Effects Of Meloxicam On Lipopolysaccharide Induced Oxidative Stress And Inflammatory Response In Bovine Endometrial Epithelial Cells

Postpartum uterine infection in cows often causes metritis and endometritis,accompanied by inflammation and oxidative injury,impairs the fertility of cows,and increases the elimination rate,thereby affecting the development of dairy breeding.The common clinical causes are mostly peripartum metabolic disorders,abnormal calving,and microbial contamination.Escherichia coli is one of the main pathogenic bacteria affecting the bovine uterus through lipopolysaccharide(LPS).The recognition of LPS by Toll like receptor 4(TLR4)activates nuclear factor kappa-B(NF-κB)signaling,resulting in the expression and release of downstream inflammatory factors,accompanied by the production of reactive oxygen free radicals.When ROS production far exceeds the body’s antioxidant clearance capacity,it destroys lipids,proteins,and DNA,leading to oxidative stress.Meloxicam(MEL)is a selective cyclooxygenase-2(COX-2)inhibitor and has been used in combination with antibiotics to alleviate postpartum uterine inflammation and provide analgesia.Studies have shown that MEL has antioxidant and anti-inflammatory effects.However,the link between MEL and uterine inflammation and oxidative stress in dairy cows has not been studied.The purpose of this study was to explore the mechanism of MEL(0.5 or 5 μM)on 1 μg/mL LPSinduced oxidative stress and inflammation of primary bovine endometrial epithelial cells(BEEC).This study provided a theoretical basis for clinical rational use of MEL to alleviate cow uterine inflammation.1.The effects of MEL on LPS-induced oxidative stress of BEECIn order to explore the influence of MEL on LPS-induced BEEC oxidative damage,the cells were treated with different concentrations of MEL(0.5 or 5 μM)alone or cotreated with LPS for 12 h.The contents of reactive oxygen species(ROS)and malondialdehyde(MDA),the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px),and total antioxidant capacity(T-AOC)were detected by the corresponding kits.As a result,MEL alone showed no effect on cell oxidative damage indexes and antioxidant enzyme activity(P>0.05).LPS stimulation leads to the increased ROS and MDA contents(P<0.05 or P<0.01).Compared with the LPS group,the addition of MEL reduced ROS and MDA contents(P<0.05 or P<0.01),and the effect of 5 μM MEL was more significant.These results suggested that MEL protected BEEC from oxidative damage induced by LPS through reducing ROS and MDA contents and improving antioxidant enzyme activities.In order to explore the changes of MEL to LPS-induced BEEC nuclear factor erythroid 2-related factor 2(Nrf2)pathway and oxidation-related cytokine expression,the cells were treated with different concentrations of MEL(0.5 or 5 μM)and LPS for 6,12 and 24 h to detect the gene expression of CAT,GPX1,SOD1,nitric oxide synthase 2(NOS2)and prostaglandin-endoperoxide synthase 2(PTGS2)were detected by RT-qPCR.The cells were treated with different concentrations of MEL(0.5 or 5 μM)alone or cotreated with LPS for 90 min to determine the key protein levels in Nrf2 pathway by Western Blot.The nuclear Nrf2 was observed by immunofluorescence.As a result,compared with the control group,the gene expression of CAT and GPXl in LPS group decreased at 6,12 and 24 h(P<0.05 or P<0.01),the SOD1 expression decreased at 6 h(P<0.01),and the NOS2 and PTGS2 expression increased at 6,12 and 24 h(P<0.05 or P<0.01).Compared with LPS group,MEL and LPS co-treatment group up-regulated(P<0.05 or P<0.01)the gene expression of CAT,GPX1 and SOD1 at 6,12 and 24 h,and down-regulated(P<0.05 or P<0.01)the NOS2 expression at 6,12 and 24 h,and PTGS2 expression at 12 and 24 h.MEL alone had no significant effect on the protein level of Nrf2 pathway(P>0.05).LPS stimulation reduced the protein levels of Nrf2,HO-1 and NQO1(P<0.05),and increased(P<0.05)Keap1 level.Compared with the LPS group,cotreatment of LPS and MEL increased(P<0.05 or P<0.01)the protein levels of Nrf2,HO-1 and NQO1,reduced(P<0.05 or P<0.01)the Keap1 level,and promoted(P<0.01)Nrf2 to entry the nucleus.These results indicated that MEL resisted LPS-induced oxidative damage of BEEC by activating Nrf2 pathway,promoting the gene expression of related antioxidant enzyme,and inhibiting the gene expression of NOS2 and PTGS2.2.The effects of MEL on LPS-induced inflammatory response of BEECIn order to explore the impact of MEL on NF-κB pathway and inflammatory cytokine gene expression in BEEC stimulated with LPS,the cells were cotreated with different concentrations of MEL(0.5 or 5 μM)and LPS for 3,12 and 18 h.The gene expression of TLR4,myeloiddifferentiationfactor88(MYD88),interleukin(IL)-1B,IL6,C-X-C motif chemokine ligand 8(CXCL8)and tumor necrosis factor(TNF)was detected by RT-qPCR.The cells were treated with different concentrations of MEL(0.5 or 5 μM)alone or cotreated with LPS for 90 min to detect the key protein levels in NF-κB pathway by Western Blot and the level of nuclear p65 by immunofluorescence.As a result,LPS stimulated gene expression of TLR4,MyD88,IL-1B,IL6,CXCL8,TNF at 3,12 and 18 h(P<0.05 or P<0.01).MEL inhibited(P<0.05 or P<0.01)LPS-induced gene expression of MYD88,IL1B,IL6,CXCL8 and TNF,but did not affect TLR4(P>0.05).MEL alone had no effect(P>0.05)on the phosphorylation level of p65 and IκBα.LPS induced(P<0.01)p65 and IκBα phosphorylation and promoted(P<0.01)p65 to enter the nucleus.compared with the LPS group,the LPS and MEL cotreatment groups reduced(P<0.05 or P<0.01)the phosphorylation levels of p65 and IκBα,and the amount of nuclear p65.These results revealed that MEL resisted LPS-induced inflammatory responses by inhibiting NF-κB pathway and downstream inflammatory cytokine gene expressions in BEEC.In conclusion,MEL inhibited LPS-induced oxidative stress and inflammation of BEEC,by activating the Nrf2 pathway and inhibiting the NF-κB pathway,respectively.