| Background:The pro-inflammatory cytokine tumor necrosis factor-a(TNF-?)plays a crucial role in the progression of rheumatoid arthritis(RA)patients and the pain production in RA patients.Recombinant human type II tumor necrosis factor receptor antibody fusion protein(RTNFRII),which has been newly developed and applied to clinical use against the pro-inflammatory response of TNF-?,has been proven to be effective in relieving the condition and pain of RA patients.Although clinical trials and clinical application have achieved positive therapeutic effects,there is no research on whether RTNFRII affects the expression of different types of neuropeptides in dorsal root ganglion(DRG)neurons by anti-inflammatory effects against TNF-?.The pro-inflammatory neuropeptides and anti-inflammatory neuropeptides synthesized and released from DRG neurons play an important role in mediating the transmission of RA noxious stimulation information.Based on the above research background,firstly,we used cultured DRG neurons to study the effects of different concentrations of TNF-? on the expression of neuronal pro-inflammatory neuropeptide calcitonin gene-related peptide(CGRP)and anti-inflammatory neuropeptide(galanin,Gal).The effect of RTNFRII on TNF-a-induced CGRP and Gal expression alteration was also studied.Secondly,we used the type II collagen-induced RA rat animal model to study the expression of the pro-inflammatory neuropeptide CGRP and the anti-inflammatory neuropeptide Gal,and the relationship between the pain-relieving effect of RTNFRII and the changes of CGRP and Gal expression.Finally,the effect of RTNFRII on laboratory test indicators of RA patients was investigated,and the mechanism of RTNFRII remission of RA was analyzed.Part ? The effects of RTNFRII on CGRP and Gal expression of cultured DRG neurons with TNF-? induced neurotoxicityObjective:TNF-? is significantly up-regulated after nerve injury,and up-regulation of TNF-? causes nerve damage.TNF-? mediates neuropathic pain in a variety of diseases or conditions of injury,and this has been confirmed by studies in various animal models.TNF-? plays an important role in the progression of disease and pain of RA patients.The dorsal root ganglion(DRG)is located on the dorsal root of the spinal nerves.It is composed of highly specialized sensory neurons that conduct normal stimulation and noxious stimulation to the spinal cord.Neurons can transmit pain,temperature,touch,pressure,proprioception,and visceral sensation.Due to the complexity of DRG neuron types,the complexity of conduction information,and the complexity of neurotransmitters,it is necessary to study the role of neurotransmitters involved in RA pain transmission in mediating RA pain.Calcitonin gene-related peptide(CGRP)is considered to be an important neurotransmitter and pro-inflammatory neuropeptide expressed in DRG neurons which mediates the transmission of nociceptive information.CGRP is a major neurogenic inflammatory molecule,and increased expression of CGRP in sensory neurons is associated with peripheral inflammatory pain.CGRP is released by capsaicin-sensitive sensory nerves,which can lead to local neurogenic inflammatory reactions and neuropathic pain,and the regulation of its production and release is unclear.Capsaicin-mediated CGRP release is a classical sensory neuron CGRP release mechanism,but its significance in the endogenous release mechanism has not been determined.CGRP receptor activation has central and peripheral sensitization.Therefore,inhibition of the release of the noxious stimulator CGRP is a promising therapeutic strategy for the treatment of chronic or pathological pain.Galanin(Gal)acts mainly as an anti-inflammatory neuropeptide.In normal adult animals,Gal is expressed at a very low level in sensory neurons,but in peripheral nerve injury and inflammation,Gal is significantly up-regulated in DRG neurons and is involved in the development,progression,and transmission of neuropathic pain.Targeting afferent stimuli in primary afferent neurons,finding a therapeutic target from the onset of pain signaling is a logical strategy for developing new analgesics.Studies have shown that capsaicin,which has a stimulating effect on primary afferent neurons,can increase the expression of Gal in DRG neurons cultured in vitro,indicating that Gal is closely related to the process of nociceptive stimulation of primary sensory neurons.Stimulated by the pro-inflammatory cytokine TNF-?,whether the expression of Gal in DRG neuron changes,and thus whether to mediate RA-related pain,is a meaningful research direction.Methods:The present study established a culture model of DRG neurons in vitro,using different concentrations of TNF-? to detect the effects of different concentrations of TNF-? on the expression of CGRP and Gal in DRG neurons.Furthermore,RTNFRII was used to incubate DRG neurons with TNF-? toxicity,and the influence effect of RTNFRII on TNF-a-induced CGRP and Gal expression was examined to study on the mechanism of action of TNF-? inhibitors to alleviate RA pain by targeting neuropeptides.Results:After incubation with low concentrations of TNF-?