism,which plays an important role in ocular cell growth and development and some pathological processes.The trabecular meshwork is the main regulator of the aqueous outflow channel in the eye.However,the trabecular meshwork tissue aging as the age increases.The dysfunction of trabecular meshwork tissue will lead to the pathological increase in intraocular pressure,which will further induce serious eye diseases such as glaucoma.In order to study the activation of autophagy in trabecular meshwork,this study collected 40 specimens of trabecular meshwork from healthy donors,and examined the expression of autophagy-related indicators LC3B and P62,and analyzed their correlation with age and gender.This will help to deepen the understanding of the degenerative diseases of trabecular meshwork,and also provide a certain experimental and theoretical basis for the future study of glaucoma living specimens.Nuclear factor-kappa B(NF-kappa B)is an evolutionary conservative transcription factor,which plays an important role in the development of glaucoma.Among them,P65 is the most active and effective subtype of NF-kappa B.In addition,recent studies have found that there is a mutual regulation mechanism between NF-kappa B and autophagy.In this study,human trabecular meshwork(HTM)with P62 and P65 gene silencing was established by lentiviral vector-mediated interference technique,and oxidative stress was induced in HTM cells by hydrogen peroxide.The effects of P62 and P65 on the activity of HTM cells were investigated.Lentivirus interference system has high efficiency and stability,which can be used to explore the biological role of other key proteins in autophagy and NF-kappa B signaling pathway in subsequent experiments.It also helps to explore the correlation and specific intermal mechanism of autophagy,NF-kappa B and glaucoma,and provides new ideas for clinical prevention and treatment of glaucoma.Methods:1.This study collected 40 cases of healthy comeal donors from January 2014 to June 2018 in the First Affiliated Hospital of Zhejiang University:divided into groups of?50 years old and ?40 years old according to age;divided into male group and female according to gender group.The expression and distribution of LC3B and P62 in trabecular meshwork tissues were detected by immunohistochemistry(immunoenzymatic labeling).The activation of autophagy was semi-quantitatively analyzed by Image-Pro Plus 6.0 software.The correlation between autophagy and age and sex was analyzed by Student's t test.2.The shRNA fragments of P62 and P65 genes were inserted into the lentivirus interference vector;the 293T cells and the lentiviral packaging plasmid system were used for virus packaging and concentration.3.HTM cells were infected by virus.The infection efficiency was measured by fluorescence microscopy and flow cytometry.The interference effect was detected by Q-PCR and Western-Blot,the HTM cell system silenced by P62 and P65 proteins was established(P62-shRNA-HTM,P65-shRNA-HTM).4.Wild type HTM cells(WT-HTM),negative control HTM(NC-HTM),P62-shRNA-HTM and P65-shRNA-HTM were stimulated by hydrogen peroxide to establish oxidative stress injury model.Cell viability was measured by CCK8.Results:1.The average optical density of P62 in normal TM tissues were significantly higher in ?40-year-old group than that in?50-year-old group(p<0.01);The expression of LC3B protein was weaker than that of P62,and the expression of LC3B protein in age?50 years old group was higher than that in ?40 years old group(p<0.05);There was no statistical difference in gender and P62 and LC3B protein expression.2.The plasmids of P62 and P65 lentiviral interference vectors were successfully constructed:P62-shRNA 1,P62-shRNA 2,P62-shRNA 3,P62-shRNA 4,P65-shRNA 1,P65-shRNA 2,P65-shRNA 3 and P65-shRNA 4.The viruses were packaged and concentrated in 293T cells.3.Flow cytometry showed that more than 90%of HTM cells were infected with lentiviruses;P62-shRNA 1,P65-shRNA 1 has the best silencing effect,and the interference efficiency is over 75%;In the control group,the expression of lentiviruses was not significantly affected.4.Oxidative stress damage in WT-HTM cells can be induced by 200?M concentration of H2O2(p<0.05);Under the stimulation of 200?M H2O2,cell death in the P62-shRNA-HTM group was increased 44.6%compared with the WT-HTM group(p<0.001),the P65-shRNA-HTM group increased 65.5%(p<0.0001);whereas the activity of NC-HTM cells was not significantly different from that of WT-HTM cells(p>0.05).Conclusion:1.The degree of autophagy activation in normal TM tissues was positively correlated with age and had no significant correlation with gender.2.The silencing system of P62 and P65 proteins in HTM cells was successfully established by using lentivirus vector mediated interference technology.3.Silencing P62 and P65 proteins under oxidative stress can increase HTM cell death.