Research On The Effect And The Mechanism Of Intrathecal Endothelin A Receptor Antagonist On Bone Cancer Pain In Mice

BackgroundWith the increasing population,the intense aging and the universality of carcinogenic factors,the incidence and the mortality rate of cancer are rising.It is reported that there were approximately 14.1 million new cancer cases and 8.2 million deaths worldwide in 2012.About 60%-90%of terminal cancer individuals suffered from different degrees of pain,and 30%of them sustained severe pain.Bone cancer pain(BCP)is the most common type of cancer pain and is often caused by primary bone tumours or bone metastasis from tumours.Patients with primary bone tumours and about one-third of patients with bone metastases secondary to tumours have varying degrees of bone pain.About two-thirds of terminal cancers tend to be bone metastasis,often to the posterior lumbar,pelvis,long bones,and ribs.Although bone is not a primary organ,many common tumours are prone to bone metastases.Bone metastases of tumours are known as the most common reason of bone cancer pain.The fact that the tumour metastasizes to the bone and spreads into surrounding tissues causes pain.The severe pain along with the hypercalcemia,susceptibility to infection,pathological fractures,and poor stability of the spine,not only affect the quality of the life,but also cause depression and anxiety of the patients,resulting in a heavy burden on the families,as well as the society.The existing analgesic drugs or therapies,including opioids,non-steroidal anti-inflammatory drugs,bisphosphonates,and radiotherapy,often fail to realize sufficient analgesia or accompanied with severe adverse reactions.Therefore,to clarify the mechanism of the formation and maintenance of the BCP and to find effective treatment is of certain significance in improving the quality of life of patients and lessening the burden on the families and the society.Endothelin-1(ET-1)is a polypeptide chain consisting of twenty-one amino acids and it is one of the three most abundant endothelins in the human body.Endothelin-1(ET-1)is involved in a large variety of pain processes,as it functions as a neuromodulator.It regulates pain mainly through the activation of endothelin A receptor(ETAR)and endothelin B receptor(ETBR),both of which are ET-1 specific G-protein-coupled receptors.ET-1 plays an important role in pain regulation,and intrathecal injection of ET-1 can reduce pain in the animal model of pain.Similarly,in the inflammatory and neuropathic pain models,astrocytes over-expressing ET-1 mice showed pain relief,whereas neuron specific ET-1 knockout mice showed the opposite results.In neuropathic pain,the up-regulated ET-1,ETAR and ETBR are together involved in the regulation of pain.BCP has the characteristics of neuropathic pain,and the changes in the expression of ET-1,ETAR and ETBR in BCP are not clear.Protein kinase B(Akt)is an important signalling pathway that mediates cell proliferation,survival,and differentiation in a variety of cell types.It is widely expressed in laminae I-IV of the spinal dorsal horn that modulate nociceptive information,and it is involved in central sensitization triggered by both inflammatory and neuropathic nociceptive stimuli.Apart from Akt,the extracellular-regulated kinase(ERK)signalling pathway also plays an important role in the regulation of synaptic and neuronal plasticity,as well as the modulation of peripheral and central sensitization induced by noxious stimuli or nociceptive information.In addition,crosstalk between the Akt and ERK signalling pathways is involved in the pathophysiology and neurochemistry alteration of neuropathic pain.BCP has the characteristics of neuropathic pain,while the role of the spinal Akt and ERK pathways in BCP are not clear.Although ET-1 regulates pain by activating ETAR and ETBR,it is mainly mediated by