| BackgroundAchilles tendon injury is a disruption in the conjoined tendon of the gastrocnemius and soleus muscles,usually about 2-6cm above the calcaneal insertion where the blood supply is relatively poor,which is usually ascribable to the retraction and shortening of the gastrocnemius-soleus musculotendinous unit.According to previous studies,as the strongest tendon in the body,Achilles tendon has the traits of low oxygen consumption and poor blood supply,and that predispose it to a slow process of healing.Clinical outcomes show that it is hard for injured tendons to recover to normal ones,but easy to palindromic rapture.At the same time,Incidence of Achilles tendon injury has risen with the sports participation being increasingly popular in recent years.Refer patients with complete rupture to a surgeon for advice about treatment options in order to accompli sh more quick and better functional recovery,but it seems to be hard to come true.Thus,Achilles tendon repairing is one of the biggest challenges in sports medicine,which demands more in-depth studies.According to our clinical observations and statistical data,one of the possible intrinsic factors related to Achilles tendon injury and healing is estrogen.Hammar et al.found that the incidence of Achilles tendon injury rose as estrogen level decreased;One study by Esra et al.showed that estrogen could promote Achilles tendon healing of rats by stimulating proliferation of fibroblasts.Estrogen works by binding its receptors and one of them is estrogen receptor ?(ER?).ER? was shown to be prevalent in the tissue of tendons in 2010 for the first time,which was proved to have positive effects during healing process of rats Achilles tendon.Thus,we assume that ER? should be closely associated with both Achilles tendon injury and repairing.The natural healing process of tendons was described to three phases: inflammation,proliferation and matrix remodeling,among which the first two are classified as early stage of healing when so abundant cells and extracellular matrix components are produced that is of great importance to the whole healing process.Extracellular matrix mainly composed of collagen type I was remodeled during remodeling stage whose corrected composition and ordered arrangement are essential for the recovery of biomechanics and function of injured tendons.Therefore,clear and definite what and how ER? plays a role in these two stages respectively contributes to clinical treatments of Achilles tendon injury.Based on all the above problems and research status,we deduce that as an important intrinsic regulating factors,ER? may participate in early stage and remodeling stage of Achilles tendon healing.In order to prove this,we established mouse Achilles tendon injury model to observe recovery outcomes after 7 and 28 days presented early stage and remodeling stage respectively by using ER?-/-mice and their corresponding WT controls,and then evaluate them by morphological observations,behavioral test and biomechanical test.Finally,try to explore and explain the mechanism by m RNA-seq,PCR and WB.1 The effect and mechanism of ER? during early stage of Achilles tendon healingThis chapter is designed to observe how the absence of ER? affects mouse Achilles tendon early healing and explore the mechanism behind it.1.1 Methods1.1.1 Establish mouse Achilles tendon injury model: 6-month-old ER?-/-mice and their WT controls are performed surgeries transecting the Achilles tendons and then re-adapting the cut ends.Restricting the movement for 7 days.1.1.2 Hematoxylin-eosin(HE)staining,Oil Red O staining,TUNEL staining,immunohistochemistry staining of ER?,Ki67,immunofluorescence staining of DAPI,CD34,Perilipin were used to observe the morphology of tendon tissues.1.1.3 PCR and WB was performed to detect expression of VEGFA/VEGFR,ERK,PTEN/AKT/p53,CD36,PPAR?,FABP4,Adiponectin,Lpl and RBP4 of tendon tissues.1.1.4 Isolation,identification and culture of rats tendon-derived stem cells(TDSCs).LY3201 concentration gradient was set at 10-9?10-7?10-5 M respectively and treated with TDSCs for 24 h or 48 h.CCK-8 assay and Immunofluorescence of PCNA and Brd U were used to observe cell proliferation of TDSCs.Oil Red O staining and PCR used to detect expression of PPAR? and FABP4 was performed to examine adipogenic differentiation of TDSCs treated with LY3201.1.1.5 10 ?M ROSI and LY3201 were used to treat TDSCs,Oil Red O staining and immunofluorescence staining of PPAR? were performed to detect adipogenic differentiation of TDSCs.PCR and WB were carried out to examine expression of CD36,PPAR? and FABP4 of TDSCs treated with LY3201 and ROSI.1.2 Results1.2.1 From histological staining,ER? expressed in abundance in tendon tissues during early healing stage in WT mice.ER?