虎潜丸诱导肩袖撕裂损伤模型大鼠血清外泌体中白细胞介素1受体拮抗剂释放促进肩袖腱骨愈合
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摘要
背景:腱骨愈合不良是导致肩袖修补术后再撕裂的最主要原因。目的:研究虎潜丸促进肩袖腱骨愈合的机制。方法:SPF级雄性SD大鼠50只,购自北京维通利华动物技术有限公司。对SD大鼠行单侧肩关节肩袖撕裂造模,造模后1个月重建肩袖,重建后分为2组:对照组和虎潜丸组(灌胃虎潜丸水煎液)。重建4周取肩关节行冈上肌破坏拉力载荷测试;重建4周后取肩关节石蜡切片,行番红固绿染色观察蛋白聚糖表达和Ⅰ型胶原蛋白和Ⅲ型胶原蛋白免疫组织化学染色观察;取重建后虎潜丸灌胃48 h大鼠血清提取外泌体,Western blotting检测外泌体水平及白细胞介素1受体拮抗剂蛋白表达。结果与结论:①虎潜丸组冈上肌拉力破坏载荷明显高于对照组;②番红固绿染色发现虎潜丸组大鼠腱骨移行区面积和蛋白聚糖沉积增加;③免疫组织化学发现虎潜丸组Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达明显增加;④虎潜丸组大鼠血清外泌体水平明显增加,白细胞介素1受体拮抗剂蛋白表达增加;⑤结果说明:虎潜丸通过诱导大鼠体内外泌体和白细胞介素1受体拮抗剂的分泌促进冈上肌止点部腱骨愈合,促进了肩袖撕裂的修复。
BACKGROUND: Nonunion of tendon-bone healing is the main reason of rotator cuff re-tear after reconstruction. OBJECTIVE: To investigate the mechanism of rotator cuff tendon-bone healing induced by Huqian Pill. METHODS: Fifty male Sprague-Dawley rats, SPF grade(provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used to construct unilateral rotator cuff tear model, and were reconstructed the rotator cuff after 1 month. Then the rats were randomly divided to control and Huqian Pill(water decoction via gavage) groups. At 4 weeks, the shoulder joint was removed for supraspinatus failure load test. The paraffin sections of shoulder joints were taken for Safranin O-fast green staining to detect the expression of proteoglycan and for immunohistochemical staining to detect the expression of collagen types Ⅰ and Ⅲ. Exosomes were extracted from the serum of the rats at 48 hours after Huqian Pill treatment, and the quantity of exosomes and protein expression of interleukin-1 receptor antagonist were detected by western blot assay. RESULTS AND CONCLUSION:(1) The supraspinatus failure load in the Huqian Pill group was higher than that in the control group.(2) The transitional area and proteoglycan deposition appeared larger in the Huqian Pill group compared with the control group.(3) Immunohistochemical staining revealed significantly high expression of collagen types Ⅰ and Ⅲ in the Huqian Pill group.(4) The serum exosomes content and expression level of interleukin-1 receptor antagonist in the Huqian Pill group were significantly increased.(5) So, Huqian Pill promotes tendon-bone healing of supraspinatus end point through inducing the secretion of exosomes and interleukin-1 receptor antagonist in rats, contributing to the repair of rotator cuff tears.
BACKGROUND: Nonunion of tendon-bone healing is the main reason of rotator cuff re-tear after reconstruction. OBJECTIVE: To investigate the mechanism of rotator cuff tendon-bone healing induced by Huqian Pill. METHODS: Fifty male Sprague-Dawley rats, SPF grade(provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used to construct unilateral rotator cuff tear model, and were reconstructed the rotator cuff after 1 month. Then the rats were randomly divided to control and Huqian Pill(water decoction via gavage) groups. At 4 weeks, the shoulder joint was removed for supraspinatus failure load test. The paraffin sections of shoulder joints were taken for Safranin O-fast green staining to detect the expression of proteoglycan and for immunohistochemical staining to detect the expression of collagen types Ⅰ and Ⅲ. Exosomes were extracted from the serum of the rats at 48 hours after Huqian Pill treatment, and the quantity of exosomes and protein expression of interleukin-1 receptor antagonist were detected by western blot assay. RESULTS AND CONCLUSION:(1) The supraspinatus failure load in the Huqian Pill group was higher than that in the control group.(2) The transitional area and proteoglycan deposition appeared larger in the Huqian Pill group compared with the control group.(3) Immunohistochemical staining revealed significantly high expression of collagen types Ⅰ and Ⅲ in the Huqian Pill group.(4) The serum exosomes content and expression level of interleukin-1 receptor antagonist in the Huqian Pill group were significantly increased.(5) So, Huqian Pill promotes tendon-bone healing of supraspinatus end point through inducing the secretion of exosomes and interleukin-1 receptor antagonist in rats, contributing to the repair of rotator cuff tears.
