Cultivation of RBFs
After 24 h of primary culture, the cell morphology of RBFs was triangular or nearly spindle-shaped, and the cells adhered to the culture bottle by enzyme-coupled digestion. On the third day, practically all the cells turned spindle-shaped, with a few polygonal cells. On the fifth day, the cytoplasm of the cells expanded and merged into a single layer. Then the cell structure became spindle-shaped with copious cytoplasm and clustered growth. The vast majority of nuclear divisions were visible. On the seventh day, fibroblast fusion caused cells to proliferate rapidly, with a convergence rate of up to 90% and tight connections. The cells become thin and undulates in a linear, radial, or palisade shape, with certain parts stacked with many layers of development (Fig. 1).
On the fifth day of primary cell culture using the tissue block adhesion method, the cells adhering to the wall were primarily polygonal, with irregular and long spindle shapes being uncommon. On the seventh day, the cells formed spindles, and the number of cells was reduced. Around the 14th day, nearly all the cells became spindle-shaped with branching protrusions and copious cytoplasms. The cells were organized in a single layer parallel to the radial or paliform pattern (Fig. 2). They can be handed down for approximately 14 days.
The optimal intervention concentration and time for RBFs by TGF-β1
CCK8 detection data indicated that increasing TGF-β1 concentrations (5, 10, and 20 ng/mL) resulted in significant increases in RBF OD450 values after 24 h (P < 0.05). Among them, when the concentration of TGF-β1 was 20 ng/mL, the OD450 value of RBFs reaches its peak and cell proliferation was at the highest level detected (Fig. 4). Therefore, we considered the optimal concentration of TGF-β1 as 20 ng/mL for subsequent experiments. As the intervention time increased, The OD450 of the RBFs continued to increase with the extended intervention time (P < 0.05). The intervention duration was excessively long, resulting in rapid cell multiplication and affecting subsequent trials. Consequently, we determined that 2 h was the most effective intervention time (Table 1).
Table 1
Optimal time of TGF-β1 on RBF proliferation
Entry
|
Concentration of TGF-β1(ng/mL)
|
OD450 value of RBFs
|
2 h
|
4 h
|
8 h
|
12 h
|
A
|
-
|
-
|
-
|
-
|
-
|
B
|
-
|
0.45 ± 0.08
|
0.45 ± 0.08
|
0.45 ± 0.08
|
0.45 ± 0.08
|
B
|
20
|
0.87 ± 0.12*
|
1.38 ± 0.63*
|
1.58 ± 0.26*
|
1.74 ± 0.52*
|
A: Control group: Containing CCK-8, without RBF cells and TGF-β1 culture medium; B: Experimental group: containing CCK-8 and RBFs cells. "-" indicates no addition. *P<0.05(± s, n = 6)..
The optimal intervention concentration of rapamycin on the response time of RBFs
The CCK8 results showed that intervention with 1, 5, 10, and 20 nmol/mL of rapamycin after 24 hours, the cell survival rates were 94.36% ± 0.55%, 85.38% ± 0.13%, 70.89% ± 0.21%, and 31.93 ± 0.12%, respectively. As the concentration increased, the survival rate decreased, and the higher the concentration, the stronger was the inhibition of cells. When the concentration of rapamycin was 10 nmol/mL, the inhibitory effect on cells was more stable than that at other concentrations after 24 h of intervention. The cell survival rate satisfied the experimental requirements and differed significantly from that of the control group (P < 0.05). Therefore, 10 nmol/mLM was selected as the most effective rapamycin concentration in this study (Table 2).
Table 2
Effect of rapamycin on the inhibition rate of RBFs
Entry
|
Concentration of rapamycin (nmol/mL)
|
Inhibition rate (%)
|
Survival rate (%)
|
A
|
-
|
-
|
-
|
B
|
-
|
0
|
100
|
B
|
1
|
5.64 ± 0.55*
|
94.36 ± 0.55*
|
B
|
5
|
14.62 ± 0.13*
|
85.38 ± 0.13*
|
B
|
10
|
29.11 ± 0.21*
|
70.89 ± 0.21*
|
B
|
20
|
68.17 ± 0.12*
|
31.93 ± 0.12*
|
A: Control group, Containing CCK-8 without RBF cells and rapamycin culture medium; B: Experimental group, containing CCK-8 and RBFs cells. Inhibition rate = [(ODA - ODB)/ODA] × 100%; survival rate = 1- inhibition rate. "-" indicates no addition. *P<0.05 (± s, n = 6).
The CCK8 assay results revealed that after intervention with rapamycin at a concentration of 10 nmol/mL for 2, 4, 8, 12, and 24 h, the OD450 value of RBFs decreased as the rapamycin stimulation period increased. RBFs had significantly higher OD450 values than the control group at different time points at the same concentration (P < 0.05). The results showed that longer rapamycin exposure at 10 nmol/mL of RBFs, the stronger the inhibition of cells (Table 3). Therefore, 2 h was selected as the most effective intervention time.
