Osthole Alleviates LPS-induced Mastitis In Mice By Regulating PI3K/AKT/mTOR Signaling Pathway

Mastitis in dairy cows is an inflammatory reaction caused by infection of the mammary gland tissue of cows,which is characterized by complex pathogenesis and difficult treatment,and is a serious threat to the development of dairy farming and dairy industry.Osthole is the main active ingredient of Osthole,a plant of the family Umbelliferae,with anti-inflammatory,antibacterial and antitumor effects.Preliminary laboratory studies have shown that osthole can significantly alleviate lipopolysaccharide(LPS)-induced mastitis in mice,but its mechanism of action has not yet been clarified.In this study,we propose to investigate the anti-inflammatory effect of serpentine and its effect on the autophagic PI3K/ AKT /mTOR signaling pathway to provide a theoretical basis for the treatment of mastitis in dairy cows with serpentine.Methods: Female mice at 6 days postpartum were randomly divided into four groups:blank group,model group,serpentine treatment group and dexamethasone treatment group.Except for the blank group,the other groups were established as a mouse mastitis model according to the method of LPS injection into the milk duct,and the blank control group was injected with equal amount of saline.1 h before and 12 h after modeling,serpentine was injected intraperitoneally(30 mg/kg)in the serpentine treatment group,an equal amount of saline was injected intraperitoneally in the blank group and the model group,and an equal amount of dexamethasone(5 mg/kg)was injected intraperitoneally in the dexamethasone treatment group.The morphological changes of mammary tissues were observed by dissecting the female rats 24 h after modeling,and the reactive oxygen species(ROS)content,total antioxidant capacity(T-AOC)and myeloperoxidase(MPO)activity in mammary tissues were detected by using kits,and the pathological changes of mammary tissues were observed by H&E staining,LC3-II expression in mammary tissues was detected by immunofluorescence,and apoptosis in mammary tissues was detected by TUNEL.Westernblot was used to detect the expression levels of autophagy marker proteins LC3-II/Ⅰ and p62,as well as the expression levels of PI3 K,AKT and mTOR phosphorylated proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway in breast tissues.Results:(1)Inflammatory changes in the mammary glands of mice: anatomical observation of the mammary glands of mice showed that the mammary glands of mice in the blank group were milky white,morphologically and structurally intact and smooth;H&E staining showed that the mammary glands of mice in the blank group had well-developed and structurally intact alveoli,without inflammatory cell infiltration.Compared with the blank group,the mammary glands of the mice in the model group was obviously red,a large number of inflammatory cells infiltrated in the glandular follicles,and the structure of the glandular follicles was destroyed;the MPO activity was significantly increased(P<0.01).Compared with the model group,the inflammation was significantly reduced in the dextran and dexamethasone treatment groups,and the inflammatory cells in the glandular alveoli were significantly reduced and the structure was more intact;compared with the model group,the MPO activity was highly significantly reduced in the dextran and dextran treatment groups(P<0.01).(2)Changes in ROS content and total antioxidant capacity(T-AOC)in mammary tissue of mice: compared with the blank group,the ROS content was highly significantly increased(P<0.01)and the total antioxidant capacity was highly significantly decreased(P<0.01)in the model group,compared with the model group,the ROS content was highly significantly decreased(P<0.01)in the dexamethasone-treated and serpentine treated groups;the total antioxidant capacity was highly The total antioxidant capacity was significantly increased(P<0.01).(3)Apoptosis in mammary tissue of mice: TUNEL staining showed no significant apoptosis in the mammary tissue of the blank group;a large number of TUNEL staining positive cells with green fluorescence were seen in the model group,and apoptotic cells were significantly increased,indicating that LPS modeling led to apoptosis in mammary cells of mice;compared with the model control group,the staining positive cells in the serpentine and dexamethasone treatment groups compared with the model control group.(4)Autophagy in mouse mammary gland tissue: Compared with the blank group,the autophagy activity expression of LC3-Ⅱ,the number of autophagosomes,and the expression level of LC3-Ⅱ/Ⅰ protein in the model group were significantly down-regulated(P<0.01),the expression level of p62 protein was significantly increased(P<0.05).Compared with the model group,the expression of autophagy activity of LC3-Ⅱ,the number of autophagosomes,LC3-Ⅱ/ The expression level of Ⅰ protein was significantly increased,and the expression level of p62 protein was extremely significantly down-regulated(P<0.01).(5)Expression levels of PI3K/AKT/mTOR pathway-related proteins in mouse mammary gland tissue: Comp ared with the control group,the protein expression levels of p-PI3 K and p-AKT in the cells of the model group were significantly increased(P<0.01),the protein expression level of p-mTOR increased significantl y(P<0.05);compared with the model group,p-PI3 K,p-mTOR and p-AKT,the expression level of the protein was significantly decreased(P<0.01).Conclusion: osthole can effectively reduce the inflammatory damage of LPS-induced mastitis in mice,reduce the r ate of apoptosis and enhance the antioxidant capacity of cells;serpentine can reduce LPS-induced mastitis in mice by inhibiting PI3K/Akt/mTOR signaling pathway to activate autophagy.