引文
[1]Gulotta LV,Nho SJ,Dodson CC,et al.Prospective evaluation of arthroscopic rotator cuff repairs at 5 years:part II--prognostic factors for clinical and radiographic outcomes.J Shoulder Elbow Surg. 2011;20(6):941-946.
[2]Ko S,Choi C,Kim S,et al.Prevalence and Risk Factors of Neuropathic Pain in Patients with a Rotator Cuff Tear. Pain Physician.2018; 21(2):E173-E180.
[3]Yamaguchi K,Ditsios K,Middleton WD,et al.The demographic and morphological features of rotator cuff disease. A comparison of asymptomatic and symptomatic shoulders. J Bone Joint Surg Am.2006;88(8):1699-1704.
[4]Harryman DT 2nd,Hettrich CM,Smith KL,et al.A prospective multipractice investigation of patients with full-thickness rotator cuff tears:the importance of comorbidities, practice, and other covariables on self-assessed shoulder function and health status.J Bone Joint Surg Am.2003; 85-A(4):690-696.
[5]Colvin AC,Egorova N,Harrison AK,et al.National trends in rotator cuff repair.J Bone Joint Surg Am.2012; 94(3):227-233.
[6]Iyengar JJ,Samagh SP,Schairer W,et al.Current trends in rotator cuff repair:surgical technique, setting, and cost. Arthroscopy.2014; 30(3):284-288.
[7]Park MC,ElAttrache NS,Tibone JE,et al.Part I:Footprint contact characteristics for a transosseous-equivalent rotator cuff repair technique compared with a double-row repair technique. J Shoulder Elbow Surg.2007; 16(4):461-468.
[8]Duquin TR,Buyea C,Bisson LJ.Which method of rotator cuff repair leads to the highest rate of structural healing? A systematic review.Am J Sports Med.2010; 38(4):835-841.
[9]Bishop J,Klepps S,Lo IK,et al.Cuff integrity after arthroscopic versus open rotator cuff repair:a prospective study.J Shoulder Elbow Surg.2006; 15(3):290-299.
[10]Tashjian RZ, Hollins AM, Kim HM, et al. Factors affecting healing rates after arthroscopic double-row rotator cuff repair.Am J Sports Med.2010;38(12):2435-2442.
[11]Kim HM,Caldwell JM,Buza JA,et al.Factors affecting satisfaction and shoulder function in patients with a recurrent rotator cuff tear.J Bone Joint Surg Am.2014; 96(2):106-112.
[12]Eljabu W,Klinger HM,von Knoch M.The natural history of rotator cuff tears:a systematic review. Arch Orthop Trauma Surg.2015; 135(8):1055-1061.
[13]Ye C,Zhang W,Wang S,et al.Icariin Promotes Tendon-Bone Healing during Repair of Rotator Cuff Tears:A Biomechanical and Histological Study. Int J Mol Sci. 2016;17(11). pii:E1780.
[14]Trevino EA,McFaline-Figueroa J,Guldberg RE,et al.Full-thickness rotator cuff tear in rat results in distinct temporal expression of multiple proteases in tendon, muscle, and cartilage. J Orthop Res. 2018 Nov20.
[15]Lu LY,Ma M,Cai JF,et al.Effects of Local Application of Adipose-Derived Stromal Vascular Fraction on Tendon-Bone Healing after Rotator Cuff Tear in Rabbits. Chin Med J(Engl).2018; 131(21):2620-2622.
[16]Phinney DG,Di Giuseppe M,Njah J,et al.Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.Nat Commun.2015;6:8472.
[17]Bernardo ME, Fibbe WE. Mesenchymal stromal cells:sensors and switchers of inflammation. Cell Stem Cell.2013;13(4):392-402.
