ABSTRACT
Objective: To investigate the effect of Inula britannica flower total flavonoids (IBFTF) on the expression of lncRNA TERRA in the senile skin fibroblasts and its mechanism. Methods: High performance liquid chromatography (HPLC) was used to analyze the composition and content of IBFTF. The human skin fibroblasts cell line was divided into control group, IBFTF group (cells were treated with IBFTF alone), cell senescence model group [cell senescence model was induced by D-galactose (D-gal)] and cell senescence model + IBFTF group (cell senescence model was treated with IBFTF). The cell viability was detected by CCK-8. The senescence-associated β-galactosidase (SA-β-Gal) staining was used to detect the positive rate of human skin fibroblasts cells, and the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were detected by enzyme microplate reader. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA TERRA, hTERT mRNA and the relative length of telomere in the cells. The protein expression of agingrelated proteins such as p53, p16 and human telomerase reverse transcriptase (hTERT) were detected by Western blotting. Results: The contents of quercetin, isorhamnetin, kaempferol and rutin in IBFTF were 6.447, 2.044, 1.272, 0.781 mg/g, respectively. CCK-8 and SA-β-Gal staining showed that IBFTF increased the viability of cells in senescence model group, and reduced the proportion of SA-β-Gal positive cells (P<0.05). Enzyme labeling results showed that IBFTF increased the activity of SOD and GSH-Px, and reduced the MDA level of senescent cells (P<0.05). qRT-PCR showed that IBFTF decreased the expression of lncRNA TERRA, increased the expression of hTERT mRNA and prolonged the telomere length in senescent cells (P<0.05). Western blotting results showed that, compared with cell senescence model group, the protein expressions of p53, p16 decreased, and the hTERT increased in cell senescence model + IBFTF group (P<0.05). Conclusions: IBFTF can decrease the expression of lncRNA TERRA in D-galinduced senescent human skin fibroblasts cells, and its mechanism may be related to the recovery of telomerase activity and the prolongation of telomere length, so as to achieve the effect of delaying senescence.
ABSTRACT
Objective To investigate the protective effect of Inula britannica flower total flavonoids (IBFTF) on aging L929 cells induced by advanced glycation end products (AGEs), and to explore the mechanism of the expression of receptor advanced glycation end products (RAGE). Methods The senescence L929 cells model was established by using 0.100 g/L AGEs for 48 h culture in vitro, and then different concentration of IBFTF (0.1, 0.2, and 0.4 g/L) was given to the treatment group for 6 h culture, and 0.1 g/L aminoguanidine hemisulphate (AG) was given to the positive group for another 6 h culture, and while blank group was cultured with common culture medium. Senescence aging index of β-galactosidase staining cell numbers and cell cycle analysis in L929 cells were measured. The levels of reactive oxygen species associated with oxidative stress in the cells, and aging indicators such as superoxide dismutase (SOD), malondialdehyde (MDA) were also examined. The fluorescent intensity of RAGE was detected by immunofluorescence. The expression of RAGE protein and mRNA was detected by Western blotting and qPCR, respectively. Results IBFTF significantly inhibited AGEs-induced L929 cells senescence and decreased RAGE protein and mRNA expression. Different concentrations of IBFTF could significantly increase the SOD activity and reduce the MDA content and eliminate the reactive oxygen species in aging L929 cells in a dose-dependent manner. Conclusion Protective effect of IBFTF on aging L929 cells induced by AGEs may be related to its effect on the surpressing the expression of RAGE.