Forsythiate Glycoside A Is Regulated Based On AMPK/mTOR/ULK1 Pathway Autophagy And Apoptosis Of MAC-T Cells Induced By LPS

Dairy mastitis seriously harms the health of dairy cows,causing damage to the mammary gland,and thus affecting milk yield and milk quality.Escherichia coli is one of the most common pathogenic microorganisms that cause mastitis in dairy cows.Its main virulence factor,lipopolysaccharide(LPS),can cause mammary inflammatory damage and enter the blood to harm the health of multiple organs in the body.At present,antibiotics are the most widely used therapeutic drugs in clinical practice.However,there are problems such as bacterial resistance and drug residue,so it is urgent to develop antibiotic substitutes that are more in line with animal food safety and environmental and ecological safety.Forsythia is a traditional Chinese medicine with functions of clearing heat and detoxifying,eliminating carbuncle and clearing knot,and evacuating wind and heat.Forsythiate side A(FTA)is one of the main active components of forsythia.Modern pharmacological studies have shown that FTA has antiinflammatory,antiviral,antioxidant and other biological activities.In this study,FTA was used as the main object of study,and LPS-induced autophagy and apoptosis of dairy mammary epithelial cells(MAC-T)were used as the model to explore the effect and mechanism of FTA regulation on autophagy and apoptosis of MAC-T cells,providing experimental basis and theoretical basis for the research and development of new drugs.1.Effects of LPS on autophagy and apoptosis of MAC-T cellsIn order to explore the effects of LPS on autophagy and apoptosis of MAC-T cells,MAC-T cells were treated with four concentrations of LPS(0 μg/ml,50 μg/ml,100 μg/ml,200 μg/ml)for 12 h,and the effects of different concentrations of LPS on the survival rate of MAC-T cells were detected by CCK-8kit.The cell damage was detected by lactate dehydrogenase kit(LDH),the expressions of autophagy and apoptosis-related genes and proteins were detected by q PCR and Western Blot,the number of autophagy spots was detected by immunofluorescence,the cell nucleus morphology was observed by Hoechst33342 staining,and the apoptosis rate was detected by flow cytometry.The results of CCK-8 and LDH showed that LPS inhibited the proliferation of mammary epithelial cells,and the content of LDH in the supernatant increased with the increase of LPS concentration.The results of q PCR and Western blot showed that compared with the control group,LPS(50-100 μg/ml)increased the expressions of autophagy related genes and proteins LC3 B,ATG5,ATG7,Beclin-1,p62(p <0.01).LPS can induce apoptosis of dairy mammary epithelial cells and improve the expression of BAX and Caspase-3 gene and protein(p <0.01),decreased the expression of BCL-2 gene and protein(p <0.01).Immunofluorescence results showed that LPS increased the number of autophagy spots compared with the control group.Flow cytometry and Hoechst 33342 staining showed that LPS significantly increased the apoptosis rate of MAC-T cells and the number of abnormal cell morphology increased.These results indicated that LPS increased the autophagy level of MAC-T cells,promoted apoptosis,inhibited proliferation of MAC-T cells,and aggravated cell damage.2.LPS regulates autophagy and apoptosis of MAC-T cells based on AMPK/m TOR/ULK1 signaling pathwayIn order to identify the mechanism of AMPK/m TOR/ULK1 signaling pathway in LPS-induced autophagy and apoptosis of MAC-T cells,Compound C(CC)was used to pretreat MAC-T cells for 2 h,and then LPS(100 μg/ml)was added for 12 h.The expression levels of AMPK/m TOR/ULK1 signaling pathway,autophagy and apoptosis related proteins were detected by Western Blot,and the apoptosis rate was detected by flow cytometry.The results showed that compared with the control group,the ratio of PAMPK and AMPK,and P-ULK1 and ULK1 protein expression levels in MAC-T cells treated with LPS significantly increased(p <0.01);After LPS(50,100 