[18]李茹,孟毅,郜妞妞,等.虎潜丸的临床应用现况[J].中医临床研究,2017,9(16):137-139.
[19]王鑫,孙滨,张婧婧.虎潜丸对骨髓基质干细胞BMSCs增长分化的影响[J].世界最新医学信息文摘,2016, 16(83):140.
[20]Mardpour S,Hamidieh AA,Taleahmad S,et al.Interaction between mesenchymal stromal cell-derived extracellular vesicles and immune cells by distinct protein content. J Cell Physiol. 2018 Oct 30.
[21]Toh WS,Zhang B,Lai RC,et al.Immune regulatory targets of mesenchymal stromal cell exosomes/small extracellular vesicles in tissue regeneration. Cytotherapy.2018; 20(12):1419-1426.
[22]Seo N,Akiyoshi K,Shiku H.Exosome-mediated regulation of tumor immunology. Cancer Sci. 2018; 109(10):2998-3004.
[23]Omi R,Gingery A,Steinmann SP,et al.Rotator cuff repair augmentation in a rat model that combines a multilayer xenograft tendon scaffold with bone marrow stromal cells.J Shoulder Elbow Surg[J]. 2016; 25(3):469-477.
[24]孙国荣,尤冬春,石鑫,等.经皮椎体后凸成形术联合虎潜丸治疗骨质疏松性胸腰椎压缩骨折45例[J].中医药导报,2015,21(02):67-69.
[25]谢义松,尤冬春,朱建华,等.虎潜丸对去势大鼠骨密度及BMP-2水平影响的研究[J].中国骨质疏松杂志,2014,20(12):1407-1410.
[26]Lee KW, Lee JS, Kim YS, et al. Effective healing of chronic rotator cuff injury using recombinant bone morphogenetic protein-2 coated dermal patch in vivo.J Biomed Mater Res B Appl Biomater.2017; 105(7):1840-1846.
[27]Dinarello CA.Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood. 2011; 117(14):3720-3732
[28]Yang G,Im HJ,Wang JH.Repetitive mechanical stretching modulates IL-1beta induced COX-2, MMP-1 expression, and PGE2 production in human patellar tendon fibroblasts. Gene.2005;363:166-172
[29]Attur MG,Dave MN,Leung MY,et al.Functional genomic analysis of type II IL-1beta decoy receptor:potential for gene therapy in human arthritis and inflammation.J Immunol.2002;168(4):2001-2010
[30]Genevay S,Finckh A,Mezin F,et al.Influence of cytokine inhibitors on concentration and activity of MMP-1 and MMP-3 in disc herniation.Arthritis Res Ther.2009;11(6):R169.
[2]Ko S,Choi C,Kim S,et al.Prevalence and Risk Factors of Neuropathic Pain in Patients with a Rotator Cuff Tear. Pain Physician.2018; 21(2):E173-E180.
[3]Yamaguchi K,Ditsios K,Middleton WD,et al.The demographic and morphological features of rotator cuff disease. A comparison of asymptomatic and symptomatic shoulders. J Bone Joint Surg Am.2006;88(8):1699-1704.
[4]Harryman DT 2nd,Hettrich CM,Smith KL,et al.A prospective multipractice investigation of patients with full-thickness rotator cuff tears:the importance of comorbidities, practice, and other covariables on self-assessed shoulder function and health status.J Bone Joint Surg Am.2003; 85-A(4):690-696.
[5]Colvin AC,Egorova N,Harrison AK,et al.National trends in rotator cuff repair.J Bone Joint Surg Am.2012; 94(3):227-233.
[6]Iyengar JJ,Samagh SP,Schairer W,et al.Current trends in rotator cuff repair:surgical technique, setting, and cost. Arthroscopy.2014; 30(3):284-288.
[7]Park MC,ElAttrache NS,Tibone JE,et al.Part I:Footprint contact characteristics for a transosseous-equivalent rotator cuff repair technique compared with a double-row repair technique. J Shoulder Elbow Surg.2007; 16(4):461-468.
[8]Duquin TR,Buyea C,Bisson LJ.Which method of rotator cuff repair leads to the highest rate of structural healing? A systematic review.Am J Sports Med.2010; 38(4):835-841.
[9]Bishop J,Klepps S,Lo IK,et al.Cuff integrity after arthroscopic versus open rotator cuff repair:a prospective study.J Shoulder Elbow Surg.2006; 15(3):290-299.
