摘要
目的 比较骨重塑能力正常和骨重塑能力缺陷大鼠肩袖撕裂(RCT)修复术后色氨酸代谢的差异,探讨色氨酸代谢是否与骨长入有关.方法 共选取50只成年雌性Sprague-Dawley(SD)大鼠进行研究,随机选择18只大鼠接受双侧卵巢摘除手术(OVX),设为OVX组;余32只大鼠接受双侧卵巢摘除假手术,设为假手术组.术后3个月,OVX组和假手术组各自随机选择4只大鼠,采用显微计算机断层扫描(Micro-CT)鉴定肱骨头的骨量流失情况.OVX组剩余大鼠进行RCT造模,并接受急性冈上肌横断修复术,设为OVX+RCT组(n=14);假手术组剩余大鼠分别选取14只接受急性冈上肌横断修复术和RCT假手术,分别设为RCT组(n=14)和SO组(n=14).修复手术后2周检测大鼠抓力,并取出冈上肌腱骨复合体,通过实时荧光定量多聚酶链式反应(qPCR)检测、苏木素伊红(HE)染色和免疫组化(IHC)染色评估新骨生成和骨长人情况,采用超高效液相色谱(UPLC)分析检测色氨酸代谢.结果 抓力测试结果表明,SO组大鼠抓力最大,RCT组次之,OVX+RCT组抓力最小,三组间差异有统计学意义(P＜0.05).qPCR分析结果表明,SO组腱骨复合体的骨硬化素相对表达量显著高于OVX+RCT组和RCT组,IHC染色显示RCT组的骨织素相对表达量显著高于SO组和OVX+RCT组,以上项目组间差异均有统计学意义(P＜0.05).UPLC检测结果表明,SO组、RCT组、OVX+RCT组肩袖腱骨复合体色氨酸含量相近,差异无统计学意义(P＞0.05).qPCR结果表明,犬尿酸代谢途径的吲哚胺2,3-双加氧酶2的表达量在SO组、RCT组、OVX+RCT组呈递增趋势:OVX+RCT组显著高于SO组,OVX+RCT组的3-羟基邻氨基苯甲酸3,4-双加氧酶的表达量显著高于RCT组和SO组,5-羟色胺代谢途径的单胺氧化酶A亚型和B亚型在SO、RCT、OVX+RCT三组间递增,OVX+RCT组吲哚代谢途径的多巴脱羧酶的表达量显著高于SO组,而细胞色素P450氧化还原酶的表达量在SO、RCT、OVX+RCT三组间递增,以上项目组间差异有统计学意义(P＜0.001).结论 大鼠正常肩袖和RCT后修复的肌腱复合体中色氨酸代谢以犬尿酸代谢为主,5-羟色胺代谢次之;在RCT骨重塑中,5-羟色胺代谢途径改变最大,吲哚途径次之.犬尿酸代谢途径的烟酸、黄尿酸含量和5-羟色胺代谢途径的5-羟色氨酸、5-羟色胺、羟基吲哚乙酸、褪黑素及吲哚代谢途径的色氨醇、吲哚乳酸均与骨长入有关.因此,色氨酸代谢与骨长入有关,为RCT寻找潜在治疗靶点.
Abstract
Objective To explore whether tryptophan metabolism is related to bone growth by com-paring the differences in tryptophan metabolism after rotator cuff tear(RCT)repair between rats with normal bone remodeling capability and those with defective bone remodeling capability.Methods Of the 50 adult female Sprague Dawley(SD)rats used for this study,18 were randomly selected to undergo bilateral ovariecto-my(OVX)(OVX group),and the remaining 32 underwent bilateral OVX sham surgery(sham surgery group).All rats were maintained in standard conditions.Three months after surgery,4 rats were randomly selected from OVX group and the sham surgery group,respectively,for Micro CT identification of bone loss at the rat humeral head.The remaining rats in OVX group underwent RCT modeling and surgical repair of acute supraspinatus muscle transection(OVX+RCT group)(n=14);the remaining rats in the sham surgery group were subjected to either surgical repair of acute supraspinatus muscle transection(RCT group,n=14)or RCT sham surgery(SO group,n=14).The grip power of the rats was tested 2 weeks after surgery.The supraspinatus ten-don-bone complex was harvested for evaluation of new bone formation and growth through real-time fluorescence quantitative polymerase chain reaction(qPCR),hematoxylin eosin(HE)staining,and immunohistochemistry(IHC)staining.The tryptophan metabolism was analyzed using ultra high performance liquid chromatography(UPLC).Results The grip power test showed that SO group had the greatest grip power,followed by RCT group and OVX+RCT group,with statistically significant differences between the 3 groups(P＜0.05).The qPCR showed that the relative expression of osteosclerosis in the tendon-bone complex in SO group was significantly higher than that in OVX+RCT group and RCT group;IHC staining showed that the relative expression of osteocal-cin in RCT group was significantly higher than that in SO group and OVX+RCT group(P＜0.05).The UPLC showed that the content of tryptophan in the rotator cuff tendon-bone complex was similar among SO,RCT,and OVX+RCT groups,showing no significant difference(P＞0.05).The qPCR showed that the expression of in-doleamine 2,3-dioxygenase 2 in the Kynurenic acid metabolism pathway showed an increasing trend from SO group to RCT group to OVX+RCT group,with OVX+RCT group significantly higher than SO group(P＜0.05).The ex-pression of 3-hydroxybenzoate 3,4-dioxygenase in OVX+RCT group was significantly higher than that in RCT group and in SO group(P＜0.001).The subtypes A and B of monoamine oxidase in the 5-hydroxytryptamine metabolism pathway increased from SO group to RCT group to OVX+RCT group.The subtypes A and B of monoamine oxidase in the 5-hydroxytryptamine metabolism pathway increased among SO,RCT,and OVX+RCT groups(P＜0.001).The expression of dopamine decarboxylase in the indole metabolism pathway in OVX+RCT group was significantly higher than that in SO group(P＜0.01).The expression of cytochrome P450 oxidoreductase increased from SO group to RCT group to OVX+RCT group(P＜0.001).Conclusions The tryptophan metabolism in the supraspinatus tendon-bone complex after RCT in rats is mainly dominated by kynurenic acid metabolism,followed by 5-hydroxytryptamine metabolism.In RCT bone remodeling,the 5-hydroxytryptamine metabolic pathway changes the most,followed by the indole pathway.The contents of niacin and xanthurenic acid in the kynurenic acid metabolism pathway are related to bone growth;the 5-hydroxytryptamine,hydroxyindoleacetic acid,and melatonin in the 5-hydroxytryptophan metabolic pathway are related to bone growth;the tryptophan and indole lactate in the indole metabolism pathway are related to bone growth.Therefore,tryptophan metabolism is re-lated to bone growth,providing potential therapeutic targets for RCT repair.
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