Therapeutic effect of low intensity pulsed ultrasound on painful gait in mice with Achilles tendon injury and its possible mechanism / 中华创伤杂志

ABSTRACT

Objective:To investigate the therapeutic effect and possible mechanism of low intensity pulsed ultrasound（LIPUS）on painful gait in mice with Achilles tendon injury.Methods:Male C57BL/6 mice were selected to establish the Achilles tendon injury model by surgically full-thinkness tear of the right Achilles tendon. The mice were divided into Achilles tendon injury group and Achilles tendon injury+LIPUS group according to the random number table method，with 7 mice per group. The Catwalk gait analysis system was used to evaluate the gait function of the mice by measuring the following five parameters 14 days after operation，including print area，standing time，step cycle，max intensity and stride length. Morphological changes of the Achilles tendon were observed by HE staining. Immunofluorescence staining was used to detect the expression level of nitric oxide synthase（iNOS）in the Achilles tendon tissues. At the same time，Achilles tendon cells were isolated and cultured in vitro. The cells were induced by 100 ng/ml lipopolysaccharide（LPS）for 12 hours to establish in vitro model（LPS group），and treated with LIPUS（LPS+LIPUS group）. The control group was set as well（without any treatment）. The nuclear translocation of nuclear transcription factor-κB（NF-κB）P65 was detected by immunofluorescence staining，and the expression of iNOS and phosphorylation（p）-NF-κB p65 protein was detected by Western blotting. Results:Compared with Achilles tendon injury group［（0.14±0.10）cm 2］，the print area of the affected limb in Achilles tendon injury+LIPUS group［（0.28±0.13）cm 2］was increased（ P<0.05）. Compared with Achilles tendon injury group［（0.11±0.04）seconds］，the standing time of the affected limb in Achilles tendon injury+LIPUS group［（0.21±0.03）seconds］was increased（ P<0.05）. Compared with Achilles tendon injury group［（0.25±0.05）seconds］，the step cycle of the affected limb in Achilles tendon injury+LIPUS group［（0.40±0.05）seconds］was increased（ P<0.05）. There was no significant difference in the max intensity and stride length between Achilles tendon injury group and Achilles tendon injury+LIPUS group（ P>0.05）. HE staining showed obvious hyperplasia of Achilles tendon tissues in Achilles tendon injury group，with loosely and randomly arranged fibers，accompanied by neovascularization and inflammatory cell infiltration. Achilles tendon injury+LIPUS group showed more orderly arranged fibers in Achilles tendon tissues，and the degree of neovascularization and inflammatory cell infiltration were reduced. Compared with Achilles tendon injury group［（5.70±0.81）%］，the expression level of iNOS in Achilles tendon injury+LIPUS group［（2.84±0.94）%］was decreased 14 days after operation（ P<0.05）. Immunofluorescence results of Achilles tendon cells showed that NF-κB p65 protein entered the nucleus in LPS group compared with control group，and that LIPUS treatment inhibited nuclear translocation. In control group，Western blotting showed that iNOS was not expressed，and that the expression of phosphorylated NF-κB p65 was 0.63±0.16. Compared with control group，the expression levels of iNOS（0.99±0.22）and p-NF-κB P65（1.02±0.19）in LPS group were significantly increased（ P<0.05）. Compared with LPS group，the expression levels of iNOS（0.62±0.10）and p-NF-κB P65（0.65±0.21）in LPS+LIPUS group were decreased（ P<0.05）. Conclusion:LIPUS treatment can alleviate pain gait in mice with Achilles tendon injury and inhibit iNOS expression in vivo and in vitro，which may be related to inhibition of NF-κB signaling pathway.