Effect Of β-hydroxybutyric Acid On The Oxidative Stress And Expression Of Contraction Related Protein In Bovine Abomasum Smooth Muscle Cell In Vitro

The displaced abomasum of dairy cows is a common clinical perinatal medicaldisease, which pathology foundation is abomasum retardation and extension.β-hydroxybutyric acid has correlation with DA and Ketosis in perinatal period ofdairy cows, while Ketosis accompany with Oxidative Stress, this prompt β-HB mayhave correlation with Oxidative Stress. In this study, bovine abomasum smoothmuscle cell were treated with β-HB cultured in vitro. MTT, Immunofluorescentstaining, RT-qPCR and Western-Blotting methods were used to research the effects ofβ-HB on the cell viability, oxidant/antioxidant enzyme activities and the change ofprotein that accommodate smooth muscle contract, explore the correlation betweenβ-HB and Oxidative Stress as well as the effect of β-HB on contraction proteinexpression in BSMCs, provide theoretical basis for prevention and cure of DA.In this study, enzymatic digestion and tissue explants methods were combinedused to upgrade the method of cultivating BSMCs in vitro. The primary abomasalsmooth muscle cells were isolated, cultured, transfer of cultured and assayed, obtainedhigh purity BSMCs that fit for the following experiments.In this study, the BSMCs cultured in vitro were transferred of culture, thentreated with different concentrations of β-HB(0,0.6,1.2,2.4and4.8mmol/L) for24h.The cell viability was determined by MTT method, the intracellular MDA and ROSlevel were determined with biochemistry detection method, the enzymes activities andmRNA transcription level of cellular redox enzymes CAT, SOD and GSH-Px weredetermined with spectrophotometric and RT-qPCR methods, The transcriptional andtranslational levels of the constriction related enzymes p-MLC, MLCK, MLC, RhoA and ROCK were determined with the RT-qPCR and Western Blotting methods,respectively.The results showed that:①β-HB can make the cell viability decrease,and take on dose-dependent change.②β-HB can make the concentration ofintracellular MDA and ROS increase, and take on dose-dependent change.③β-HBcaused the decrease in the activities of CAT, SOD and GSH-Px, and thetranscriptional levels of cellular redox enzymes.④The transcriptional andtranslational levels of the constriction related enzymes were decreased with increasingconcentration of β-HB significantly.In conclusion: high concentration of β-HB can destroy the intracellularoxidation/anti-oxidant balance, lead the BSMCs in Oxidative Stress state. Highconcentration of β-HB reduces the transcriptional and translational levels ofconstriction related mazymes, which would decrease the contracted ability of BSMCs.