The Effect of Drugs on Cell Viability
The results of the effect of drugs on the viability of HT-29 cells were shown in Fig. 1a-c. HT-29 cells were incubated with Different concentrations of LPS, RSV, and Jagged-1 for 24 h and 48 h, and the effect of the drugs on the viability of HT-29 cells were detected. The results of CCK-8 showed that HT-29 cells were incubated with drugs for 24 h, and the cell viability was not affected at the highest concentration (the highest concentrations of LPS, RSV, and Jagged-1 were 100 µg/mL, 100 µmol/L, and 10 µmol/L, respectively); However, after HT-29 cells were incubated with drugs for 48 h, cell viability could be affected to varying degrees. To ensure that the subsequent experimental results were not caused by drug-induced cell proliferation or apoptosis, the selected drug intervention concentration and intervention time did not affect the viability of HT-29 cells.
LPS Inhibits the Expression of Tight Junction Proteins in HT-29 Cell Inflammation Model
LPS can reduce the expression of tight junction proteins, so we detected the expression levels of tight junction proteins occludin, ZO-1, and claudin-1 after HT-29 cells were exposed to different concentrations of LPS (0-100 µg/mL) for 24h by Western Blot. The results were shown in Fig. 2a-d: when the concentrations of LPS were lower than 10 µg/mL, it did not affect the expression levels of occludin, ZO-1, and claudin-1. When the concentration of LPS was 100 µg/mL, it down-regulated the expression levels of occludin, ZO-1, and claudin-1. Therefore, the concentration of LPS in subsequent experiments was 100 µg/mL.
RSV Inhibits the Expression of TACE in HT-29 Cell Inflammation Model
The activation of IL-6 and TNF-α are regulated by TACE. Therefore, we detected the inhibition of RSV on LPS-stimulated TACE in the HT-29 cell inflammation model by qRT-PCR, the results were shown in Fig. 3: Compared to control cells, higher levels of TACE were observed after 24 h incubation with LPS. RSV prevented this increase in a dose-dependent manner in the HT-29 cell inflammation model, suggesting RSV can inhibit the expression of TACE.
RSV Inhibits the Expression of IL-6 and TNF-α in HT-29 Cell Inflammation Model
Inflammatory factors IL-6 and TNF-α are important indicators for judging the degree of cellular inflammation. Therefore, we detected the inhibition of RSV on LPS-stimulated IL-6, and TNF-α in the HT-29 cell inflammation model by qRT-PCR. The results were shown in Fig. 4a-b: Compared to control cells, LPS increased the expression levels of inflammatory factors IL-6 and TNF-α in HT-29 cells. After HT-29 cells were incubated with RSV, the expression levels of inflammatory factors IL-6 and TNF-α in the HT-29 cell inflammation model were significantly decreased. These results indicated that RSV has a good anti-inflammation effect.
RSV Up-regulates the Expression of Tight Junction Proteins in HT-29 Cell Inflammation Model
Occludin, ZO-1, and claudin-1 are important components of tight junction proteins, which are involved in the maintenance of the structure and function of the intestinal epithelial barrier. Therefore, we detected the expression of occludin, ZO-1, and claudin-1 by Western Blot and qRT-PCR. The results were shown in Fig. 5a-g: Compared to control cells, LPS down-regulated the expression levels of tight junction proteins occludin, ZO-1, and claudin-1. On the contrary, the expression of occludin, ZO-1, and claudin-1 in the HT-29 cell inflammation model were up-regulated after being incubated with RSV.
We further observed the tight junction structure by transmission electron microscope. The results were shown in Fig. 6: the normal tight junction structure between HT-29 cells was narrow and continuous strips under the transmission electron microscope. After HT-29 cells were incubated with LPS, the tight junction structure was loose, the intercellular space was widened, and the continuity of bands of the structure was interrupted. When the HT-29 cell inflammation model was incubated with RSV, the tight junction structure became tighter, the gap between cells was narrowed, and the disruption of tight junctions was improved.
Altogether, these results suggested that RSV can up-regulate tight junction proteins to protect the integrity of tight junction structures.
RSV Inhibits the Activity of Notch1 Pathway in HT-29 Cell Inflammation Model
The Notch1 pathway is involved in the signal transduction of cell differentiation, apoptosis, and survival, and plays an important role in the maintenance of the structure and function of the intestinal epithelial barrier. Therefore, we detected the expression of Notch1 pathway-related indicators by Western Blot and qRT-PCR. The results were shown in Fig. 7a-e: LPS led to the activation of Notch1 pathway that was prevented by RSV in a dose-dependent manner.
RSV Up-regulates the Expression of Tight Junction Proteins in HT-29 Cell Inflammation Model by Inhibiting Notch1 Pathway Activity
To verify the mechanism of RSV regulating the expression of tight junction proteins in the HT-29 cell inflammation model, we verified its mechanism by the Notch1 pathway activator Jagged-1. The results were shown in Fig. 8a-e: Jagged-1 abolished the inhibition effect of RSV against LPS-induced the activation of Notch1 pathway.
After Jagged-1 abolished the inhibition effect of RSV against LPS-induced the activation of Notch1 pathway, we detected the expression of tight junction proteins occludin, ZO-1, and claudin-1 in the HT-29 cell inflammation model incubated by RSV via Western Blot and qRT-PCR. The results were shown in Fig. 9a-g: Compared to cells incubated by RSV, the expression levels of tight junction proteins occludin, ZO-1, and claudin-1 in the HT-29 cell inflammation model incubated with RSV and Jagged-1 were decreased.
We further observed the tight junction structures by transmission electron microscope, and the results were shown in Fig. 10: Compared to cells incubated by RSV, the tight junction structure of cells incubated with RSV and Jagged-1 was loose, and the gap between cells became wider.
All these results supported that RSV can up-regulate the expression of tight junction proteins by inhibiting the activity of the Notch1 pathway.
Activation of Notch1 Pathway Promotes the Expression of TACE in RSV-Incubated HT-29 Cells Inflammation Model
The expression levels of TACE in the RSV-incubated HT-29 cell inflammation model were detected by qRT-PCR after the Notch1 pathway was activated by Jagged-1. The results were shown in Fig. 11: Compared to cells incubated by RSV, the expression levels of TACE in the HT-29 cell inflammation model incubated with RSV and Jagged-1 were increased.
Activation of Notch1 Pathway Promotes the Expression of IL-6 and TNF-α in RSV-Incubated HT-29 Cells Inflammation Model
The expression of inflammatory factors IL-6 and TNF-α in the RSV-incubated HT-29 cell inflammation model was detected by qRT-PCR after the Notch1 pathway was activated by Jagged-1. The results were shown in Fig. 12a-b: Compared to cells incubated by RSV, the expression levels of IL-6 and TNF-α in the HT-29 cell inflammation model incubated with RSV and Jagged-1 were increased.