The Mechanism Of Spinal Dorsal Horn MHC-? Promoting In Cancer Induced Bone Pain In Rats

BackgroundAccording to the last statistic in journal of CA:A Cancer Journal for Clinicians,almost 429.16 million people were suffering from cancer in China in 2015.Cancer pain is one of the most common clinical symptoms in patients with advanced cancer.Cancer induced bone pain(CIBP)is a severe cancer pain,which originated from bone sarcomas or other malignant tumors that metabolize to bone.Once tumor cells have metastasized to the skeleton,75%patients would suffer moderate to severe bone pain and pathological fractures.Clinically,nonsteroidal anti-inflammatory drugs(NSAIDs)are the main therapy,usually associated with potent opioids,radiotherapy,and assistant agents which can decrease osteoclast activity for example bisphosphonates and denosumab.However,due to the complicated underlying mechanisms of CIBP,the drug resistance and dose-dependent adverse drug reactions,the treatment effect is not ideal.Thus it is necessary to discussing the pathology of CIBP and exploring targeted therapies.The mechanism of CIBP is complex,including the peripheral and central aspect.Tumors may contain a variety of inflammatory cells,and both inflammatory cells and tumor cells secrete multiple types of pain-related signal molecules that stimulate sensory nerve.Tumors can also gradually make the microenvironment acidic,inducing activation of pH sensitive nerve terminals and pain.Moreover,osteoclast activity spoils the osseous and contributes to pain by mechanoreceptors.While in the central part,the dorsal horns of spinal cord receive the peripheral nociceptive information and then project such noxious stimulation to the superior central nervous system.The crosstalk between neurons and glias,the activation of neurons in wide dynamic range,synaptic plasticity and the stimulation of central descending facilitatory system would participated in the formation of central sensitization,which would further contribute to CIBP.Recent studies indicated that the decline of tonic and phasic inhibitory control or"disinhibition" in the dorsal horn accounts for the amplification of pain information which then cause chronic pain.y-aminobutyric acid(GABA)is a predominant inhibitory neurotransmitter in the central nervous system(CNS),the downregulation of the GABA-releasing interneurons in the spinal cord has been regarded as significant factors causing chronic pain.It has been estimated that intrathecal injection of GABA analogue gabapentin or GABA could relieve chronic pain.CIBP was one of the most component of chronic pain,thus,we speculate that the decrease of GABAergic interneurons may be involved in mediating CIBP.Meanwhile,accumulating evidence demonstrated neurons could express high levels of MHC-? mRNA and/or protein in response to axotomy,cytokines treatments,and changes in electrical activities.The high expression of neuronal MHC-? could make neurons susceptible to cytotoxic CD8+ T cells and finally lead to neurons apoptosis.Thus we proposes hypothesis that tumor cells inoculation induced the high expression of MHC-? on GABAergic interneurons in spinal dorsal horn and the expression of MHC-? could then promote the apoptosis of GABAergic interneurons and finally lead to CIBP.To test this notion,a rat model of Walker 256 tumor cells intratibial injection was constructed to mimic the pathological process of CIBP.RNAi lentivirus was built to knockdown the expression of MHC-? in spinal dorsal horn.Western blot,immunofluorescence,behavioral study would be applied to investigate the roles of MHC-I and underlying mechanisms in CIBP.The preset study would provide a theoretical basis for the development of targeted therapies of CIBP.Methods and Results1.The role of spinal dorsal horn MHC-I in the development of CIBPMethodsFemale adult Sprague-Dawley(.SD)rats were randomly sorted into four groups(Sham,Tumor,Tumor + NC-LV,Tumor + RNAi-LV).Nontargeting control lentivirus(NC-LV)or MHC-I RNAi-lentivirus(RNAi-LV)were injected into the lumbar spinal cord of Tumor + NC-LV or Tumor + RNAi-LV rats respectively three days before the building of bone cancer rat model.Pain related behaviors were measured every three days after intratibial implantation of either tumor cells or boiled cells.Real-time PCR,western blot and immunostaining were applied to measure the