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

Huqian Pill accelerates rotator cuff tendon-bone healing through inducing interleukin-1 receptor antagonist release from exosomes of rat serum

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.