Effect Of Quercetin On The Expression Of CYP450 In Carrageenan-Induced Hoof Dermal Inflammatory Cells Of Dairy Cows

Laminitis is a common disease in dairy cows.It is easy to cause systemic symptoms in dairy cows,reduce milk production and bring economic losses to dairy farming.There are many hypotheses and speculations about the pathogenesis and principles of laminitis,but the real mechanism is not very clear.In order to further clarify this problem,this experiment chose to use carrageenan to treat hoof dermal cells,induce and establish an inflammatory model,and then treat inflammatory cells with quercetin.To examine the effects on the expression of inflammatory mediators and related pathway proteins in cells,as well as the effects on cytochrome P450 enzymes,from the level of cells and molecules as an entry point,to explore what changes in the cells during the pathogenesis of laminitis.In this study,two methods of tissue block adherence and enzyme digestion were used to separate and culture cow’s hoof dermis cells to obtain well-grown cells.The hoof cells were treated with carrageenan,the cell viability was determined by MTT assay,the cell culture medium was collected,the levels of IL-1β and TNF-ɑ in the inflammatory mediators were detected by ELISA,and the expression of p65 NF-κB and p38 MAPK were detected by Western Blot.The results showed that the inhibition rate of cell growth increased with the increase of carrageenan concentration.With 200 μg/mL carrageenan,the inhibition rate of cell growth was closest to 10%.After treatment with carrageenan for 48 h,the expression of p65 NF-κB and p38 MAPK protein was increased,and the levels of TNF-α and IL-1β in the supernatant were significantly higher than those in the control group.(P<0.05).Therefore,treatment of cells with 200 μg/mL carrageenan for 48 h enables the production of inflammatory cell models.The cytotoxicity of quercetin was detected by MTT assay,and it was detected by PCR whether it directly affected the expression of CYP450 enzyme in cells.The results showed that the treatment of hoof dermis cells with quercetin at a concentration of not more than 100 mmol/L for 48 h did not cause toxicity to the cells,nor did it affect the expression of CYP1A1 and CYP3A4 mRNA.The carcinogenesis model was established with carrageenan,and the inflammatory cells of the hoof dermis were treated with several different concentrations of quercetin.The cells were divided into two treatment groups by controlling the time when quercetin was added to the cells,Collect cell supernatants and compare changes of IL-1β and TNF-ɑn in cells,lysing cells extract proteins,and detecting quercetin in p65 NF-κB and p38 MAPK proteins and cytochrome P450 enzymes.According to the results,quercetin can reduce the secretion of IL-1β and TNF-α in inflammatory cells;inhibit the expression of p65 NF-κB and p38 MAPK protein;and also inhibit the inhibition of CYP450 enzyme protein expression by carrageenan.Treatment with 30 mmol/L concentration is the best.Conclusion: Both well-developed primary dermal cells were obtained by double enzyme digestion and tissue adherence.The cow dermal cells were treated with carrageenan at 200 μg/mL for 48 hours to induce an inflammatory model.For carrageenan-induced IL-1β and TNF-α produced by inflammatory cells,quercetin can significantly reduce its production,and also inhibit the expression of p65 NF-κB and p38 MAPK proteins,and reduce carrageenan.The optimal therapeutic concentration for cytochrome P450 enzyme is 30 mmol/L.