The Role And Mechanism Of IRF8 In Hyperalgesia Induced By Nicotine Dependence And Withdrawal

BackgroundTobacco is one of the biggest public health problems in the world today.Smoking has been defined as the leading cause of preventable death globally.It is estimated that with the progress of medical technology,60 million smokers around the world receive major surgery every year.Given that smoking is an independent risk factor for perioperative complications,smoking cessation can significantly reduce the risk of the perioperative cardiovascular,respiratory and trauma-related complications.Therefore,all smokers who need to undergo elective surgery should quit smoking perioperative,and the relationship between nicotine dependence or withdrawal and postoperative pain has aroused widespread concern among anesthesiologists.Although accumulating studies have shown that long-term smoking cessation can reduce the pain status of patients with chronic pain and improve the prognosis,experimental pain response increases in the early stage of nicotine withdrawal,which is defined as hyperalgesia.Moreover,the pain intensity score is positively correlated with nicotine withdrawal symptoms.It has been repeatedly confirmed in animal pain models caused by hot plate method,tail flick method and plantar stimulation method.Later clinical studies also showed that compared with non-smokers,smokers were more sensitive to laboratory pain in the early stages of smoking cessation,and the pain intensity score was also positively correlated with nicotine withdrawal symptoms.Perioperative smoking cessation can increase postoperative pain score and the dosage of opioid analgesics,resulting in an increased incidence of postoperative respiratory depression,nausea and vomiting,skin itching and other opioid analgesic-related side effects.Therefore,it is an urgent problem to formulate an individualized postoperative analgesia scheme for smokers.In recent years,based on the current clear mechanism of nicotine dependence and withdrawal induced hyperalgesia,perioperative pain treatment for smoking patients has been improved,but the effect is not ideal.Therefore,it is necessary to further explore the possible mechanism of nicotine dependence or withdrawal hyperalgesia in order to provide ideal postoperative analgesia for smokers.It is currently recognized that neurons are not the only cells involved in pain response.Swan cells,satellite cells and immune cells(microglia,macrophages and T cells)are all involved.In particular,microglia is activated earlier than other cells in the early stage of injury response,and the interaction between microglia and neurons is critical for the formation of pain sensitization.A large number of studies have shown that activated microglia are involved in the occurrence and maintenance of neuropathic pain,cancer-related pain and hyperalgesia induced by morphine tolerance by up-regulating the expression of cell surface receptors,secreting inflammatory factors and activating pain related signaling pathways.Previous studies Long-term nicotine exposure and nicotine withdrawal can cause microglia activation,and intrathecal injection of minocycline can reduce hyperalgesia induced by nicotine withdrawal by inhibiting microglia activation.It was concluded that microglia activation was also involved in the development of hyperalgesia induced by nicotine withdrawal.Then researchers looked into the mechanisms and confirmed the importance of microglia P2X4R,brain-derived neurotrophic factor(BDNF)and interleukin-1?(IL-1?)by deeply understanding the mechanism of microglia activation regulating hyperalgesia.However,the effects and mechanisms of nicotine dependence or withdrawal on microglia activation and the expression of these pain related molecules are unknown,which needs to be further studied and determined.Interferon regulatory factor 8(IRF8)is a nuclear transcription factor,also known as interferon consensus sequence binding protein.It is specifically expressed in the microglia of the central nervous system and plays an important role in the activation and movement of microglia.Under physiological conditions,IRF8 deficiency can lead to abnormal microglia morphology,decreased