S2 Data -

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.

泪液基质金属蛋白酶9（matrix metalloproteinase, MMP-9）是干眼症（dry eye, DE）患者体内的炎性信号分子。本研究为阐明益生菌LB101（植物乳杆菌NK151与两歧双歧杆菌NK175按4:1比例混合的制剂）对抗干眼症的作用机制，针对单侧眶外泪腺摘除/阿托品-苯扎氯铵造模（EB）小鼠以及接受EB模型小鼠粪便菌群移植（eFMT）的小鼠，探究了该制剂对泪液分泌量及炎症标志物表达水平的影响。经口灌胃LB101可提升EB模型小鼠被抑制的泪液分泌量，并降低其诱导的眨眼次数。进一步研究发现，LB101可下调EB模型小鼠结膜组织中EB诱导的肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）及MMP-9的表达水平，减少TNF-α阳性与核因子κB（NF-κB）阳性CD11c+细胞的浸润数量，并缓解结膜水肿；同时可恢复EB模型小鼠被抑制的IL-10与闭合蛋白（occludin）的表达。LB101同样可下调结肠组织中EB诱导的TNF-α与IL-1β的表达水平，以及NF-κB阳性CD11c+细胞的浸润数量。粪便菌群移植（eFMT）也会降低受体小鼠的泪液分泌量，并增加其眨眼次数。eFMT可上调结膜组织中TNF-α、IL-1β及MMP-9的表达水平，以及TNF-α阳性与NF-κB阳性CD11c+细胞的浸润数量；同时上调结肠组织中TNF-α与IL-1β的表达水平，以及NF-κB阳性CD11c+细胞的浸润数量。经口灌胃LB101可提升eFMT受体小鼠被抑制的泪液分泌量，并降低其诱导的眨眼次数。进一步研究发现，LB101可下调eFMT受体小鼠结膜组织中TNF-α、IL-1β及MMP-9的表达水平，减少TNF-α阳性与NF-κB阳性CD11c+细胞的浸润数量，并缓解结膜水肿；同时下调结肠组织中TNF-α与IL-1β的表达水平，以及NF-κB阳性CD11c+细胞的浸润数量，并恢复eFMT所抑制的IL-10与闭合蛋白的表达。此外，LB101可提升eFMT受体小鼠肠道菌群中被抑制的鼠杆菌科（Muribaculaceae）、普雷沃菌科（Prevotellaceae）与乳杆菌科（Lactobacillaceae）的丰度，同时降低eFMT诱导升高的拟杆菌科（Bacteroidaceae）的丰度。本研究结果表明，干眼症可能引发肠道菌群失调，而肠道菌群失调或为干眼症的风险因素；LB101可通过调控肠道菌群参与的核因子κB信号通路，下调MMP-9及促炎细胞因子TNF-α、IL-1β的表达，从而缓解干眼症伴随的肠道炎症。