分泌鲢鱼cystatin C的布拉迪酵母构建、特性鉴定及其对炎症性骨质疏松的调节作用

Saccharomyces boulardii strains secreting Hypophthalmichthys molitrix cystatin C: construction, characterization, and investigation of their regulatory effects on inflammatory osteoporosis

摘要:

炎症性骨质疏松是一种由慢性炎症导致骨稳态失衡的代谢性骨病，为开发能有效调节炎症相关骨微环境的干预策略，缓解疾病症状，本研究采用基因工程手段构建分泌鲢鱼cystatin C (HmCys C)的重组布拉迪酵母，并初步评估不同剂量酵母在炎症性骨质疏松调节中的效果。设计了含4种启动子-信号肽组合的重组质粒，即pSF-TEF1-αMF-HmCys C、pSF-TEF1-STA1-HmCys C、pSF-TDH3-SED1-HmCys C、pSF-TDH3-CL-HmCys C，经电转化、PCR鉴定、测序分析确定成功构建4株对应重组酵母菌株MC、TC、EC、LC。Tricine-SDS-PAGE及Western blotting分析表明，菌株MC、EC可分泌HmCys C蛋白，表观分子量为12.44 kDa，对应抑制活性分别为(56.07±3.82)%、(35.85±1.15)%；相对定量结果表明，MC、EC菌株表达的HmCys C蛋白占比分别为(24.31±0.40)%、(15.15±0.94)%。菌株MC、EC的体外特性分析结果显示，相较野生型，重组菌株呈现细胞塌陷、表面粗糙和轮廓不规则的形态学变化，但生长性能变化较小(P>0.05)；菌株MC的细胞疏水性显著提升至[(21.54±4.18)%, P<0.05]，而EC的自聚性降至[(49.72±2.91)%, P<0.05]。在不同条件下(pH 1.0?7.0、胆盐浓度0.1%?2.0%、温度37?60 ℃)，菌株MC、EC均表现出优异的pH [存活率≥(88.18±3.80)%]及胆盐[存活率≥(86.95±0.39)%]耐受性；而对高温的耐受性较低，在60 ℃时菌株MC、EC的存活率分别为(37.50±1.91)%、(37.16±0.22)%。经模拟唾液、胃液、肠液连续消化，各菌株存活率呈现下降趋势，但均保持在(78.71±4.33)%以上，具有较强胃肠道压力耐受能力。溶血实验证实分泌HmCys C蛋白的重组菌株及野生型酵母均无溶血效应。最后，构建脂多糖诱导的炎症性骨质疏松小鼠模型，经3种剂量的MC菌株灌胃后，中剂量组骨密度显著提升(P<0.05)。本研究成功构建了分泌HmCys C的布拉迪酵母并明确了其体外特性，中剂量组菌株在炎症性骨质疏松中表现出调控潜力，实验结果为后续深入探究调控效果及机制奠定基础，为建立预防骨质疏松的益生菌干预策略提供了实验依据。

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.