| Objective: Multiple sclerosis(MS)is a chronic autoimmune disease of the central nervous system(CNS)that mainly occurs in young adults,with a female predominance.Multiple niduses and recurrent attacks are two important features of MS patients.The characteristic pathologic changes in MS patients are multifaceted,including axonal injury,demyelinating plaques in the white matter and chronic neuroinflammation.Despite a great deal of effort,there are still difficulties in the diagnosis and differential diagnosis of MS,which has also limited the development of treatment strategies.Inflammasome is a multi-protein complex associated with the initiation process of various inflammatory responses.NLRP3 inflammasome,one of the most meticulously studied inflammasomes,is highly expressed in CNS and is closely related to many CNS diseases.Pyroptosis is a newly discovered form of inflammatory cell death which can be triggered by activation of the NLRP3 inflammasome,it is also strongly linked to abnormal microglia activation.Previous studies have revealed that NLRP3 inflammasome signaling pathway and pyroptosis could be one of the underlying pathogenesis of MS.Ghrelin,a gastrointestinal hormone composed of 28 amino acids,acts as an endogenous ligand of the growth hormone secretagogue receptor.Ghrelin has a variety of endocrine regulatory functions,including regulation of growth hormone secretion,dietary cravings and maintaining energy homeostasis.Accumulating studies have shown that ghrelin can also play a neuroprotective role as a neuropeptide in CNS.In this study,rats were immunized with guinea pig spinal cord homogenates to develop an animal model of MS,namely experimental autoimmune encephalomyelitis(EAE).We aimed to examine the neuroprotective effects of ghrelin in EAE.Besides,we also studied the molecular changes involving the NLRP3 inflammasome pathway before and after ghrelin administration and explored the effects of ghrelin on pyroptosis.This study will provide novel information on the effects of ghrelin as a potential therapeutic drug for MS patients.Methods: In this study,guinea pig spinal cord homogenate was used as immunogen to build the animal model of MS.Clinical manifestations of SD rats were recorded to evaluate whether the model was successfully established.All SD rats were randomly divided into four groups: normal control group,EAE group,EAE + ghrelin group,and ghrelin control group.Clinical behavioral scores and weight changes of experimental animals in each group were blindly recorded.H&E and LFB staining were used to assess the degree of inflammatory cell infiltration and spinal demyelination,respectively.Based on the method introduced above,we explored the neuroprotective effects of ghrelin in MS preliminarily.Moreover,using PCR,ELISA and immunohistochemical(IHC)staining,we detected levels of some inflammatory factors in spinal cord tissue and serum as well as microglial activity,so as to further clarify the anti-inflammatory effect of ghrelin in EAE.Finally,PCR,ELISA and western blot were used to measure expression levels of related proteins and cytokines involved in the NLRP3 signaling pathway and pyroptosis process before and after ghrelin treatment,which helped us delve deeper into the underlying mechanism of ghrelin's treatment effects in EAE.Results: 1?After the establishment of EAE animal model with guinea pig spinal cord homogenate,we observed that disease onset appeared on day 9 and the peak of disease was on day 13 or day 14,which met the experimental requirements and laid the foundation for the following studies.After ghrelin treatment,weight loss occurred later and neurobehavioral scores were significantly improved compared with the untreated EAE group.In addition,H&E and LFB staining results showed that inflammatory infiltration of brain tissue and demyelination injury of spinal cord were also alleviated after ghrelin treatment.2?Experimental results revealed that mRNA levels of TNF-?,IL-6,COX-2,and iNOS were markedly expressed in the spinal cord of EAE rats compared with levels in healthy control rats.Marked increases in TNF-?,IL-6,and NO expressions were also observed in serum of EAE rats.Nevertheless,ghrelin treatment dramatically reduced these changes,which convincingly demonstrated the anti-inflammatory effect of ghrelin.Results of IHC showed that iba-1 and CD68 was upregulated in EAE rats compared with the healthy control group rats,which represented that more microglia were activated after EAE.Ghrelin treatment decreased positive rates of both iba1 and CD68 expressions,which indicated that ghrelin could suppress microglia aggregation and abnormal activation.Therefore,the regulation of microglial activity is a potential mechanism of ghrelin in the treatment of EAE.Besides,expressions of CD68,iba-1 and related cytokines were at low levels,which revealed that ghrelin had no side effects on immune status in healthy rats.3?Experimental results showed that levels of NLRP3?ASC?caspase-1?IL-1? and IL-18 were markedly expressed in EAE rats compared with levels in healthy control rats.Ghrelin treatments significantly reduced these changes,which indicated that ghrelin could inhibit NLRP3 signaling pathway.Additionally,LDH level in serum and GSDMD level in spinal cord tissue were all decreased by ghrelin,which indicated that ghrelin could inhibit pyroptosis process in EAE.Level of NF-?B p-P65 in ghrelin-treated rats was relatively low,which revealed that ghrelin could inhibit the activation of NF-?B by regulating phosphorylation,thereby inhibiting the NLRP3 inflammasome signaling pathway.Conclusion: 1?Ghrelin has neuroprotective effects on EAE rat model immunized by guinea pig spinal cord homogenate and pertussis toxin.Ghrelin could delay the onset and relieve the clinical symptoms of EAE rats,improve the neurological deficits,reduce the inflammatory brain infiltration and demyelination injury of spinal cord.The dosage of ghrelin used in this study did not exert unconcerned effects on healthy rats.2?Ghrelin may decrease the severity of EAE by controlling microglia activity.Ghrelin reduced the expression levels of related inflammatory markers in CNS tissues and serum,inhibited the aggregation and activation of microglia in brain parenchyma.Besides,the dosage of ghrelin used in this study had no effect on immune status of healthy rats.3?The therapeutic effects of ghrelin on EAE rats were closely related to its function on NLRP3 inflammasome signaling pathway and pyroptosis.Ghrelin could inhibit phosphorylation level of NF-?B P65,which may be a potential mechanism accounting for its regulation of NLRP3 inflammasome signaling pathway. |
PDF Full Text Request