原花青素对磷酸三钙磨损颗粒所致小鼠颅骨溶解的干预作用及其机制*

doi:10.12047/j.cjap.5765.2019.053

摘要/Abstract

摘要： 目的:研究原花青素对磷酸三钙(TCP)磨损颗粒诱导小鼠颅骨溶解的保护作用,并探讨其机制。方法:48只雄性ICR小鼠,随机分为假手术(Sham)组、TCP磨损颗粒(TCP)组、原花青素(0.2 mg/kg, 1 mg/kg, 5 mg/kg)组,每组12只。将TCP磨损颗粒30 mg包埋于小鼠颅骨顶部构建假体周围模型,于术后第2日颅顶局部注射原花青素,隔日1次。2周后处死小鼠采血、取颅骨。抗酒石酸酸性磷酸酶(TRAP)染色和HE染色观察假体周围骨溶解和破骨细胞生成情况;实时荧光定量PCR检测假体周围骨组织中破骨细胞调控基因TRAP、capthesin K、c-Fos和NFATc1的mRNA水平;化学比色法检测血清中丙二醛(MDA)和总抗氧化能力(T-AOC)含量及超氧化物歧化酶(SOD)活性;Western blot法检测小鼠假体周围骨组织中自噬标志蛋白Beclin-1和微管相关蛋白1轻链3(LC-3)表达变化。结果:与Sham组比较,TCP组假体周围骨溶解面积、破骨细胞生成及其调控基因mRNA水平显著增加(P＜0.05),血清MDA含量均明显升高、T-AOC水平和SOD活性明显降低(P＜0.05),假体周围骨组织中Beclin-1和LC-3均表达显著上调、LC-3I向LC-3II转换明显增加(P＜0.05)。与TCP组比较,原花青素组假体周围骨溶解面积、破骨细胞生成及其上述调控基因、血清MDA含量明显减少(P＜0.05),血清T-AOC含量和SOD活性显著增加(P＜0.05)且Beclin-1和LC-3等蛋白表达及LC-3I向LC-3II转换也明显下调。结论:原花青素对TCP磨损颗粒所致的假体周围骨溶解具有明显保护作用,其机制可能与减轻氧化应激反应和自噬的活化密切相关。

关键词: 原花青素, 磷酸三钙磨损颗粒, 骨溶解, 破骨细胞, 氧化应激, 自噬, 小鼠

Abstract: Objective: To investigate the protective effects of procyanidin on periprosthetic osteolysis caused by tricalcium phosphate (TCP) wear particles in the mouse calvaria and its mechanism. Methods: Forty-eight male ICR mice were randomly divided into sham group, TCP group, and procyanidin (0.2 mg/kg, 1 mg/kg, 5 mg/kg)-treated group (n=12). A periprosthetic osteolysis model in the mouse calvaria was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2^nd day post-operation, procyanidin (1 mg/kg, 5 mg/kg) was locally injected to the calvaria under the periosteum every other day. After 2 weeks, all the mice were sacrificed to collect the blood samples and the calvaria. Periprosthetic osteolysis and osteoclastogenesis in the mouse calvaria were observed by tartrate resistant acid phosphatase (TRAP) staining and HE staining. mRNA levels of TRAP, capthesin K, c-Fos and NFATc1 in the periprosthestic bone tissue were examined by real-time fluorescence quantitative PCR. Serum contents of total anti-oxidation capacity (T-AOC) and MDA, and superoxide dismutase (SOD) activity were determined by chemical colorimetry. Protein expressions of autophagic biomarkers such as Beclin-1 and LC-3 in periprosthetic bone tissue of the calvaria were examined by Western blot. Results: Compared with sham group, periprosthetic osteolysis, osteoclastogenesis, mRNA levels of TRAP, capthesin K, c-Fos and NFATc1, and serum MDA content were increased significantly in the TCP group (P＜0.05), whereas serum T-AOC level and SOD activity were decreased. The protein expressions of Beclin-1 and LC-3, and the conversion of LC3-II from LC3-I were both up-regulated markedly in the mouse calvaria of TCP group (P＜0.05). Compared with TCP group, osteolysis, osteoclastogenesis, mRNA levels of TRAP, capthesin K, c-Fos and NFATc1 and serum MDA content were decreased obviously in the procyanidine group (P＜0.05), serum T-AOC level and SOD activity were increased, the expressions of Beclin-1 and LC-3, and the conversion of LC3-II from LC3-I were down-regulated obviously in the mouse calvaria of procyanidin group (P＜0.05). Conclusion: Procyanidin has a protective effect of periprosthetic osteolysis caused by TCP wear particles in the mouse calvaia, its mechanism may be mediated by inhibition of oxidative stress and autophagy.

Key words: procyanidin, tricalcium phosphate wear particles, osteolysis, oxidative stress, autopahgy, mouse

林琨, 陈佳濠, 方泽浩, 叶铖龙, 韩超杰, 严明, 方剑, 张云. 原花青素对磷酸三钙磨损颗粒所致小鼠颅骨溶解的干预作用及其机制^*[J]. 中国应用生理学杂志, 2019, 35(3): 250-255.

LIN Kun, CHEN Jia-hao, FANG Ze-hao, YE Cheng-long, HAN Chao-jie, YAN Ming, FANG Jian, ZHANG Yun. The effect of procyanidin on periprosthetic osteolysis caused by TCP wear particles in the mouse calvaria and its mechanism[J]. CJAP, 2019, 35(3): 250-255.

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原花青素对磷酸三钙磨损颗粒所致小鼠颅骨溶解的干预作用及其机制^*

The effect of procyanidin on periprosthetic osteolysis caused by TCP wear particles in the mouse calvaria and its mechanism

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