Feghhi Zahra, Khorasgani Mohammad Rabbani, Hadjighassem Mahmoud Reza, Esmaeili Hossein, Esfahani Sayyed Hamid Zarkesh
Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
Department of Neuroscience, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Probiotics Antimicrob Proteins. 2025 Jan 7. doi: 10.1007/s12602-024-10438-6.
Heat-killed lactobacilli seem to have protective effects against oxidative stress and neurotoxicity. This study aimed to evaluate the antioxidant properties of specific heat-killed lactobacilli extracts and determine their neuroprotective effects against the neurotoxicity induced by blood plasma from people with multiple sclerosis (MS). The antioxidant activity of the three heat-killed lactobacilli was measured using the DPPH assay. For neuroprotective evaluations of lactobacilli, human neuroblastoma cells (SK-N-SH) were exposed to plasma from individuals with relapsing-remitting MS (RRMS) and healthy controls, with or without pre-treatment of heat-killed lactobacilli including Lactiplantibacillus plantarum (L. plantarum), Levilactobacillus brevis (L. brevis), and Lacticaseibacillus rhamnosus (L. rhamnosus). The morphological changes of SK-N-SH cells associated with plasma-induced apoptosis were observed using an inverted microscope. The neurotoxic effects of plasma samples were assessed using flow cytometry as the percentage of apoptosis in neuronal cells treated with plasma from RRMS patients and healthy controls. The neuroprotective effects of the lactobacilli were also evaluated using flow cytometry, which showed an increased viability percentage in cells pretreated with heat-killed lactobacilli extracts compared to those without pre-treatment. Compared to plasma from healthy controls, plasma from RRMS patients caused morphological changes characteristic of apoptosis such as rounding, detachment, and shrinkage in SK-N-SH cells on microscopy observations. Significant apoptosis in MS plasma-treated neuronal cells was identified by flow cytometry analysis compared to cells treated with plasma from healthy controls (p < 0.01). Heat-killed lactobacilli extracts showed antioxidant activity above 50% in the DPPH radical scavenging assay. Pre-treatment of cells with heat-killed lactobacilli significantly reduced the morphological changes and apoptosis percentage in neuronal cells induced by MS plasma samples. L. plantarum and L. rhamnosus had considerable neuroprotective effects (p < 0.001), followed by L. brevis (p < 0.05). These findings demonstrate that heat-killed lactobacilli extracts as bacterial fractions free of live microorganisms, are suitable safe candidates for adjunctive therapy with potential antioxidant and neuroprotective properties in MS.
热灭活乳杆菌似乎对氧化应激和神经毒性具有保护作用。本研究旨在评估特定热灭活乳杆菌提取物的抗氧化特性，并确定它们对多发性硬化症（MS）患者血浆诱导的神经毒性的神经保护作用。使用DPPH法测定三种热灭活乳杆菌的抗氧化活性。为了对乳杆菌进行神经保护评估，将人神经母细胞瘤细胞（SK-N-SH）暴露于复发缓解型MS（RRMS）患者和健康对照者的血浆中，同时或不进行热灭活乳杆菌的预处理，这些热灭活乳杆菌包括植物乳杆菌（L. plantarum）、短乳杆菌（L. brevis）和鼠李糖乳杆菌（L. rhamnosus）。使用倒置显微镜观察与血浆诱导的细胞凋亡相关的SK-N-SH细胞的形态变化。使用流式细胞术评估血浆样品的神经毒性，以RRMS患者和健康对照者血浆处理的神经元细胞中的凋亡百分比表示。还使用流式细胞术评估乳杆菌的神经保护作用，结果显示与未预处理的细胞相比，用热灭活乳杆菌提取物预处理的细胞的活力百分比增加。与健康对照者的血浆相比，RRMS患者的血浆在显微镜观察下导致SK-N-SH细胞出现凋亡特征性的形态变化，如细胞变圆、脱离和收缩。与健康对照者血浆处理的细胞相比，流式细胞术分析确定MS血浆处理的神经元细胞中存在明显的凋亡（p < 0.01）。在DPPH自由基清除试验中，热灭活乳杆菌提取物的抗氧化活性高于50%。用热灭活乳杆菌预处理细胞可显著减少MS血浆样品诱导的神经元细胞的形态变化和凋亡百分比。植物乳杆菌和鼠李糖乳杆菌具有相当大的神经保护作用（p < 0.001），其次是短乳杆菌（p < 0.05）。这些发现表明，作为不含活微生物的细菌组分的热灭活乳杆菌提取物，是MS辅助治疗中具有潜在抗氧化和神经保护特性的合适安全候选物。
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