| [Objective]The study is aimed to evaluate whether Tuina can promote the proliferation of SC and inhibit the formation of nerve scar.From the perspective of behavior,morphology and molecular biology,this experiment comprehensively evaluated whether the three-handing-three-point Tuina could affect the proliferation of SC and the formation of nerve scar at the injured in site of sciatic nerve in sciatic nerve injury(SNI)model rats by regulating the expression of TGF-?1/Smad2 pathway protein,so as to promote the recovery of motor function in SNI rats.The aim is to further reveal the mechanism in repairing sciatic nerve injury,enrich the theoretical basis of peripheral nerve injury,and provide scientific evidence of treating peripheral nerve injury for Tuina.[Method]1 Animal grouping and model preparation 78 healthy male SD rats were randomly divided into three groups:sham operation group,model group and Tuina group.SNI model rats were made by sciatic nerve crushing injury.From the 8th day after modeling,Tuina group intervened Yinmen(BL37),Chengshan(BL57)and Yanglingquan(GB34)of rats with dialing-manipulation,plucking-manipulation and kneading-manipulation,each point was used for 1min.The intervention lasted for 20 times.2 Behavioral test methodSix rats in each group were randomly selected for behavioral observation.At 7,14,21 and 28 days after surgery,the oblique plate test was evaluated.SFI was used to evaluate the recovery of fine motor in rats 7 and 28 days after surgery.3 Morphological detection methodAt each time of each group,4 rats were randomly selected.HE staining was used to observe the number of SC cells,the thickness of nerve scar and the depth of staining at the site of sciatic nerve injury 28 days after surgery.4 Protein quantitative detection methodAt each time point of each group,4 rats were randomly selected.The expression of S100,TGF-?1 and Smad2 in the injured site of sciatic nerve was observed at 7 and 28 days after the establishment of the model,the expression of collagen ?/? and FN in the injured site of sciatic nerve was observed 28 days after surgery by immunofluorescence method.6 rats were randomly selected at each time point in each group.Western-blotting was used to detect the expression changes of the key protein TGF-?1/Smad2,Smad2 and p-Smad2 in the TGF-?1/Smad2 pathway three times(7,14 and 28 days after surgery),and to explore whether the Tuina can affect the expression of TGF-?1/Smad2 pathway protein.5 Statistical methodsImmunofluorescence and Western-blotting images were analyzed by Image J 1.48u software SAS 8.0 software was used to analyze the data.[Result]1 Tuina can promote the recovery of motor function of SNI rats1.1 Sciatic nerve function index(SFI)The SFI of rats in model group was significantly lower than that in sham group(P<0.01).On the 28th day after surgery,compared with sham group,SFI of rats in model group decreased significantly(P<0.01).Compared with model group,SFI of rats in Tuina group was significantly higher(O<0.01),and close to the level of sham group.1.2 Inclined plate testOn the 7th day after modeling,the inclined angle of rats in model group was significantly lower than that of sham group(P<0.05).14 days and 28 days after surgery,the angle of rats in model group was still significantly lower than that of sham group(P<0.05),and there was no significant difference between Tuina group and model group(P>0.05).Compared with the model group,the inclined angle in Tuina group increased(P>0.05).On the 28th day after surgery,the inclined angle of model group was still significantly lower than that of sham group(P<0.05).2 The effect of Tuina on SC proliferation of sciatic nerve in SNI rats The results of HE staining showed that the axons and myelin of nerve fibers in the sham operation group were complete and closely arranged,and SC was embedded outside the tunica vaginalis.In the model group,the nerve fibers were sparse and scattered,and the number of SC nucleus outside the tunica vaginalis was less than that in the sham operation group.In Tuina group,there were regenerated nerve fibers around the sciatic nerve with thick myelin sheath and clear axons,and the number of SC nucleus outside the sphingomyelin increased significantly compared with the model group.The results of S100 immunofluorescence staining showed that,compared with the sham operation group,the average optical density of S100,the SC marker of SNI rats' sciatic nerve injury site in model group,was significantly lower(P<0.05)after 7 days of modeling.After 20 days of intervention,the average optical density of S100 on the injured site of SNI rats in the model group was significantly lower than that in the sham operation group(P<0.05).Compared with the model group,there was a significant difference in the average optical density of S100 of the sciatic nerve in Tuina group(P<0.05).These results suggest that Tuina can up regulate the expression of S100,which is the SC marker of SNI rat sciatic nerve,but it can not explain if Tuina can promote the proliferation of SC at the injured site of sciatic nerve.3 Tuina can alleviate the scar around sciatic nerve in SNI ratsThe HE staining results showed that after 20 days of intervention,the scar tissue around the sciatic nerve in the sham group was deeply stained and thin,and the scar tissue around the sciatic nerve in the model group was deeply stained and thick.The sciatic nerve in Tuina group was closely arranged,the scar tissue around the nerve was thick,and the staining color was significantly paler than that in model group.The results of immunofluorescence of ?,? collagen and FN showed that 20 days after the intervention of Tuina,the area of collagen around the sciatic nerve in the injured site of SNI rats in sham group was very thin,the area of FN was limited,and the edge was neat.The area of collagen around nerve in model group was significantly thickened,the area of FN was limited,and the edge was not neat.Compared with model group,the area of collagen in Tuina group was significantly reduced,while the area of FN in the nerve was not obvious.4 The effect of Tuina on SC proliferation is not related to TGF-?1/Smad2 pathway Western-blotting results showed that TGF-?1,Smad2 and p-Smad2 in the injured sciatic nerve of SNI rats in model group increased significantly(P<0.05).After 7 days of Tuina,TGF-?1,Smad2 and p-Smad2 in the injured sciatic nerve of SNI rats in model group were still significantly different from those in sham group(P<0.05).TGF-?1,Smad2 and p-Smad2 in Tuina group were down regulated compared with those in model group(P>0.05).After 20 times of Tuina,there was no significant difference in TGF-?1,Smad2 and p-Smad2 in the injured sciatic nerve between model group,sham group and Tuina group(P>0.05).It is suggested that the effect of Tuina on TGF-?1/Smad2 pathway is not related to the promotion of SC proliferation,but may be related to the inhibition of scar formation.[Conclusion]Tuina can inhibit the nerval scar formation by slightly down regulating TGF-?1/Smad2 pathway after sciatic nerve injury. |
PDF Full Text Request