| BackgroundsAngiostrongylus cantonensis(AC)was first discovered and named in 1935 by Professor Chen Xintao in Guangzhou.AC was identified as a human pathogen in 1945,and its larvae can break the blood-brain barrier into the brain,causing sernous central nervous system infectious diseases such as eosinophilic meningitis or Eosinophilic meningitis(EM).Angiostrongylus cantonensis is distributed in tropical and subtropical regions and is mainly prevalent in Southeast Asia,Pacific Islands,Japan and the United States.China is mainly popular in Taiwan,Hong Kong,Guangdong,Zhejiang,Fujian,Hainan,Tianjin,Heilongjiang,Liaoning,Shanghai,Hunan,Beijing and Yunnan.The terminal host of A.cantonensis is rodent,and the intermediate host is mainly mollusc,such as brown agate snail or snail.The firststage larvae of Angiostrongylus cantonensis can develop into infected larvae in the intermediate host.Humans and mice are umsuitable hosts that can be infected by eating raw or undercooked infected intermediates or the contaminated vegetables.So far,more than 3,000 cases have been reported in the world,most of which are scattered,but thereare also reports of group outbreaks.In recent years,people's eating habits have changed,and the regional spread of freshwater snails in southern China,angiostrongylus cantonensis has become one of the most potentially dangerous foodbome parasitic diseases in some parts of China.Angiostrongylus cantonensis is larva migrans that can cause multiple organ damage.The larvae migrate in the body and cause a series of mechanical damage through the intestinal wall,liver,lungs and brain.In addition,their secretions and metabolites have toxic effects.The most serious is that stage 3 larvae can invade the central nervous system,causing eosinophilic meningoencephalitis or meningitis.The disease is characterized by a marked increase in eosinophils in the cerebrospinal fluid.The lesions can occur in the brain,meninges,and can also affect the cerebellum,brainstem and spinal cord,and the cranial and spinal nerves can also be affected.Studies have shown that after the worm enters the central nervous system,microglia are activated,down-regulate the immune system,secrete anti-inflammatory factors,and regulate its own apoptosis.Further study of the role of microglia in eosinophilic meningitis or meningoencephalitis will provide a new strategy for the clinical treatment of Angiostrongylus cantonensis.MicroRNAs are a very rich and conserved small non-coding RNA that regulates post-transcriptional expression of genes by inhibiting binding to messenger RNA or accelerating the degradation of messenger RNA.It has been reported that in infectious diseases(including parasitic diseases),some miRNAs play a role in regulating apoptosis in the host immune response.In our previous study,a total of 25 miRNAs were differentially expressed between the control group and the Angiostrongylus cantonensis infection group,with miR-181a-5p being the only down-regulated.miR-181a-5p,a highly conserved miRNA in most vertebrates,is abundant in the brain and increases in expression during hippocampal neuronal maturation.miR-181a-5p is involved in many processes in cell biology,such as cellfate determination and cell invasion.The study showed that the expression of miR-181a-5p in the mouse brain tissue infected with A.C and microglia stimulated with the excretory secreted protein(ESP)of the fifth stage larvae of Angiostrongylus cantonensis was down-regulated and the expression of target gene bcl-2 was up-regulated.By flow cytometry,we found that ESP promoted apoptosis of microglia and was inversely correlated with the ratio of bcl-2/bax.Transfection of miR-181a-5p antagomir increases the expression of bcl-2 in microglia and attenuates apoptosis induced by ESP.ObjectivesStudying how the excretory and secretory products of A.cantonensis stimulates mouse microglia to regulate its target genes through miR-181a-5p,thereby regulating apoptosis and affecting the progression of infectious diseases.To provide a basis for a better understanding of the molecular mechanism of the pathogenesis of A.cantonensis infection.Methods1.Preparation of experimental animals and ESPSD rats infected with A.cantonensis in the laboratory were circulated in the laboratory through two generations of snails and rats to become stable laboratory strains.6-8 