The Molecular Mechanisms Of Cell Death Induced By Non-esterified Fatty Acid In Bovine Abomasal Smooth Muscle Cell

Displaced abomasum (DA) is a typical gastric disorder disease, which frequentlyoccurred in high-yielding dairy cows. Recently, with the rapid development of dairyfarming, the incidence of DA has been rising. Due to this disease has attracted greatattention of researchers, its etiology and pathogenesis is a hot spot under research.Envidences suggested that hypotony or atony of abomasal muscles was postulated asaprerequisite for the occurrence of DA. At the same time, epidemiological studiespointed out that the occurrence of DA is usually accompanied by high concentrationsof non-esterified fatty acid (NEFA) and beta-hydroxybutyrate in blood, suggestingthat NEFA or beta-hydroxybutyrate plays some possible role in the process of DA.An in vitro model of primary cultured bovine abomasum smooth muscle cell(BSMC), which was isolated from newborn calf, was used in the present study.BSMC were exposed to different concentrations of NEFA, ranging fromphysiological to pathological concentrations, to investigate the effects of NEFA onBSMC and to explore the potential mechanisms. By MTT assay, we found that the0.1-0.9mmol/L NEFA had no detrimental effects on cell vitality of BSMC, however,when the concentration of NEFA was over1.2mmol/L, up to3.3mmol/L, asignificant reduction of cell vitality was observed. Next, apoptosis assay wasperformed and the results showed that0.6mmol/L NEFA had no significant effect onapoptosis rate, while after the treatment of1.2mmol/L or1.8mmol/L NEFA, BSMCexhibited a higher apoptosis rate. These results further confirmed that NEFA hascytotoxicity on BSMC.In order to explore whether oxidative stress participated in the NEFA-inducedBSMC cell death, several oxidative/antioxidative indexes were detected, including thelevels of intracellular reactive oxygen species level (ROS) with the DCFH-DAfluorescent probe, the activities of antioxidant enzymes superoxide dismutase (SOD)and catalase (CAT), as well as the content of malonaldehyde (MDA) and glutathioneby biochemistry kits. The results showed that high concentrations of NEFAeffectively reduced intracellular antioxidant capacities and promoted the levels of oxidative factors, indicating that NEFA-induced BSMC cell death is mediated viaoxidative stress.Further, we tried to study the mechanisms how NEFA caused the cell apoptosisBSMC. It is commonly believed that oxidative stress mediates apoptosis by activatingthe mitochondrial apoptotic pathway. Therefore, the factors which are related tomitochondrial apoptotic pathways were examined: the change of mitochondrialmembrane potential (with Rhodamine123probe), the distribution of cytochrome C(by Western blot), the activation of apoptosis-associated proteins caspase-9, caspase-3,PARP activation (by Western blot), the expression of apoptosis promoting/suppressing proteins Bax/Bcl-2(by Western blot), as well as the translocation ofapoptosis inducing factor AIF (by immunofluorescence). From the results, it can befound that high concentrations of NEFA significantly caused a decline inmitochondrial membrane potential, cytochrome C release, activation of caspase-9/caspase3/PARP, increased expression of Bax and decreased expression of Bcl-2,translocation into nuclear of AIF, suggesting that NEFA induced cell apoptosis wasassociated with the activation of the mitochondrial apoptosis pathway.Considering all the results above, it can be confirmed that more than1.2mmol/LNEFA, which was observed in clinical DA cases, significantly decreased the cellvitality and increased cell apoptosis rate, reduced intracellular antioxidant capacitiesand resulted in the augment of content of ROS and MDA, as well as the activation ofapoptosis proteins. In addition, when BSMC were pretreated with antioxidants NAC,the above events caused by NEFA were suppressed effectively, further confirming theNEFA-caused cell apoptosis was assoicatied with oxidative stress-mediatedmechanisms. This study discusses the detrimental effects of NEFA on BSMC, andprovides the theory basis for the understanding of the physiological functions ofNEFA. Meanwhile, it also helps elucidate the pathogenesis of DA and provides thenovel understandings for the prevention and control of this disease.