The Effect And Mechanism Of Bone-derived Neuropeptide Y On The Healing Of The Rotator Cuff In Mice

Background:Rotator cuff tears are common injuries to the bone-tendon interface（BTI）in orthopedics and sports medicine,resulting in persistent shoulder pain and movement limitation,thus requiring surgical treatment.Furthermore,the healing of the RC tears is poor after surgery and is prone to re-tear.Therefore,it is necessary to explore the key molecules that affect the healing process of BTI injury.Neuropeptide Y（NPY）is a small molecule polypeptide widely expressed in the peripheral musculoskeletal system and is involved in the metabolism and regeneration of peripheral bone tissue.However,its effect on the repair of the BTI and related mechanisms have not been explored in the literature yet.Objectives:The purpose of this study was to investigate the role of NPY in the repair of BTI injury and its relevant cellular and molecular mechanisms.Methods:（1）A mouse model of RC injury was constructed,and immunofluorescence and western blot（WB）were used to detect the expression of NPY and its Y-type receptors at the bone-tendon healing site after RC injury.Further local administration of Y1R antagonist BIBO3304 or NPY recombinant protein intervention,comprehensive evaluation of the role of NPY/Y1R in the repair of BTI injury from the aspects of histology,imaging,and biomechanics at 4 and 8 weeks after surgery.（2）The expression of bone-derived NPY at the BTI was analyzed by co-staining immunofluorescence,and bone-derived NPY knockout mice(DMP1-i Cre;NPY^fl/fl)with rotator cuff injury were further constructed.Subsequently,the effect of bone-derived NPY knockout on the healing of the enthesis around the rotator cuff was comprehensively evaluated from the aspects of histology,imaging,and biomechanics at 2 weeks,4 weeks and 8 weeks after operation;In addition,immunohistochemical staining was performed to observe the expression of fibrous scars-and bone/fibrocartilage-related molecules at the healing site of mice RC in each group.（3）Macrophages are closely related to the formation of fibrous scars in the early stages of BTI healing.To explore the relationship between bone-derived NPY and macrophages,a large number of osteocyte culture supernatants(NPY^fl/fl-CM and DMP1-i Cre;NPY^fl/fl-CM)were collected for ELISA analysis to detect the NPY content in the supernatant.The supernatant of different mouse-derived osteocytes was used to intervene macrophages in vitro,and scanning electron microscopy,flow cytometry,and cellular immunofluorescence staining were used to detect the phenotypic changes of M1/M2 macrophages;based on the mouse rotator cuff injury model,immunohistochemical staining was used to detect the polarization of macrophages at the repaired bone-tendon insertion around the mice RC in each group.（4）Bone marrow mesenchymal stem cells（BMSCs）are closely related to bone/cartilage regeneration in the middle and late stages of BTI healing.To explore the relationship between bone-derived NPY and BMSCs,different osteocyte culture supernatants were collected,and NPY^fl/fl-CM and DMP1-i Cre;NPY^fl/fl-CM were used to intervene in BMSCs in vitro.After that,a low-and high-concentration NPY-BMSCs intervention system was established,and transcriptome RNA-seq sequencing was performed to find key differential signaling pathways,then verified by WB,immunohistochemistry and q RT-PCR in vitro and in vivo.Finally,different groups of intervention experiments were established based on different osteocyte culture supernatants,Y1R antagonists and signaling pathway agonists to clarify the regulatory effect of NPY/Y1R on the osteogenic and chondrogenic differentiation of stem cells through the selected key differential signaling pathway.Results:（1）The results of immunofluorescence and WB showed that the characteristic expression of NPY first increased and then decreased at the healing site after RC injury in mice,and Y1R was widely expressed in the repaired insertion of the mouse RC,while Y2R was not expressed.After administration of the Y1R antagonist BIBO3304,the results of histological staining,imaging detection and biomechanical tests were improved at 4 and 8 weeks after surgery,while the above results became worse after local NPY intervention.（2）The results of co-staining immunofluorescence confirmed that bone-derived NPY was the important source of NPY at the repaired enthesis.Subsequently,the model of rotator cuff injury was created in bone-derived NPY knockout mouse,and it was confirmed that after bone-derived NPY knockout,the inflammatory cell infiltration at the healing site was reduced 2 weeks after surgery,and the healing morphology of BTI was better at 4 weeks and 8 weeks after surgery,and related parameters of imaging and biomechanical indicators are also better.In addition,the results of immunohistochemical staining showed that after bone-derived NPY knockout,the formation of fibrous scar at the healing site was reduced in the early postoperative period,while the formation of bone and cartilage at the healing site increased in the middle and late postoperative period.（3）The results of ELISA showed that the supernatant of DMP1-i Cre;NPY^fl/fllacked NPY expression;When the macrophages were intervened after NPY deletion in the supernatant of osteocytes,the macrophages showed a long spindle shape using the scanning electron microscopy,and the expression of M2-type marker CD206 increased,and the expression of M1-type marker CD86 decreased by the flow cytometry and cellular immunofluorescence.In addition,the results of immunohistochemical staining showed that after bone-derived NPY deletion or local administration of BIBO3304,the expression of CD206 increased and the expression of CD86 decreased at the healing site of RC in mice,while the expression of CD206 and CD86 were the opposite trend at the healing site of RC after local administration of NPY.（4）The results of BMSCs induction differentiation experiments showed that the osteogenic and chondrogenic differentiation of BMSCs increased after NPY was depleted in osteocyte supernatant.After establishing low and high NPY intervention concentration-BMSCs system in vitro,RNA-seq sequencing results showed that the Wnt/β-Catenin pathway was significantly differentially expressed,then verified by WB.Additionally,the results of q RT-PCR,WB and immunohistochemistry showed that the expression ofβ-Catenin increased at the healing site of BTI after bone-derived NPY deletion or local administration of BIBO3304.Next,it was further confirmed by BIBO3304 or Wnt/β-Catenin pathway agonist intervention in vitro,bone-derived NPY deletion,Y1R antagonist intervention or Wnt/β-Catenin pathway agonist intervention could promote the osteogenic and chondrogenic differentiation of BMSCs,compared with NPY-containing osteocyte supernatant.Conclusion:Bone-derived NPY-Y1R promotes the polarization of macrophages to M1 and inhibits their polarization to M2 in the early postoperative period,affecting the osteogenic and chondrogenic differentiation of stem cells by down-regulating the Wnt/β-Catenin signaling pathway in the middle and late postoperative period,thus delaying the healing of BTI in mouse RC.