phagocytosis and low expression of Ionized calcium binding adaptor molecule-1(IBA-1).Under pathological conditions,IRF8 is involved in regulating the expression of genes related to microglia activation and transforming it into a reactive phenotype.In addition,IRF8 gene knockout can change the molecular protein expression profile of microglia under different external stimuli.Studies in animal models have shown that up regulation of IRF8 expression in the spinal cord is involved in the development of tactile pain in mice subjected to repeated cold stress.It has been found that IRF8 can enhance the expression of P2X4R through IRF5,thereby promoting the activation of P38 mitogen-activated protein kinase(p38MAPK)and the release of BDNF in microglia,and promoting the development and maintenance of neuropathic pain.As mentioned above,previous animal model studies have identified the important role of microglia activation and the P2X4R-BDNF pathway in hyperalgesia induced by chronic nicotine dependence and nicotine withdrawal.However,the mechanisms of microglia activation and up-regulation of P2X4R expression remain unclear.Based on the similarities between the mechanisms of nicotine withdrawal induced hyperalgesia and neuropathic pain,we investigated whether IRF8 also plays an important role in the formation of hyperalgesia induced by nicotine withdrawal,and further explored its related mechanisms.In this study,In this study,we first constructed a nicotine dependence BV2 microglia model to observe the effect of nicotine dependence on Microglia activation,and then explored the key role of IRF8 through gene intervention.Then,by establishing the nicotine withdrawal mice model,the changes of IRF8 expression in the spinal dorsal horn were observed.Then,down-regulated IRF8 expression by intrathecal injection of IRF8shRNA lentivirus vector,the changes of thermal withdrawal latency and microglia activation were observed in mice with nicotine withdrawal.In addition,the molecules expression of P2X4R-BDNF pathway were observed.This study will further clarify the mechanism of hyperalgesia induced by nicotine dependence and withdrawal,and provide a new target and theoretical basis for postoperative pain management in smokers.Part I Effects of chronic nicotine exposure on activation of BV2 microgliaObjective The aim of this study was to investigate the effects of chronic nicotine exposure on BV2 cells activation and the role of IRF8 in it.Methods ?BV2 microglia were cultured with different concentrations of nicotine(the final concentrations were 0,10,50,100?mol/L).After 72 hours of intervention,the levels of BDNF and IL-1? in the supernatant of culture medium were detected by ELISA.The protein expression of IRF8 and P2X4R in BV2 microglia were detected by Western blot.In addition,the mRNA expression of IRF8,P2X4R,BDNF and IL-1? were detected by Real time-PCR.?To further determine the effects of chronic nicotine exposure on the secretion of BDNF and IL-1? in BV2 cells,the experiment was divided into five groups:control group,ATP group,low-dose nicotine+ATP group,medium-dose nicotine+ATP group,high-nicotine dose+ATP group.Specifically,ATP group,low-dose nicotine+ATP group,medium-dose nicotine+ATP group and high-dose nicotine+ATP group were stimulated by adding ATP(final concentration 50?mol/L)for 2h after chronic nicotine exposure for 72h.The levels of BDNF and IL-1? in the supernatant of culture medium after ATP stimulation were detected by ELISA.?To further determine the role of P2X4R in regulating the secretion of BDNF and IL-1? in nicotine dependence BV2 cells,the experiment was divided into six groups:control group,5-BDBD group,ATP group,ATP+5-BDBD group,nicotine+ATP group,nicotine+ATP+5-BDBD group.Purine receptor agonist ATP(final concentration 50 ?mol/L)and antagonist 5-BDBD(final concentration 20?mol/L)were intervened respectively.The levels of BDNF and IL-1?in the supernatant of culture medium were detected by ELISA.?In order to further clarify the role of IRF8 in regulating the expression of P2X4R,BDNF and IL-1?,we first constructed lentviruses targeting the IRF8 gene interference or overexpression,in which three interference sequences of IRF8shRNA were