木犀草素对大鼠股骨骨折愈合及Wnt/β-catenin信号通路影响

Effects of Luteolin on Femoral Fracture Healing and Wnt/β-catenin Signaling Pathway in Rats

目的 探究木犀草素对大鼠股骨骨折愈合的影响以及对Wnt/β-连环蛋白(β-catenin)信号通路的调控作用。方法 SD大鼠随机分为正常对照组、模型组、木犀草素低剂量组(50 mg/kg)、木犀草素高剂量组(100 mg/kg)、木犀草素高剂量+Wnt通路抑制剂(IWR-1)组(100 mg/kg+5 mg),每组18只。除正常对照组外,其余4组建立大鼠左股骨骨折模型。造模成功24 h后各治疗组进行相应的药物干预,正常对照组和模型组大鼠灌胃等体积0.9%氯化钠溶液,每日1次,持续干预14 d。干预周期结束后,检测大鼠血清炎症因子[白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)]和骨形成因子[骨碱性磷酸酶(BALP)、骨钙素(OC)]水平;对大鼠进行左侧股骨骨折愈合影像学检查和骨密度(BMD)、骨矿物质含量(BMC)测定;苏木素-伊红染色观察大鼠左侧股骨组织病理形态;蛋白免疫印迹法检测大鼠左侧股骨组织Wnt/β-catenin信号通路相关蛋白的表达。结果与正常对照组比较,模型组大鼠左侧股骨骨组织之间可见大面积骨损伤,断端形成大量骨小梁,以及大量纤维组织生成和炎症细胞浸润,血清IL-6、IL-1β、TNF-α及左侧股骨组织Wnt3a、β-catenin蛋白表达水平显著升高,血清BALP、OC及左侧股骨BMD、BMC水平显著降低(P<0.05)。与模型组比较,木犀草素低、高剂量组和木犀草素高剂量+IWR-1组大鼠骨折线逐渐消失,左侧股骨组织骨损伤面积逐渐减少,骨小梁、纤维组织生成逐渐减少,血清IL-6、IL-1β、TNF-α水平降低,血清BALP、OC,左侧股骨BMD、BMC及Wnt3a、β-catenin蛋白表达水平升高(P<0.05);且木犀草素高剂量组大鼠上述指标变化优于木犀草素低剂量组(P<0.05)。与木犀草素高剂量组比较,木犀草素高剂量+IWR-1组大鼠骨折愈合情况较差,左侧股骨组织骨损伤面积较大,断端新生较多骨小梁,纤维组织增生,有较多炎症细胞浸润,血清IL-6、IL-1β、TNF-α水平升高,血清BALP、OC,左侧股骨BMD、BMC及Wnt3a、β-catenin蛋白表达水平降低(P<0.05)。结论 木犀草素可以通过激活Wnt/β-catenin信号通路,刺激骨形成因子的表达,减轻炎症反应,促进大鼠股骨骨折愈合。

Objective To explore the effect of luteolin on the healing of femoral fracture in rats and its regulation on Wnt/β-catenin signaling pathway.Methods SD rats were randomly divided into normal control group,model group,luteolin low dose group(50 mg/kg),luteolin high dose group(100 mg/kg),luteolin high dose+Wnt pathway inhibitor(IWR-1)group(100 mg/kg+5 mg),with 18 rats in each group.In addition to the normal control group,the other four groups established the rat model of left femoral fracture.After 24 hours of successful modeling,each treatment group received corresponding drug intervention,normal control group and model group rats were given equal volume of physiological saline by gavage,once a day for 14 days.After the intervention period,the levels of serum inflammatory factors[interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)]and bone morphogenetic factor[bone alkaline phosphatase(BALP),osteocalcin(OC)]of rats were measured;imaging examination of left femoral fracture healing in rats and bone mineral density(BMD),bone mineral content(BMC)of rats were measured;hematoxylin eosin staining to observe the pathological morphology of left femur tissue in rats;the expression of Wnt/β-catenin signal pathway related proteins in rat left femur tissue was detected by Western blot.Results Compared with the normal control group,a large area of bone damage was observed between the left femoral bone tissue of the model group,a large number of bone trabeculae were formed at the broken end,a large number of fibrous tissue formation and inflammatory cell infiltration,and serum IL-6,IL-1β,TNF-α,the expression levels of Wnt3a,β-catenin protein in left femur tissue was significantly increased,and the levels of serum BALP,OC,BMD,BMC in left femur were significantly decreased(P<0.05).Compared with the model group,the fracture lines of rats in the luteolin low and high dose groups and the luteolin high dose+IWR-1 group gradually disappeared,the bone damage area of left femoral tissue gradually decreased,the production of bone trabecula and fibrous tissue gradually decreased,and the levels of serum IL-6,IL-1β,TNF-αwere decreased,the expression levels of serum BALP,OC,BMD,BMC,Wnt3a,β-catenin protein in left femur were increased(P<0.05);and the changes of the above indexes in the luteolin high dose group were better than those in the luteolin low dose luteolin group(P<0.05).Compared with the luteolin high dose group,the fracture healing of rats in the luteolin high dose+IWR-1 group was poor,the bone damage area of left femur tissue was large,more bone trabeculae were born at the broken end,fibrous tissue proliferated,there were more inflammatory cell infiltration,the levels of serum IL-6,IL-1β,TNF-αwere increased,the expression levels of serum BALP,OC,BMD,BMC,Wnt3a,β-catenin protein in left femur were decreased(P<0.05).Conclusion Luteolin can activate the Wnt/β-catenin signaling pathway to stimulate the expression of bone morphogenetic factor,reduce inflammation,and promote femoral fracture healing in rats.