μg/ml)treatment,the expression ratio of P-Mtor and m TOR protein in MAC-T cells was significantly decreased(p <0.01),but there was no significant difference in p-m TOR and m TOR protein expression ratio under LPS treatment of 200 μg/ml.Compared with the control group,pretreatment with CC significantly decreased P-AMPK /AMPK and P-ULK1/ULK1(p <0.01),compared with LPS group,the protein expression levels of P-AMPK /AMPK,PULK1 /ULK1,LC3 B and BCL-2 in CC + LPS group were significantly decreased(p <0.01),significantly increased the protein expression levels of P-Mtor /m TOR and BAX(p <0.01).Flow cytometry showed that compared with LPS group,the apoptosis rate of CC + LPS group was significantly increased(p <0.01).These results indicated that LPS regulated autophagy of MAC-T cells through AMPK/m TOR/ULK1 signaling pathway.CC inhibited AMPK activity,reduced the autophagy level of LPS-induced MAC-T cells,and promoted apoptosis.3 Forsythiolate glycoside A regulates LPS-induced autophagy and apoptosis of MAC-T cells based on AMPK/m TOR/ULK1 pathwayIn order to clarify the protective effect of FTA on the autophagy and apoptosis of MAC-T cells,MAC-T cells were pretreated with FTA for 12 h,LPS(100 μg/ml)for another 12 h,and CCK-8 was used to detect the proliferation effect of FTA on MAC-T cells and the effect of FTA pretreatment on the proliferation of MAC-T cells.The number of autophagy spots was detected by immunofluorescence,the expressions of autophagy and apoptosis-related genes and proteins were detected by q PCR and Western Blot,and the apoptosis rate was detected by flow cytometry.The results of CCK-8 showed that FTA pretreatment could promote the proliferation of MAC-T cells,and could resist the decrease of the activity of MAC-T cells by LPS.Western Blot and q PCR results showed that LC3 B and Beclin-1 gene and protein levels in LPS + F group were significantly increased compared with LPS group(p <0.05 or p<0.01),p62 gene and protein levels were significantly decreased(p <0.01),the levels of apoptosisrelated genes and protein BAX were significantly decreased(p <0.01),BCL-2 gene and protein levels were significantly increased(p <0.05),and significantly increased the protein expression levels of PAMPK /AMPK and P-ULK1 /ULK1(p <0.01),significantly down-regulated P-mtor /m TOR protein expression levels(p <0.05);Immunofluorescence results showed that compared with the control group, FTA pretreatment significantly increased the number of autophagy spots.Flow cytometry showed that compared with LPS group,FTA pretreatment significantly reduced the apoptosis rate.These results suggest that FTA can regulate autophagy and apoptosis through AMPK/m TOR/ULK1 pathway,and play a protective role in MAC-T cells.In order to verify that the regulation effect of FTA on autophagy and apoptosis of MAC-T cells was realized through AMPK/m TOR/ULK1 pathway,MAC-T cells were pretreated with CC for 2 h,treated with FTA for 12 h,and cultured with LPS for 12 h.The expressions of autophagy,apoptosis and inflammatory proteins were detected by Western Blot.The results showed that LC3 B and BCL-2 were significantly decreased in LPS +F+ CC group compared with LPS +F group(p<0.01),p62,BAX,IL-1β,IL-6,p-NFκB/ NFκB,P-IKB /IKB were significantly increased(p <0.01 or p <0.05).These results indicated that CC silences AMPK pathway could block FTA to relieve p62 protein accumulation in LPS-induced MAC-T cells,inhibit autophagy,aggravate apoptosis and inflammatory damage.In conclusion,LPS can increase the level of fine autophagy and apoptosis of MAC-T,cause the arrest of autophagy flow and promote apoptosis.LPS can deregulate autophagy and apoptosis of MAC-T cells through AMPK/m TOR/ULK1 pathway.FTA can reduce the inhibition of autophagy flow induced by LPS,promote the smooth autophagy flow,and play a protective role in the process of autophagy and apoptosis of MAC-T cells induced by LPS.FTA regulates autophagy and apoptosis-related processes of MAC-T cells mainly through AMPK pathway.