The Protective Effect Of Naringenin On LPS-induced Inflammatory Injury Of Mammary Epithelial Cells In Dairy Cows

Dairy cow mastitis is one of the three major diseases that affect the development of dairy farming industry,and it often brings greater economic losses to pastures.Bovine mammary epithelial cells(BMECs),as an important part of dairy cow mammary gland immunity,are important materials for studying the pathogenesis of dairy cow mastitis and screening effective drugs for prevention and treatment.Naringenin(NAR)is a kind of natural flavonoids.Studies have shown that it has anti-oxidation,antibacterial,anti-inflammatory,anti-cancer and other physiological activities.It is widely regarded as a substitute for future antibiotics.In this study,bovine mammary epithelial cells and ICR mice were used as the research objects,and an E.coli-type dairy cow mammary inflammation model was constructed through lipopolysaccharide(LPS):induction to study the inflammatory damage of LPS-induced dairy cow mammary epithelial cells by NAR.The main experimental results are as follows:1、Different concentrations of LPS(0,30,50,100,500 ng/mL)were used to treat cow mammary epithelial cells for 12 h.The CCK-8 test was used to detect cell viability.The results showed that LPS of 100 ng/mL and 500 ng/mL could be significant reduce the cell viability of BMECs;After treating BMECs with different concentrations of NAR(0,0.3,0.5,1,5 μM)for 6 h,the results show that low concentration of NAR(<1 μM)has no significant effect on the cell viability of BMECs,and high concentration of NAR(>5 μM)significantly inhibited the cell viability of BMECs.2、EdU test showed that LPS significantly reduced the cell proliferation activity of BMECs,and the cell proliferation activity of BMECs in the 1 μM NAR+LPS group was significantly higher than that in the LPS group,indicating that NAR can resist the inhibitory effect of LPS on cell proliferation.The results of flow cytometry showed that the apoptotic rate of the LPS group was 12.49%,and the 1 μM NAR+LPS group had the lowest apoptotic rate of 11.56%,indicating that NAR has a protective effect on LPS-induced apoptosis.3、Fluorescence quantitative PCR test results showed that LPS significantly increased the expression of inflammatory genes(IL6,IL8,TNF-α,NF-κB),but decreased in the NAR+LPS group,and the 1 μMNAR treatment group decreased the most.It is speculated that NAR can increase the resistance of BMECs to LPS by reducing the expression level of BMECs inflammatory factors.4、Transcriptome sequencing results showed that the differential genes in the LPS and NAR+LPS treatment groups were mainly enriched in biological processes such as signal transduction and cellular stress response;KEGG analysis found that multiple differential genes were enriched in the HIF-1 signal transduction pathway.It is preliminarily inferred that naringenin may protect dairy cow mammary epithelial cells through the HIF-1 signaling pathway.5、The results of the murine mastitis model show that NAR can reduce the stimulation of LPS on dairy cow mammary epithelial cells by inhibiting the expression of inflammatory factors(IL6,IL8,TNF-α,NF-κB),thereby achieving the effect of protecting mouse mammary glands.This experiment was conducted in dairy cow mammary epithelial cells and mice to verify whether naringenin can inhibit the inflammatory symptoms caused by LPS.The cell test results are consistent with the mouse test results,and both confirm that naringenin can effectively slow down LPS.The damage of cells and body provides a theoretical basis for the further development of naringenin and its application in production practice.