1. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325027, China
2. Zhejiang Provincial Key Laboratory of Orthopaedics, Wenzhou 325027, China
3. The Second Clinical Medical College of Wenzhou Medical University, Wenzhou 325027, China
4. School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China
5. Department of Orthopedic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
6. Department of Clinical Laboratory, The First Affiliated Hospital of Wannan Medical College, Wuhu 241001, China
7. Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China
#Haojie Zhang, Wenfei Ni and Gaoxiang Yu are co-first authors who equally contributed to this manuscript
Background: Caloric restriction mimetics (CRMs) mimic the favourable effects of caloric restriction (CR) and have been shown to have therapeutic effects in neuroinflammatory disease. However, whether CRMs improve the functional recovery from spinal cord injury (SCI) and the underlying mechanism of action remain unclear. In this study, we used the CRMs 3,4-dimethoxychalcone (3,4-DC) to evaluate the therapeutic value of CRMs for SCI.
Methods: HE, Masson and Nissl staining; footprint analysis; and the Basso mouse scale were used to determine the functional recovery from SCI after 3,4-DC treatment. RNA sequencing was used to identify the mechanisms of 3,4-DC in SCI. Western blotting, qPCR and immunofluorescence were used to detect the levels of pyroptosis, necroptosis, autophagy and the AMPK-TRPML1-calcineurin signalling pathway. We employed a dual-luciferase reporter assay in vitro and applied AAV vectors to inhibit TFEB in vivo to explore the mechanism of 3,4-DC.
Results: 3,4-DC reduced glial scar area and motor neuron death and improved functional recovery after SCI. RNA-sequencing results indicated that oxidative stress, pyroptosis, necroptosis, and autophagy may be involved in the ability of 3,4-DC to improve functional recovery. Furthermore, 3,4-DC inhibited pyroptosis and necroptosis by enhancing autophagy. We also found that 3,4-DC enhances autophagy by promoting TFEB activity. A decrease in the TFEB level abolished the protective effect of 3,4-DC. In addition, 3,4-DC partially regulated TFEB activity through the AMPK-TRPML1-calcineurin signalling pathway.
Conclusions: 3,4-DC promotes functional recovery by upregulating TFEB-mediated autophagy and inhibiting pyroptosis and necroptosis after SCI, which may have potential clinical application value.
Keywords: 3, 4-dimethoxychalcone, spinal cord injury, TFEB, autophagy, pyroptosis, necroptosis