ETAR.When applied epineurally onto the exposed rat sciatic nerve,ET-1 produced unilateral hind paw flinching,an effect blocked by pre-emptive i.p.morphine,and completely prevented,or reversed,by an epineural ETAR antagonist,with no effect from an ETBR antagonist.In the thermal pain model,the analgesic effect of central administration of ET-1 can be blocked by ETAR antagonists,whereas ETBR antagonists are ineffective,indicating that the analgesic effect of ET-1 in the central nervous system is mediated through ETAR rather than ETBR.In the neuropathic pain model of spinal cord injury,intrathecal injection of the ETAR antagonist BQ-123 alleviated the pain,whereas the ETBR antagonist BQ-788 was ineffective.This result indicates that ET-1 regulates pain through ETAR but not ETBR.Upregulation of spinal Akt and ERK is involved in the formation and maintenance of neuropathic pain,while inhibition of the Akt and ERK or their upstream proteins can reduce pain.Intrathecal injection of the ETAR receptor antagonist BQ-123 can down-regulate the spinal Akt and ERK and reduce neuropathic pain.Since BCP has the characteristic of neuropathic pain,the effect and mechanism of intrathecal ETAR antagonists on BCP in mice has not been investigated.On the one hand,this study aims to clarify the changes of spinal ET-1 and its receptors,and to assess the role of Akt and ERK pathways in the condition of BCP to provide a basis for possible therapeutic targets as well as understanding the mechanisms of BCP.On the other hand,by evaluating the effect of intrathecal injection of ETAR on BCP in mice,the possible molecular mechanisms for the reduction of pain was clarified,which could provide a new direction for the study of BCP treatment.This study is divided into two parts.Part I:Changes of spinal ET-1 and its receptor,Akt and ERK pathways in the BCP;Part Ⅱ:Intrathecal ETAR antagonist attenuates BCP in mice by inhibiting the spinal Akt and ERK pathways.ObjectivesPart I:After successful preparation of mouse BCP model,by detecting the pain behaviours,histology and the expression of spinal ET-1,ETAR,ETBR,Akt and ERK,clear the changes of ET-1 and its receptors in the BCP,as well as the Akt and ERK pathways in the formation and of maintenance of BCP.Part II:After intrathecal injection of the ETAR antagonist BQ-123,the effects of BQ-123 on BCP in mice and its possible mechanism were determined by measuring the pain behaviours and the expression of spinal Akt and ERK.MethodsPart I:The NCTC 2472 tumour line was cultured in NCTC 135 medium with 10%horse serum and was kept at 37℃ with an atmosphere of 5%CO2 and 95%O2.The cells were passaged twice per week.Four-to six-week-old male SPF C3H/HeJ mice,weighing 20-25 g were used in this study.The murine model of BCP was induced by implantation of tumour cells into the medullary cavity of the femur.The mice were randomly divided into two groups:sham operation group(Sham group)and cancer pain group(BCP group).Eight mice in each group were randomly selected for behavioural testing,including number of spontaneous flinches(NSF)and pain withdrawal threshold(PWT).On postoperative days-1,4,7,10,14 and 21 after tumour cell implantation,the 8 selected mice were removed for NSF and PWT.The blind method were used,the pain behaviour testers did not know how the mice were handled.At the same time,5 mice,excluding the 8 selected for behaviour test,were sacrificed randomly in each group on postoperative days-1,7,14 and 21,and L4-6 segments of the spinal cord were removed to detect the expression of ET-1 mRNA,ETAR mRNA and ETBR mRNA by real-time quantitative PCR,and the expression of ET-1,ETAR,ETBR,phosphorylate Akt(p-Akt),total Akt(t-Akt),phosphorylate ERK-1/2(p-ERK-1/2)and total ERK-1/2(t-ERK-1/2)by Western blot.p-Akt/t-Akt and p-ERK-1/2/t-ERK-1/2 were calculated.On day 21 after tumour cells inoculation,histology was used to