| PurposeSkeletal muscle injury has a high incidence in sports medicine,which can be divided into acute and chronic injuries depending on the time of injury.According to the muscle injury,the muscle atrophy,muscle spasm,dysfunction,etc.,it has serious effects on the normal work and the quality of life of the patients.At present,there are traditional Chinese medicine,and non-steroidal anti-inflammatory drug therapy and physical therapy(electricity,laser,thermal,ultrasonic)for the treatment of chronic skeletal muscle injury treatment.The above methods are effective,but there are some side effects and they can not treat the injury thoroughly.At present,studies show that skeletal muscle injury can be regenerated by muscle cell and BMSCs.Low intensity ultrasound has been shown to promote stem cell migration,proliferation and differentiation,and has been used by researchers to treat skeletal muscle injury,but the mechanism still lacks in-depth research.This study adopted can focus on low intensity ultrasound(low-intensity focused ultrasound,LIFU)instruments-is ultrasonic therapeutic apparatus.The ultrasonic energy was focused on New Zealand rabbits and SD rats chronic muscle lesions treatment,observed its alone or with the effect of BMSCs to repair damage and study its mechanism.The results of this study will provide a solid theoretical basis for the clinical application of ultrasound therapy instrument.Methods1.The damage models of chronic muscle tissue of New Zealand rabbits were made by gravity strike.Five validation models and normal groups were detected the located values to verify the success of the models.The other models were divided into ultrasound group(n=30)and control group(n=30).Ultrasound group was treated continuously with LIFU for10 days and the control group was subjected to sham exposure,set the 1st,3rd,7th,10th day in treatment and 17th,24th day measured the two groups of local tissue pain threshold,the NF-?B mRNA expression levels and muscle tissue inflammation factors PGE2,IL-1?,5-HT concentration.The changes in the concentration of beta-endorphin before and after treatment were measured on day 1,3,7,a 10 of two groups.2.The animal model of chronic skeletal muscle injury was produced by using the free falling gravity strike method.The normal group and model group were used to make HE staining to observe the result of the models.The rest of the damage models are divided into Control group(n=30),Ultrasound group(n=30),cells group(n=30),ultrasonic combined cell group(n=30)four groups.Each group had six points,including the treatment of the 3rd day,7th day,10th day,14th day,and the 21st day,28th day after the treatment begining.The ultrasound group was treated by LIFU for 14 consecutive days.The combination group was treated for 14days with LIFU after injection of BMSCs,which the treatment method and time were the same as that of the ultrasound group.The cell group was subjected to sham exposure after the injection of BMSCs 10 x 10~6after the successful models.At each point time,the serum SDF-1 content was detected by ELISA.The local tissue was used to paraffin sections for HE staining.and?-Actinin,Myf-5 and Vimentin immunohistochemistry were made,and the average optical density(AOD)value of images was analyzed with IPP 6.0.The MyoD and Myogenin immunofluorescence slices were made for damaged tissue.After the local tissue was frozen,quantitative local MyoD mRNA expressions were detected by RT-qPCR.Results1.NF-?B and pro-inflammatory mediators(5-HT,PGE2,and IL-1?)were significantly up-regulated,and pain threshold in the model group was significantly reduced compared with normal control values(P<0.05).There was no significant difference between the model and normal groups for?-endorphin levels.The value of pain threshold were increased at the3rd,7th and 10th day and the 17th,24th day(P<0.05).?-endorphin levels were significantly increased after focused ultrasound treatment at 1,3,7,and 10 days(P<0.05).NF-?B gene expression was significantly reduced in the ultrasound group(P<0.05).The mRNA quantitation of NF-?B,5–HT and PGE2 levels were reduced largely in the LIFU group from the3rd treatment to 17th day(P<0.05).IL-1?levels showed a decreasing trend until 24th day after treatment(P<0.05).2.HE staining showed muscle fiber breakage,a small amount of inflammatory cell infiltration and fibrosis,indicating the success of chronic skeletal muscle injury models.The skeletal muscle HE staining showed that there were also fewer inflammatory cells and less fibrosis in ultrasound group and combination group.The AOD value of?-Actinin,Myf-5immunohistochemicalpictures,MyoDandMyogenin immunofluorescence images and MyoD mRNA expression level suggested that new muscle cells of ultrasound group and combined group at all time points were higher than the control group,the difference was statistically significant compared with controls during treatment(P<0.05).The number of newborn muscle cells was significantly higher in the 3,7 and 10days than in the control group(P<0.05).The combined group was significantly higher than the cell group(P<0.05)in the treatment of 3,7,10,and 10 days,and significantly higher than the ultrasound group in the7th and 10th days.The AOD value of Vimentin immunohistochemical images showed that the hyperplasia of fibrous tissue in the control group was higher than that of the ultrasound group,the combined group and the cell group at various points in time.The ultrasound group and the combined group were significantly lower than the control group(P<0.05)in the 3rd,7th,10th and 21st days.The cell group was significantly lower than the control group(P<0.05)in the 3rd,7th and 10th days.The AOD value of the combined group Vimentin images showed that at all time points were lower than that of the ultrasound group and the cell group,but the difference was not significant(P>0.05).The serum SDF-1concentration of ultrasound group was significantly higher than the control group(P<0.05)in the 3rd,7th and 14th day during the treatment.The combination group was higher than the control group at all time points,except for the 10th day of treatment,the differences in the remaining time points were statistically significant(P<0.05).Cell group at 3rd,14th,21th,28th day SDF-1 levels were lower than the control group,which at 3rd,14th,21th day the difference was statistically significant(P<0.05).The cell group was higher than the control group at the 7th,10th day,and there was significant difference at the 7th day(P<0.05).The SDF-1 level of the combination group was higher than that of the ultrasound group,and the difference was statistically significant(P<0.05)in the 14th,21st day.Any time except in the treatment of 7th day,the joint group were higher than that of cell groups,and at 3rd,14th,21st,28th day the difference was statistically significant(P<0.05).Immunofluorescence images showed that ultrasound group and the combined cell group of MyoD and Myogenin were both positive.Conclusions1.LIFU can reduce the local inflammatory level of chronic skeletal muscle injury in rabbits,and stimulate the release of analgesic substances in the body,including the release of endorphins into the blood,which can relieve the pain symptoms.2.LIFU significantly increased the number of new muscle cells and decreased the proliferation of fibrous tissue of rats'muscle injury,which was conducive to the repair of damaged tissue structures.LIFU combined with BMSCs in the treatment of chronic skeletal muscle injury,the serum SDF-1 level showed a high level,suggesting that combined treatment is conducive to the continuous mobilization of BMSCs homing,which is conducive to tissue repair and is worth further discussion.3.This topic provides a solid theoretical basis for the future application of LIFU as a new technique for alleviating pain and promoting chronic muscle injury repair. |
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