Table 3
Optimal inhibition time of rapamycin on RBFs
Entry
|
Concentration of rapamycin (nmol/mL)
|
OD450 value of RBFs
|
2 h
|
4 h
|
8h
|
12h
|
A
|
-
|
-
|
-
|
-
|
-
|
B
|
-
|
0.35 ± 0.16*
|
0.35 ± 0.16*
|
0.35 ± 0.16*
|
0.35 ± 0.16*
|
B
|
10
|
0.87 ± 0.12*
|
0.31 ± 0.08*
|
0.29 ± 0.09*
|
0.26 ± 0.03*
|
A: Control group: Containing CCK-8, without RBF cells and rapamycin culture medium; B: Experimental group: containing CCK-8 and RBFs cells. "-" indicates no addition. *P<0.05 (± s, n = 6).
The effect of TGF-β1 for rapamycin induced autophagy on the expression of related proteins in RBFs
Autophagy is a process by which lysosomes break down organelles and macromolecules, recycle them, and create new cellular components. This mechanism is crucial for the maintenance of intracellular homeostasis [21]. Autophagic activity was evaluated by autophagy-related proteins such as LC3-II, Beclin-1, and P62 [28–31]. Western blot analysis was performed to determine the expression levels of LC3II, Beclin1, and P62 proteins in rapamycin-induced RBFs after autophagy (Fig. 5). The results show that the addition of rapamycin to normal cells increased the expression of LC3-II and Beclin-1 in RBFs and decreased the expression of P62 protein (P < 0.05). However, adding TGF-β1 and intervention for 2 hours decreased the expression of LC3-II and Beclin-1 proteins, while increasing the expression of P62 protein (P < 0.01).
The effect of TGF-β1 for starvation induced autophagy on the expression of related proteins in RBFs
After EBSS-induced autophagy, LC3-II and Beclin-1 expression levels increased significantly compared to those in the control group (P < 0.05). After 2 h of EBSS induction, the expression levels of LC3-II and Beclin-1 also increased. Protein expression peaked after 12 h of fasting and began to decline after 24 h (Fig. 6A, 6C). After adding TGF-β1 for 2 or 4 h, no significant difference in LC3-II expression levels was observed compared to the simple starvation group (P > 0.05) (Fig. 6B). The TGF-β1 intervention group had lower LC3-II expression levels than the basic starvation group at 8–24 hours (P < 0.05). Beclin-1 protein expression increased after 2 h of fasting, peaked at 12 h, and decreased at 24 h (P < 0.05; Fig. 6C). Figure 6D shows that TGF-β1 lowered Beclin-1 expression levels across groups (P < 0.05). After 2 h of starvation, the P62 protein expression level did not differ significantly from that in the control group (P > 0.05). After 4 h of starvation, P62 protein expression decreased, reaching its lowest level after 24 h (P < 0.05; Fig. 6E). Adding TGF-β1 increases P62 expression levels (P < 0.05; Fig. 6F).
The effect of TGF-β1 for rapamycin induced autophagy on the expression of MMP-1 and TIMP-1 levels in RBFs
During pathological fibrosis, RBFs activate and move to the injured region, secreting ECM and TGF-β1. When combined with its endogenous growth factor domain, it directly induces profibrotic signals, increases collagen synthesis, and decreases MMP activity, resulting in a decrease in collagen degradation ability [32]. TIMP, a functional peptide that inhibits MMP proteolytic enzyme activity, can selectively prevent ECM breakdown, causing a dynamic imbalance between MMPs and TIMPs, and hastening the process of airway fibrosis. MMP-1 and TIMP-1 are commonly used markers of collagen breakdown [33, 34]. We used ELISAs to measure MMP-1 and TIPM-1 expression in the cell supernatant following rapamycin-induced autophagy in RBFs. The rapamycin and TGF-β1 groups had higher MMP-1 levels than the control group, while the rapamycin-TGF-β1 group had lower levels. In comparison to the control group, the rapamycin and rapamycin-TGF-β1 groups had lower TIMP-1 levels, while the TGF-β1 group had higher MMP-1 levels. MMP-1 levels decreased compared to the rapamycin group, while TIMP-1 levels increased in the rapamycin-TGF-β1 and TGF-β1 groups. These changes were statistically significant (P < 0.05) (Fig. 7).
The effect of TGF-β1 for starvation induced autophagy on the expression of MMP-1 and TIMP-1 levels in RBFs
We used ELISA to measure MMP-1 and TIMP-1 expression in the supernatants of RBFs during starvation-induced autophagy. Starvation increased MMP-1 levels compared with those in the control group. After 12 h, MMP-1 expression peaked in starved cells lacking TGF-β1 (Fig. 8A). MMP-1 protein expression did not change significantly at 2–8 h, but decreased at 12–24 h with the addition of TGF-β1 (Fig. 8B). MMP-1 expression decreased over time in starved cells, reaching its lowest level at 24 h with TGF-β1 (Fig. 8C). TIMP-1 expression in the cell supernatant following starvation-induced autophagy in RBFs diminished as starvation time increased, with the lowest expression occurred after 24 h of starvation (Fig. 9A). TIMP-1 expression increased and peaked at 12 h after adding TGF-β1 (Fig. 9B). TIMP-1 expression in starved cells increased with increasing stimulation duration with TGF-β1 (Fig. 9C).