The Molecular Mechanism Of Geniposide Against Inflammatory Injury Of LPS-induced Mammary Gland

Mastitis, an inflammatory reaction of the mammary gland that is usually causedby microbial infection, is recognized as the most costly disease in dairy cattle.Decreased milk production accounts for approximately70%of the total cost ofmastitis. Mammary tissue damage reduces the number and activity of epithelial cellsand consequently contributes to decreased milk production. Geniposide is widely usedfor its anti-inflammatory，antioxidant and other diverse activities. In the present study,we used primary bovine mammary epithelial cells, primary mouse mammaryepithelial cells and a mouse mastitis model to study the protective effect of geniposideon LPS-induced mammary inflammatory damage. Maybe it could provideexperimental basis for its application to bovine mastitis clinical treatment.Firstly, we took tissue cultivationmethod and differential digestion method toisolate and purify primary bovine mammary epithelial cells. After pretreatment withgeniposide, the cells were stimulated with LPS. MTT was taken to measure the effectof geniposide on cell viability. qRT-PCR was used to detect the effect of geniposideon the levels of TNF-α, IL-1β and IL-6mRNA. The effect of geniposide on cellapoptosis was measured by LIVE/DEAD. The results showed that cell viability wasnot affected by geniposide at the concentrations used (50,100, and200μg/ml).Geniposide significantly inhibited LPS-induced elevated production of TNF-α, IL-1βand IL-6mRNA; declined the number of dead cells in a dose-dependent manner.Secondly, we isolated and purified primary mouse mammary epithelial cells byusing mixed enzyme digestion method. The cells were stimulated with LPS followingwith geniposide pretreatment. The effect of geniposide on cell viability was measuredby MTT. The effect of geniposide on the production of TNF-α, IL-1β and IL-6wasdetermined by ELISA. Western Blot was used for detecting the effect of geniposideon NF-κB, MAPKs signal pathways, Bax, Bcl-2, Caspase-3, p53and TLR4. qRT-PCRwas taken to measure the effect of geniposide on the mRNA of TNF-α, IL-1β, IL-6,Bax, Bcl-2, Caspase-3, p53and TLR4. The results showed that geniposide had noeffect on cell viability at the concentrations of20,50, and100μg/ml; inhibited the release of TNF-α, IL-1β and IL-6; reduced LPS-induced apoptosis; decreased theexpression of TLR4; suppressed the activation of NF-κB and MAPKs signal pathways;downregulated the expression of Bax; upregulated the expression of Bcl-2; inhibitedthe cleavage of Caspase-3and the phosphorylation of p53.Finally, to further verify the protective effects of geniposide on LPS-inducedinflammatory damage, we established mouse mastitis model by infusion of LPS inmammary gland. The levels of TNF-α, IL-1β and IL-6in mammary tissues weremeasured by ELISA, the histopathological changes of mammary tissues wereobserved by H&E staining, and the apoptosis of mammary tissues was detected byTUNEL. The results showed that geniposide significantly decreased the levels ofTNF-α, IL-1β and IL-6in mammary gland; protected the normal and integrity ofmammary gland structure; alleviated LPS-induced apoptosis.In summary, the present study demonstrated that geniposide showed itsprotective effect and good results by decreasing the expression of TLR4, suppressingthe activation of NF-κB and MAPKs signal pathways, downregulating the expressionof Bax; upregulating the expression of Bcl-2and inhibiting the cleavage of Caspase-3and the phosphorylation of p53. Geniposide is a potential drug for the treatment of E.coli-induced mastitis. It may be applied in the treatment of bovine mastitis in thefuture.