expression of MHC-I and its cellular localization in spinal lumbar enlargements on days 7,14d,and 21d post tumor cells inoculation.ResultsThe behavior tests showed a persistent decrease of PWT in tumor-bearing rats 9 days post tumor cells inoculation.RNAi-LV,rather than NC-LV,attenuated mechanical allodynia induced by tumor cells injection.Quantification analysis of ?2-microglobulin mRNA(represent for MHC-I mRNA)and MHC-I protein indicated that MHC-I was significantly increased in the spinal dorsal horn of tumor-bearing rats.Pretreatment with RNAi-LV inhibited the increase of MHC-I mRNA and protein in the spinal cord.Immunostaining results showed that MHC-I predominantly expressed on GABAergic interneurons in the dorsal horn of tumor-bearing rats.2.MHC-I promotes apoptosis of GABAergic interneurons in the spinal dorsalhorn and contributes to cancer induced bone painMethodsFemale adult Sprague-Dawley(SD)rats were randomly sorted into four groups(Sham,Tumor,Tumor+NC-LV,Tumor+RNAi-LV).Nontargeting control lentivirus(NC-LV)or MHC-I RNAi-lentivirus(RNAi-LV)were injected into the lumbar spinal cord of Tumor+NC-LV or Tumor+RNAi-LV rats respectively three days before the building of bone cancer rat model.Pain related behaviors were measured every three days after intratibial implantation of either tumor cells or boiled cells.Western blot was used to detect the expression of GAD(glutamic acid decarboxylase)and cleaved caspase 3(Casp-3a)on days 7,14d,and 21d post tumor cells inoculation.Immunofluorescence was performed to show the co-expression of spinal Casp-3a and GAD in spinal dorsal horn.In Situ Apoptosis Fluorescein Detection Kit was applied to detect the apoptosis of GABAergic interneurons in spinal cord.ResultsQuantitative analysis showed that the GAD protein decreased in a time dependent manner while the Casp-3a protein increased in spinal of tumor-bearing rats.Knockdown of MHC-I with RNAi-LV inhibited the decrease of GABAergic interneurons and the increase of Casp-3a in tumor-bearing rats.TUNEL labeling revealed the apoptosis of GABAergic interneurons in the spinal dorsal horn of tumor-bearing rats which was prevented by pretreatment of RNAi-LV.Immunofluorescence results showed the Casp-3a immunoreactivity was localized in GABAergic interneurons in the dorsal horn of bone cancer rats.3.Apoptosis of GABAergic interneurons induced by MHC-I was closely relatedto CD8+ T cellsMethodsFemale adult Sprague-Dawley(SD)rats were randomly sorted into Sham and Tumor group.Flow cytometry and western blot was used to detect the expression of CD8+ T cells on days 7,14d,and 21d post tumor cells implantation.Immunofluorescence was performed to show the co-expression of spinal MHC-I and CD8+ T cells as well as the co-localization of CD8+ T cells and GABAergic neurons in spinal dorsal horn.ResultsFlow cytometry data showed the number of CD8+ T cells increased in a time dependent manner in the spinal cord of tumor-bearing rats,which agreed with the result of western blot.Immunofluorescence results indicated that CD8+ T cells were colocalized with MHCI and GABAergic neurons in the spinal dorsal horn.4.Statistical analysesAll data were analyzed using SPSS(V16.0)and expressed as mean ± s.e.m.To analyze the differences among groups,one-way repeated measures analysis of variance or two-way analysis of variance were used followed by the Bonferroni post-hoc test.Significance was indicated by P<0.05.Conclusion1.The increase of MHC-I expression on GABAergic interneurons in the spinal dorsal horn of bone cancer pain.2.Increased expression of MHC-I can remove GABAergic interneurons in the spinal cord by promoting its apoptosis via caspase-3 signal pathway.3.Knockdown of MHC-I with RNAi-LV can alleviate the apoptosis of GABAergic interneurons in the spinal cord and reversed abnormal behavioral responses of bone cancer pain rats.4.MHC-I induced the apoptosis of GABAergic interneurons is related to CD8+ T cells in the CNS.SignificanceIn this study,we provided evidence that MHC-I inhibited GABAergic interneurons by promoting its apoptosis and contributed to the development of CIBP.And the apoptosis of GABAergic interneurons induced by MHC-I was closely related to CD8+ T cells.These results have demonstrated a new immunologically-based molecular mechanism of CIBP,which can provide theoretical basis for seeking effective therapies in CIBP.