Anti-inflammatory effect and mechanism of Chlorogenic acid extract from Taraxacum officinale on LTA-induced mastitis in dairy cows

Abstract

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. Mastitis is one of the costliest diseases affecting dairy cattle. Direct economic costs associated with mastitis include reduced milk yield and quality, increased veterinary costs, discarded milk (during the course of treatment) and somatic cell count (SCC) penalties. When analyzing literary sources, we established that mastitis still remains a pressing problem for many countries, including China and Ukraine. And extracts from plant have obvious therapeutic effects and play an inportent role in the treatment of mastitis in dairy cows. They can not only avoid the production of drug-resistant bacteria caused by the application of antibiotics, but also avoid problems such as drug residues. Therefore, the application of plant extracts has always been high hopes in the prevention and treatment of dairy cow mastitis. Therefore, research needs to understand the antibacterial mechanism of plant extracts against pathogenic bacteria such as Staphylococcus aureus and Escherichia coli, and reveal the protective mechanism of plant extracts against mastitis in dairy cows. It has long been known that mastitis resistance or susceptibility is a complex trait that is comprehensively affected by the environmental, physiological and genetic factors. Analyzing plans for carrying out treatment and preventive measures in dairy farms of Ukraine and China, we determined that at present, antibiotics are mainly used for the treatment of cow mastitis. However, the abuse of antibiotics has lead to increased resistance to pathogenic bacteria and serious drug residues. Chlorogenic acid (3-O-caffeoylquinic acid) is the main active ingredient of an extract from Dandelion (Taraxacum officinale), which has played an important pharmacological role in the study of many inflammatory diseases. However, the molecular mechanism underlying the formation of resistance is poorly understood. This research takes chlorogenic acid as the research object, which is a natural ingredient in the Chinese herbal medicine Taraxacum officinale. Firstly, the optimal extraction conditions of chlorogenic acid in dandelion were determined by orthogonal experiment. A high performance liquid chromatography (HPLC) method for the detection of chlorogenic acid in dandelion was established. Secondly, detection of the antibacterial mechanism of phenolic acid extract (include chlorogenic acid) against Staphylococcus aureus. Lastly, the mammary epithelial cells were isolated and cultured from milk, and obtained pure primary cells; BMEC inflammatory model was established by Lipoteichoic acid (LTA); CCK-8, ELISA, qRT-PCR and WB were used to analyze the protective and anti-inflammatory effect of chlorogenic acid on LTA-induced inflammatory lesions. In order to provide a reference for the Chinese herbs treatment of dairy cow mastitis, it provides scientific basis for the research and development of Chinese herbs and related basic research. In the study of detection and extraction process of chlorogenic acid extract from Taraxacum officinale was conducted. The extraction technology of chlorogenic acid was researched from multiple factors, the best parameters of the technology were obtained that the ultrasonic temperature was 80 0C, the solid-liquid ratio was 1 : 30, the solvent concentration was 50%, and the ultrasonic time was 40 min, then the extraction rate can reach 1.921%. 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. In the process of the antibacterial experiment, it was found that phenolic extract from T. Officinale has an inhibitory effect on S. aureus, and the mechanism of action was to destroy the integrity of its cell walls and cell membranes. The minimum inhibitory concentration (MIC) of dandelion phenolic extract exhibited against S. aureus was 12.5 mg/mL. The antibacterial kinetic curve analysis showed that the inhibitory effect of dandelion phenolic extract on S. aureus was mainly in the exponential growth phase. After the action of dandelion phenolic extract, the growth of S. aureus was obviously inhibited entering into the decay phase early. Furthermore, after the action of dandelion, the extracellular AKP (alkaline phosphatase) contents of S. aureus, the electrical conductivity and the extracellular protein contents were all increased. The phenolic extract also affects the normal reproduction of S. aureus. When analysis the content of phenolic extract from T. Officinale, the dandelion was extracted by ultrasonic assisted extraction method with 70% methanol-water (v/v) as a solvent. Subsequent identification and quantification of phenol in extract was carried out using High Performance Liquid Chromatography (HPLC). The results showed that chlorogenic acid (1.34 mg/g) was present in higher concentration, followed by luteolin (1.08 mg/g), ferulic acid (0.22 mg/g), caffeic acid (0.21 mg/g), and rutin (0.19 mg/g) in the dandelion phenolic extract. When studying the establishment of bovine mammary epithelial cells (BMEC) from the milk of Ukraine Holstein dairy cows in order to develop a general in vitro model. The whole growth process is different when culturing epithelial cells from tissue. Single adherent cells appear on the culture dish after culturing for 3-4 days. Bovine mammary epithelial cells formed islands when cultured at low density after 5-7 days. Primary epithelial cells were elongated after culturing for 8-10 days. Mammary epithelial cells developed into different shapes, including oval, typical cobblestone, and irregular polygon. Most of the isolated cells that extended from the milk had an irregular polygon shape. The cells obtained after freezing and thawing maintained normal morphology and growth characteristics. In the selection of frozen storage medium, the frozen solution containing 90% FBS and 10% DMSO was more conducive to the survival of cryopreserved BMECs than DMEM/F12: FBS: DMSO= 5:4:1. For subsequent research, FBS containing 10% DMSO was used for preservation of frozen cells. 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. We recommend using the materials of the dissertation work when studying the courses " Veterinary microbiology", "Veterinary internal medicine" and Veterinary sanitary examination" for masters of the Faculty of Veterinary Medicine of Sumy NAU. And for the courses "Veterinary internal medicine" and "Chinese traditional veterinary science" for masters of the Henan Institute of Science and Technology (HIST). We can recommend that at farm level, dandelion extract may use as a new type of feed additive in production. And new therapies with CGA as a drug should be developed in future.