The Effect Of Paeonol Against LPS-Induced Inflammatory Injury In Mammary Tissue And Epithelial Cells

Dairy cow mastitis is a common and frequent disease in the process of breeding,it frequently results in a drop in milk production from dairy cows,an increase in feeding expenses,and a rise in the elimination rate of dairy cows,all of which have a negative impact on the growth of the dairy industry.Pathogenic microorganism infection is the main cause of cow mastitis,antibiotics are used to treat dairy mastitis in veterinary clinic.The issues of bacterial resistance and medication residue are progressively worse with the widespread use of antibiotics.Therefore,it is urgent to find an effective and less toxic antibiotic substitute.Paeonol is an extract of traditional Chinese medicine which has rich pharmacological effects,such as anti-inflammatory,anti-oxidative stress,anti-tumor,etc.In this study,the inflammatory models of BMECs and mouse mammary tissue were established by LPS induction to explore the protective effect of Paeonol on mammary epithelial cells and tissue to provide basis for subsequent clinical application.Before the formal trial,BMECs were observed after resuscitation,and the effect of Paeonol at gradient concentrations on the proliferation of BMECs were detected by CCK-8.The results showed that the BMECs grew well and the shapes looked like paving stones.The cells were fusiform and the nuclei were bluish-purple by Jimsa staining.By the detection of CCK-8,it showed that within 200 mg/L Paeonol had no significant toxic effect on cell proliferation,10 mg/L,50 mg/L and 100 mg/L Paeonol were selected as follow-up tests.In this study,the inflammatory model of BMECs in vitro was established by 20 mg/L LPS,the mRNA expressions of IL-1β,IL-6,TNF-α,iNOS and COX2 in BMECs were detected by qPCR;The cell apoptosis was detected by Annexin V-FITC apoptosis detection kit;The activities of SOD and GSH-Px,and the content of MDA were detected by the assay kit;Western blot was used to find evidence of NF-κB,MAPK,and Nrf2 protein expression.The findings demonstrated that following Paeonol treatment,the mRNA expression levels of IL-1,IL-6,TNF-,iNOS,and COX2 in BMECs significantly decreased as compared to the LPS group(P<0.05),and the number of cell apoptosis and the content of MDA decreased significantly(P<0.05).The activities of SOD and GSH-Px increased significantly in BMECs(P<0.05).The phosphorylation levels of IB,p65,jnk,p38,and erk,as well as the expression levels of Keap1,increased significantly(P<0.05)in the LPS treatment group,while the expression levels of HO-1,NQO1,and Nrf2 decreased significantly(P<0.05)in BMECs.Paeonol treatment reversed these changes.Among them,100 mg/L Paeonol treated BMECs had the most significant effect.In this study,the mouse model of mastitis was established by 200 mg/L LPS.The pathological changes of mammary gland were analyzed,and the contents of IL-6,IL-1β,TNF-α and MPO activity in the tissues were detected by the assay kit.The results showed that the mammary tissue of mice in the LPS group showed obvious bleeding and swelling,the mammary acinus wall was clearly thickened,and the acinus cavity was heavily infiltrated with macrophages and neutrophils,and Paeonol treatment alleviated the above pathological changes.Paeonol treatment significantly reduced the contents of IL-6,IL-1β,TNF-α and the activity of MPO in mammary tissue(P<0.05).The gavage dose of 100 mg/kg showed the most significant anti-inflammatory effect on mouse mammary tissue.In conclusion,Paeonol had no toxic effect on BMECs activity within 200 mg/L.100 mg/L Paeonol can inhibit inflammatory response and oxidative stress induced by LPS,inhibit the activity of NF-κB and MAPK pathways,and improve the activity of Nrf2 pathway in BMECs.This concentration of Paeonol has the best protective effect on dairy mammary epithelial inflammatory cells.Moreover,100 mg/kg gavage dose of Paeonol can attenuate the pathological injury induced by LPS and reduce the inflammatory response in mouse mammary tissue.