CXCL13/CXCR5 Signaling Activation In Spinal Cord Contributes To The Pathogenesis Of Cancer-induced Bone Pain In Rats

Background and ObjectiveCommon types of cancer,such as breast,lung,and prostate cancer,tend to form bone metastases in bones such as the femur,tibia,ribs,and vertebrae.Tumor formation in the bone often leads to anemia,increased risk of fracture,infection,and in many cases severe pain conditions that impair the patients’ quality of life.Pain caused by bone cancer is a complex state of pain that causes persistent pain,exercise-induced pain,and severe sudden onset of pain that are difficult to control.Although progress has been made in the treatment of bone cancer pain in recent years,current treatment methods are still insufficient,which may be partly due to insufficient understanding of the mechanism of bone cancer pain.It is well known that chemokines are significantly associated with the pathogenesis of neuropathic pain,and the relationship between chemokines and bone cancer pain has received increasing attention in recent years.A growing number of studies have confirmed that chemokines can promote tumor metastasis,and inhibition of these chemokines shows antitumor activity.The expression level of chemokines is up-regulated in the bone metastasis state,and chemokines regulate tumor metastasis,which determines the development of cancer in the tumor microenvironment.It has been confirmed that multiple chemokines and their receptors(such as CXCL12/CXCR4,CXCL1/CXCR2,CCL2/CCR2,CCL5/CCR5,CX3CL1/CX3CR1 and CXCL10/CXCR3)are involved in the development of bone cancer pain.More and more researches have proved that chemokines play an important role in the development and maintenance of bone cancer pain.Several chemokines involved in bone cancer pain have been identified,such as CCL2/CCR2,CX3CL1/CX3CR1,CCL5/CCR5,CXCL12/CXCR4,CXCL10/CXCR3,and CXCL1/ CXCR2.The expressions of these chemokines are upregulated in a model of bone cancer pain,and the function of blocking the signaling pathway by a receptor antagonist or a neutralizing antibody can inhibit the hyperalgesia induced by bone cancer in animals.The chemokine CXCL13 is effective in chemotaxis of B cells,which is been also known as B cell chemokine.It is originally found in stromal cells in B cell follicles,regulating the homing of B cells and T cell subsets.Under physiological conditions,CXCL13 is not only expressed in the central nervous system,but also in pathological conditions such as: neurosoftening,autoimmune demyelination and primary central nervous system lymphoma.The expression of CXCL13 is upregulated in the brain and spinal cord.In a mouse experiment simulating autoimmune encephalomyelitis,CXCL13 was found in infiltrating dendritic cells in the inflammatory meninges and spinal cord.CXCR5 is the sole receptor for CXCL13 and is expressed in all lymphocytes,blood T cell subsets,lymphoid tissues and cerebrospinal fluid.Studies have shown that CXCL13 levels in brain tissue and cerebrospinal fluid are directly related to the number of B cells in the cerebrospinal fluid.Successful treatment of multiple sclerosis is often accompanied by a decrease in the number of CXCL13 and B cells in the cerebrospinal fluid.However,it has been reported that animals lacking CXCL13 are characterized by normal B cell recruitment and mild self-limiting experimental immunoencephalitis.It is shown that CXCL13/CXCR5 regulates neurogenic inflammation in the central nervous system and does not depend on the B cell mechanism.In the model of neuropathic pain,the study found that CXCL13 and CXCR5 are rarely expressed in normal rats and humans.CXCL13 expression increased by 47 times 10 days after modeling neuropathic pain,far exceeding CCL7 and CCL2.And other chemokines such as CXCL1,the only receptor CXCR5 expression is also rapidly up-regulated,and the CXCL13/CXCR5 signaling pathway plays an important role in neuropathic pain.However,whether the spinal CXCL13/CXCR5 signaling could play a role in bone cancer pain needs to be further studied.This study would firstly investigate the role of CXCL13/CXCR5 in spinal cord in the development and maintenance of cancer-induced bone pain in rats.Then the molecular mechanisms of CXCL13/CXCR5 signaling contributing to bone cancer pain would be further studied.This finding might provide a new target for the treatment of bone cancer pain.Methods 1 The role of spinal cord CXCL13/CXCR5 signaling pathway in the development and maintenance of rat bone cancer painThe rat model of bone cancer pain was established,and then the expressions of CXCL13 and CXCR5 in the spinal dorsal horn of rats in the L4-5 segment at 7,14 and 21 day after inoculation of tumor cells were detected by Western Blot.The effect of CXCL13 overexpression on pain behavior in normal rats was observed by intrathecal injection of CXCL13 recombinant protein(rrCXCL13).The mechanical withdrawal response threshold(PWT)was measured by continuous intrathecal injection of CXCL13 neutralizing antibody(starting 1st day after inoculation,once a day,continuous injection for 14 days)at 1,3,5,7,10 and 14 days after inoculation,to verify the role of CXCL13 in the development of bone cancer pain in rats.PWT was tested by pre-intrathecal injection of CXCR5 siRNA(starting 4 days before inoculation,once a day,continuous injection for 3 days),to verify the role of CXCR5 in the development of bone cancer pain in rats.Rats were injected intrathecally with CXCL13 neutralizing antibody at 14 day after inoculation,and PWT was measured at 30 