Probiotic LB101 alleviates dry eye in mice by suppressing matrix metalloproteinase-9 expression through the regulation of gut microbiota-involved NF-κB signaling

Abstract

Tear matrix metalloproteinase (MMP)-9 is an inflammatory signal in patients with dry eye (DE). In the present study, to understand the action mechanism of probiotic LB101 (Lactobacillus plantarum NK151 and Bifidobacterium bifidum NK175 [4:1] mix) against DE, we investigated its effect on tear amount and inflammatory marker expression levels in mice with unilateral exorbital lacrimal gland excision/atropine-benzalkonium chloride application (EB) or fecal microbiota transplantation from mice with EB (eFMT). Oral gavage of LB101 increased EB-suppressed tear amount and decreased EB-induced blinking number. Furthermore, LB101 decreased EB-induced TNF-α, IL-1β, and MMP-9 expression, TNF-α+ and NF-κB+CD11c+ cell populations, and edema in the conjunctiva, while EB-suppressed IL-10 and occludin expression increased. LB101 also decreased EB-induced TNF-α and IL-1β expression and NF-κB+CD11c+ cell population in the colon. eFMT also decreased tear amount and increased blinking number in the transplanted mice. eFMT increased TNF-α, IL-1β, and MMP-9 expression and TNF-α+ and NF-κB+CD11c+ cell populations in the conjunctiva and TNF-α and IL-1β expression and NF-κB+CD11c+ cell populations in the colon. Oral gavage of LB101 increased eFMT-suppressed tear amount and decreased eFMT-induced blinking number. Furthermore, LB101 decreased TNF-α, IL-1β, and MMP-9 expression, TNF-α+ and NF-κB+CD11c+ cell populations, and edema in the conjunctiva and TNF-α and IL-1β expression and NF-κB+CD11c+ cell population in the colon, while eFMT-suppressed IL-10 and occludin expression decreased. Furthermore, LB101 increased eFMT-suppressed Muribaculaceae, Prevotellaceae, and Lactobacillaceae populations in the gut microbiota, while eFMT-induced Bacteroidaceae population decreased. These findings suggest that DE may cause gut dysbiosis, which may be a risk factor for DE, and LB101 may alleviate DE with gut inflammation by suppressing the expression of MMP-9 and proinflammatory cytokines TNF-α and IL-1β with the regulation of gut microbiota-involved NF-κB signaling.

Fig 1. Effect of LB101 on EB-induced dry eye in mice.

(a) Effect on tear amount. (b) Effect on blinking number. Effect on the IL-1β (c), TNF-α (d), and IL-10 expression (e), IL-1β to IL-10 expression ratio (f), TNF-α to IL-10 expression ratio (g), and MMP-9 expression (h), assessed by ELISA. (i) Effect on the TNF-α⁺ and NF-κB⁺CD1c⁺ cell populations in the conjunctiva, assessed by immunofluorescence assay. (j) Effect on occludin expression in the conjunctiva, assessed by immunoblotting. NC, vehicle; vehicle in normal control group; Sham, vehicle in mice operated without the resection of ELG; DE, vehicle in mice with the resection of ELG; LB101, probiotic LB101 (5×10⁸ CFU/mouse/day) in mice with the resection of ELG. Data values are described as mean ± SD (n = 6). ^#p<0.05 vs. sham. *p<0.05 vs. EB.

Fig 2. Effect of LB101 on EB-induced colitis in mice.

(a) Effect on myeloperoxidase (MPO, a), IL-1β (b), TNF-α (c), and IL-10 expression (d) in the colon, assessed by ELISA. Effect on IL-1β to IL-10 expression ratio (e) and TNF-α to IL-10 expression ratio (f). (g) Effect on the NF-κB⁺CD1c⁺ cell populations in the colon, assessed by immunofluorescence assay. NC, vehicle; vehicle in normal control group; Sham, vehicle in mice operated without the resection of ELG; DE, vehicle in mice with the resection of ELG; LB101, probiotic LB101 (5×10⁸ CFU/mouse/day) in mice with the resection of ELG. Data values are described as mean ± SD (n = 6). ^#p<0.05 vs. sham. *p<0.05 vs. EB.

Fig 3. Effect of LB101 on eFMT-induced dry eye in mice.

(a) Effect on tear amount. (b) Effect on blinking number. Effect on the IL-1β (c), TNF-α (d), and IL-10 expression (e), IL-1β to IL-10 expression ratio (f), TNF-α to IL-10 expression ratio (g), and MMP-9 expression (h), assessed by ELISA. (i) Effect on the TNF-α⁺ and NF-κB⁺CD1c⁺ cell populations in the conjunctiva, assessed by immunofluorescence assay. (j) Effect on occludin expression in the conjunctiva, assessed by immunoblotting. NC, vehicle; vehicle in normal control group; hFMT, vehicle in mice transplanted with healthy mouse gut microbiota; eFMT, vehicle in mice with eFMT-induced dry eye; LB101, probiotic LB101 (5×10⁸ CFU/mouse/day) in mice with eFMT-induced dry eye. Data values are described as mean ± SD (n = 6). ^#p<0.05 vs. hFMT. *p<0.05 vs. eFMT.

Fig 4. Effect of LB101 on eFMT-induced colitis in mice.

(a) Effect on myeloperoxidase (MPO, a), IL-1β (b), TNF-α (c), and IL-10 expression (d) in the colon, assessed by ELISA. Effect on IL-1β to IL-10 expression ratio (e) and TNF-α to IL-10 expression ratio (f). (g) Effect on the NF-κB⁺CD1c⁺ cell populations in the colon, assessed by immunofluorescence assay. NC, vehicle; vehicle in normal control group; hFMT, vehicle in mice transplanted with healthy mouse gut microbiota; eFMT, vehicle in mice with eFMT-induced dry eye; LB101, probiotic LB101 (5×10⁸ CFU/mouse/day) in mice with eFMT-induced dry eye. Data values are described as mean ± SD (n = 6). ^#p<0.05 vs. hFMT. *p<0.05 vs. eFMT.

Fig 5. Effect on LB101 on fecal Bacterioidaceae, Muribaculaceae, Desulfovibrionaceae, Prevotellaceae, and Lactobacillaceae populations in mice with eFMT-induced DE.

Data values are described as mean ± SD (n = 6). ^#p<0.05 vs. hFMT. *p<0.05 vs. eFMT.