Effects Of Hydrogen Sulfide On Lipopolysaccharide-induced Inflammation And Autophagy In Bovine Mammary Epithelial Cells

Mastitis seriously impairs animal health and results in economical loss.For scientists,this is a problem to be solved in the prevention and treatment of cow mastitis.Lipopolysaccharide（LPS）may cause immune response and inflammation in the bovine mammary gland.And some studies reported that autophagy pathways and autophagy related proteins play a key role in immunity and inflammation.Hydrogen sulfide（H₂S）is released in livestock waste during livestock production,which affects livestock productivity.H₂S is the third gasotransmitter discovered after nitric oxide and carbon monoxide,which is widely distributed in cardiovascular,visceral and nervous systems.Many studies reported that H₂S acts as an anti-inflammation regulator in many cells.However,the effect of H₂S on the inflammatory and autophagy in bovine mammary epithelial cells has not been reported.Therefore,the purpose of this study was to investigate the effect and mechanism of H₂S on LPS-induced inflammatory and autophagy in bovine mammary epithelial cells.In the present study,with NaHS as a donor of H₂S,the bovine mammary epithelial cell line（MAC-T）was applied as an in vitro model to study the role of H₂S on LPS-induced bovine mammary epithelial cells.These results proved that the cell viability was reduced by LPS（50,100,200μg/ml）and the expression of IL-6,IL-1β,TNF-αand IL-8 mRNA was increased.We selected 100μg/ml as the concentration for subsequent LPS addition.Treatment with 30μM NaHS for 24 h significantly reversed the decrease in cell viability caused by LPS.The content of H₂S in supernatant was detected.The results showed that the content of H₂S was significantly reduced in LPS group.Meanwhile,treatment with NaHS significantly reduced the increase of inflammatory factors IL-6,IL-1β,TNF-αand IL-8 mRNA induced by LPS.Besides,NaHS alone could also reduce the expression of inflammatory factors.The results of ROS test indicated that the addition of NaHS could significantly reduce the rise of ros induced by LPS.In addition,H₂S inhibited the mRNA expression of TLR4 and the activation of NF-κB signaling pathway.Next,we explored whether the anti-inflammatory mechanism is related to autophagy.The results showed that H₂S activated the mTOR signaling pathway,inhibited the increase of LC3B-I to LC3B-II and Beclin-1 protein expression induced by LPS.And H₂S promoted the expression of p62 protein.We also found that the addition of NF-κB inhibiter（PDTC）also inhibited the expression of LC3B-II/LC3B-I and Beclin-1 protein,and promoted the expression of p62 protein.Taken together,H₂S plays an anti-inflammatory role by blocking the TLR4/NF-κB pathway and activates the mTOR signaling pathway to inhibit autophagy on the LPS-induced bovine mammary epithelial cells.Our finding might throw light on new prophylactic approaches for mastitis.