Clodronate liposomes may biases MSC differentiation toward adipogenesis through activation of NLRP3

Introduction: Clodronate-Liposomes (Clod-Lipo) injection after hematopoietic stem cell transplantation (HSCT) has been shown to be detrimental to hematopoietic reconstitution after transplantation, and our previous study showed that Clod-Lipo injection after HSCT increased adipocytes in the bone marrow cavity of mice after HSCT, but the reason for the large increase in adipocytes has not been clearly explained. The aim of this study was to investigate the source and mechanism of bone marrow cavity adipocytes after HSCT injection of Clod-Lipo. Methods: BALB/c mice received 7.5 Gy of total body irradiation followed by infusion of 5x106 bone marrow mononuclear cells from C57BL/6 via the tail vein. Clod-Lipo were injected through the tail vein on the first day after HSCT and every 5 days for the rest of the day. BALB/c mice were then divided into three groups: BMT, BMT + Clodronate-Liposomes (BMT + Clod-Lipo), and BMT + PBS-Liposomes (BMT + PBS-Lipo). Bone marrow pathological changes were detected by H&E staining, Western blot was used to detect the expression of NLRP3 and Caspase-1 in mouse bone marrow cells, and RT-qPCR was used to detect the expression levels of the key transcription factors peroxisome proliferator-activated receptor γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBPα) mRNA in bone marrow cells. Mouse mesenchymal stem cells (MSC) cultured in vitro were identified by flow cytometry, and adipocyte induction assays were performed using Clod-Lipo action for 24 h, Oil red staining was used to identify adipogenesis. Western blot was performed to detect NLRP3 and caspase-1 expression in MSC after Clod-Lipo action. Caspase-1 was blocked with Ac-YVAD-cmk (Ac-YV), followed by adipogenesis assay after 24 h of Clod-Lipo action to observe the change in the amount of adipogenesis. Results: Compared with the other two groups, a significant increase in adipocytes was found in the Clod-Lipo group by HE staining, and increased expression of NLRP3 and Caspase-1 in mouse bone marrow cells was found by western Blot. By culturing MSC in vitro and performing adipogenesis assay after 24 h of Clod-Lipo action, it was found that adipogenesis was increased in the Clod-Lipo group, while the expression of NLRP3 and Caspase-1 was increased in MSCs, and adipogenesis assay was performed after 2 h of action using Caspase-1 inhibitor, and it was found that adipocytes was reduced. Conclusions: The results of this study suggest that MSC are biased towards adipocyte generation in response to Clod-Lipo, a process that may be associated with activation of the NLRP3/caspase-1 pathway.