Based on scientific demonstration and experimental research, this thesis is devoted to studying Anti-inflammatory effect and mechanism of Chlorogenic acid extract from Taraxacum officinale on LTA-induced mastitis in dairy cows. The HPLC detection method of chlorogenic acid from Taraxacum officinale was 5% methanol elution condition 0 ~ 5 min, 5 - 15% methanol elution condition 5 ~ 15 min, 15 - 5% methanol elution condition 15 ~ 20 min, 5 % methanol elution condition 20 ~ 25 min, and the buffer salt was 1‰ phosphoric acid aqueous solution, and the wavelength was 350 nm. The method was stable and reliable. The characteristics of BMECs were examined using real time cell assay (RTCA), immunocytochemistry (ICC), reverse transcription- polymerase chain reaction (RT-PCR), and Western blot (WB). RTCA provides a remarkable method for real time monitoring of cell viability. The result showed that the best seeding density for the proliferation of BMECs was 1 × 104 cells. The BMECs culture slowly grew within the first 3 days and cells entered the stable phase in the best seeding density. Cells exhibited strong positive staining for cytokeratin 18, which is specific for epithelial cells, indicating that the cultured cells possessed the properties of epithelial cells. RT-PCR was used to determine the mRNA expression and WB was used to determine the milk proteins expression in the BMEC. And the results confirmed the ability of the isolated cells to synthesize milk proteins. It is very important that MECs express milk proteins and can mimic the in vivo system.
CCK-8 assay carried out to examine the viability of cells. The viability of BMECs infected with LTA were lower than that of the normal BMECs. RTAC was used to detect the effect of different concentrations of LTA on the proliferation of BMECs, the results were shown that with the increase of LTA concentration, the proliferation activity of BMEC cells was inhibited. According to the change of cell index value and different proliferation curves, the dynamic detection after LTA treatment of cells was found. The effects of LTA at different concentrations (0, 10, 20, 40, and 80 μg / mL) on BMEC were observed at different time points (12, 24 and 48 h); It was found that with the increase of LTA dose, the inhibition rate of cell vitality increased gradually, indicating that LTA of certain concentration would cause the damage of BMEC. According to 24 h of LTA action cells of different concentrations, the inhibition rate of concentration of 20 μg / mL and 40 μg / mL was significantly increased. The results of ELISA and qRT-PCR showed that the treatment of BMEC with LTA at the concentration of 20 μg / mL for 24 h obviously induced TNF-α and IL-6 protein and gene expression levels.
In the last, the anti-inflammatory effect and mechanism of chlorogenic acid extract were detected in LTA-stimulated bovine mammary epithelial cells. The effect of chlorogenic acid extract on the viability of bovine mammary epithelial cell was evaluated using the CCK-8 and RTCA assay. The effects of chlorogenic acid extract on NF-κB activation was detected by Western blotting.
The effects of chlorogenic acid extract on LTA-induced inflammatory cytokines expression were detected by ELISA and qRT-PCR. The results showed that the cell viability was not affected by chlorogenic acid extract at the dose of 12.5, 25, 50, 100, 200, and 400 μg/mL. The IC50 of the CCK-8 and RTCA analysis methods were 326.8 and 320.4 ug/mL.
Meanwhile, chlorogenic acid extract inhibited LTA-induced TNF-α, IL-1β, and IL-6 expression. Western blot analysis showed that chlorogenic acid extract suppressed LTA-induced NF-κB activation in a dose-dependent manner.
This paper investigates the anti-inflammatory mechanism of chlorogenic acid extract from Taraxacum officinale in inflammatory signaling pathways. It provides a basis for reference to the study of drug prevention and treatment of mastitis in dairy cows.
