Identification of inflammatory regulation roles of thalidomide/ruxolitinib in nucleus pulposus and construction of polyelectrolyte nanocomplexes-impregnated injectable hydrogels for synergistic intervertebral disk degeneration treatment

Abstract

Degeneration of the intervertebral discs (IVD) is an important underlying etiology of degenerative diseases contributor to lower back and neck pain. Alleviation of inflammation is recognized as the most direct way for the treatment of IVD. Repurposing of approved drugs is a viable alternative strategy to de novo drug discovery and development. The aim of this study is to identify the inflammatory regulation roles of the small molecule thalidomide (an immunomodulatory and TNF-α inhibitor) and ruxolitinib (a selective JAK1/2 inhibitor) in nucleus pulposus (NP) cells and investigate the combinational therapeutic potentials of thalidomide and ruxolitinib for ameliorating IVD degeneration. RNA-sequencing (RNA-seq) was used to detect differentially expressed genes (DEGs) after treatment of human NP cells with individual thalidomide and ruxolitinib, and relevant signaling pathways and functions of DEGs were detected by enrichment analysis. Western blot and immunofluorescence were applied to explore the mechanisms by which thalidomide and ruxolitinib regulated inflammatory factors in human NP cells. Moreover, we developed ruxolitinib and thalidomide co-delivered polyelectrolyte nanocomplexes (RTNPs) by the assembly of chitosan derivatives of positively charged chitosan hydrochloride (CS) and negatively charged carboxymethyl chitosan (CMCS). In vitro studies revealed that RTNPs could efficiently deliver drugs into NP cells and corresponding extracellular matrix (ECM)-related genes and proteins were adjusted. Furthermore, The RTNPs were assembled with Pluronic F127 to form a RTNPs/F127 injectable thermoresponsive hydrogels to maintain resident time within the nucleus pulposus, achieve sustained drugs release and achieve long-term therapy effects. The caudal disc degeneration model of SD rats was established by acupuncture, and X-ray, magnetic resonance scanning, μCT scanning, and histological methods were performed to detect the therapeutic effects of RTNPs/F127 hydrogel on IVD in vivo. As was demonstrated in the in vivo studies, in situ intradiscal injection of RTNPs/F127 composite displayed a relatively slower disc degenerating progression in a puncture-induced IVD rat model compared with the monotherapy pattern.