| Cow mastitis is one of the most common diseases on cattle farms,which causing huge losses to the cattle industry.So far,antimicrobial therapy is still the first choice for most farms to treat breast infections and mastitis.However,antibiotic abuse and antibiotic residues pose a major threat to human and animal health.China has a long history of Chinese medicine treatment about mastitis.Traditional Chinese medicine monomers have a wider range of biological activities and are more difficult to develop drug resistance.Therefore,the selection of traditional Chinese medicine monomer molecules to treat mastitis is expected to be an alternative antibiotic for cow mastitis treatment.Gambogic acid(GA)is the main active ingredient of Garcinia Cambogia,which isolated and extracted from Garciniaceae plants.GA has anti-inflammatory,antibacterial,and neuromodulation functions.GA is known for its anti-cancer effects.It is reported that in clinical studies,it can effectively inhibit various cancers,such as gastric cancer and colorectal cancer.But there is no literature figured out the role of GA in the treatment of mastitis,and the role and molecular mechanism of GA on mastitis are not yet clear.In this study,mouse mammary epithelial cells were used to study the effect and mechanism of GA on lipopolysaccharide(LPS)-induced inflammation of mammary epithelial cells(MMECs)in vitro.At the same time,LPS was injected into milk ducts to mimic the in vivo mastitis model,and the effect of GA on mammary gland inflammation,apoptosis,and the blood-milk barrier was studied to explore the role and mechanism of GA in mastitis caused by LPS.The main results of this topic are as follows:1.GA concentration determinationAccording to the preliminary research conclusions of our laboratory,LPS with a final concentration of 1 μg/m L was added to the cultured wells of the extracted mouse mammary epithelial cells to establish an in vitro mastitis model.The test results show that 1μg/m L LPS can cause the proinflammatory factors IL-1β,IL-6 and TNF-α expression to be significantly increased(p<0.001).Pre-incubated mouse mammary epithelial cells with GA of 10 n M,25 n M,and 50 n M and then added LPS to incubate 24 hours to detect the expression levels of pro-inflammatory factors IL-1β,IL-6,and TNF-α m RNA.The pro-inflammatory factors had inhibition trends among GA groups,and the degree of inhibition showed a dose-dependent relationship with the concentration of GA.At the same time,the effect of different concentrations of GA,LPS and dimethyl sulfoxide(DMSO)on the activity of mouse mammary epithelial cells was tested by the CCK-8 kit.The results showed that GA,LPS and DMSO concentrations of 10 n M,25 n M and 50 n M does not affect the cell viability of mouse mammary epithelial cells.Based on this,GA in concentrations of 10 n M,25 n M,and 50 n M was selected for follow-up experimental research.2.GA inhibited LPS-induced inflammation of mouse mammary epithelial cellsThe expression levels of pro-inflammatory factors IL-1β,IL-6 and TNF-α in mammary epithelial cells were detected by RT-q PCR.The results showed that LPS can significantly increase the expression levels of the three pro-inflammatory factors compared with the control group(P<0.001).With pretreatment different concentrations of GA,the expression levels of IL-1β,IL-6,and TNF-α decreased in a dose dependent manner.The related proteins such as TLR4 and proteins in downstream signaling pathways NF-κB and MAPKs were detected.Experimental data shows that GA can down-regulate the expression of TLR4 protein and have a certain inhibitory effect on the phosphorylation of p65 and IκB protein,which can reduce the phosphorylation level of these two proteins compared with the LPS group.Similarly,the phosphorylation levels of p38,ERK,and JNK caused by LPS in the MAPKs signaling pathway can also be inhibited by GA to varying degrees.The results of RT-q PCR showed that GA could inhibit the m RNA expression of p65,IκB,P38,JNK and ERK in NF-κB and MAPKs signaling pathways caused by LPS.Therefore,GA can inhibit mastitis by inhibiting the TLR4-NF-κB/MAPKs signaling pathway.3.GA inhibited LPS-mediated apoptosis of mouse mammary epithelial cells via ROS and MMPIn the study of GA on LPS-induced mammary epithelial cell apoptosis,the level of reactive oxygen species(ROS)expression,mitochondrial membrane potential(MMP)and apoptosis-related protein expression were detected.The results of flow cytometry showed that LPS caused a significant increase in the expression of ROS(p <0.001),and this phenomenon was reversed in GA group,with the expression of ROS decreased.In the next step,MMP,which is closely related to ROS was detected.Laser confocal microscopy was used and