(1 ng/ml),the length of neuronal processes increased;while after incubation with medium-concentration TNF-?(10 ng/ml)and high-concentration TNF-?(100 ng/ml),the length of neuronal prominence became shorter and showed dose-dependent with TNF-? concentration and TNFRII incubation can reverse the length of neurites caused by TNF-? stimulation.After incubation with different concentrations of TNF-?,the apoptotic rate of neuronal cells increased,and the increase of neuronal apoptosis rate was dose-dependent with the concentration of TNF-?.RTNFRII incubation can reverse the increase of neuronal apoptosis rate induced by TNF-? stimulation.After incubation with different concentrations of TNF-?,neuronal cell viability decreased,and neuronal cell viability decreased with increasing TNF-? concentration.RTNFR? incubation reverses the decrease in neuronal cell viability caused by TNF-? stimulation.Low concentration(1 ng/ml)and medium concentration(10 ng/ml)TNF-? incubation can up-regulate the expression level of CGRP in DRG neurons,which is a reactive manifestation of DRG neurons in response to TNF-? stimulation.High concentration(100 ng/ml)TNF-? incubation can down-regulate the expression level of CGRP in DRG neurons,indicating that high concentration of TNF-? causes severe damage to DRG neurons and impairs their ability to express CGRP,causing the expression level of CGRP to be down-regulated,and RTNFRII can resist the up-regulation of CGRP expression induced by TNF-?;Low concentration(1 ng/ml)and medium concentration(10 ng/ml)TNF-? incubation can up-regulate the expression level of Gal in DRG neurons,which is a reactive expression of DRG neurons in response to TNF-?stimulation.High concentration(100 ng/ml)TNF-? incubation down-regulated the expression of Gal in DRG neurons,suggesting that high concentration of TNF-?stimulated DRG neurons to severely damage,impairing their ability to express Gal This indicates that high concentration of TNF-? stimulated DRG neurons to severely damage,and weakened its ability to express Gal,resulting in down-regulation of Gal expression.RTNFRII can resist the up-regulation of Gal mRNA expression induced by TNF-?.Conclusions:TNF-a-mediated neurogenic inflammation is closely related to the expression of pro-inflammatory neuropeptide CGRP and anti-inflammatory neuropeptide Gal.After stimulation with appropriate concentration of TNF-?,not only the expression of pro-inflammatory neuropeptide CGRP was up-regulated,but also the expression of anti-inflammatory neuropeptide Gal was up-regulated.This indicates an adaptive response of DRG neurons to the pro-inflammatory cytokine TNF-? stimulation.Two different types of neuropeptides have been up-regulated,which is an effective response of DRG neurons under certain stimulation conditions to maintain the balance of neuronal homeostasis and an effective way to fight against external noxious stimuli.Part ? The effects of RTNFRII on relieving inflammatory pain in type ? collagen induced arthritis by modulating neuropeptide expressionObjective:TNF-? is a major pro-inflammatory factor in the pathogenesis of RA.Up-regulation of TNF-? expression in RA disease state triggers a series of changes in inflammatory response molecules,and the disease state progresses in a serious direction.Up-regulation of TNF-? expression not only mediates increased secretion and activation of pro-inflammatory cytokines in the immune system,but also affects reactive changes in the nervous system.Up-regulation of TNF-?expression in primary afferent neurons is one of the important mechanisms that induce joint pain.In neuropathological conditions,changes in the expression of neuropeptides associated with inflammatory responses may be a new mechanism for the development of RA pain.Since the pro-inflammatory effect of TNF-? will correspondingly cause changes in the expression of pro-inflammatory neuropeptides and anti-inflammatory neuropeptides,it plays a role in the development of RA pain.Whether RTNFRII further inhibits the expression of neuropeptides with different biological effects by inhibiting the pro-inflammatory response of TNF-? is an important subject worthy of discussion.After decades of long-term exploration and recent research,CGRP has been established as an important neurochemical phenotype of DRG neurons,which can serve as an important peptide neurotransmitter that mediates nociceptive information transmission under neuropathological conditions.The role of Gal is more complicated.According to a series of research evidence,Gal may play a role in the adaptive response of the peripheral nervous system to injury and the regulation of pain transmission.Under normal circumstances,Gal plays only a minor role in the process of nociceptive stimulation.However,in the neuropathological state,Gal plays an important role in the regulation of noxious stimulation.In neuropathic pain,Gal has an effective effect against noxious stimuli and a role in the treatment of chronic pain.Different concentrations of Gal have different regulatory effects,and Gal is a two-way regulator of peripheral pain information.Different stimuli,as