摘要
目的利用同位素相对和绝对标记定量(isobaric tags for relative and absolute quantification,iTRAQ)技术筛选正常大鼠和膝骨关节炎模型大鼠关节组织中差异表达蛋白,进一步研究右归丸对膝骨关节炎模型鼠差异表达蛋白组织蛋白酶K(cathepsin K,CtsK)、凝血酶敏感蛋白-2(thrombospondin-2,TSP-2)、Rho蛋白依赖性肌球蛋白磷酸酶(Rho protein-dependent myosin phosphatase,Mprip)和钙调蛋白-1(calmodulin-1,CaM-1)等蛋白表达的影响,以揭示右归丸治疗膝骨关节炎的可能机制。方法60只SD大鼠分为假手术组、模型组、硫酸氨基葡萄糖组(0.17 g/kg)及右归丸高(4.8 g/kg)、中(2.4 mg/kg)、低(1.2 g/kg)剂量组,每组10只。除假手术组外,采用改良Hulth法建立KOA大鼠模型,模型制备成功6周后分别给予干预,假手术组和模型组给予0.9%(质量分数)氯化钠注射液、硫酸氨基葡萄糖组给予硫酸氨基葡萄糖,右归丸干预组给予不同浓度的右归丸水煎剂,各组大鼠连续灌胃2个月,处死大鼠取膝关节。iTRAQ技术筛选假手术组和模型组大鼠的差异表达蛋白,并利用生物信息学软件进行数据分析。HE法观察各组大鼠软骨组织病理形态改变并进行Mankin评分,免疫组化法验证各组大鼠软骨组织差异表达蛋白CtsK、TSP-2、Mprip和CaM-1等表达。结果假手术组和模型组共筛选出有意义的差异蛋白1000个(以差异倍数≥1.2或≤0.83为筛选标准),其中上调蛋白442个,下调蛋白558个。基因本体论(Gene Ontology,GO)分析显示这些差异表达蛋白可分为59种功能类型,17种参与细胞成分,16种参与基因的分子功能,26种参与生物学过程,Pathway通路分析显示这些差异蛋白主要集中在核糖体、酪氨酸、Th17细胞分化、Th1和Th2细胞分化、戊糖磷酸化通路等。与假手术组比较,模型组关节软骨边缘严重破坏,软骨细胞排列紊乱;其Mankin评分、TSP-2、CtsK等蛋白表达均明显升高,Mprip、CaM-1等蛋白表达均明显降低(P<0.05)。与模型组比较,右归丸高剂量组软骨结构趋于正常,软骨细胞分布偶见不均,关节软骨表面欠光滑;其软骨组织Mankin评分、TSP-2、CtsK等蛋白表达均明显降低,Mprip、CaM-1等蛋白表达均明显增强(P<0.05)。结论膝骨关节炎的形成是多基因共同作用的结果,其中CtsK、TSP-2、Mprip和CaM-1对膝骨关节炎的发生、发展起着至关重要的作用,右归丸治疗效果可能通过下调TSP-2、CtsK和上调Mprip、CaM-1蛋白表达而实现。
Objective To screen differentially expressed proteins between normal rats and rats with knee osteoarthritis through iTRAQ technology and to study the effects of youguiwan on the expression levels of cathepsin K(CtsK),thrombin-2(TSP-2),Rho protein-dependent myosin phosphatase(Mprip)and calmodulin-1(CaM-1).Methods Sixty SD rats were randomly divided into sham operation group,model group,glucosamine sulfate group,and high,medium,and low dose groups of youguiwan(10 rats in each group).Except the sham operation group,knee osteoarthritis model rats were induced by modified Hulth method for 6 weeks.The sham operation group and model group were given physiological saline,glucosamine sulfate group was given glucosamine sulfate and youguiwan groups were given different concentration decoction of youguiwan.The rats were treated by intragastric administration once a day for 2 months.After killing the rats,the knee joint was take,the differential proteins between sham operation group and knee osteoarthritis(KOA)group were identified through iTRAQ technology,and the analysis was conducted using biological informatics software.The morphological changes of articular cartilage was determined by HE staining,and the levels of differentially expressed proteins CtsK,TSP-2,Mprip,and CaM-1 were tested by immunohistochemical method.Results In reference to the screening criteria,the differential multiples≥1.2 or≤0.83,the results of differential expression showed that a total of 1000 differential proteins were screened between sham operation group and model group.Among them,the expressions of 442 proteins were up-regulated and 558 proteins down-regulated.Based on the gene ontology,it was indicated that the differential proteins could be divided into 59 functional types,17 involved cell components,16 participaed molecular functions of genes,26 participater in biological processes.The analysis of KEGG pathway showed that the differential proteins were mainly related to ribosome,tyrosine,Th17 cell differentiation,Th1 and Th2 cell differentiation,pentose phosphorylation pathway,etc.Compared with the sham group,articular cartilage was seriously damaged,and chondrocytes were arranged in disorder;the Mankin score and the expression levels of TSP-2,and CtsK were all raised obviously,while the expression levels of Mprip and CaM-1 showed down-regulated in the model group(P<0.05).Compared with the model group,cartilage structure was tended to be normal,the chondrocytes distribution was uneven,and articular cartilage surface was not smooth,the Mankin score and the expression levels of TSP-2 and CtsK were all declined obviously,the protein expressions of Mprip and CaM-1 were evidently raised in high-dose group of youguiwan(P<0.05).Conclusions Multiple gene interactions contribute to the formation of KOA.Among them,CtsK,TSP-2,Mprip,and CaM-1play a pivotal role in the pathogenesis of KOA.Youguiwan has therapeutic effect on knee osteoarthritis rat,its mechanism could be related to the down-regulating the expression of TSP-2,CtsK and up-regulating the expression of Mprip and CaM-1.
作者
安方玉
颜春鲁
刘永琦
张艳霞
蒋国凤
邓婕
吕学颖
张蕾蕾
AN Fang-yu;YAN Chun-lu;LIU Yong-qi;ZHANG Yan-xia;JIANG Guo-feng;DENG Jie;LYU Xue-ying;ZHANG Lei-lei(Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Key Laboratory of Dunhuang Medicine,Ministry of Education,Lanzhou 730000,China;Key Laboratory of Medicinal Chemistry and Quality Research,Colleges of Gansu Province,Lanzhou 730000,China;Provincial-Level Key Laboratory for Molecular Medicine of Major Diseases and the Study on Prevention and Treatment of Traditional Chinese Medicine,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Lanzhou for Disease Control and Prevention,Lanzhou 730000,China)
出处
《中华骨质疏松和骨矿盐疾病杂志》
CSCD
北大核心
2021年第1期49-59,共11页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
甘肃省自然科学基金(20JR5RA185)
甘肃省高等学校创新能力提升项目(2019A-080)
甘肃省高等学校科研项目(2018A-043)
敦煌医学与转化省部共建教育部重点实验室开放基金(DHYX17-10)
甘肃省高校中(藏)药化学与质量研究省级重点实验室开放基金(zzy-2018-01)
甘肃省高校重大疾病分子医学与中医药防治研究重点实验室开放基金(FZYX17-18-10)。
