Nicotinamide Adenine Dinucleotide Mediates Bone Cancer Pain By Activating A1AR

Bone cancer pain(BCP)is among the most common and severe cancer pain conditions.Because the pathogenesis of BCP remains unclear,there is a lack of targeted clinical treatments.Therefore,it is pertinent to explore the cellular and molecular mechanisms of BCP to develop new analgesics.Nicotinamide adenine dinucleotide(NAD~+)is a neuroprotective agent that inhibits the release of presynaptic glutamate from hippocampal neurons,thereby suppressing neuronal hyperexcitability.NAD~+also inhibits glutamatergic excitotoxicity in neurons to exert a neuroprotective effect.The increased excitability of neurons in the spinal cord dorsal horn(SDH)is one of the essential mechanisms underlying the development of BCP.However,it is unclear whether NAD~+is involved in the development of BCP and whether it relives pain.In this study,a BCP model was constructed by injecting 4T1mouse mammary cancer cells to observe the analgesic effect of NAD~+on BCP and to explore its molecular mechanisms.The mechanical allodynia,thermal hyperalgesia,cold allodynia,and spontaneous pain were induced in BCP mice.The trabecular bone was severely destroyed,accompanied with proliferation of tumor cells in the femoral cavity.Microglia and astrocytes were activated in the SDH,and the expression of c-fos and p-CREB in SDH neurons were significantly elevated,suggesting that the BCP mouse model was successfully established.The NAD~+content in the dorsal root ganglia,SDH,and cerebrospinal fluid of BCP mice were significantly reduced.Further investigation revealed that the m RNA levels of nicotinamide phosphoribosyl transferase(Nampt),the rate-limiting enzyme for NAD~+synthesis,were significantly downregulated in DRG and SDH.Additionally,the activation/upregulation of NAMPT in SDH significantly relieved mechanical allodynia,thermal hyperalgesia,and cold allodynia in BCP mice.Moreover,the intrathecal administration of NAD~+significantly relieved both evoked and spontaneous pain in BCP mice.Fortunately,NAD~+did not induce craving behavior,indicating that mice did not display drug dependence on NAD~+.Furthermore,the results showed that NAD~+did not affect the pain thresholds and locomotor ability of na(?)ve mice through pain behavior,open-field,and rotarod tests.These results suggest that the decrease of NAMPT in SDH of BCP mice leads to a decrease in the NAD~+content,which may be the main cause of pain in BCP mice.The perfusion of NAD~+(8μM)in spinal cord slices immediately inhibited the spontaneous excitatory postsynaptic current(s EPSC)in SDH neurons but did not change the amplitude of s EPSC,whereas NAD~+significantly inhibited the NMDA and AMPA currents of SDH neurons.NAD~+inhibited the firing frequency of the wide-dynamic-range(WDR)neuron in SDH.The results suggest that NAD~+reduces the central sensitization of the spinal cord by inhibiting the excitability of SDH neurons in vivo and in vitro electrophysiology.DPCPX,the adenosine(ADO)A1 receptor antagonist,could reverse the inhibitory effect of NAD~+on s EPSC,WDR neuron firing of SDH neurons,and analgesic effect of NAD~+on BCP model mice.In the BCP mice model,the m RNA levels of Enpp1 and CD73,key enzymes that metabolize NAD~+into ADO,were significantly decreased in SDH,and the m RNA levels of Enpp1 and CD73 in SDH were significantly increased after intrathecal administration of NAD~+.The ADO in SDH of mice was significantly increased compared with that in the control group after intrathecal administration of NAD~+.Finally,the intrathecal administration of ADO significantly alleviated mechanical allodynia,thermal hyperalgesia,and cold allodynia behaviors in the BCP mice model.What’s more,ADO inhibited the firing frequency of neuronal s EPSC and WDR neurons in SDH,suggesting that NAD~+is metabolized to ADO to inhibit central sensitization in the spinal cord via ADO A1 receptors,thereby producing analgesic effects.In summary,this study first found that a decrease of NAMPT in the spinal cord of BCP mice caused a decrease in the level of NAD~+and that the upregulation of NAMPT or intrathecal injection of NAD~+produced significant analgesic effects in BCP mice.NAD~+can exert an analgesic effect by directly inhibiting the firing frequency of s EPSC,NMDA,and AMPA currents of neurons and the firing of WDR neurons in SDH,thereby inhibiting the central sensitization in SDH.Finally,both the analgesic effect on BCP mice and the inhibitory effect on the excitability of SDH neurons could be reversed by DPCPX.The intrathecal administration of NAD~+upregulated Enpp1 and CD73,which is important for ADO metabolism,and ADO levels were also upregulated.Therefore,we hypothesize that NAD~+acts on ADO A1 receptors directly or is metabolized to ADO to act on ADO A1 receptors to alleviate pain behavior in BCP mice by inhibiting the central sensitization of spinal cord.This study elucidates the modulatory effects and mechanisms of NAD~+on BCP and provides a theoretical basis for the clinical development of new analgesic drugs to address BCP.