well as the different stages of noxious stimuli,affect the effects of Gal,making it effective in promoting or inhibiting noxious stimuli.Whether Gal is also involved in mediating the analgesic effect of RA needs to be further clarified.Methods:The present study established a type ? collagen-induced RA rat animal model to investigate the expression changes of the pro-inflammatory neuropeptide CGRP and the anti-inflammatory neuropeptide Gal.We further studied the effect of RTNFRII on the expression of CGRP and Gal,and analyzed the new mechanism of RTNFRII in relieving RA pain.Results:The inflammatory response induced by type II collagen can trigger tactile allodynia and thermal hyperalgesia in rats,while the TNF-?inhibitor RTNFRII can significantly increase the mechanical stimulation threshold and the thermal stimulation withdrawal time threshold.RTNFRII is an important marker for relieving neuropathic pain;Type ? collagen stimulation can trigger up-regulation of CGRP expression in DRG neurons,which may be a mechanism for its induction of tactile allodynia and thermal hyperalgesia,while TNF-? inhibitor RTNFRII inhibits type ? collagen-induced upregulation of CGRP.This may be a pathway by which RTNF? produces an analgesic effect by inhibiting the expression of the pro-inflammatory neuropeptide CGRP;Type ? collagen stimulation can trigger up-regulation of DRG neuronal Gal expression,which may be a reactive change in the body's response to type ? collagen-induced inflammation.The TNF-? inhibitor RTNFRII inhibits the type ? collagen-induced Gal expression from up-regulating.This may be because RTNFR? reverses the expression of Gal in DRG tissue by blocking the activity of TNF-?.Conclusions:The up-regulation of pro-inflammatory response neuropeptide CGRP and anti-inflammatory neuropeptide Gal expression in DRG neurons induced by type II collagen in RA model rats is consistent with the pain behavior of rats.This may be an adaptive response of DRG neurons to neurogenic inflammation.After continuous treatment with RTNFRII,DRG neurons showed a decreasing trend of CGRP and Gal,which was consistent with the actions of RTNFRII in relieving pain behavior in RA model rats,which may be a new mechanism for RTNFRII in relieving RA pain.The exact molecular mechanism by which RTNFRII down-regulates the expression of CGRP and Gal in DRG neurons remains to be further clairfied.Targeting the pro-inflammatory neuropeptide CGRP and the anti-inflammatory neuropeptide Gal as a therapeutic strategy for relieving RA pain will be a new development direction.Part ? The effects of RTNFRII on laboratory test indicators of active rheumatoid arthritis patientsObjective:TNF-? is a multifunctional cytokine that acts as a central regulatory factor in the regulation of key immune functions.The key role of TNF-? in the progression of RA disease has long been recognized.Therefore,inhibitors targeting TNF-? are first used as biological reagents for RA treatment.RTNFRII has recently been used clinically as an important TNF-? inhibitor for RA treatment,and plays an effective role in alleviating the condition of RA by counteracting the inflammatory effect of TNF-?.The reliability,effectiveness,good tolerance,and rapid onset of TNF-? inhibitors make it the most commonly used biological reagent for the treatment of RA.In the previous clinical trials,our research found that RTNFRII can effectively alleviate the condition of RA.Methods:This study will further analyze the actions of RTNFRII on erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),white blood cell(WBC),neutrophile granulocyte(N),lymphocyte(L),red blood cell(RBC),hemoglobin(HGB),platelet(PLT),rheumatoid factor(RF),alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),serum creatinine(Cr).Results:RTNFRII can improve the key laboratory indicators ESR and CRP,which is an important mechanism of action for its therapeutic effect.However,some laboratory test indicators have not changed much,indicating that RTNFRII does not have a substantial impact on these indicators.In some individual RA patients,RTNFRII has an adverse effect on these laboratory tests,indicating that RTNFRII has a risk of side effects.Conclusions:Improvement of the key laboratory indicators ESR and CRP reflects the therapeutic actions of RTNFRII.According to the findings of this study,a small number of laboratory test indicators have abnormal changes in individual RA patients,indicating that the risk of side effects is not severe,and serious side effects(such as tuberculosis,serious infections,etc.)have not been found in this study.This may be related to the insufficient number of cases observed in this study.Further large-scale observational studies and long-term application of treatment to more complex RA patients are necessary in order to obtain more comprehensive data to guide clinical practical applications.The results of this study provide not only meaningful guidance for RTNFRII in the relief of RA,but also provide guidance for the determination of RTNFRII in laboratory test indicators for RA efficacy. |
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