weeks of BALA/c mice were intragastrically administered 30 three-stage(infected)larvae,and some of them obtained the fifth-stage larvae from the infected mouse brain 21 days later,and obtained the ESP of Angiostrongylus cantonensis.The other part was sacrificed after infection for 0 days,7 days,14 days,21 days,and 24 days,and brain tissue was obtained and RNA and protein were extracted.2.miR-181a-5p agomir/antagomir transfectionmiR-181a-5p agomir/antagomir and its negative control were transfected into mouse microglia MG-N9 using transient transfection.3.ESP stimulates microgliaThe secretory antigen ESP of the fifth stage larvae was obtained by culture,and the microglia cells were cultured in a medium containing 50?g/ml ESP,and simultaneously transfected,and the cells after different stimulation times were collected to extract total cellular RNA and protein.4.Real-time PCR detection of gmiR-181a-5p expression levelIn this experiment,SYBR Grenn staining method was used to perform relative quantitative Real-time PCR on the Applied Biosystem 7500 detection system,and U6 was used as an internal reference.5.Real-time PCR detection of Bcl-2 expression levelThis experiment uses the GoScriptTM Reverse Transcription System synthesis kit for reverse transcription,and uses SYBRTM Select Master Mix to detect the expression level of Bcl-2.GAPDH is used as an internal reference.6.Western Blot detection of Bcl-2 and Bax protein expression levelsThe mouse brain tissue protein was obtained by liquid nitrogen milling.The microglia total protein was obtained by cell lysis method,and the expression amount was detected by Western Blot,and GAPDH was used as an internal reference.7.Cellular immunochemistry to detect the expression level of bcl-2 protein in cellsThe cells after ESP stimulation were collected,and the expression level of bcl-2 protein in microglia was detected by cell immunochemistry.8.Flow cytometry to detect microglia apoptosisThe microglia after transfection and ESP stimulation were collected,and the apoptosis rate was detected by flow cytometry.9.Data analysisThe results of this study were at least 3 separate experiments with three replicates per experiment.Statistical analysis was performed using two-tailed t-test and kruskal-wallis ANOVA using SPSS 20.0 statistical software.Pearson correlation and linear regression were used to analyze the correlation of miRNA-181a and bcl-2 mRNA in brain tissue and microglia after infection and ESP stimulation.P<0.05 was a significant difference.Results1.mir-181a-5p negatively regulated bcl?2 after angiostrongylus cantonensis infection and ESP stimulationmir?181a-5p expression was down-regulated and bcl-2 up-regulated in mice infected with angiostrongylus cantonensis.We used ESP to stimulate microglia as an in vitro validation model for angiostrongylus cantonensis infection.After the infection of angiostrongylus cantonensis and the stimulation of ESP,the expression of mir-181a-5p in brain tissue and microglial was down-regulated,while the expression of bcl-2 was up-regulated.When we up-regulated the expression of mir-181a-5p in microglial,the expression level of bcl-2 in the cells decreased.When we down-regulated the expression of mir-181a-5p,the expression of bcl-2 in the cells was increased.2.Effects of ESP stimulation and transfection on microglial apoptosisBcl-2 is an anti-apoptotic protein,and we collected microglia after transfection and ESP stimulation.Flow cytometry showed that the apoptosis rate increased after ESP stimulation compared with the control group.The apoptosis rate increased after ESP stimulation and transfection of agomir,but decreased after transfection of antagomir.Meanwhile,the expression of apoptotic protein Bax was detected and the bcl-2/Bax ratio was calculated.The results showed that the bcl-2/Bax ratio was negatively correlated with the apoptosis rate of microglias.Conclusion1.Bcl-2 is the target gene of miR-181a-5p in microglia stimulated by ESP,and miR-181a-5p can negatively regulate the expression of bcl-2.2.ESP can promote the apoptosis of microglia cells,and transfection of miR-181a?5p antagomir can increase the expression of microglia cells bcl-2 and reduce the apoptosis caused by ESP,so it may be a potential therapeutic target for eosinophilic meningitis or meningititis caused by angiodoliasis. |
PDF Full Text Request