Probiotic Bifidobacterium breve MCC1274 Protects against Oxidative Stress and Neuronal Lipid Droplet Formation via PLIN4 Gene Regulation

Abstract

Consumption of Bifidobacterium breve MCC1274 has been shown to improve memory and prevent brain atrophy in populations with mild cognitive impairment (MCI). Preclinical in vivo studies using Alzheimer's disease (AD) models indicate that this probiotic protects against brain inflammation. There is growing evidence that lipid droplets are associated with brain inflammation, and lipid-associated proteins called perilipins could play an important role in neurodegenerative diseases such as dementia. In this study, we found that B. breve MCC1274 cell extracts significantly decreased the expression of perilipin 4 (PLIN4), which encodes a lipid droplet docking protein whose expression is known to be increased during inflammation in SH-SY5Y cells. Niacin, an MCC1274 cell extract component, increased PLIN4 expression by itself. Moreover, MCC1274 cell extracts and niacin blocked the PLIN4 induction caused by oxidative stress in SH-SY5Y cells, reduced lipid droplet formation, and prevented IL-6 cytokine production. These results offer a possible explanation for the effect of this strain on brain inflammation.

Keywords: Bifidobacterium breve; PLIN4; inflammation; lipid droplets; niacin; oxidative stress.

Figure 1

The effects of Bifidobacterium breve MCC1274 extract on perilipin gene expression in SH-SY5Y cells. The SH-SY5Y cells were exposed to B. breve MCC1274 heat-killed supernatant (HKS) or sonicated supernatant (Sonic) for 1 h at a 1% v/v final concentration. The expression of human perilipin mRNA genes relative to human GAPDH by RT-PCR (A–E). The data are shown as the mean +/− standard deviation (SD) of six replicates. * p < 0.05, vs. Control (non-repeated measures ANOVA with the Bonferroni post hoc test).

Figure 2

Bifidobacterium breve MCC1274 extracts and niacin reduce PLIN4 mRNA expression in SH-SY5Y neuroblastoma cells. The SH-SY5Y cells were exposed to HKS (B. breve MCC1274 heat-killed cell extracts, 1% final v/v), niacin (NA), or niacinamide (NAM) (final concentration of 30 or 100 nM) for 1 h. Human PLIN4 mRNA expression relative to human GAPDH was determined by RT-PCR. The data are shown as the mean + SD of three replicates. * p < 0.05, ** p < 0.01 (non-repeated measures ANOVA with the Bonferroni post hoc test).

Figure 3

Bifidobacterium breve MCC1274 extracts and niacin protect SH-SY5Y cells against oxidative stress caused by exposure to H₂O₂. The SH-SY5Y cells were exposed to HKS (B. breve MCC1274 heat-killed cell extracts, 1% v/v) or niacin (NA, 100 nM) with or without H₂O₂ (200 µM) for 24 h. Human PLIN4 mRNA expression relative to human GAPDH expression was determined by RT-PCR. The data are shown as the mean + SD of four replicates. * p < 0.05 (Student’s t-test for two-group or non-repeated measures ANOVA with the Bonferroni post hoc test for multiple-group comparison, respectively).

Figure 4

Bifidobacterium breve MCC1274 extracts and niacin suppress lipid droplet formation in SH-SY5Y cells. The SH-SY5Y cells were exposed to siRNA (random siRNA or PLIN4 siRNA), HKS (B. breve MCC1274 heat-killed cell extracts, 1% final v/v), or NA (100 nM) in the presence of H₂O₂ (200 µM) for 24 h and then stained with BODIPY (2 µM). Untreated cells were used as a control. Oleic acid (60 µM) was used as a control to confirm the induction of lipid droplets. The y-axis shows the FITC signal as measured by flow cytometry. The data are shown as the mean + SD of three replicates. * p < 0.05 (Student’s t-test for two-group or non-repeated measures ANOVA with the Bonferroni post hoc test for multiple-group comparison, respectively).

Figure 5

Bifidobacterium breve MCC1274 extracts and niacin prevent the increase in IL-6 expression induced by oxidative stress. The SH-SY5Y cells were exposed to HKS (B. breve MCC1274 heat-killed cell extracts at 1% v/v final concentration) or niacin (NA, 100 nM), with or without H₂O₂ (200 µM), for 24 h. Human interleukin-6 (IL-6) mRNA expression relative to human GAPDH expression was determined by RT-PCR. The data are shown as the mean + SD of four replicates. * p < 0.05 (Student’s t-test for two-group or non-repeated measures ANOVA with the Bonferroni post hoc test for multiple-group comparison, respectively).