大鼠骨关节炎尿生物标志物靶向代谢组学研究

Targeted metabolomics of urinary biomarkers in rats with osteoarthritis

目的建立不同实验周期的大鼠骨关节炎(OA)模型,分析其尿中小分子代谢物,探讨具有疾病判别和/或病情指示作用的OA生物标志物和/或生物标志物簇。方法选用60只雄性SPF级SD大鼠,体重为300~350 g,按体重采用随机数字表法分为模型组和对照组,每组30只,实验周期分别为4、8、12周。采用改良Hulth法建立大鼠OA模型。实验期满采集大鼠膝关节组织和尿液。膝关节组织常规制片、HE染色后光镜下观察造模情况,用高效液相色谱四级杆离子阱串联质谱[HPLC-(Q-TRAP)-MS/MS]定量检测大鼠尿液中候选物质。采用SPSS 20.0软件对计量资料进行数据处理和分析,组间比较采用Wilcoxon rank-sum检验;采用Python 3.0软件进行受试者工作特征(ROC)曲线检验。P<0.05为差异有统计学意义。结果组织学观察可见,模型组大鼠关节间隙变窄或消失,软骨变薄、破损或大面积剥脱,软骨细胞变性、坏死、缺失,随实验周期延长,病变加重。靶向代谢组学研究发现,4周时尿液中筛选出7种差异代谢物,分别为富马酸、琥珀酸、草酰乙酸、苹果酸、顺乌头酸、α-酮戊二酸、磺基丙氨酸,其中模型组磺基丙氨酸低于对照组,其余6种有机酸均高于对照组(P均<0.05);8周时尿液中筛选出12种差异代谢物,包括组氨酸、赖氨酸、酪氨酸、色氨酸、苏氨酸、缬氨酸、亮氨酸、天冬氨酸、肌酸酐、α-酮戊二酸、草酰乙酸、丙二酰肉碱,模型组均高于对照组(P均<0.05);12周时尿液中筛选出4种差异代谢物,其中模型组磺基丙氨酸、半胱氨酸和肌氨酸均低于对照组,琥珀酸高于对照组(P均<0.05)。结论模型组大鼠出现典型OA病理及病程改变,模型建立成功。不同病程大鼠尿液小分子差异代谢物谱不同,主要为有机酸和氨基酸,尿液三羧酸循环相关代谢物和必需氨基酸可作为OA生物标志物簇。

Objective Rat osteoarthritis(OA)model with different experimental cycles was established,analysis of small molecule metabolites in urine were carried out,in order to study the OA biomarkers and/or biomarker clusters with disease and/or the severity of disease indicator function.Methods Using random number table method,sixty male SPF sprague-dawley(SD)rats weighing 300-350 g were randomly divided into model group and control group according to their body weights with 30 rats in each group.The experimental cycles were 4,8 and 12 weeks,respectively.Rat OA model was established by modified Hulth method.At the end of each experiment,knee joint tissue and urine samples were collected.The knee joint histopathological slides were used to observe modeling situation under light microscope.High performance liquid chromatography quadrupole ion trap tandem mass spectrometer[HPLC-(Q-TRAP)-MS/MS]was used to quantitatively detect the candidate substance in the urine of rats.SPSS 20.0 was used to process and analyze the measurement data.Wilcoxon rank-sum test was used for comparison between groups.Python 3.0 was used to plot the receiver operator characteristic(ROC)curve.P<0.05 was considered to be statistically significant.Results Histological observation showed that in model group,the joint space narrowed or disappeared,the cartilage became thinner,damaged or extensively exfoliated.The chondrocytes were degenerated,necrotic or absence.With the extension of the experimental cycles,the lesions worsened.Targeted metabolomics study found that 7 different metabolites were screened at 4 weeks,namely fumaric acid,succinic acid,oxaloacetic acid,malic acid,cis-aconite acid,α-ketoglutaric acid and sulfoalanine.Sulfoalanine was low expressed in model group and other 6 organic acids were high expressed(P<0.05).At 8 weeks,a total of 12 different metabolites were screened,including histidine,lysine,tyrosine,tryptophan,threonine,valine,leucine,aspartic acid,creatinine,α-ketoglutaric acid,oxaloacetic acid and malonyl carnitine,all of them were highly expressed in model group(P<0.05).At 12 weeks,a total of 4 different metabolites were screened,among which sulfoalanine,cysteine and sarcosine were low expressed in model group,and succinic acid was high expressed(P<0.05).Conclusions Typical OA pathology changes and disease progress in the rats of model group are exhibited,the model is established successfully.The urinary small molecular metabolite profiles of OA rats with different disease progress are different,mainly organic acids and amino acids.The metabolites related to urinary tricarboxylic acid cycle and essential amino acids can be used as biomarker clusters of OA.