Pathway of programmed cell death and oxidative stress induced by β-hydroxybutyrate in dairy cow abomasum smooth muscle cells and in mouse gastric smooth muscle

Abstract

The administration of exogenous β-hydroxybutyrate (β-HB), as well as fasting and caloric restriction, is a condition associated with β-HB abundance and decreased appetite in animals. Increased β-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of β-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous β-HB on the stomach, mice were injected intraperitoneally with β-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of β-HB. We found that β-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. β-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. β-HB also promoted AIF, EndoG release and p53 expression. β-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. β-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with β-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that β-HB causes ROS generation through a Ca2+-dependent mechanism and that intracellular Ca2+ levels play a critical role in β-HB -induced apoptotic cell death. The impact of β-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of β-HB on smooth muscle. We propose that β-HB is a possible cause of some stomach diseases, including bovine LDA.

Figure 1. Effect of β-HB on cell death.

Cells were incubated 48β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium (A and C) or medium with 10% FBS (B and D). A) and B) Cell viability was determined with a MTT assay. Viability was calculated as the percentage of living cells in treated cultures compared to those in control cultures. C) and D) Mortality analysis. The mortality of BSMCs were assayed for with annexin V-FITC by flow cytometry. The mean of three independent experiments is shown. *p<0.05, **p<0.01 versus control group.

Figure 2. Effect of β-HB on intracellular ROS level and intracellular calcium level in BSMCs.

Intracellular ROS level was measured with DCFH-DA by confocal microscopy. Intracellular calcium level was measured with Fura-3/AM by confocal microscopy. A) and D) Cells were incubated 12 h with β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. B) and E) Cells were incubated 12 h with β-HB (0, 2.4 mM) in serum-free medium with or without pretreatment with NAC (1 mM) or BAPTA/AM (15 µM). C) and F) Cells were incubated 12 h with β-HB (0, 2.4 mM) in medium with 10% FBS with or without pretreatment with NAC (1 mM) or BAPTA/AM (15 µM). The mean of three independent experiments is shown. *p<0.05, **p<0.01 versus control group.

Figure 3. Effect of β-HB on phosphorylation of MAPKs in BSMCs.

Cells were incubated 48β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. Phosphorylated-p38 and total p38, phosphorylated-JNK and total JNK, phosphorylated-ERK1/2 and total ERK1/2 were detected by Western blot.

Figure 4. Effect of β-HB on the expression of p53 in BSMCs.

Cells were incubated 48β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. A) p53 mRNA was detected by Q-PCR, and the ratio of p53 to β-actin was calculated. B) p53 protein was detected by Western blot, and the ratio of p53 to β-actin was calculated. C) Cells were incubated 48 h with β-HB (0, 2.4 mM) in serum-free medium with or without pretreatment with NAC (1 mM) or BAPTA/AM (15 µM). The mean of three independent experiments is shown. *p<0.05, **p<0.01 versus control group.

Figure 5. Effect of β-HB on the expression of cleaved caspase-12, -9, -3 Bax, Bcl-2 in BSMCs.

A) and B) Cells were incubated 48 h with β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. Cleaved caspase-12, -9, -3, Bax and Bcl-2 were detected by Western blot. C) Cells were incubated 48 h with β-HB (0, 2.4 mM) in serum-free medium in the absence or presence of NAC (1 mM) or BAPTA/AM (15 µM). Cleaved caspase-12, -9, -3 were detected by Western blot.

Figure 6. Effect of β-HB on AIF and EndoG expression and translocation in BSMCs.

Cells were incubated 48β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. A) AIF and EndoG mRNA were detected by Q-PCR, and the ratios of AIF and EndoG to β-actin were calculated. B) AIF and EndoG protein were detected by Western blot. C) and D) Protein localization of AIF and EndoG was detected by immunofluorescence. The mean of three independent experiments is shown. *p<0.05, **p<0.01 versus control group.

Figure 7. Effect of β-HB on PCNA induction.

Cells were incubated 48β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. A) PCNA mRNA was detected by Q-PCR, and the ratio of PCNA to β-actin was calculated. B) The percentage of PCNA was measured from the immunofluorescence in figure 7C. C) Protein localization of PCNA was detected by immunofluorescence. The mean of three independent experiments is shown. *p<0.05, **p<0.01 versus the control group.

Figure 8. Effect of β-HB on cell cycle.

Cells were incubated 48β-HB (0, 0.6, 1.2, 2.4 or 4.8 mM) in serum-free medium. A) Cell cycle analysis was performed by flow cytometry to determine the percentage of cells in the G1, S, and G2 phases. B) The mRNAs of P21, P27, cyclin D1, CDK2 and cyclin E1 were detected by Q-PCR, and the ratios of these mRNA levels to β-actin were calculated. The mean of three independent experiments is shown. *p<0.05, **p<0.01 versus control group.

Figure 9. Effects of β-HB on the expression of apoptosis proteins in vivo.

Mice were intraperitoneally given β-HB or PBS (control). Cleaved caspase-12, -9, -3, Bax, Bcl-2, AIF and EndoG protein were detected by Western blot.