-/-tendon scars had inferior gross appearance,lower histological scores,more cell apoptosis but less cell proliferation paralleled with more blood vessel and adipose accumulation with activated PPAR? signaling pathway during early healing.1.2.2 10-7 M LY3201 could activate expression of ER? but not ER? and GPER in TDSCs and promote proliferation.10-7 M LY3201 could inhibit adipogenic differentiation of TDSCs and decrease the expression of PPAR? signaling pathway.1.2.3 ROSI could reverse the inhibition effect of adipgenic differentiation by LY3201.1.3 Summary1.3.1 We explicit the effects of ER? during early healing stage: The absence of ER? leaded to an inferior morphological outcome characterized by adipocytes accumulation and accompanied by reduced cell proliferation,increased cell apoptosis and more blood vessels.1.3.2 We explore the mechanism ER? affecting early healing stage: Because of the interaction between ER? and PPAR?,the absence of ER? leaded to activation of PPAR? signaling pathway so that there were more adipocytes accumulation.At the same time,we verified that ER? affected angiogenesis,cell proliferation and cell apoptosis respectively through VEGFA/VEGFR,ERK and PTEN/AKT/p53 pathway.1.3.3 We explained one approach how the absence of ER? induced adipose infiltration: ER? could affected adipogenic differentiation of TDSCs through PPAR? signaling pathway.At the same time,we concluded a relative safe and effective dosage of LY3201 for cell examination.2 The effect and mechanism of ER? during remodeling stage of Achilles tendon healingThis chapter is designed to observe how the absence of ER? affects mouse Achilles tendon remodeling and explore the mechanism behind it.2.1 Methods2.1.1 Open filed behavioral tests were performed to detect sports ability of mice.2.1.2 Biomechanical tests were carried out to examine the biomechanical properties of tendons including length,cross-section area,load to failure and stiffness.2.1.3 HE staining,Sirius Red staining,immunofluorescence of collagen I(Col I)and collagen III(Col III)was used to observe the morphology of tendon tissues;PCR was used to examined expression of Col I,Col III,TNMD and FMOD of tendon tissues.2.1.4 m RNA-seq was performed to screen significant genes,and WB was used to verify it;Swiss Regulon Portal and JASPAR were used to predict possible binding site of ER? and IFR5,and immunofluorescence of ER? and IRF5 was carried out to detect co-expression of them.2.1.5 Immunofluorescence of IRF5 and CCL3 was performed to observe the expression of them in tendon tissues;CCL3 was used to treat TDSCs to verify its activation effects of Col I.2.2 Results2.2.1 Open field tests showed that ER?-/-mice worse sport ability characterized by decrease of travel distance and speed;Biomechanical tests showed that ER?-/-tendons was inferior manifested as decrease of cross-sectional area and load to failure.2.2.3 From histological staining,we found that the absence of ER? leaded to abnormal ECM composition characterized by aberrant Col I content.2.2.4 m RNA-seq found a significantly decrease of nuclear transcription factor IRF5,WB of IRF5 of tendon tissues verified it.Confocal laser scanning found the co-expression of ER? and IRF5;Immunofluorescence of IRF5 and CCL3 found a significant decrease expression of both.CCL3 could activate Col I expression in TDSCs.2.3 Summary2.3.1 We explicit the effects of ER? during remodeling stage: The absence of ER? leaded to a significant reduction in sports ability and weakness in biomechanical properties.Behind them is the abnormal deposition of Col I.2.3.2 We explore the mechanism ER? affecting remodeling stage: m RNA-seq,bioinformatical prediction and immunofluorescence found that the absence of ER? leaded to reduction of Col I through interaction between ER? and IRF5/CCL3 axis. |
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