[10]Tashjian RZ, Hollins AM, Kim HM, et al. Factors affecting healing rates after arthroscopic double-row rotator cuff repair.Am J Sports Med.2010;38(12):2435-2442.
[11]Kim HM,Caldwell JM,Buza JA,et al.Factors affecting satisfaction and shoulder function in patients with a recurrent rotator cuff tear.J Bone Joint Surg Am.2014; 96(2):106-112.
[12]Eljabu W,Klinger HM,von Knoch M.The natural history of rotator cuff tears:a systematic review. Arch Orthop Trauma Surg.2015; 135(8):1055-1061.
[13]Ye C,Zhang W,Wang S,et al.Icariin Promotes Tendon-Bone Healing during Repair of Rotator Cuff Tears:A Biomechanical and Histological Study. Int J Mol Sci. 2016;17(11). pii:E1780.
[14]Trevino EA,McFaline-Figueroa J,Guldberg RE,et al.Full-thickness rotator cuff tear in rat results in distinct temporal expression of multiple proteases in tendon, muscle, and cartilage. J Orthop Res. 2018 Nov20.
[15]Lu LY,Ma M,Cai JF,et al.Effects of Local Application of Adipose-Derived Stromal Vascular Fraction on Tendon-Bone Healing after Rotator Cuff Tear in Rabbits. Chin Med J(Engl).2018; 131(21):2620-2622.
[16]Phinney DG,Di Giuseppe M,Njah J,et al.Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs.Nat Commun.2015;6:8472.
[17]Bernardo ME, Fibbe WE. Mesenchymal stromal cells:sensors and switchers of inflammation. Cell Stem Cell.2013;13(4):392-402.
[18]李茹,孟毅,郜妞妞,等.虎潜丸的临床应用现况[J].中医临床研究,2017,9(16):137-139.
[19]王鑫,孙滨,张婧婧.虎潜丸对骨髓基质干细胞BMSCs增长分化的影响[J].世界最新医学信息文摘,2016, 16(83):140.
[20]Mardpour S,Hamidieh AA,Taleahmad S,et al.Interaction between mesenchymal stromal cell-derived extracellular vesicles and immune cells by distinct protein content. J Cell Physiol. 2018 Oct 30.
[21]Toh WS,Zhang B,Lai RC,et al.Immune regulatory targets of mesenchymal stromal cell exosomes/small extracellular vesicles in tissue regeneration. Cytotherapy.2018; 20(12):1419-1426.
[22]Seo N,Akiyoshi K,Shiku H.Exosome-mediated regulation of tumor immunology. Cancer Sci. 2018; 109(10):2998-3004.
[23]Omi R,Gingery A,Steinmann SP,et al.Rotator cuff repair augmentation in a rat model that combines a multilayer xenograft tendon scaffold with bone marrow stromal cells.J Shoulder Elbow Surg[J]. 2016; 25(3):469-477.
[24]孙国荣,尤冬春,石鑫,等.经皮椎体后凸成形术联合虎潜丸治疗骨质疏松性胸腰椎压缩骨折45例[J].中医药导报,2015,21(02):67-69.
[25]谢义松,尤冬春,朱建华,等.虎潜丸对去势大鼠骨密度及BMP-2水平影响的研究[J].中国骨质疏松杂志,2014,20(12):1407-1410.
[26]Lee KW, Lee JS, Kim YS, et al. Effective healing of chronic rotator cuff injury using recombinant bone morphogenetic protein-2 coated dermal patch in vivo.J Biomed Mater Res B Appl Biomater.2017; 105(7):1840-1846.
[27]Dinarello CA.Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood. 2011; 117(14):3720-3732
[28]Yang G,Im HJ,Wang JH.Repetitive mechanical stretching modulates IL-1beta induced COX-2, MMP-1 expression, and PGE2 production in human patellar tendon fibroblasts. Gene.2005;363:166-172
[29]Attur MG,Dave MN,Leung MY,et al.Functional genomic analysis of type II IL-1beta decoy receptor:potential for gene therapy in human arthritis and inflammation.J Immunol.2002;168(4):2001-2010
[30]Genevay S,Finckh A,Mezin F,et al.Influence of cytokine inhibitors on concentration and activity of MMP-1 and MMP-3 in disc herniation.Arthritis Res Ther.2009;11(6):R169.