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

doi:10.3969/j.issn.2095-4344.1247

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Huqian pill, rotator cuff tear, exosomes, interleukin-1 receptor antagomist, tendon healing

CLC Number:

R496
R318

Huang Yao1, He Bing1, Shu Hao1, Zhang Chunsheng1, Zhang Fucheng1, Peng Chenjian1, Sun Luning2. Huqian Pill accelerates rotator cuff tendon-bone healing through inducing interleukin-1 receptor antagonist release from exosomes of rat serum [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(19): 3007-3012.

Figures/Tables 2

2.1 实验动物数量分析实验选用SD大鼠50只，分为2组，实验过程中对照组大鼠死亡5只，虎潜丸组死亡4只，最终实验组20只，虎潜丸组21只进入结果分析。 2.2 虎潜丸组冈上肌拉力破坏载荷明显高于对照组在冈上肌重建术后4周取材后测试冈上肌破坏载荷。发现虎潜丸组破坏载荷明显高于对照组；虎潜丸组的拉力破坏载荷在16.5-23.9 N，而对照组的拉力破坏载荷在11.1-16.7 N，两组数据差异有显著性意义(P < 0.01)，见图2C。 2.3 虎潜丸促进腱骨结合区域的蛋白聚糖、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达基于拉力破坏载荷的差异，考虑虎潜丸是否促进腱骨愈合。腱骨愈合的关键在于肌腱-骨之间的移行区，腱骨移行区面积和蛋白聚糖是反应腱骨愈合的重要指标。番红固绿染色发现在虎潜丸组中，腱骨移行区的面积大于对照组，并且蛋白聚糖表达强于对照组，见图 3a，b，均有明显统计学意义，见图3c，番红固绿染色表明虎潜丸促进了腱骨移行区的生长愈合。虎潜丸组冈上肌破坏载荷大于对照组也同时说明冈上肌肌腱弹性模量大于对照组，肌腱主要由胶原组成，95%的胶原是Ⅰ型胶原蛋白，Ⅰ型胶原蛋白和Ⅲ型胶原蛋白是维持肌腱正常结构和弹性最重要的组分，行免疫组织化学发现在虎潜丸组中，Ⅰ型胶原蛋白和Ⅲ型胶原蛋白的表达量增加，见图4，5。结果证实虎潜丸组冈上肌肌腱生长愈合优于对照组。将冈上肌远端3 mm腱性部分行Western blotting半定量分析，发现虎潜丸促进Ⅰ型和Ⅲ型胶原蛋白的表达，见图6。 2.4 虎潜丸促进大鼠体内外泌体及白细胞介素1受体拮抗剂分泌大鼠在重建肩袖术后灌胃虎潜丸水煎液48 h，提取血清外泌体后，行电镜及Western blotting证实外泌体，见图7A。虎潜丸灌胃48 h检测大鼠血清中外泌体表达，发现外泌体表面标志物CD63、CD9和CD81的表达明显增加，差异有统计学意义，见图7 B，C，由此可知虎潜丸促进了大鼠体内外泌体的表达。基于外泌体对细胞的免疫和新陈代谢调控，推测外泌体携带有调控炎症因子的蛋白，因此检测对白细胞介素有重要调控作用的白细胞介素1受体拮抗剂，发现虎潜丸组外泌体中白细胞介素1受体拮抗剂表达增加，见图8，针对大鼠重建后4周肩关节免疫组织化学检测白细胞介素1受体拮抗剂，同样发现虎潜丸组腱骨移行区的白细胞介素1受体拮抗剂表达较对照组的明显增加，差异有统计学意义，见图9。"

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