evaluate the bone destruction.Part II:Mice were randomly assigned into 7 groups:Sham+NS,Sham+BQ-123,Sham+BQ-788,BCP,BCP+NS,BCP+BQ-123 and BCP+BQ-788 groups.Eight mice were randomly selected for NSF and another 8 mice were selected for PWT in each group.On the day 14 after tumour cell implantation after pain-related behaviour tests,BQ-123(30 nmol in 7 μl per dose)or BQ-788(10 nmol in 7 μl per dose)was intrathecally administered(30 nmol in 7 μl per dose)at a speed of 1 μl/s in Sham+BQ-123,BCP+BQ-123 groups or Sham+BQ-788,BCP+BQ-788 groups,whereas the same volume of normal saline was injected in the Sham+NS and BCP+NS groups.The behaviour tests were performed 30 min before intrathecal injection and half hourly until 180 min after intrathecal administration.At the time point when the behaviour testing changed most significantly in the BCP+BQ-123 group,5 mice were sacrificed randomly in each group,and L4-6 segments of the spinal cord were removed to detect the levels of p-Akt,t-Akt,p-ERK-1/2 and t-ERK-1/2 by western blot,and p-Akt/t-Akt and p-ERK-1/2/t-ERK-1/2 were calculated.ResultsPart I:Compared with-1 d,there was no significant difference in the changes of NSF and PWT in Sham group in various time points(P>0.05),whereas the NSF increased and PWT decreased at 7,10,14 and 21 d after tumour cells implanted in BCP group(P<0.01 or 0.001).From the repeated measurements taken at-1,4,7,10,14,and 21 days after tumour cell implantation,BCP mice showed mechanical hyperalgesia,as indicated by a significantly higher NSF and a lower PWT from day 7,respectively,than sham mice(P<0.01 or 0.001).Meanwhile,the expression of p-Akt and p-ERK-1/2,together with the ratio of p-Akt/t-Akt and p-ERK-1/2/t-ERK-1/2 were significantly up-regulated in BCP group compared with Sham group(P<0.05,0.01 or 0.001).Histological results indicated the tumour infiltrated and eroded cortical bone significantly in BCP group whereas no obvious bone destruction was observed in sham mice.Compared with-1d,there was no significant difference in the expression of mRNA and proteins of ET-1,ETAR and ETBR in various time points(P>0.05),whereas these indicators up-regulated at 14 d and 21 d in BCP group(P<0.05,0.01,or 0.001),and no significant difference at 7 d(P>0.05).Compared with Sham group,the expression of mRNA and proteins of ET-1,ETAR and ETBR up-regulated in BCP mice at 7,14 and 21 d(P<0.05,0.01 or 0.001).Part II:Tumour-bearing BCP mice showed a significantly higher NSF and a lower PWT score than Sham mice(P<0.001),and the score remained constant during the 180 min time window.However,BQ-123 intrathecal administration evidently affected the magnitude and duration of pain in BCP animals.BQ-123-treated BCP mice showed a "V shaped" pain response curve in terms of NSF and PWT.The NSF score of the BCP+BQ-123 group decreased steeply and significantly between 0 and 90 min and reached the lowest value at 90 min;however,the NSF score increased 90 min after intrathecal injection.The changes in PWT in the BCP+BQ-123 group were exactly the opposite of the changes in NSF.Therefore,90 min after intrathecal injection,5 mice were randomly selected in each group to sacrifice for L4-6 segments of spinal cord,and p-Akt,t-Akt,p-ERK-1/2 and t-ERK-1/2 were detected by Western blot,and the p-Akt/t-Akt and p-ERK-1/2/t-ERK-1/2 were calculated.The results indicated the expression of p-Akt and p-ERK-1/2,together with the ratio of p-Akt/t-Akt and p-ERK-1/2/t-ERK-1/2 were significantly down-regulated in group BCP+BQ-123 compared with those in group BCP and BCP+NS(P<0.001).ConclusionPart Ⅰ:Spinal ET-1 and its receptors are involved in the maintenance of BCP.The spinal Akt and ERK pathways play an important role in the formation and maintenance of BCP.Part Ⅱ:Intrathecal injection of BQ-123 attenuates bone cancer pain in mice probably by inhibiting the spinal Akt and ERK signalling pathways.