designed,and screened out the sequence with the highest interference efficiency for subsequent experiments.lentivirus targeting the IRF8 gene were constructed to infect BV2 cells and then down-regulate or up-regulate the expression of IRF8.?After BV2 cells were successfully infected by IRF8 gene interference or overexpression of lentivirus,chronic nicotine exposure was performed for 72h.The protein expression of P2X4R in BV2 microglia was detected by Western blot and the mRNA expression of BDNF and IL-1? were detected by Real time-PCR.Results?Real-time PCR results showed that chronic nicotine exposure increased the mRNA expression of IRF8,P2X4R and BDNF in a dose-dependent manner,and the final concentration of 50?mol/L and 100?mol/L nicotine significantly increased the mRNA expression of IL-1?(P<0.05).However,the expression of IL-1? mRNA was increased by 10?mol/L nicotine,but the difference was not statistically significant(P>0.05).Western Blot results showed that chronic nicotine exposure increased the expression of P2X4R protein in BV2 cells in a dose-dependent manner,and the final concentration of 50?mol/L and 100?mol/L nicotine significantly increased the expression of IRF8 protein(P<0.05),while the trend of the increase of IRF8 protein expression induced by 10?mol/L nicotine was not obvious(P>0.05).ELISA results showed that BDNF and IL-1? secretion were not induced by nicotine at final concentrations of 10?/mol/L and 50?mol/L compared with the control group(P>0.05).Even 100 ?mol/L nicotine promoted only small amounts of BDNF(67.22±3.79pg/mL vs.42.3 1±2.95pg/mL)and IL-1?(33.78±1.25pg/mL vs.26.39±1.03pg/mL)(P<0.05).?Compared with the control group,the concentrations of BDNF and IL-1? in the supernatant of BV2 cells in the low-dose,medium-dose and high-dose nicotine+ATP groups were significantly increased,and the increased levels were positively correlated with nicotine dose.The concentrations of BDNF and IL-1? in the medium supernatant of high-dose nicotine+ATP group were 3.1 times(159.56±5.32pg/mL and 41.19±3.97pg/mL)and 2.4 times(64.37±1.93pg/mL and 26.32±1.03pg/mL)higher than those of the control group respectively(P<0.05).?Compared with the nicotine+ATP group,the concentrations of BDNF and IL-1? in the medium supernatant of nicotine+ATP+5-BDBD group were decreased by 52%and 55%respectively(P<0.05).?After screening,IRF8shRNA2 can be used as the first choice to interfere with lentivirus in subsequent cell and animal experiments.Lentivirus infection in BV2 cells could successfully up-regulate or up-regulate the expression of IRF8.?Down-regulation of IRF8 partially inhibited the expression of P2X4R protein and the synthesis of IL-1? mRNA and BDNF mRNA in nicotine-dependent BV2 cells,and conversely,Up-regulation of IRF8 increased the expression of P2X4R protein and the synthesis of IL-1? mRNA and BDNF mRNA in nicotine-dependent BV2 cells.Conclusion Chronic nicotine exposure can up-regulate the expression of IRF8,P2X4R and the synthesis of BDNF and IL-1? in BV2 cells,and maintain the ability of BV2 cells to release high levels of IL-1? and BDNF under appropriate stimulation.IRF8 regulates the expression of BDNF,IL-1? and P2X4R in nicotine dependence BV2 cells.Part II The effects of nicotine dependence withdrawal on pain threshold and IRF8 expression in spinal cord of miceObjective To observe the changes of pain behavior and the expression of IRF8 in spinal cord of nicotine dependent-withdrawal mice.Methods Forty healthy male C57BL/6 mice were randomLy divided into normal saline group(Control group)and nicotine withdrawal group(NTW group),with 20 mice in each group.Mice in NTW group were injected subcutaneously with nicotine(3mg/kg)three times per day for 7 consecutive days and mice in Control group were injected with normal saline of the same volume,followed by injection with mecamylamine to induce NTW.Nicotine withdrawal symptoms and their scores of all mice were observed and recorded within 30 minutes after injection of mecamylamine,and judge whether the nicotine dependence-withdrawal model is established successfully.The thermal withdrawal latency(TWL)were measured longitudinally by using Hargreaves Thermal radiation method on the Id,4d,7d,10d