Anti-inflammation Of Apigenin And Its Molecular Pathways In LPS-stimulated MAC-T Cells

Bovine mastitis has caused serious damage to health of cows,which continually increases treatment costs of bovine mastitis.Mastitis is the immunoreaction to prevent mammary gland from external stimulus,and moderate inflammation in mammary gland is significant to maintain the health and integrity of mammary gland.But chronic inflammation occurs if long-term irritation lasts in the bovine mammary gland,which leads to decline in cattle utilization ratio and product quality.Nuclear factor ?B(NF-?B)is one of classical signal pathway in controlling inflammation.The subunit of p65 and the regulatory factors who directly or indirectly affect p65 are reported to play a key role in control the activation bovine mastitis.when the irritant signaling such as LPS is transducted into cell via toll like receptor 4(TLR4)and after a series of signaling transduction,I?B kinase(IKK)is activated and further leads to degradation of inhibitor of NF-?B(I?B)through phosphorylation and ubiquitination.The p65,released after being phosphorylated and acetylated,translocates into nuclear where it binds to ?B sequence to promote expression of inflammatory factors,proinflammation factors and chemotactic factors,etc.It means IKK,I?B and p65 proteins are the critical targets in regulating NF-?B,and controlling of NF-?B is significant to intervene in inflammatory response.Besides,MAPK signaling pathway involves oxidative stress of inflammation reaction via regulating of expression of iNOS,COX-2,etc.MAPK signaling consists of three MAP kinases including ERK1/2,JNK1/2 and p38 existing in all the eukaryocyte.Phosphorylated MAP kinases promote production of inflammatory factors(iNOS,COX-2,etc.)by phosphorylating ERK1/2,JNK1/2 and p38,which can affect oxidative stress of inflammation reaction.In this research,optimum treatment concentrations of apigenin were determined by cytotoxicity examination using MTT assays in MAC-T cells.The optimal concentrations of LPS for inducing inflammatory response in MAC-T cells were also determined by testing expression of IL-6 using real-time PCR.Furthermore,effects of apigenin on expression of IL-1?,IL-6,TNF-?,COX-2 and iNOS were investigated in MAC-T cells treated with different combination of LPS and apigenin concentrations.In addition,inhibition of apigenin on NF-?B and MAPK pathways was revealed by phosphorylation detection of p65,I?B?,ERK1/2,JNK1/2 and p38 using Western blot and immunofluorescence assays.The results were as follow: 1.LPS is a category of ordinary matters which is the composition of Germ-negative bacteria cell wall and can promote inflammatory response.After treating with different concentration of LPS(0,1,5,10,20 ?g/m L)and varying treating time(1,12,24 h),the relative level of IL-6 and IL-1? measured by quantitative PCR revealed a significant increase with the increase of LPS concentration and a remarkable up-regulation by the increase of treatment time.2.Apigenin is extracted from natural plant which has low cytotoxicity.MTT test reveal that an increased cytotoxicity appeared by the increase of apigenin concentration(1,3,5,7,10,15,20,50,100 ?g/mL)and the growth of MAC-T cells was significantly inhibited at the apigenin concentration of 20 ?g/mL(p<0.05).3.After treating with variety of apigenin(1,7,15 ?g/mL)to LPS(1 ?g/mL)activated MAC-T cells for 24 h and the results showed remarkable decline in relative mRNA level of COX-2,iNOS,IL-1? and IL-6 in compared with control group(p<0.01).4.Western blot and immunofluorescence reflected apigenin significantly suppressed the phosphorylation level of p65 and I?B?(p<0.05)and inhibited translocation of p65 in LPS-induced MAC-T cells.Besides,the phosphorylation level in p38,JNK1/2 and ERK1/2 were also decreased after treating with apigenin in LPS-induced MAC-T cells(p<0.01).Conclusion: 1.LPS can significantly up-regulate IL-6 under the treatment condition of 1 ?g/mL LPS for 1 h.The optimal concentration of apigenin to MAC-T cells was 20 ?g/mL.2.Apigenin exert a protection effect to inflammation via suppressing the relative level of COX-2,iNOS,IL-1? and IL-6 in LPS-stimulated MAC-T cells.3.The anti-inflammatory effect and anti-oxidative function of apigenin are exerted through down-regulating the NF-?B and MAPK signaling pathways.The results all above indicate apigenin can use for preventing and therapy of bovine mastitis.