Authors
Yuwei Jiang, Ran Li, Changhe Yang, Xinyu Zhao, Lisi Cheng, Haonan Tong, Xiaofeng Wu, Meiliang Li, Shuhong Li
Published in
Sheng wu gong cheng xue bao = Chinese journal of biotechnology. Volume 42. Issue 7. Pages 3059-3078. Jul 25, 2026.
Abstract
Inflammatory osteoporosis is a metabolic bone disease characterized by chronic inflammation-induced disruption of bone homeostasis, and its alleviation requires intervention strategies capable of effectively modulating the inflammation-associated bone microenvironment. This study employed genetic engineering techniques to construct recombinant Saccharomyces boulardii strains capable of secreting Hypophthalmichthys molitrix cystatin C (HmCys C), and preliminarily evaluated the efficacy of different dosages of strains in modulating inflammatory osteoporosis. Four recombinant plasmids, designated pSF-TEF1-αMF-HmCys C, pSF-TEF1-STA1-HmCys C, pSF-TDH3-SED1-HmCys C, and pSF-TDH3-CL-HmCys C, incorporating distinct promoter-signal peptide combinations, were designed. Following electroporation, four corresponding recombinant yeast strains (MC, TC, EC, and LC) were successfully constructed, as confirmed by PCR identification and sequencing analysis. Tricine-SDS-PAGE and Western blotting results demonstrated that strains MC and EC successfully secreted HmCys C with an apparent molecular weight of 12.44 kDa. The corresponding inhibitory activities were (56.07±3.82)% and (35.85±1.15)%, respectively. Relative quantification results indicated that the expressed HmCys C accounted for (24.31±0.40)% and (15.15±0.94)% of the total protein in strains MC and EC, respectively. Characterization of the in vitro characteristics of strains MC and EC revealed morphological alterations-including cell collapse, surface roughness, and irregular contours-compared with the wild-type strain. However, the growth profiles showed minimal changes (P>0.05). The cell surface hydrophobicity of strain MC increased to (21.54±4.18)% (P<0.05), whereas the auto-aggregation of strain EC decreased to (49.72±2.91)% (P<0.05). Under various stress conditions (pH 1.0‒7.0, bile salt concentration of 0.1%‒2.0%, and 37‒60 ℃), both MC and EC strains exhibited excellent tolerance to acidic pH [survival rate≥(88.18±3.80)%] and bile salts [survival rate≥(86.95±0.39)%]. In contrast, their thermotolerance was comparatively lower, and strains MC and EC showed the survival rates of (37.50±1.91)% and (37.16±0.22)%, respectively, after exposure to 60 ℃. After sequential exposure to simulated salivary, gastric, and intestinal fluids, the survival rates of all the strains declined progressively but remained above (78.71±4.33)%, indicating robust tolerance to gastrointestinal stresses. Hemolysis assays confirmed that neither the recombinant strains secreting HmCys C nor the wild-type strain induced hemolytic activity. Finally, a murine model of lipopolysaccharide (LPS)-induced inflammatory osteoporosis was established. Oral gavage with strain MC at the medium dosage resulted in an increase in bone mineral density (P<0.05). In conclusion, this study successfully constructed S. boulardii strains secreting HmCys C and revealed their in vitro characteristics. The medium-dosage group demonstrated potential regulatory effects on inflammatory osteoporosis. These findings provide a foundation for further probing into the underlying mechanisms and efficacy, contributing experimental data towards developing probiotic-based intervention strategies for the prevention of osteoporosis.
PMID:
42522617
Bibliographic data and abstract were imported from PubMed on 29 Jul 2026.
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