The Anti-inflammatory Effects And Molecular Mechanism Of Apigenin And Acupuncture On Lipopolysacchride-induced Mastitis In Lactating SD Rats

Mastitis is prevalent in mammals,pets and lactating women around the world,endangering the development of animal husbandry and human health.Apigenin is an important component of mastitis treatment and has good anti-inflammatory effects.It is a real non-toxic,no genotoxic flavonoids.Acupuncture treatment of disease safe and convenient,the method has been widely used in the treatment of inflammatory diseases,but the effects of apigenin or/ and acupuncture treatment of mastitis have not been reported.In this study,SD rats were used as subjects to establish a model of mastitis to study the effects of apigenin or/and acupuncture on mastitis.In addition,SD mammary epithelial cells were isolated and cultured to further study the protective effects and mechanism of apigenin on LPS-induced inflammatory injury,so as to provide the scientific theory basis for the clinical application of apigenin and acupuncture in the treatment of mastitis.At first,we established LPS-induced model of mastitis in lactating SD rats.30 SD rats were control group and 5 experimental groups(5 SD rats in each group)at 72 h postpartum.The rats were perfused with different doses(10?g/side,20?g/side,30?g/side,40?g/side,50?g/side)LPS by the fourth papilla,the control group perfusion equal volume of PBS.After 24 h of perfusion,the clinical symptoms and histopathological changes of the rats were recorded.The results showed that different levels of inflammatory response were observed in the fourth pair of breasts in each test group and were proportional to the LPS dose.The results showed that the LPS-induced model of mastitis in lactating SD rats was successfully established.Secondly,the use of mastitis model to explore the cure effect of the apigenin or/and acupuncture treatment of mastitis.In this study,SD rats with induced mastitis were divided into four groups: LPS stimulation group(LPS),apigenin treatment group(Api),acupuncture treatment group(Acu),apigenin and acupuncture combined treatment group(Api+Acu),another group of normal SD rats as the control group(Con),each group of 5 rats.Treatment was performed 24 h after LPS perfusion,once a day,for 5 days,and the rats were sacrificed at 24 h after the last treatment to collect breast tissue.Observe the pathological changes of breast tissue.the levels of TNF-?,IL-1? and IL-6 in breast tissue were detected by q RT-PCR and ELISA.Western blot was used to detect the expression of MPO and TLR4/NF-?B signaling pathway.The results showed that the levels of TNF-?,IL-1? and IL-6 mRNA and protein were significantly decreased in the threetreatment groups compared with the LPS group,and the activation of TLR4/NF-?B signaling pathway was significantly inhibited.These results suggest that apigenin/acupuncture can treat LPS-induced mastitis in SD rats by inhibiting TLR4/NF-?B signaling pathway.Finally,the mammary epithelial cells of SD rats were isolated and cultured,and the protective effect and mechanism of apigenin on LPS-induced inflammatory injury of mammary epithelial cells were investigated by in vitro experiments.Toxicity of apigenin to mammary epithelial cells in SD rats was detected by MTT assay and the effects of apigenin on the expression of TNF-?,IL-1? and IL-6 in mammary epithelial cells of SD rats were detected by q RT-PCR and ELISA.Effect of apigenin on TLR4/NF-?B signaling pathway in mammary epithelial cells of SD rats was detected by western blot and indirect immunofluorescence.The results showed that apigenin at 0 ~ 10?g/m L concentration does not produce toxic effects on rat mammary epithelial cells.Compared with LPS group,cells pretreatment with apigenin can significantly reduce the level of TNF-?,IL-1? and IL-6 mRNA and protein.The results of western blot showed that apigenin exerts its anti-inflammatory effect by inhibiting the activation of TLR4/NF-?B signaling pathway in mammary epithelial cells.And indirect immunofluorescence results further proved that apigenin can inhibit NF-?B p65 phosphoric acid and into the nucleus.In this study,we carried out a preliminary exploration on the effect and mechanism of apigenin/acupuncture treatment of mastitis.It was found that apigenin/acupuncture could down-regulate the expression of TLR4 and inhibit the activation of NF-?B signaling pathway downstream resulting in a significant decrease in mRNA and protein expression levels of TNF-?,IL-1? and IL-6 in the breast.