A Study On Role And Mechanism Of TRPC1 Regulation Of Macrophage Polarization Affecting Osteoporosis

Objective:Osteoporosis（OP）is a systemic bone disease characterized by decreased bone mass,impaired bone tissue microarchitecture,increased bone fragility,and susceptibility to fracture,which results in progressive bone loss due to imbalance in bone homeostasis caused by increased bone resorption and decreased bone formation.Chronic inflammation induced by macrophage polarization was found to lead to enhanced osteoclast activity and diminished osteoblast activity,which play an important regulatory role in the pathological progression of OP.With the increasing aging of the population and the increasing incidence of OP in China,it is estimated that the number of OP patients will reach 120 million by 2050,and the mortality of hip fracture in OP patients reached 20-24%within 1 year,and the disability rate was as high as 40%.In contrast to the high morbidity and disability rates,the current treatment for OP is still limited,therefore,it is urgent to explore new therapeutic targets and strategies and find new drugs.Transient receptor potential canonical channels 1（TRPC1）were found to be expressed in a variety of cells in bone tissue,and their mediated Ca²⁺-related pathways were strongly associated with bone disease processes.Moreover,it was experimentally confirmed that elevated[Ca²⁺]i in the cytoplasm promotes macrophage polarization toward M1.However,no study has reported whether TPRC1 is involved in the development of OP by regulating macrophage polarization.In this study,we used TRPC1 as a target to investigate the anti-OP effect and molecular mechanism of TRPC1at the animal,tissues,cells,and molecular levels,with the aim of providing new targets and theoretical basis for the development of anti-OP drugs.\Methods:Osteoporosis induced by ovariectomy（OVX）was established in C57BL/6J mice and TRPC1 knockout(Trpc1^-/-)mice.Primary bone marrow macrophages from C57BL/6J mice and Trpc1^-/-mice were extracted and induced polarization to construct macrophage polarization model.Micro-CT analysis of the femoral epiphysis was performed to evaluate the effect of TRPC1 on OP.The bone tissues were pathologically examined for the number of osteoblasts and osteoclasts to explore the role of TRPC1on bone resorption and bone formation.Fluorescence co-localization was performed to detect the distribution of TRPC1 expression in OP tissues to find the main effector cells of TRPC1.Immunohistochemistry and Western blot were used to investigate the effect of TRPC1 on the polarization of macrophages at the tissue and cell levels,which to confirm that TRPC1 mediated the macrophage polarization in regulating OP.Dual-wavelength ion imaging assay,Western blot,nuclear plasma separation,immunofluorescence and Elisa were used to further investigate the molecular mechanisms by which TRPC1 regulates Ca²⁺levels in macrophages and thus affected cell polarization at the cellular level.Results:1.The expression of TRPC1 among the TRPCs was elevated and changed most significantly in bone tissue of OVX mice.2.TRPC1 knockout mice showed increased bone volume,increased number of trabeculae,and decreased trabecular spacing,and TRPC1 knockout reversed the decrease in bone volume and microstructure damage caused by OVX.3.TRPC1 knockout inhibited the attenuated bone formation and enhanced bone resorption induced by OVX.4.TRPC1 was mainly expressed and distributed in bone marrow-derived macrophages in the bone tissue of OVX mice.5.TRPC1 knockout reversed OVX-induced increase in M1 polarization and decrease in M2 polarization in macrophages.6.TRPC1 knockout inhibited LPS/IFN-γ-induced M1 polarization and promoted IL-4-induced M2 polarization in macrophages.7.TRPC1 knockout inhibited LPS/IFN-γinduced Ca²⁺influx in macrophages,inhibited M1 polarization in macrophages induced by activation of the Ca N/NF-κB signaling pathway,and inhibited macrophage secretion of inflammatory cytokines.Conclusion:In this study,we found that TRPC1 promoted M1 polarization of bone marrow macrophages,enhanced bone resorption,inhibit bone formation,led to imbalance in bone homeostasis,and promoted OP by promoting Ca²⁺inward flow in macrophages and affecting intracellular Ca²⁺levels,while activating Ca N/NF-κB signaling pathway.TRPC1 knockout reduced OVX-induced high Ca²⁺levels in macrophages to further inhibit the Ca N/NF-κB signaling pathway and slowing OP.These findings suggested that TRPC1 was expected to be a potential target for OP treatment,and blocking the mechanism of action of OP by inhibiting TRPC1 function might also be a new strategy for the prevention and treatment of such diseases.