A Preliminary Study On The Functional Mechanism Of Vitamin A On Respiratory Mucosal Epithelial Cell

Objective: Vitamin A(VA)is closely related to respiratory infection in children.It can promote the proliferation and differentiation of respiratory epithelial cells,but the underlying mechanism is still unclear.This study aimed to explore the effect of VA on normal human tracheal epithelial cells(HNTEC)stimulated with or without lipopolysaccharide(LPS),and whether it was involved in respiratory tract infections in children by regulating mitochondrial function and affecting cell metabolism.Methods: CCK-8 was used to detect the effects of VA and LPS on HNTEC proliferation and toxicity,and the treatment concentration of VA and LPS was determined.HNTEC and HNTEC stimulated by LPS were cultured with medium without or with 0.1mg/L,0.5mg/L and 2mg/L of VA.CCK-8 was used to detect the effect of VA on survival rate of HNTEC stimulated by LPS.Cell energy metabolism analyzer was used to analyze the effect of VA on the aerobic metabolism of cellular mitochondria.qPCR was used to detect expression level of p62,LC3 B,PINK1 and mitochondrial DNA copy number in VA-treated HNTEC stimulated with or without LPS.Flow cytometry was used to detect the changes of mitochondrial membrane potential and apoptosis of cells stimulated by LPS after VA treatment,and the expression of LC3 B and Bcl2 was detected by Western Blot.Results:(1)VA treatment concentration was 0.1mg/L,0.5mg/L,2mg/L,and LPS treatment concentration was 200μg/m L.(2)After VA treatment,HNTEC stimulated by LPS for 24 h showed that the cell survival rate increased with the increase of VA concentration.At 48 h,the cell survival rate of 2mg/L VA+LPS group was decreased.Compared with the control group,at 24 h and 48 h,ATP production,the maximum respiratory potential and proton leakage were increased in 0.1mg/L VA group and 0.5mg/L VA group,but the above indexes decreased in 2mg/L VA group.(3)Compared with LPS group,In HNTEC stimulated by LPS for 24 h and 48 h after VA treatment,the percentage of cells with reduced mitochondrial membrane potential in VA treatment group was increased,and it increased with the increase of VA concentration(P<0.05).Compared with the control group,the expression level of p62 and cell mitochondrial DNA copy number of HNTEC after VA treatment for 24 h was increased,and it increased with the increase of VA concentration(P<0.05).At48 h,p62 expression level was significantly increased(P<0.001),but p62 level in2mg/L VA group was lower than that in 0.5mg/L VA group(P<0.05).Mitochondrial DNA copy number was increased only in 0.1mg/L VA group(P<0.05).Compared with the LPS group,in HNTEC stimulated by LPS for 24 h after VA treatment,PINK1 expression level was decreased in 0.1mg/L VA+LPS group and 0.5mg/L VA+LPS group(P < 0.05).The copy number of mitochondrial DNA was significantly increased in 0.1mg/L VA+LPS group(P<0.05).At 48 h,PINK1 expression level presented a decreasing tendency in VA treatment group.LC3 B gene and protein expression was decreased with the increase of VA concentration(P<0.05),but the copy number of mitochondrial DNA was increased only in 0.5mg/L VA+LPS group(P<0.001).(4)Compared with the LPS group,in HNTEC stimulated by LPS for 24 h after VA treatment,Bcl2 expression was significantly increased in VA treatment group(P<0.001),while at 48 h,Bcl2 expression showed a decreasing tendency only in the0.1mg/L VA+LPS group.The apoptosis rate in VA treatment group was lower than that in LPS group.The number of early apoptosis cells was the least in 0.1mg/L VA+LPS group.The number of late apoptosis cells was the least in 0.5mg/L VA+LPS group(P<0.05).But in 2mg/L VA+LPS group,the apoptosis rate was increased compared with other groups.Conclusion: VA can affect the metabolism of human main bronchial epithelial cells by regulating mitochondrial function and quantity,and play a certain role in the anti-infection process of respiratory cells.