min,1 h,2 h,3 h,and 4 h after injection to investigate the spinal CXCL13 in the maintenance of bone cancer pain in rats.Rats were continuously intrathecally injected with CXCR5 siRNA(once a day,3 days)at 7 day after inoculation,and PWT was measured at 7,10,14 and 21 days after inoculation to investigate the role of the spinal CXCR5 in the maintenance of bone cancer pain in rats.2 The role of spinal astrocytes in the pathogenesis of rat bone cancer induced by CXCL13/CXCR5 signaling pathwayImmunofluorescence double staining was used to detect the colocalization of CXCR5 s with neuronal marker NeuN,astrocyte marker GFAP and microglia OX-42,in the spinal cord of bone cancer pain rats.The expression of GFAP mRNA in the spinal cord was detected by intrathecal injection of CXCR5 siRNA at 14 days after inoculation of tumor cells into bone cancer pain rats.The pain behavior of was observed by intrathecal injection of microglia inhibitor minocycline or astrocyte inhibitor LA-α-aminoadipate(L-α-AA)in CXCL13-induced bone cancer pain rats.3 The mechanisms of spinal CXCL13/CXCR5 contributing to bone cancer pain by activating ERK signaling pathway in astrocytes in ratsThe expression of p-ERK in the spinal cord was observed by Western blotting 1 h after intrathecal injection of rrCXCL13 in normal rats.Immunofluorescence double staining was used to observe the colocalization of p-ERK and astrocyte marker GFAP in the spinal cord at 14 day after inoculation in bone cancer pain rats.Western Blot was used to detect the effect of astrocyte inhibitor L-α-AA on the expression of p-ERK in the spinal cord of rats with bone cancer induced by rrCXCL13.RT-PCR and pain behavioral tests were used to observe the effects of ERK inhibitor U0126 on the activation of spinal astrocytes and pain behavior in middle-grade bone cancer pain induced by rrCXCL13 in rats.Results 1 CXCL13/CXCR5 signaling pathway in the spinal cord is involved in the development and maintenance of bone cancer pain in ratsIoculated tumor cells caused allodynia in the bilateral hind paws and up-regulation of CXCL13 and CXCR5 expressions in the spinal cord of rats.The pain behavioral results showed that the PWT of the bilateral hind paws began to decrease at 7 d after inoculation and continued to 21 d after the inoculation in bone cancer pain rats.Western blot results confirmed that compared with the sham group,the expressions of CXCL13 and CXCR5 in the spinal cord was up-regulated at 7 d after inoculation and continued to 21 d in rats.Compared with the na?ve group and the saline group,PWT began to decrease at 15 min after intrathecal injection of rrCXCL13 and continued for 120 min in normal rats.Intrathecal injection of CXCL13 neutralizing antibody or CXCR5 siRNA can alleviate hyperalgesia in the development and maintenance of bone cancer in rats.2 The role of spinal astrocytes in the pathogenesis of bone cancer pain mediated by CXCL13/CXCR5 signaling in ratsImmunofluorescence double staining showed that CXCR5 in the spinal cord of rats with bone cancer pain co-localized with the neuronal marker NeuN and the astrocyte marker GFAP,and did not co-localize with the microglia marker OX-42.RT-PCR data confirmed that compared with the CIBP-vehicle group,intrathecal injection of CXCR5 siRNA can up-regulate the expression of GFAP mRNA in the spinal cord of rats with bone cancer pain.Intrathecal injection of the microglia activation inhibitor minocycline had no effect on rrCXCL13-induced hyperalgesia in rats.Pre-intrathecal injection of the astrocyte inhibitor L-α-AA partially attenuated rrCXCL13-induced hyperalgesia in normal rats.Intrathecal injection of L-α-AA relieved the pain behavior of bone cancer pain in rats,but LA could not alleviate the pain induced by CXCL13 recombinant protein in the early stage of bone cancer pain.Intrathecal injection of L-α-AA not only relieved the pain behavior in the middle and late stage of bone cancer pain,but also alleviated the pain induced by CXCL13 recombinant protein in the late stage of bone cancer pain in rats.3 CXCL13/CXCR5 in the spinal cord contributed to bone cancer pain by activating ERK signaling pathway in astrocytes in ratsWestern Blot data showed that compared with the Vehicle group,p-ERK expression in the spinal cord was upregulated in rrCXCL13 group,while CXCR5 siRNA-rrCXCL13 group by pre-intrathecal injection of CXCR5 siRNA inhibited up-regulation of p-ERK expression.The results of immunofluorescence double staining confirmed that p-ERK in the spinal cord was co-expressed with astrocyte marker GFAP in the middle-late stage of bone cancer pain in rats.Western Blot results showed that compared with the vehicle-rrCXCL13 group,intrathecal injection of astrocyte inhibitor L-α-AA inhibited the up-regulation of p-ERK expression in the spinal cord of rats with bone cancer pain induced by CXCL13 recombinant protein.RT-PCR data confirmed that compared with the vehicle-rrCXCL13 group,intrathecal injection of ERK inhibitor U0126 inhibited the up-regulation of GFAP mRNA expression in the spinal cord of rats with bone cancer pain induced by CXCL13 recombinant protein.Behavioral test results showed that intrathecal injection of U0126 not only reduced the pain sensitization induced by CXCL13 in normal rats,but also alleviated the hyperalgesia in rats with bone cancer pain.ConclusionThese results suggest that the upregulated CXCL13 in the spinal cord via acting on the specific receptor CXCR5 evoked ERK signaling pathway activation in astrocytes contributing to bone cancer pain in rats.