通过渗透性输液泵局部常山酮给药抑制软骨下骨转化生长因子-β信号通路的实验研究

Local administration of halofuginone via osmotic infusion pumps inhibits transforming growth fac- tor-β signaling in subchondral bone

目的探讨渗透性输液泵局部常山酮(HF)给药能否抑制软骨下骨转化生长因子-β(TGF-β)信号介导的间充质干细胞(MSCs)异常招募进而延缓骨关节炎(OA)。方法通过大鼠膝关节前交叉韧带切断(ACLT)诱导OA模型。30只3月龄SD大鼠随机数字表法分为3组(n=10):Sham组(不予特殊处理)、Vehicle+ACLT组(软骨下骨局部给药常山酮溶剂二甲基亚砜+磷酸盐缓冲液)及HF+ACLT组(软骨下骨局部给药常山酮)。将渗透性输液泵植入软骨下骨,以实现软骨下骨定量、持续和稳定的局部给药。采用micro-CT、免疫荧光、免疫组化、Safranin O and fastgreen染色等检测软骨下骨软骨下骨骨体积与组织体积比值(BV/TV)、松质骨模式因子(Tb.Pf)、松质骨数量(Tb.N)及软骨下骨板厚度(SBP.Th)及关节软骨相关蛋白(pSmad2/3、Nestin)变化、关节软骨国际骨关节炎研究协会(OARSI)评分。结果成功建立小鼠膝关节OA模型及软骨下骨给药装置。Sham组和HF+ACLT组软骨下骨BV/TV[(0.381±0.060)mm3和(0.322±0.060)mm3]、SBP.Th[(0.570±0.042)mm和(0.521±0.122)mm]、Tb.N[(4.871±0.214)mm-1和(4.364±0.466)mm-1]均大于Vehicle+ACLT组[(0.229±0.063)mm3、(0.324±0.165)mm、(2.978±0.804)mm-1],Sham组和HF+ACLT组软骨下骨Tb.Pf[(-0.880±0.210)mm-1和(-0.377±0.259)mm-1]、pSmad2/3表达(90.2±40.0和90.8±34.5)、Nestin表达(16.9±5.8和18.5±4.7)、OARSI评分[(1.2±0.7)分和(2.5±1.9)分]均小于Vehicle+ACLT组[(0.057±0.535)mm-1、142.7±37.0、25.9±7.4、(5.4±2.8)分],差异有统计学意义(P<0.05);Sham组软骨下骨BV/TV、Tb.Pf、Tb.Pf、SBP.Th、Tb.N、pSmad2/3、Nestin、OARSI评分和HF+ACLT组比较差异均无统计学意义(P>0.05)。结论通过软骨下骨常山酮给药能够抑制软骨下骨TGF-β信号介导的MSCs异常招募进而延缓OA的进展。

ObjectiveTo explore the impact of inhibition of transforming growth factor-β(TGF-β) signaling by local administration of halofuginone (HF) via osmotic infusion pumps on osteoarthritis (OA) pathogenesis and its underlying mechanism.MethodsKnee OA models were induced by the anterior cruciate ligament transection (ACLT) in 30 3-month-old male SD rats. They were randomized by random number table into 3 equal groups (n=10): Sham, Vehicle+ACLT and HF+ACLT ones. Specific admin-istration of drugs was achieved via osmotic infusion pumps directly implanted in subchondral bone. Safranin O and fast green, H&E, immunofluorescence staining, CT-based microangiography and bone micro-CT (μCT) were used to measure alterations in articular cartilage and subchondral bone [BV(bone volume)/TV (tissue volume), Tb.Pf (trabecular pattern factor), Tb.N (trabecular number), SBP.Th(subchondral bone plate.Th), pSmad2/3, Nestin, and OARSI (Osteoarthritis Research Society International) scoring].ResultsKnee OA models and drug administration devices in subchondral bone were successfully established in rats. Sham and HF+ACLT groups had greater subchondral BV/TV(0.381±0.060 mm3 and 0.322±0.060 mm3), SBP.Th (0.570±0.042 mm and 0.521±0.122 mm) and Tb.N (4.871±0.214 mm-1 and 4.364±0.466 mm-1) than Vehicle+ACLT group did (0.229±0.063) mm3, 0.324±0.165 mm and 2.978±0.804 mm-1, respectively);Sham and HF+ACLT groups had less subchondral Tb.Pf (-0.880±0.210 mm-1 and -0.377±0.259 mm-1), lower expression of pSmad2/3 (90.2±40.0 and 90.8±34.5) and Nestin (16.9±5.8 and 18.5±4.7) and OARSI scores (1.2±0.7 and 2.5±1.9) than Vehicle+ACLT group did (0.057±0.535 mm-1, 142.7±37.0, 25.9±7.4 and 5.4±2.8, respectively). All the above differences were statistically significant (P<0.05). There were no significant differences between Sham and HF+A-CLT groups in subchondral BV/TV, Tb.Pf, Tb.Pf, SBP.Th, Tb.N, expression of pSmad2/3 or Nestin, or OARSI scores (P>0.05).ConclusionSubchondral administration of HF can inhibit TGF-β induced erroneous recruitment of mesenchymal stem cells in subchondral bone, thus attenuating OA progression.