目的 研究苦杏仁苷(AD)对骨质疏松(OP)大鼠骨折愈合的影响及其可能的作用机制.方法 将SD大鼠随机分为假手术组、模型组和低、中、高剂量实验组及抑制药组,每组12只.用双侧卵巢切除手术构建OP大鼠模型,造模成功后,低、中、高剂量实验组大鼠腹腔注射0.1、0.5、1.0 mg·kg-1 AD,抑制药组腹腔注射5.0 mg·kg-1H-89[环腺苷酸/蛋白激酶A/环腺苷酸反应元件结合蛋白(cAMP/PKA/CREB)通路抑制药]+1.0 mg·kg-1 AD,假手术组和模型组腹腔注射等量的0.9％NaCl,每天1次,连续90 d.用显微计算机断层扫描技术观察大鼠股骨微结构;通过骨科生物力学测试评估大鼠股骨生物力学状况;用酶联免疫吸附测定(ELISA)法检测大鼠血清骨钙素(OC)和Ⅰ型胶原交联C-末端肽(CTX-Ⅰ)、骨形态发生蛋白2(BMP-2)、cAMP的含量;用试剂盒检测大鼠血清中氧化应激产物的水平;用蛋白质印迹法检测大鼠骨组织中碱性磷酸酶(ALP)及 cAMP/PKA/CREB 信号通路蛋白(p-PKA/PKA、p-CREB/CREB)表达.结果 假手术组、模型组和低、中、高剂量实验组及抑制药组的骨密度分别为(251.54±15.41)、(135.82±10.92)、(173.57±12.65)、(204.31±14.48)、(235.62±11.37)和(187.83±13.64)mg·cm-3;cAMP 含量分别为(0.85±0.06)、(0.20±0.03)、(0.34±0.07)、(0.48±0.09)、(0.81±0.12)和(0.57±0.06)μmol·L-1;p-PKA/PKA 分别为 0.96±0.08、0.06±0.02、0.35±0.04、0.67±0.07、0.94±0.09 和 0.37±0.05;p-CREB/CREB 蛋白表达水平分别为 0.92±0.12、0.04±0.01、0.28±0.03、0.59±0.06、0.91±0.10 和0.29±0.04.以上指标,假手术组与模型组比较,低、中、高剂量实验组与模型组比较,抑制药组与高剂量实验组比较,差异均有统计学意义(均P＜0.05).结论 AD具有减弱氧化应激反应、促进骨折愈合、减轻大鼠的OP症状的作用,其作用机制可能与激活cAMP/PKA/CREB信号通路有关.
Objective To investigate the effect of amygdalin(AD)on fracture healing in osteoporotic(OP)rats and its possible mechanism.Methods SD rats were randomly divided into sham group,model group,inhibitor group and low,medium,high dose experimental groups,12 rats in each group.The OP rat model was established by bilateral ovariectomy.After the model was successfully established,rats in low,medium and high dose experimental groups were intraperitoneally injected with 0.1,0.5 and 1.0 mg·kg-1 AD;the inhibitor group was intraperitoneally injected with 5.0 mg·kg-1 H-89[cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate response element binding protein(cAMP/PKA/CREB)pathway inhibitor]+1.0 mg·kg-1 AD;sham group and model group were intraperitoneally injected with the same amount of 0.9％NaCl,once a day for 90 days.Micro-computer tomography was applied to observe the microstructure of the epiphysis in rats;the biomechanical status of femur was evaluated by orthopedic biomechanical test;the contents of serum osteocalcin(OC),C-terminal telopeptides of type Ⅰ collagen(CTX-Ⅰ),bone morphogenetic protein 2(BMP-2)and cAMP were detected by enzyme-linked immunosorbent assay(ELISA);the level of oxidative stress products in rat serum was detected by kit;Western blot was used to detect the expression of alkaline phosphatase(ALP)and cAMP/PKA/CREB signaling pathway protein(p-PKA/PKA,p-CREB/CREB)in rat bone tissue.Results The bone mineral density of sham group,model group and low,medium,high dose experimental groups,inhibitor group were(251.54±15.41),(135.82±10.92),(173.57±12.65),(204.31±14.48),(235.62±11.37)and(187.83±13.64)mg·cm-3;the contents of cAMP were(0.85±0.06),(0.20±0.03),(0.34±0.07),(0.48±0.09),(0.81±0.12)and(0.57±0.06)μmol·L-1;the expression of p-PKA/PKA were 0.96±0.08,0.06±0.02,0.35±0.04,0.67±0.07,0.94±0.09 and 0.37±0.05;p-CREB/CREB protein were 0.92±0.12,0.04±0.01,0.28±0.03,0.59±0.06,0.91±0.10 and 0.29±0.04,respectively.There were significant differences in the above indexes between sham group and model group,between low,medium,high dose experimental groups and model group,between inhibitor group and high dose experimental group(all P＜0.05).Conclusion AD can reduce oxidative stress,promote fracture healing and alleviate OP symptoms in rats.The mechanism may be related to the activation of cAMP/PKA/CREB signaling pathway.
王文娟;王海明;王胜军;黄亚超;曹剑
赤峰学院附属医院骨外一病区,内蒙古赤峰 024000赤峰学院附属医院医务科,内蒙古赤峰 024000赤峰学院附属医院疼痛科,内蒙古赤峰 024000
中医学
苦杏仁苷;环腺苷酸/蛋白激酶A/环腺苷酸反应元件结合蛋白;骨质疏松;骨折愈合
amygdalin;cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate response element binding protein;osteoporosis;fracture healing
《中国临床药理学杂志》 2024 (008)
1198-1202 / 5
内蒙古自治区2021年自然科学基金资助项目(2021-131)
10.13699/j.cnki.1001-6821.2024.08.023
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