PTH Promotes Rabbit Tibial Fracture Healing Via the Notch Signaling Pathway Q.-H
Total Page：16
File Type：pdf, Size：1020Kb
European Review for Medical and Pharmacological Sciences 2020; 24: 1616-1623 PTH promotes rabbit tibial fracture healing via the Notch signaling pathway Q.-H. LIU1, L.-M. LIAO1, H. WU2, Y.-P. LIN1, S. YU1 1Department of Orthopedics, First People’s Hospital of Fuyang District, Hangzhou, China 2Department of Orthopedics, Zhejiang Cancer Hospital, Hangzhou, China Abstract. – OBJECTIVE: To explore the effect Introduction of parathyroid hormone (PTH) on the expression of Jagged1 in the rabbit tibial fracture healing, and Fracture healing is a very important, complex, its function and mechanism in this process via the slow and long-term pathological process after Notch signaling pathway. MATERIALS AND METHODS: A total of 60 fracture, involving chemotaxis and accumulation New Zealand white rabbits were randomly divid- of mesenchymal stem cells (MSCs), differentia- ed into control group (n=30) and experimental tion and maturation of osteoblasts, and formation group (n=30). Then, a rabbit tibial fracture model of extracellular matrix (ECM) and angiogene- was established. After surgery, the rabbits in ex- sis1. Besides, fracture healing is a self-repairing perimental group were given 10 μg/kg PTH (1-34) process of damaged tissues, and such a process once a day for 5 days a week, while those in con- may be slowed down or lead to bone nonunion trol group were given an equal volume of normal 2 saline. Six rabbits were randomly selected from by interventions with various factors . Given this, each group at 1, 2, 3, 4, and 6 weeks after sur- clarifying the pathogenesis of fracture healing gery to collect right tibia specimens. Next, X-ray in depth is of great significance for the fracture examination, bone mineral density (BMD) test, his- treatment. tological detection, and serum biochemical test Parathyroid hormone (PTH), a hormone se- were performed. Additionally, the messenger ribo- creted by the parathyroid glands, regulates the nucleic acid (mRNA) expression levels of Notch1 Ca-P balance in the human body. Intermittent and Jagged1 in the Notch signaling pathway were 3 measured via polymerase chain reaction (PCR) administration with PTH (1-34) triggers endo- assay. Their protein levels were detected through chondral bone formation and facilitates osteo- Western blotting analysis. genic differentiation of MSCs, thus accelerat- RESULTS: The healing and BMD in experi- ing fracture healing. At present, mechanisms of mental group were better than those in control PTH underlying fracture healing remain to be group since cortical and medullary bridging was clarified. The Notch signaling pathway plays a observed in the rabbits of experimental group at the 6th week after surgery. Plasma level of alkaline crucial role in cell survival and self-renewal. phosphatase (ALP), P content, and the product Jagged1, a Notch ligand, is an important partic- of Ca and P significantly increased (p<0.05) in ipant in matrix interaction with hepatic stellate experimental group. The pathological morphology cells (HSCs)4. The activation of PTH1 receptor of the calluses stained with hematoxylin-eosin (PTH1R) in osteoblasts leads to matrix-mediat- (HE) in experimental group was overtly superior ed proliferation of HSCs. In mice with activat- to that in control group. The PCR results revealed that both mRNA and protein levels of Notch1 and ed PTH1R only in osteoblasts (Col1-caPTH1R Jagged1 were lower in control group than those mice), the number of osteoblasts increases, and in experimental group (p<0.05). high-level Jagged1 is produced, while Notch is CONCLUSIONS: PTH (1-34) promotes the rab- inactivated. In vitro HSC proliferation is blocked bit tibial fracture healing by regulating Jagged1 by γ-secretase inhibitor5. ligand molecules in the Notch signaling pathway. A rabbit tibial fracture model was established in this study to investigate the effects of PTH on Key Words: PTH, Notch signaling pathway, Jagged1, Tibial Jagged1 and the Notch signaling pathway during fracture, Fracture healing, Rabbit. the fracture healing, so as to make a contribution to the treatment of fracture healing. 1616 Corresponding Author: Qunhua Liu, BM; e-mail: [email protected] PTH promotes rabbit tibial fracture healing Materials and Methods er by layer. Subsequently, X-ray films were tak- en under anesthesia to observe the postoperative Materials fixation of fracture. The rabbits recovered from A total of 60 healthy New Zealand white rab- anesthesia were placed in cages for feeding. They bits (Laboratory Animal Center of Lanzhou Uni- intramuscularly injected with 200,000 U of pen- versity) aged 6 months old and weighing 2.8-3.4 kg icillin for 3 d, and then randomly divided into 2 were adaptively fed in separate cages (60×51×35 groups (experimental group and control group, cm, W×D×H) at (22±2)°C and (40±20)% humid- n=30 in each group). The rabbits in experimen- ity for one week. Rabbits were given free