山姜素调节cAMP/PKA/CREB信号通路促进骨质疏松性骨折大鼠骨折愈合

Alpinetin promotes fracture healing in rats with osteoporotic fracture by regulating the cAMP/PKA/CREB signaling pathway

背景:山姜素具有抗炎、抗肿瘤、抗菌等作用,已被证实能够缓解骨质疏松症,但山姜素对骨质疏松性骨折的影响及机制仍不清楚。目的:探讨山姜素调节环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应元件结合蛋白信号通路对骨质疏松性骨折大鼠的改善作用。方法:采用双侧卵巢切除手术构建骨质疏松症骨折大鼠模型,将成功造模大鼠依据随机数字表法分为山姜素低、中、高剂量组、抑制剂组和模型组,另选12只大鼠作为假手术组。骨折造模当天,山姜素低、中、高剂量组大鼠灌胃7.5,15,30 mg/kg的山姜素+腹腔注射等量的生理盐水,抑制剂组灌胃30 mg/kg的山姜素+腹腔注射5 mg/kg的H-89(通路抑制剂),模型组与假手术组给予(灌胃+腹腔注射)等量生理盐水,1次/d,连续8周。放射性检查评估大鼠骨折愈合情况并进行愈合评分;骨密度扫描仪测定骨折处骨密度;通过三点弯曲实验和压缩实验评估大鼠股骨生物力学状况;苏木精-伊红染色观察大鼠骨折处病理损伤;酶联免疫吸附(ELISA)法检测血清碱性磷酸酶、骨钙素和Ⅰ型胶原交联C-末端肽、环磷酸腺苷水平的变化;Western blot检测股骨组织中骨形态发生蛋白2和环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应元件结合蛋白通路蛋白表达。结果与结论:①相较于假手术组,模型组大鼠骨折愈合评分、骨密度、最大负荷、最大应力、碱性磷酸酶、骨钙素、环磷酸腺苷水平、骨形态发生蛋白2、磷酸化蛋白激酶A/蛋白激酶A、磷酸化环磷酸腺苷反应元件结合蛋白/环磷酸腺苷反应元件结合蛋白表达下降,Ⅰ型胶原交联羧基末端肽水平增加(P<0.05);与模型组比较,山姜素各剂量组大鼠上述各项指标呈现相反的变化(P<0.05);与山姜素高剂量组比较,抑制剂组大鼠上述指标变化均被逆转(P<0.05)。②结论:山姜素可能通过激活环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应元件结合蛋白信号通路加速骨质疏松症骨折大鼠的骨折愈合。

BACKGROUND:Alpinetin has anti-inflammatory,antitumor and antibacterial effects and has been proven to alleviate osteoporosis,but the effect and mechanism of alpinetin on osteoporotic fractures are still unclear.OBJECTIVE:To investigate the improvement effect of alpinetin in rates with osteoporotic fracture by regulating the cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cAMP-responsive element binding protein(CREB)signaling pathway.METHODS:A rat model of osteoporotic fracture was constructed using bilateral ovariectomy.The successfully modeled rats were randomly separated into model group,low-,medium-,and high-dose alpinetin group,and inhibitor group,with 12 rats per group.Another 12 rats were regarded as the sham operation group.On the day of fracture,rats in the low-,medium-,and high-dose alpinetin groups were gavaged with 7.5,15,and 30 mg/kg alpinetin+intraperitoneally injected with an equal amount of normal saline,respectively;the inhibitor group was gavaged with 30 mg/kg alpinetin+intraperitoneally injected with 5 mg/kg H-89(a pathway inhibitor);and the model and sham operation groups were given(gavaged+intraperitoneally injected)an equal amount of normal saline,once a day for 8 consecutive weeks.Radiological examination was applied to evaluate fracture healing and assess healing scores;bone mineral density scanners were used to measure bone mineral density at the fracture site;three-point bending experiment and compression experiment were conducted to evaluate the biomechanical status of the femur;pathological damage at the fracture site in rats was observed by hematoxylin-eosin staining;enzyme linked immunosorbent assay(ELISA)was used to detect changes in serum alkaline phosphatase,osteocalcin,type I collagen cross-linked C-terminal peptide,and cAMP levels;western blot was applied to detect the expression of bone morphogenetic protein 2 and cAMP/PKA/CREB pathway proteins in femoral tissue.RESULTS AND CONCLUSION:Compared with the sham operation group,the fracture healing score,bone mineral density,maximum load,maximum stress,alkaline phosphatase,osteocalcin,cAMP levels,bone morphogenetic protein 2,p-PKA/PKA,p-CREB/CREB expression in the model group were decreased,the level of type I collagen cross-linked C-terminal peptide was increased(P<0.05).The above indicators showed opposite changes in all the alpinetin groups compared with the model group(P<0.05).Compared with the high-dose alpinetin group,the changes in the above indicators were reversed in the inhibitor group(P<0.05).To conclude,alpinetin may accelerate fracture healing in osteoporotic fracture rats,by activating the cAMP/PKA/CREB signaling pathway.