Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (29): 6187-6197.doi: 10.12307/2025.772
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Chai Jinlian1, Sun Tiefeng2, Li Wei3, Zhang Bochun3, Li Guangzheng4, Zhou Zhongqi4, Liang Xuezhen3, 5, Wang Ping2
Received:
2024-09-18
Accepted:
2024-11-12
Online:
2025-10-18
Published:
2025-03-06
Contact:
Liang Xuezhen, MD, Associate professor, Master’s supervisor, First College of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China; Department of Orthopedic Microsurgery, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China
Corresponding author: Wang Ping, PhD, Researcher, Master’s supervisor, Shandong Provincial Research Institute of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China
About author:
Chai Jinlian, MS, College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China
Supported by:
CLC Number:
Chai Jinlian, Sun Tiefeng, Li Wei, Zhang Bochun, Li Guangzheng, Zhou Zhongqi, Liang Xuezhen, Wang Ping. Therapeutic effect of Cornus Cervi Colla on steroid-induced osteonecrosis of the femoral head in rat models: fecal metabolomics analysis[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(29): 6187-6197.
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2.1 实验动物数量分析 30只SD大鼠全部进入结果分析。 2.2 各组大鼠基本状态 对照组大鼠毛色和光泽正常,饮食、排泄和精神状态良好,体质量随时间逐渐增加。在甲泼尼龙琥珀酸钠注射期间,模型组部分大鼠精神状态不佳,食欲下降,毛色灰暗稀释,大便稀软,尿量多,体质量出现下降,停止注射后体质量上升,第3周注射完成后食欲尚可,毛色、光泽恢复正常,体质量呈现持续增长状态,但明显低于对照组。在甲泼尼龙琥珀酸钠注射期间,鹿角胶组大鼠食欲尚可,毛色略灰暗稀疏,大便轻度稀软,尿量稍多,体质量也出现一定程度的下降,停止注射后体质量上升,第3周注射完成后食欲恢复正常,毛色、光泽逐渐恢复正常,体质量呈现持续增长状态,整体增长态势介于对照组与模型组之间。各组大鼠体质量变化见图1。 2.3 各组大鼠股骨头大体形态观察结果 对照组股骨头形态规则,表面光滑圆润,呈现健康的白色或略带黄色;模型组股骨头形态较对照组无明显差异,部分股骨头关节软骨略粗糙,呈现暗且薄的软骨表面,并伴有暗红色坏死灶;鹿角胶组股骨头形态较对照组形态无明显差异,软骨表面偶见不均匀的灰白色,但骨坏死程度无模型组严重,见图2。 2.4 各组大鼠股骨头Micro-CT扫描及骨形态计量学参数分析结果 Micro-CT扫描图像显示,与对照组相比,模型组与鹿角胶组大鼠股骨头均表现不同程度的损伤,股骨头轮廓欠规整,股骨头软骨下骨中的骨小梁缺失和紊乱,部分可见囊性变结构,但鹿角胶组大鼠骨小梁损伤程度轻于模型组,见图3。 骨形态计量学参数中骨密度半定量分析结果显示,模型组和鹿角胶组大鼠骨密度均明低于对照组(P < 0.05),鹿角胶组大鼠骨密度高于模型组(P < 0.05),各组骨形态计量学参数中的其他参数(包括组织体积、骨体积、骨表面积、骨体积分数、骨表面积组织体积比值、骨表面积骨体积比值)比较差异均无显著性意义(P > 0.05),见表1。"