found that the stability of MMP in mammary epithelial cells was destroyed with the addition of LPS,and the apoptosis signal Annexinv FITC was enhanced.The steady-state of MMP in breast epithelial cells pretreated with different concentrations of GA was gradually recovered,and the apoptosis signal Annexinv FITC also gradually weakened.The related apoptotic proteins were detected by western blot.The results showed that the increase of Caspase-3 and Caspase-9 protein expression caused by LPS was suppressed in different concentrations of the GA group,and the degree of inhibition was dose-dependent on GA.Compared with the control group,the expression level of Bcl-2 protein in the LPS group was significantly suppressed,and the expression level of Bcl-2 protein in each concentration group of GA showed an upward trend.Gene expression levels of Caspase-3,Caspase-9 and Bcl-2 are similar to protein expression levels.With LPS treatment,the m RNA expression levels of Caspase-3 and Caspase-9 in mammary epithelial cells increased significantly,but with adding different concentrations of GA,the gene expression level of Caspase-3 and Caspase-9 gradually decreased.In the LPS group,the m RNA expression of Bcl-2 decreased significantly,and the m RNA expression level gradually increased by adding different concentrations of GA.4.GA inhibited the damage of LPS to mammary gland tissue and protects the blood-milk barrierA mouse mastitis model was established by injecting LPS into the nipples of the mammary glands,and different concentrations GA groups were set.Blood samples and mammary gland tissues of mice were collected after stimulated 24 hours.The expression of myeloperoxidase(MPO)and three pro-inflammatory factors IL-1β,IL-6 and TNF-α in serum were detected by ELISA.Pathological section analysis and pathological scoring of breast tissues were performed to better understand the effect of GA on mastitis.The results showed that the expression levels of MPO,IL-1β,IL-6 and TNF-α in the serum of mice in the LPS group were significantly improved and significantly different from those in the control group(p<0.001).Compared with LPS,the expression levels of MPO,IL-1β,IL-6 and TNF-α in GA groups at different concentrations were reduced to varying degrees,of which the difference was most significant at the 50μM concentration group(p<0.001),indicating that GA The inflammatory markers MPO and pro-inflammatory factors caused by LPS have a good inhibitory effect.Pathological section results showed that the structure of the acinar mammary glands in mice only treated with LPS was destroyed,the edges were swollen,and accompanied by cell swelling and neutrophil infiltration.The GA treated mammary glands have some improvements when compared with the LPS group,manifested by more complete acinar structure,clear boundaries and reduced infiltration of inflammatory cells and blood cells.Pathological scoring of breast tissue sections revealed that the GA group score was significantly lower than that of the LPS group(p<0.001),indicating that the mastitis symptoms of the GA group were alleviated to a certain extent.A permeability test was carried out on the mammary gland tissue of mice.The test results showed that the acinar structure in the mammary gland tissue after LPS treatment was destroyed,and the permeability of the blood-milk barrier increased,which led to the infiltration of a large amount of albumin into the mammary acinar.However,the acinar structure in the breast tissue of mice pretreated with GA was relatively tight,and the albumin infiltrating into the acinar cavity gradually decreased with the increase of the concentration of GA,indicating that the blood-milk barrier of the GA groups was more complete.At the same time,the expression signal of Claudin-3 protein in the blood milk barrier was detected by immunofluorescence experiments,and we found that the expression signal of Claudin-3 protein in the LPS group was greatly reduced compared with the control group,while the signal of Claudin-3 protein in the GA groups went down.With the increase of GA concentration,the signal of Claudin-3 protein gradually increased,indicating that GA has an important role in maintaining the expression of Claudin-3 in the blood milk barrier.In summary,GA can protect LPS-induced mastitis by inhibiting MAPKs and NF-κB signaling pathways.GA can maintain the stability of MMP and reduce the excessive production of ROS to inhibit the expression of LPS-induced apoptosis-related proteins and genes,while also protecting the structure of the blood-milk barrier.Our research results show that GA can be used as a potential treatment for mastitis,and provide new ideas and theoretical basis for clinical mastitis treatment. |
