Cobratoxin Alleviates Cancer-Induced Bone Pain in Rats via Inhibiting CaMKII Signaling Pathway after Acting on M4 Muscarinic Cholinergic Receptors

Cancer-induced bone pain (CIBP) is
a common pain in clinics, which
can reduce the quality of life and increase the mortality of patients,
but the treatment of CIBP is limited. This study was designed to investigate
the analgesic effect of α-cobratoxin on CIBP and further to
explore the molecular target and potential signal pathway. As shown
by the mechanical allodynia test in a CIBP rat model, administration
of α-cobratoxin produced significant analgesia in a dose-dependent
manner, and the analgesic effects were blocked by pretreatment with
an intrathecal injection of M4 mAChR-siRNA or intraperitoneal injection
of tropicamide, an antagonist of M4 muscarinic cholinergic receptor.
Whole-cell patch-clamp recording showed that α-cobratoxin can
decrease the spontaneous firing and spontaneous excitatory postsynaptic
currents of SDH neurons in CIBP rats. In primary lumber SDH neurons,
intracellular calcium measurement revealed that α-cobratoxin
decreased intracellular calcium concentration, and immunofluorescence
demonstrated that M4 muscarinic cholinergic receptor and CaMKII/CREB
were co-expressed. In the CIBP model and primary SDH neurons, Western
blot showed that the levels of p-CaMKII and p-CREB were increased
by α-cobratoxin and the effect of α-cobratoxin was antagonized
by M4 mAChR-siRNA. The quantitative polymerase chain reaction (qPCR)
results showed that α-cobratoxin downregulated the expression
of proinflammatory cytokines through M4 muscarinic cholinergic receptor
in SDH. These results suggest that α-cobratoxin may activate
M4 muscarinic cholinergic receptor, triggering the inhibition of SDH
neuronal excitability via CaMKII signaling pathway, thereby resulting
in antagonistic effects in the CIBP rat model.