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.