Regulation Of Caffeic Acid On Lipopolysaccharide-Activated BMEC And RAW264.7 Nox-ROS Signaling

CA(Caffeic acid.CA)is rich in medicinal plants(Compositae,Rosaceae,Sapindaceae etc.)and is a natural phenolic compound with anti-inflammatory and anti-oxidant effects.Bovine marmmary epithelial cells(bMEC)are important functional units for milk synthesis and secretion.They are the main cell model for the study of mammary gland growth and lactation regulation,and are susceptible to damage during cow mastitis.Previous studies have shown that CA can inhibit the damage of bMEC structure caused by lipopolysaccharide(LPS).Macrophages participate in non-specific and specific immunity in the body.When mastitis occurs,the inflammatory response is initiated when antigens and chemotactic neutrophils are presented.In this study,based on the LPS-induced bMEC structural damage model established in the previous stage,Nox(non-phagocytic cell oxidase)inhibitor(DPI)and ROS scavenger(NAC)treatment were used to investigate the Nox/ROS signal induced by LPS in bMEC injury.The role of the mouse macrophage cell line(RAW 264.7)in inflammation,oxidative stress,and the regulation of CA by this signal.In this study,bMEC and RAW 264.7 were divided into blank control group(Con),model group(Mod),ROS scavenger group(NAC+LPS),Nox inhibitor(DPI+LPS)group,and CA treatment group(CA+LPS).CA,control group(CA),flow cytometry was used to detect ROS content,mitochondrial membrane potential change,apoptosis rate.Immunoblotting was used to detect the expression of key proteins in NF-?B and Nrf2 signaling pathways.The localization of nuclear transcription factors was detected by laser confocal technique.The expression of inflammatory factors was detected by RT-PCR,and the expression of MDA and SOD activity were detected by ELISA.The research results are as follows:Optimization of preparation conditions for primary mammary gland epithelial cells.In this experiment,bMEC with good cell viability was cultured quickly and easily by collagenase one-step digestion.CA inhibits LPS-activated bMEC Nox-ROS signaling.LPS(50 ?g/mL)stimulated bMEC for 0.5 h:Compared with Con group,there was no significant difference in ROS content between Mod group(P>0.05),ROS decreased significantly in CA+LPS group and CA group(P<0.01);LPS stimulated for 0.5 h did not cause a significant decrease in mitochondrial membrane potential,and the mitochondrial membrane potential decreased significantly at 2 h,4 h,and 8 h(P<0.01).LPS(50 ?g/mL)stimulated bMEC for 0.5 h:Compared with Con group,the expression of p-I?B? and p-p65 in Mod group was significantly increased(P<0.01),and p65 nuclear translocation increased;NAC compared with Mod group The expression of p-p65 and p-l?B? protein in LPS,DPI+LPS,CA+LPS group and CA group was significantly decreased(P<0.01),and p65 nuclear translocation was decreased.When LPS(50 ?g/mL)stimulated bMEC for 12 h:compared with Con group,ROS content in Mod group was significantly increased(P<0.01),mitochondrial membrane potential was significantly decreased(P<0.01),apoptosis rate was significantly increased(P<0.01),and the Bax/Bcl-2 expression was significantly increased(P<0.01);compared with Mod group,ROS were significantly decreased in NAC+LPS group,DPI+LPS group,CA+LPS group and CA group(P<0.01),mitochondrial membrane potential increased significantly(P<0.01),apoptosis rate decreased significantly(P<0.01),and the Bax/Bcl-2 expression increased significantly(P<0.01),Nrf2 nuclear protein expression decreased significantly(P<0.0]),Nrf2 nuclear shift increased.Compared with the Con group,the expression of IL-6 and TNF-a in the Mod group were significantly increased(P<0.01)and IL-10 was significantly lower(P<0.01).Compared with the Mod group,the expression of IL-6 and TNF-a in the NAC+LPS group,DPI+LPS group and CA+LPS group were significantly decreased(P<0.01)and IL-10 was significantly increased(P<0.01).Compared with the Con group,the MDA content of the Mod group increased(P<0.01)and the SOD activity decreased significantly(P<0.01).Compared with the Mod group,the MDA content decreased significantly(P<0.01),the SOD of the NAC+LPS group,the DPI+LPS group,and the CA+LPS group increased significantly(P<0.01).CA inhibits LPS-activated RAW 264.7 Nox-ROS signaling.LPS(1 ?g/mL)stimulated RAW 264.7 for 0.5 h:Compared with the Con group,the ROS content in the Mod group increased significantly(P<0.01),the mitochondrial membrane potential decreased significantly(P<0.01),the expression of p-p65,p-I?B? protein was significantly increased(P<0.01),and the nuclear translocation of p65 was increased.Compared with the mod group,the ROS of the NAC+LPS group,the DPI+LPS group,the CA+LPS group and the CA group were significantly lower(P<0.01),the expression of p-p65 and p-I?B? protein was significantly decreased(P<0.01),and the nuclear shift of p65 was decreased.LPS(1 ?g/mL)stimulated RAW 264.7 for 12 h:Compared with Con group,mitochondrial membrane potential was significantly decreased in Mod group(P<0.01),apoptosis rate was significantly increased(P<0.01),the expression of Bax/Bcl-2 was significantly increased(P<0.01),Nrf2 nucleoprotein expression was significantly higher(P<0.01),and Nrf2 nuclear translocation was increased.Compared with Mod group,The ROS content was significantly decreased in the NAC+LPS group,DPI+LPS group,CA+LPS group and CA group(P<0.01),the mitochondrial membrane potential was significantly increased(P<0.01),the apoptosis rate was significantly decreased(P<0.01),and the Bax/Bcl-2 of expression was significantly decreased(P<0.01).Nrf2 nuclear shift is reduced.Compared with the Con group,the expression of IL-6 and TNF-? in the Mod group were significantly increased(P<0.01)and IL-10 was significantly lower(P<0.01).Compared with the Mod group,the expression of IL-6 and TNF-a in the NAC+LPS group,DPI+LPS group and CA+LPS group were significantly decreased(P<0.01)and IL-10 was significantly increased(P<0.01).Compared with the Con group,the MDA of the Mod group increased(P<0.01)and the SOD decreased significantly(P<0.01).Compared with the Mod group,the MDA content decreased significantly(P<0.01),the SOD activity of the NAC+LPS group,DPI+LPS group,CA+LPS group increased significantly(P<0.01).Studies have shown that CA can inhibit bMEC,RAW 264.7 Nox-ROS signaling,regulate cellular NF-?B,Nrf2 signaling pathway,thereby reducing LPS-induced inflammation,oxidative stress damage and exert anti-inflammatory and anti-oxidative effects.