2.5 各组大鼠股骨头苏木精-伊红染色结果 苏木精-伊红染色显示,与对照组相比,模型组和鹿角胶组大鼠股骨头软骨下骨的骨小梁稀疏或中断,空骨陷窝率和脂肪细胞侵袭增加;与模型组相比,鹿角胶组大鼠股骨头软骨下骨的空骨陷窝率和脂肪细胞侵袭有所减少,见图4。 2.6 各组大鼠粪便代谢物分析 2.6.1 多元统计分析 采用主成分分析(principal component analysis,PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA)对结果进行分析。PCA分析显示各组样本表现组内聚类及组间离散,各组样本之间具有差异;进一步对比各组OPLS-DA模型的相关评价参数表明,代谢物表达量与样品类别之间建立的关系模型可靠;OPLS-DA评分图显示了对照组、模型组、鹿角胶组之间的样本对比区分非常显著。OPLS-DA置换检验表明各组OPLS-DA模型不存在过拟合,具备较好的有效性及稳健性。各组对比的OPLS-DA模型相关验证参数见表2。PCA分析、OPLS-DA评分和置换检验见图5。"
2.6.2 差异代谢物分析 对各组之间的差异代谢物丰度进行差异倍数计算,并根据变量权重值(VIP值)≥1、P≤0.05,利用R包ggplot2绘制差异代谢物的火山图,可以看出在组间差异代谢物分布情况,见图6。 此次研究设定OPLS-DA模型中的VIP≥1、P≤0.05。经筛选,模型组与对照组相比共247种差异代谢物,其中59种上调,188种下调。鹿角胶组与模型组相比共233种差异代谢物,其中109种上调,124种下调。根据精准分子质量及数据库比对,最终确定对照组与模型组中有65个具有显著差异和明确注释的代谢物;模型组与鹿角胶组中有64个。对上述差异代谢物进行聚类分析得到图7。聚类热图显示各组组内数据重复性较好。同模型组相比,鹿角胶组粪便样本中有30个代谢物上调,34个代谢物下调,各代谢物详细信息见表3。 由表3可以看出,脂质和氨基酸是模型组与鹿角胶组最主要的几类差异代谢产物。脂质代谢中与胆汁酸分泌、初级胆汁酸生物合成、次级胆汁酸生物合成途径相关的代谢物B-鼠胆酸(log2_FC=1.865)、胆汁酸(log2_FC=1.463)以及胆酸(log2_FC=1.195),在鹿角胶组粪便样本中的含量显著高于模型组;与炎症递质对瞬时受体电位通道调节途径相关的佛波醇(log2_FC=2.679),在鹿角胶组粪便样本中的含量显著高于模型组;与丁酸代谢途径相关的丁酸(log2_FC=-0.699),与甲状旁腺激素合成、分泌和作用途径相关的骨化三醇(log2_FC=-0.719),在鹿角胶组粪便样本中的含量均显著低于模型组。 氨基酸代谢中与D-氨基酸代谢途径相关的代谢物D-苏氨酸(log2_FC=2.822)和D-鸟氨酸(log2_FC=0.658),在鹿角胶组在鹿角胶组粪便样本中的含量显著高于模型组,以及与甘氨酸、丝氨酸和苏氨酸代谢途径相关的甜菜碱(log2_FC=1.126)、肌氨酸(log2_FC=0.855),在鹿角胶组粪便样本中的含量显著高于模型组;与丙氨酸、天冬氨酸和谷氨酸的代谢途径相关的代谢物精氨酸琥珀酸(log2_FC=-1.059),在鹿角胶组粪便样本中的含量显著低于模型组。 值得注意的是,与初级胆汁酸生物合成、牛磺酸和次牛磺酸代谢途径相关的牛磺酸(log2_FC=2.887),在鹿角胶组粪便样本中的含量要显著高于模型组。 对64个代谢物的VIP值进行降序排列,可以看出胆酸、硫酸固醇脂、麻黄碱、次黄嘌呤、甜菜碱、左旋肉碱、B-鼠胆酸、胞啶、4-嘧啶酸、牛磺酸、N-乙酰-d-葡萄糖胺、丁酸是影响度最高的几种代谢物(VIP > 5),其中胆酸、左旋肉碱、B-鼠胆酸、牛磺酸均与胆汁代谢途径相关。 另外,在对照组vs. 模型组65个有显著差异和明确注释的代谢物中,有10种在鹿角胶应用后有明显的回调,见表4。 2.6.3 差异代谢物通路分析 采用KEGG数据库(http://metpa.metabolomics.ca)与MetPA数据库(http://metpa.metabolomics.ca)对鹿角胶组干预激素性股骨头坏死模型大鼠后的233种差异代谢物进行映射、富集分类,共得到73条显著富集的代谢通路。根据富集和影响因子分析得到代谢通路影响因子气泡图,见图8。其中,6条具有显著性功能的代谢通路(通路影响因子 > 0.1),分别为牛磺酸和次牛磺酸的代谢、丙氨酸,天冬氨酸和谷氨酸的代谢、类固醇激素生物合成、甘氨酸,丝氨酸和苏氨酸的代谢、精氨酸生物合成、络氨酸代谢,见表5。在代谢调控网络中的重要性最高的是牛磺酸和次牛磺酸的代谢途径(通路影响因子=0.428 57),从该通路的显著性通路图中可以看出,鹿角胶组牛磺酸的上调是影响该代谢途径的核心环节,而与其密切相关的初级胆汁酸生物合成途径当中,初级胆汁酸的上调是引起牛磺酸上调的关键因素。"
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