Effect Of Silymarin On CYP450 Expression In LPS-induced Hoof Dermal Inflammatory Cells Of Dairy Cows

Hoof disease is one of the major diseases of dairy cows,among which laminitis is the most common one.Economic losses caused by laminitis were enormous,which were severely restricted the development of dairy farming.However,the etiology and pathogenesis of laminitis have not been clarified yet,so there were no effective diagnosis and control measures.To explore this issue,an inflammatory model in hoof dermal cells of dairy cows was established by LPS,silymarin was used to treat inflammatory cells,and a series of inflammation-related factors,signaling pathways,and CYP450 expressions were studied.The pathogenesis of laminitis in dairy cows was investigated at cellular and molecular levels in this study,which will provide a basis for the prevention and treatment of laminitis in dairy cows.In this study,hoof dermal cells of dairy cows were isolated by adherent culture of hoof dermal tissue blocks.The obtained cells grew well and corresponded to the proliferation characteristics of normal cells.Dermal cells were treated with different concentrations of LPS.Cell supernatants and total protein were collected at different time intervals.The contents of IL-1? and TNF-? in the supernatant were detected by ELISA,and the protein expressions of p65 NF-?B and p38 MAPK were detected by Western Blot.The results showed that certain concentrations of LPS promoted the secretions of IL-1? and TNF-?,and the protein expressions of p65 NF-?B and p38 MAPK in the dermal cells.The proinflammatory effect of LPS for 48 h was more significant than that for 24 h.And 10 ?g/mL LPS was the optimal concentration for inducing inflammation model of hoof dermal cells.The cytotoxicity of silymarin was measured by MTT assay to determine the safe range of use in subsequent trials.The results showed that there were no obvious toxic effects on hoof dermal cells when the concentration of silymarin was under 20 ?g/mL within 48 h.Hoof dermal cells were treated with different concentrations of silymarin and 10 ?g/mL LPS.To study the anti-inflammatory effects of silymarin and its effect on expression of CYP450 in hoof dermal inflammatory cells of dairy cows induced by LPS,cell supernatants,total protein,and total cellular RNA were collected to detect the levels of IL-1?,TNF-?,the protein expressions of p65 NF-?B,p38 MAPK,and the m RNA expressions of CYP3A4,CYP1A1.The results showed that all groups of silymarin used in this experiment for 48 h can significantly inhibit the secretions of IL-1? and TNF-? in the dermal inflammatory cells(P<0.05).Certain concentrations of silymarin can inhibit the protein expressions of p65 NF-?B and p38 MAPK.Silymarin of 1,5 ?g/mL can promote the mRNA expressions of CYP3AA4 and CYP1A1,among which 1 ?g/mL was the most applicable.Conclusions: Hoof dermal cells of dairy cows can be successfully isolated and cultured by tissue adherent culture method.Certain concentrations of LPS can promote the secretions of IL-1? and TNF-?,promote the protein expressions of p65 NF-?B and p38 MAPK in the hoof dermal cell of dairy cows.10 ?g/mL LPS works best,which can be the optimal concentration to establish the inflammation model of hoof dermal cells.Certain concentrations of silymarin can markedly inhibit the secretions of IL-1? and TNF-?,inhibit the protein expressions of p65 NF-?B and p38 MAPK,and promote the mRNA expressions of CYP3AA4 and CYP1A1.1 ?g/mL silymarin works best.