3.1. Macroscopic and histological evaluation of EF
Cell counting of the HE stained sections demonstrated that as the concentration of quercetin increased, fibroblast counts decreased. This results suggested that the local application of quercetin can reduce EF by reducing the number of fibroblasts (p < 0.05, Fig. 1(a,c)).Masson staining showed that the collagen content in the 200mg/kg group was the lowest, while decreasing collagen Ⅰ and collagen Ⅲ trends were related to the rise in drug concentration. These results indicated that the collagen content in the operation area decreased along with the increase in quercetin concentration ( p < 0.05, Fig. 1(b,d,e,f,g,h)).
3.3. Quercetin affects the biological behaviors of fibroblasts such as apoptosis, proliferation and migration
In regard to the investigation of the specific effect of quercetin on fibroblasts, cytological experiments were carried out. CCK-8 showed that the viability of fibroblasts decreased with the increase in quercetin concentration (p < 0.05, Fig. 3(a)). Fibroblasts were continuously treated with 20µmol/L quercetin, and their cell viability was detected at different times. Accordingly, the viability of fibroblasts was found to decrease with time in a time-dependent manner (p < 0.05, Fig. 3(b)). Edu staining showed that the number of positive cells decreased significantly (p < 0.05, Fig. 3(e,f)), indicating that quercetin had a significant inhibition on cell proliferation, while Western blot analysis yielded similar results. The expression of PCNA and Cyclin D1 decreased (p < 0.05, Fig. 3(c,d)), which further demonstrated the inhibitory effect of quercetin on the proliferation of fibroblasts.
In order to investigate the specific effect of quercetin on fibroblast apoptosis, fibroblasts with different concentrations of quercetin were stimulated for 24h. Afterward, TUNEL staining was used to detect whether quercetin had an effect on fibroblast apoptosis (p < 0.05, Fig. 3(j)). Next, Western blot assay was performed to detect the apoptotic proteins, which demonstrated the promotional effect of quercetin on the fibroblast apoptosis. (p < 0.05, Fig. 3(g,h,i)).
Finally, immunofluorescence staining was used to detect the expression of migration protein α - SMA, collagen Ⅰ and collagen Ⅲ (p < 0.05, Fig. 3(k,l,m)). The results showed that the expression of α - SMA decreased after quercetin, and collagen Ⅰ and collagen Ⅲ content were significantly inhibited. All of the corresponding results indicated that quercetin can inhibit the proliferation, migration and collagen secretion of fibroblasts while promoting their apoptosis.
3.4. Quercetin regulates fibroblast apoptosis and proliferation through autophagy, and mTOR mediated signaling pathway is involved
In order to study the specific mechanism of quercetin on the proliferation and apoptosis of fibroblasts, further experiments were performed. First, the expression of autophagy related proteins following quercetin were detected at different concentrations. With the increase in drug concentration,the results illustrated that the expression of Beclin 1, Atg5, and LC3 Ⅱ / LC3 Ⅰ increased, while that of p62 decreased (p < 0.05, Fig. 4(a,b)). This trend was closely related to drug concentration, which exhibited a concentration dependent manner. Moreover, LC3 immunofluorescence images demonstrated the presence of a significantly increased number of LC3 dots in fibroblasts following treatment of quercetin (p < 0.05, Fig. 4(c,d)). To further explore the role of autophagy, autophagy inhibitor 3-MA was used. However, following the stimulation of 3-MA, the low expression levels of Cyclin D1 and PCNA were partly reversed after quercetin induction. Additionally, the high expression levels of cleaved-PARP and LC3-Ⅱ/LC3-Ⅰ were found to be partially inhibited (p < 0.05, Fig. 4(e,f)). Meanwhile, the results of TUNEL, EdU assay and LC3 immunofluorescence yielded the same results (p < 0.05, Fig. 4(g,i,k,h,j,l)), which indicated that autophagy was inhibited after application of 3-MA.
Finally, proteins related to the classical pathway of autophagy were then detected. To further investigate its mechanism, the expression levels of related proteins in the mTOR mediated signaling pathway were detected. As the concentration of quercetin rose, the expression levels of p-PI3K, p-mTOR, and p-AKT decreased, while that of p-AMPK increased in a concentration-dependent manner (p < 0.05, Fig. 4(m,n)). The above results demonstrated that quercetin-induced negative regulation of mTOR mediated signaling pathway played an important role in the action of quercetin.