and 14d after injection of mecamylamine.After TWL determination,the lumbar enlargement of spinal cord was taken from 3 mice in each group,and the relative levels of IRF8 expression in the spinal cord tissues were determined by Western blot.In addition,after the TWL determination was completed on the 7d after the injection of mecamylamine,the spinal cord was taken from 5 mice in each group after cardiac perfusion,and the expression of IRF8 in the dorsal horn of the spinal cord was detected by immunohistochemistry.Results ?NTW group mice had obvious nicotine withdrawal symptoms,and the withdrawal symptom score was similar to the previously reported nicotine withdrawal model caused by chronic nicotine infusion with osmotic micro pump.Therefore,it is considered that the establishment method of this nicotine dependence-withdrawal model is simple and feasible.?The results of pain behavior test showed that TWL decreased on the first day after nicotine withdrawal and decreased to the peak on the seventh day,then gradually increased,and the decrease of TWL lasted for at least 14 days.?The expression of IRF8 in spinal cord increased after nicotine withdrawal,and the expression level of IRF8 was negatively correlated with the change trend of TWL in mice.Conclusion The nicotine withdrawal mouse model can be successfully established by injecting subcutaneously with nicotine(3mg/kg)three times per day for 7 consecutive days,followed by injection with mecamylamine lmg/kg after the last injection.The increased expression of IRF8 in the spinal dorsal horn is related to the production and maintenance of hyperalgesia after nicotine dependence withdrawal.Part ? Effect and Mechanism of lentivirus-mediated shRNA interference IRF8 expression on hyperalgesia induced by nicotine withdrawal in miceObjective Observe the effect of intrathecal injection of IRF8 gene interfering lentiviral vector on nicotine dependence-withdrawal hyperalgesia in mice and its possible mechanism.Methods Thirty-six C57BL/6 mice successfully established nicotine withdrawal model were randomly divided into three groups:?Normal saline group(NS group):5?L normal saline was injected intravenously every day for consecutive 3 days;?IRF8shRNA lentivirus group(IRF8-RNAi-LV group):5?L IRF8shRNA lentivirus was injected intravenously every day for consecutive 3 days;?Negative control group(NC-LV group):5?L negative control lentivirus was injected intravenously every day for consecutive 3 days.The first intrathecal injection of all mice was performed 24h after the successful construction of nicotine withdrawal model.The expression of IRF8 mRNA and protein in spinal cord were measured by Real-time PCR and Western blot to determine the lentivirus interference efficiency in vivo.TWL values of mice in each group were measured at the same time every day 24 hours after the last intrathecal injection for 7 days.After the last TWL test on the 7th day,5 mice from each group were collected for spinal cord after cardiac perfusion.The expression of microglia marker IBA-1 in the dorsal horn of mouse spinal cord was detected by immunohistochemistry,and the activation degree of microglia was analyzed.Spinal cords of 7 mice in each group were collected,and the mRNA and protein expression of P2X4R and BDNF in spinal cord were measured by Real-time PCR and Western blot,respectively.Results?Intrathecal injection of IRF8shRNA lentivirus vector could successfully down regulate the expression of IRF8 in spinal cord;?TWL increased significantly on the 4th day after the last intrathecal injection and continued until the 7th day after the last intrathecal injection;?Intrathecal injection of IRF8shRNA lentivirus partially inhibited the activation of microglia;?Intrathecal injection of IRF8shRNA lentivirus could down regulate the mRNA and protein expression of P2X4R,BDNF in mouse spinal cord.Conclusion Intrathecal injection of IRF8shRNA lentivirus downregulates the expression of IRF8,which can significantly reduce hyperalgesia induced by nicotine dependence withdrawal.The mechanism may be related to inhibiting the activation of microglia in the spinal dorsal horn and the expression of P2X4R and BDNF after nicotine dependence withdrawal.