access tal group were injected with 10 μg/kg PTH (1-34) to water and specific granular food for laboratory once a day for 5 days every week, while those in animals. This investigation was approved by the control group were subcutaneously injected with Animal Ethics Committee of First People’s Hos- the same volume of normal saline every day. The pital of Fuyang District Animal Center. injection was continued until the rabbits were ex- ecuted. Main Reagents and Instruments Evaluation Via X-ray Agarose and radio immunoprecipitation as- Examination say (RIPA; Yeasen, Shanghai, China); β-actin At 6 weeks after surgery, 6 rats in each group (Santa Cruz Biotechnology, Santa Cruz, CA, were randomly selected and anesthetized. Then, USA); automatic biochemical analyzer (Hitachi the right tibia of the rabbits was examined by 7600-020, Hitachi, Tokyo, Japan); kirschner X-ray for small animals under the same condi- wire and triangular blade (1.2×230 mm; Kan- tions to evaluate the callus and fracture line. ghui Orthopedics, Changzhou, China); elec- tronic balance (FA2004A, Shanghai, China); Determination of Bone Mineral electric thermostatic water tank (DK-8AD, Density (BMD) Shanghai, China); desktop centrifuge (TL-4, After anesthesia with urethane at 0.8 g/kg, Hunan, China); internal cutting homogenizer rabbit right tibia was scanned at a standard po- (XHF-D, Ningbo, China); automatic microplate sition using a dual-energy X-ray absorptiometry reader (EON, Landshut, Germany); rabbit an- scanner, with the lateral surface of the bone fac- ti-mouse Jagged1 polyclonal antibody β-actin, ing the scanner plate. At 2, 4 and 6 weeks, BMD horseradish peroxidase (HRP)-goat anti-rab- value was measured, including that in proximal bit IgG, RIPA lysis buffer, bicinchoninic acid and distal old bones and calluses. (BCA) protein assay kit and protein pre-stained marker (Wuhan Boster Biological Technology Detection of Serum Alkaline Co., Ltd., Wuhan, China) and citicoline (Qilu Phosphatase (ALP), Ca, and P Levels Co., Ltd., Jinan, China). Blood was collected from rabbit ear vein at 1, 2, 3, 4, and 6 weeks after surgery and centrifuged Modeling of Laboratory at 10,000×g for 10 min. Next, plasma levels of Animals ALP, Ca, and P were determined using the cor- The rabbits were anesthetized with urethane responding kits. at 1.6 g/kg via subcutaneous administration and then placed in a lateral position on an operating Hematoxylin-Eosin (HE) Staining table. Next, an incision (about 4 cm in length) was Rabbit right tibia was placed in 10% forma- made on the skin close to the right tibia, followed lin for 48 h, decalcified with 5% nitric acid solu- by cutting of the fascia, separation of the muscles, tion for 24 h, routinely dehydrated, hyalinized, and exposure of the posteromedial surface of the and soaked and embedded in paraffin. Thereaf- tibia. After that, the periosteum was longitudinal- ter, an ultra-thin semi-automatic microtome was ly cut and retracted, and a transverse fracture in employed to slice the sagittal plane of the tibia, the middle part of the tibia was made using an and 10 serial sections (3 μm in thickness) were oscillating saw. Thereafter, the fractured end was obtained after baking at 45°C for 1 h. Afterwards, fixed with a 7-hole micro-anatomical plate and 6 the sections were stained with HE and mounted screws (with 3 screws above and below the frac- with neutral resin, followed by observation of the ture line, respectively). Afterwards, the incision morphology of tissues at the fracture site using a was washed with normal saline and sutured lay- microscope. 1617 Q.-H. Liu, L.-M. Liao, H. Wu, Y.-P. Lin, S. Yu Detection of Messenger Ribonucleic USA, Cat. No.: 500-0006). Thereafter, 25 μg (or Acid (mRNA) Expressions of Notch1 above indicated on the specific blot) of total pro- and Jagged1 Via Polymerase Chain teins were loaded onto NuPage 4-12% Bis-Tris Reaction (PCR) Assay gel (Invitrogen, Carlsbad, CA, USA, Cat. No.: The total RNAs were extracted from tissues NP0322BOX) for electrophoresis in MES run- using TRIzol (Invitrogen, Carlsbad, CA, USA), ning buffer (Invitrogen, Carlsbad, CA, USA, Cat. followed by determination of RNA concentra- No.: NP0002-02) and transferred onto a 0.2 μm tion and purity using a Thermo NanoDrop 2000. nitrocellulose membrane (Invitrogen, Carlsbad, Afterwards, complementary deoxyribonucleic CA, USA, Cat. No.: LC2000). After that, mem- acids (cDNAs) were synthesized using 1 μg of branes were blocked in 5% milk /0.1 M Tris (pH total RNAs, reverse transcriptase (Fermentas, 7.4) /0.15 M NaCl /0.1% Tween 20. After incuba- Waltham, MA, USA) and Oligo-dT primers. A 50 tion with primary antibody and HRP-conjugated μL of PCR system containing reaction buffer, Taq secondary antibody (Jackson Immuno Research, DNA polymerase, dNTPs, 1 μL of forward and West Grove, PA, USA), enhanced chemilumi- 1 μL of reverse primers, and 3 μL of cDNAs (re- nescence (ECL) kit (Thermo Fisher Scientific, sulting products of reverse transcription reaction) Waltham, MA, USA, Cat.