DISCUSSION
Recent years, the use of antibiotics (primarily the tetracycline analogues) to treat RA (van der Veen, van der Heide, Kruize, & Bijlsma, 1994) has seen a resurgence of interest as they interfere with the production of prostaglandins and leukotrienes, scavenges oxygen radicals, as well as the expression of nitrous oxide synthase (Porstmann et al., 2005), and enhances natural inhibitors of matrix metalloproteinases (Pastorino & Shulga, 2008). Consistent with previous study suggesting the potential efficacy of AZM in arthritic rats (Carevi & Djoki, 1988), in this study, we found that AZM effectively suppressed principal indices of arthritis in the CIA model, as evidenced by the decrease of joints swelling, articular cartilage and bone destruction, deformity and synovitis. These data provide further evidence to support the therapeutic potential of AZM in RA. Importantly, AZM could ameliorate the inflammatory phenotype in CIA models as effectively as TNF-α blocker, thus suggesting an alternative intervention to overcome the drug insensitivity and high price of TNF-α inhibitors in RA treatment.
Together with our previous studies, chronic ER stress could be an underlying cause of synovial cell proliferation and production of pro-inflammatory cytokines (Geng et al., 2020). Besides, RA FLSs are resistant to apoptosis induced by ER stress (Kabala et al., 2017). Therefore, the components of ER stress have become an interesting target for RA therapy. The connection between ER stress and RA began to evolve after the contribution of GRP78 to the pathogenesis of RA(Yoo et al., 2012). During ER stress, the increase of GRP78 expression could amplify the inflammatory cascade by preventing RA FLSs from apoptotic death and escalating pannus formation. GRP78 knockdown markedly reduces vascular endothelial growth factor (VEGF)-induced angiogenesis, inhibits cytokine-mediated proliferation and invasion, and induces apoptosis of FLSs in mouse models of RA, thus presenting as a cross-point for multiple pathological phenotypes in RA (Song et al., 2019). Indeed, GRP78/BiP inhibition served as an effective approach to suppress the pathogenic features of RA, particularly for those patients with treatment-resistant RA. Additionally, as recombinant human GRP78/BiP is currently in Phase I/II clinical trials in patients with RA, and the outcomes are promising. Therefore, both the inhibition and expression of GRP78/BiP can be of therapeutic benefit in RA, depending on the clinical scenario. In this study, our findings that the commonly used antibiotics drug AZM targets GRP78 and inhibits its ATPase activity, further supporting the potential of AZM in RA treatment.
Cholesterol and lipids are essential for the proper biological function of cellular membranes, whereas excess cholesterol and lipids can be toxic and even lead to apoptosis. Previous study showed that SREBP activation leads to the induction of key enzymes of the cholesterol and fatty acid biosynthesis pathways, and thus membrane homeostasis (Porstmann et al., 2005). However, in response to ER stress, SREBPs overexpression increased the expression of cholesterolgenic and lipogenic gene, resulting in cholesterol and lipid accumulation and provoking consequent apoptosis. Three potential mechanisms by which ER stress induces SREBP activation: caspase-induced SREBP cleavage (Colgan, 2007), eIF2α-phosphorylation- dependent downregulation of INSIG (Birkenfeld et al., 2011) and GRP78 dissociation from the SCAP-SREBP complex (Su et al., 2019). Previous studies showed that in vitrooverexpression of GRP78 could decrease markers of ER stress, attenuate ER-stress-induced SREBP activation and lipid accumulation (Lhoták et al., 2012). Here, we examined the changes of molecular events occurred in RA FLSs following AZM treatment and the data showed that the significantly dysregulated genes were involved mainly in cholesterol and lipid biosynthetic process. Consistently, detailed analysis showed that AZM treatment promoted the dissociation of SREBP away from GRP78 and increased the expression of SREBP targeted genes. In essence, the evidence presented here marks UPR activation as an additional effector program by which AZM exerts its anti-arthritis potential, and argues for a GRP78-mediated mechanism as the underlying mode of AZM’s molecular basis for RA treatment.
Initial characterizations of AZM have demonstrated functional roles in controlling acute and chronic inflammatory responses, both in vitro and in vivo (Banjanac et al., 2012). In these studies, anti-inflammatory activity was observed on monocytes, lymphocytes, microphage, epithelial cells and fibroblasts, all cell populations which contribute to pathology in RA and other forms of inflammatory arthritis (Kvien et al., 2004). Therefore, the therapeutic effects of AZM might be caused by immunomodulatory activities towards different cells types, even though the direct effect of AZM on these cells in CIA mice or in RAin vivo must be further explored.
In summary, the specific AZM significantly decreased the severity of CIA, reduced the production of proinflammatory cytokines, and preventedin vivo bone destruction in CIA models. Thus, our study provides AZM as an alternative intervention for RA treatment that may overcome the drug insensitivity and high price of TNF-α inhibitors, and further support the components of UPR as promising treatment targets for RA.