The Mechanism Of Up-regulation Of TGF-β1 By Low Intensity Pulsed Ultrasound (LIPUS) In Promoting Type Ⅰ Collagen Formation In The Early Stage Of Acute Tendon Injury

Objective:The reduction of mechanical properties after tendon injury healing leading to tendon re-injury and re-rupture remains a clinical challenge.Type I collagen is the main collagen component of normal tendon tissue and provides mechanical strength to tendons,so promoting type I collagen is one of the directions to improve its re-injury and re-rupture.Low-intensity pulsed ultrasound(LIPUS)can exert mechanical stimulation on acute tendon injury and promote type I collagen synthesis,however,the mechanism of action keeps to be further studied.In this study,we established the model of acute tendon injury and analyzed the molecular mechanism of type I collagen in the process of acute tendon injury repair by LIPUS,to provide the experimental and theoretical basis for the clinical treatment of acute tendon injury.Methods:Rat tendon fibroblasts were cultured in vitro.The cells were stimulated by low-intensity pulsed ultrasound with different intensity parameters in vitro,and the cell proliferation activity was detected by CCK8.The parameters of LIPUS were frequency 3 MHz,duty cycle 20%,intensity 1.0W/cm~2,5 min/d,once a day.At the end of the intervention,Lysis were collected at 6,24,and 48 hours.The expression of type I collagen,TGF-β1,Smad3,and p-Smad3 were detected by Western Blot.The m RNA expression of type I collagen,TGF-β1,and Smad7 were detected by RT-PCR.In vivo,twenty-seven8-week-old SPF male SD rats weighed 250-300g.The rat model of acute Achilles tendon injury was established 3 days after the injection of type I collagenase.After successful modeling,the rats were randomly divided into 1W model group,1W Intervention Group,2W Model Group,and 2W Intervention Group.The intervention group received LIPUS treatment.The LIPUS parameters were a duty cycle of 20%,intensity of 0.5W/cm~2,frequency of 3MHz,once a day,5 minutes per time,and continuous treatment for 2 weeks.At the end of 1 week and 2 weeks,the rats in each group were killed,the Achilles tendon tissues were taken for general observation,and the tendon healing and collagen changes were observed by HE staining and Sirius red staining,at 2weeks,imaging examination was performed to observe the tendon healing.The expression of type I collagen and TGF-β1 protein was detected by immunohistochemistry,and the expression of type I collagen,TGF-β1,Smad3,and p-Smad3 protein was detected by Western Blot.The m RNA expression of type I collagen,TGF-β1,and Smad7 were detected by RT-PCR.Results:In vitro experiments,rat tendon fibroblasts irradiated with LIPUS increased m RNA and protein synthesis of type I collagen and TGF-β1according to the intensity of CCK8 screening(1.0 W/cm~2)for LIPUS treatment parameters of rat tendon cells;Activation of Smad3 protein decreased the expression of Smad7 m RNA.In vivo experiments were carried out by gross observation,HE staining,Sirius red staining,and ultrasound imaging.The results showed that two weeks of LIPUS treatment increased collagen synthesis,and the improvement of collagen fiber arrangement is beneficial to the healing of tendon injury.LIPUS treatment increased m RNA and protein synthesis of type I collagen and TGF-β1,activated Smad3 protein,and decreased m RNA expression of Smad7.Conclusion:1.In this rat model of acute Achilles tendon injury,LIPUS treatment promoted type I collagen synthesis and accelerated tendon repair.3.LIPUS may accelerate tendon repair by up-regulating TGF-β1,activating Smad3 protein,and promoting type I collagen synthesis during acute tendon injury repair.