| Stomolophus meleagris is a kind of very dangerous Cnidarians,which has strong toxic effects including skin toxicity,cardiovascular toxicity and neurotoxicity.In recent years,jellyfish often erupts in the coastal areas of China,causing a large number of stings.Symptoms vary depending on the strength,mainly including local skin inflammation and systemic poisoning.The nematocyst mainly exists in the jellyfish tentacles,which are feeding,attacking and defending cells,and have the function of producing and releasing jellyfish toxin.Therefore,there are a lot of jellyfish toxin in the jellyfish tentacles extract(TE).High doses of jellyfish toxin can cause cardiovascular failure and cardiac arrest,causing death within hours of stings;Moderate doses of toxins inhibit central nervous system and respiratory action for minutes and hours after stings;At low doses,the venom can cause delayed damage to multiple organs and death within days to weeks of the sting.However,delayed injury of pathogenic mechanism is not clear,this article uses the means of the transcriptome studies explore the S.meleagris delay toxic injury in mice after multiple organ m RNA expression changes,this paper analyzes the reason of the multiple organ injury,and intervene with the corresponding inhibitor,in order to clear delay jellyfish toxic injury syndrome pathogenesis.1.Establishment and evaluation of animal model of delayed S.meleagris envenomation syndrome(DJES).Methods: TE poisoning mouse models with different doses were established through caudal vein administration for continuous observation for 48 h.The toxicity grade of S.meleagris stings was completed according to the toxicity dose,toxic effect and death occurrence time of TE to determine the TE dose of DJES model mice.H&E staining,second-generation sequencing and RT-PCR were used to carry out pathological examination,hematological analysis,transcriptome analysis and m RNA expression level change detection of related genes in the DEJS model.Results:(1)S.meleagris TE caused death in mice in a dose-dependent way,with a lethal dose of LD50 = 1.54 mg/kg.The toxicity grade of stings included hyperacute toxicity(≧ 5 mg/kg),acute toxicity(2.5-5 mg/kg),delayed toxicity(0.5-2.5 mg/kg)and mild toxicity(0-0.5 mg/kg).(2)It was clear that 1.54 mg/kg TE dose could cause systemic poisoning with multiple organ dysfunction and death within 2 h to 2 d,so the DJES model was established at this dose.(3)The three organs had different degrees of pathological damage,among which the liver was the most serious,followed by the heart and kidney.(4)Hematological indicators also indicated significant functional damage in the liver,heart and kidney of DJES models.(5)In the three organs of the DJES group,35 of the 40 differential genes with the highest degree of upregulation were related to inflammatory response and immune response.After KEGG enrichment analysis of the differential genes,38inflammation-related signaling pathways were obtained,and GO enrichment analysis results also focused on GO items related to inflammation and immunity.(6)The up-regulated expression levels of inflammation-related genes such as Cxcl10,Ccl4,Ifit1,Ifit2,Isg15 and Rsad2 in multiple organs in the DJES group were verified.Conclusion: DJES causes synchronous damage to multiple organs in vivo and induces similar inflammatory responses in each organ.2.Inflammatory inhibitors were used to intervene with DJES,and inflammation was identified as the main cause of DJES.Methods: Blank control group phosphate buffer solution(PBS)-PBS,internal reference control group(0.01 mg/kg,0.03 mg/kg,0.1 mg/kg,0.3 mg/kg,1mg/kg,3 mg/kg and 10 mg/kg)dexamethasone(DXMS-PBS),positive control group PBS-TE and inflammatory intervention group DXMS-TE were administered through tail vein.The survival status of mice was observed to confirm the appropriate concentration of effective intervention dose of DXMS,and the histopathological,hematological and transcriptome indexes of DJES model mice after DXMS intervention were detected by H&E staining and second-generation sequencing technology.Result:(1)After DXMS intervention,the survival rate of mice increased in a dose-dependent manner,and the IC50 was 1.005 mg/kg.(2)The degree of pathological injury of heart,liver and kidney induced by DJES decreased after DXMS intervention.(3)Blood indexes also showed a significant partial recovery trend of multi-organ function after DXMS intervention.(4)Compared with the blank control group,KEGG and GO enrichment analysis of the differential genes showed that the pathway annotation and functional annotation of the differential genes were still mainly concentrated in inflammation-related pathways,but the 32 inflammatory genes that were jointly up-regulated in multiple organs of the DJES model were significantly down-regulated after DXMS intervention.Conclusion: DXMS is an effective therapeutic agent for DJES,and inflammatory response plays an important role in DJES.3.The establishment of S.meleagris TE inflammatory macrophage model provides the basis for the follow-up in vivo mechanism study.Methods: TE was co-incubated with RAW264.7 cells(0 μg/m L,0.85 μg/m L,2.56 μg/m L,8.53 μg/m L,12.80 μg/m L,17.07 μg/m L,21.34 μg/m L,25.61 μg/m L,51.22 μg/m L,85.36 μg/m L),and the cell survival rate was detected to determine the suitable concentration range for the establishment of RAW264.7inflammatory model by S.meleagris TE.PCR and RT-PCR were used to investigate the changes in m RNA expression levels of inflammatory related genes in RAW264.7(0.5 ～ 4 h)at this concentration,so as to determine the suitable incubation time of TE in the inflammatory model of TE-RAW264.7.Then,blank control group PBS-PBS,internal reference control group DXMS-PBS,positive control group PBS-LPS,model group PBS-TE and inflammatory suppression group DXMS(25 μM)-TE group were set,respectively,and PCR and RT-PCR were used to detect the changes in the m RNA expression levels of inflammation-related genes in each group.Finally,cell survival assay(0 μg/m L,5 μg/m L,15 μg/m L,500 μg/m L,1500μg/m L,5000 μg/m L)was used to detect the antagonistic ability of DXMS against the cytotoxic effect of TE.Results:(1)The cell survival rate was inversely proportional to the concentration of TE in a dose-dependent manner,with LD50 = 17.8 μg/m L.According to the data,12.8μg/m L of TE co-incubated cells had a higher survival rate(80%)and significant changes in cell phenotype.Therefore,12.8 μg/m L was determined as the concentration for modeling.(2)RAW264.7 cells exposed to TE(including 12.8 μg/m L)within 0.5 h ～ 4 h,inducing the increasing of expression level of Cxcl10 and Ccl4,TNF alpha,Ifit1,IL-6 and p65,which the expression of TNF alpha and Ifit1 amount present within 4 h of alternate rise.When the exposure time of 1 h,the detection of all inflammatory factors m RNA expression levels were significantly increased,thus determine the model building time of 1 h.(3)DXMS(25 μM)decreased the expression levels of Cxcl10 and Ccl4,TNF alpha,Ifit1,IL-6 and p65 in the inflammation model of TE-RAW264.7.(4)DXMS could antagonize jellyfish toxin in a dose-dependent manner.Conclusion: S.meleagris TE induce cell death by inducing cellular inflammatory response,and DXMS inflammatory inhibitors reduce the cytotoxicity of TE by inhibiting S.meleagris TE mediated cellular inflammatory response.In this study,the role of S.meleagris in delaying toxic injury was explored,and brand-new animal model and cell model of DJES were constructed.It was found that systemic inflammatory response plays an important role in DJES,and inhibition of inflammatory response by DXMS is a new method for the treatment of DJES. |
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