基于Wnt/β-catenin通路研究川续断皂苷Ⅵ对胫骨骨折模型大鼠骨折的修复作用

The study on the repair effect of Dipsacus saponinsⅥon tibial fracture model rats based on Wnt/β-catenin pathway

目的观察川续断皂苷Ⅵ对胫骨骨折模型大鼠骨折的修复作用及其可能机制。方法30只SD大鼠经锯开法建立胫骨骨折大鼠模型,随机分为模型组、干预组、联合组,每组各10只。术后第2天模型组每天腹腔注射生理盐水、DMSO溶液各0.2 ml;干预组每天腹腔注射川续断皂苷Ⅵ生理盐水溶液0.2 ml(10 mg/kg),DMSO溶液0.2 ml;联合组每天腹腔注射川续断皂苷Ⅵ生理盐水溶液0.2 ml(10 mg/kg),XAV939 DMSO溶液0.2 ml(1 mg/只),均连续干预14 d。干预后2、4周采用Micro-CT扫描、X线片扫描观察骨折愈合情况;干预后4周检测胫骨湿重;苏木素-伊红(HE)染色观察骨痂组织病理学改变;Western blot法检测骨痂组织β-连环蛋白(β-catenin)、p-β-catenin、糖原合成酶激酶3β(GSK-3β)、Runt相关转录因子2(Runx2)蛋白表达量。结果干预组干预后2、4周骨体积分数(BV/TV)及骨小梁数量(Tb.N)、Lane-Sandhu评分、骨痂体积均高于模型组(均P<0.05);联合组干预后2、4周BV/TV及Tb.N、Lane-Sandhu评分、骨痂体积均低于干预组(均P<0.05)。干预组干预后4周胫骨湿重高于模型组(P<0.05);联合组胫骨湿重低于干预组(P<0.05)。HE染色结果显示,模型组有纤维组织增生,较多骨小梁生成,但成熟度不高、增粗不明显;干预组形成较多骨性骨痂,骨小梁排列有序、部分成熟,且矿化明显,与应力方向一致;联合组形成较多软骨性及纤维性骨痂,软骨性骨痂边缘矿化较多,并形成骨小梁,在间隙中可观察到丰富的毛细血管。干预组骨痂组织中Runx2及p-β-catenin/β-catenin蛋白表达量高于模型组,GSK-3β蛋白表达量低于模型组(均P<0.05);联合组骨痂组织中Runx2及p-β-catenin/β-catenin蛋白表达量低于干预组,GSK-3β蛋白表达量高于干预组(均P<0.05)。结论川续断皂苷Ⅵ可有效促进胫骨骨折模型大鼠骨折修复,可能是通过激活Wnt/β-catenin信号通路来发挥作用。

Objective To observe the repair effect and possible mechanism of Dipsacus saponinsⅥon tibial fracture model rats.Methods Thirty Sprague Dawley(SD)rats were randomly divided into model group,intervention group,and combination group,with 10 rats in each group,to establish a tibial fracture rat model using the sawing method.On the second day after surgery,the intervention group was intraperitoneally injected with 10 mg/kg of Chuanduduan saponinⅥ;The combination group received intraperitoneal injection of Dipsacus saponinsⅥ10 mg/kg and XAV9391 mg/animal;The model group was intraperitoneally injected with 0.2 ml of physiological saline solution and 0.2 ml of dimethylsulfoxide(DMSO)solution;Once a day,continuous intervention for 14 days.After 2 to 4 weeks of intervention,Micro CT scan and X-ray scan were used to observe the fracture healing status;After 4 weeks of intervention,the wet weight of the tibia was detected;Hematoxylin eosin(HE)staining was used to observe the pathological changes of callus tissue;The Western blot method was used to detect the expression level of callus tissueβ-catenin(β-catenin),p-β-catenin,glycogen synthase kinase 3β(GSK-3β)and Runt related transcription factor 2(Runx2)protein.Results After 2 and 4 weeks of intervention,the bone volume fraction(BV/TV),number of trabeculae(Tb.N),Lane Sandhu score,and callus volume in the intervention group were higher than those in the model group(all P<0.05);After 2 and 4 weeks of intervention,the BV/TV,Tb.N,Lane Sandhu score,and callus volume in the combined group were lower than those in the intervention group(all P<0.05).The wet weight of the tibia in the intervention group was higher than that in the model group at 4 weeks after intervention(P<0.05);The wet weight of the tibia in the combined group was lower than that in the intervention group(P<0.05).The HE staining results showed that the model group had fibrous tissue hyperplasia and more bone trabeculae,but the maturity was not high and the thickening was not significant;The intervention group formed more bony callus,with orderly arrangement of bone trabeculae,partially mature,and obvious mineralization,consistent with the direction of stress;The combined group formed more cartilaginous and fibrous callus,with more mineralization at the edge of the cartilaginous callus and the formation of bone trabeculae.Abundant capillaries can be observed in the gaps.The expression level of Runx2 and p-β-catenin/β-catenin protein in callus tissue of the intervention group was higher than that of the model group,the protein expression GSK-3βlevel was lower than that of the model group(all P<0.05);The expression level of Runx2 and p-β-catenin/β-catenin protein in the callus tissue of the combined group was lower than that of the intervention group;the protein expression level of GSK-3βwas higher than that of the intervention group(all P<0.05).Conclusions Dipsacus saponinsⅥcan effectively promote fracture repair in tibial fracture model rats;It is possible to plays a role by activating the Wnt/β-catenin signaling pathway.