Mechanism Of Phospholipase A₂ Toxicity Of Jellyfish Rhopilema Esculentum

Background and Objective:Jellyfish live in special habitats with high salt,high pressure,low temperature,low light and low oxygen content,and their outbreaks have led to frequent stinging events and aroused society concern recently.However,there are no specific drugs for effective treatment of jellyfish stings,which seriously threatens the life and property safety of coastal people.Among the reported toxic organisms and mammals,on the one hand,PLA₂ as the main toxic component causes inflammatory storm and lethal effect in the victim,on the other hand,PLA₂ products are involved in the metabolic activity of almost all cells and are one of the main triggers of a variety of chronic diseases and cancers in human beings.Although PLA₂ is often reported as one of the main components of jellyfish toxins,inefficient identification methods,unclear toxin components and toxicity,and the lack of specific therapeutic drugs or means are the main problems faced in the field of jellyfish poisoning,prevention and treatment.Therefore,we firstly used transcriptome and proteome technology as well as toxicity evaluation methods in vivo and in vitro to reveal the toxicity of R.esculentum.Then used mass spectrometry of protein to clarify the toxin composition and distribution of jellyfish while recombinantly expressing the important PLA₂ proteins and exploring its mechanism of mediating R.esculentum toxicity.Finally,based on the current PLA₂ inhibitors,antioxidants and "medical gas" to explore effective strategies for the treatment of R.esculentum stings and provide ideas for the research of new therapeutic drugs.Methods:Part I:The construction of transcriptome and proteome of jellyfish R.esculentum and the preparation and toxicity evaluation of tentacle extract（TE）1.Construction of transcriptome and proteome:The total RNA and protein of R.esculentum tentacles were extracted,high-throughput sequencing and mass spectrometry analysis were performed after quality control to construct the transcriptome and proteome databases of R.esculentum;Morphological comparison,sequence alignment and evolutionary tree were performed to do the species identification of this jellyfish.2.Preparation of TE:Cut off the tentacles,continuously stir at 4℃ for 72 h after filtering through a 200-mesh sieve and centrifuge the filtrate at 4℃,1,000 g for 15 min,the supernatant in 1×PBS after overnight dialysis is TE.Toxicity evaluation of TE:Mice were randomly divided into TE and Control group to inject tail vein with different concentrations of TE（0.9～8.6 mg/kg）and 1×PBS,respectively.The symptoms and death time of each mouse within 16 h were observed and recorded,and the lethal dose of 50%（LD50）was calculated;The LD50 of TE（2 mg/kg）and 1×PBS were injected into mice through tail vein,respectively,the hematologic index and pathology of heart,liver and kidney were performed after 4～6 h,respectively;TE（5～40 μg）and 1×PBS of 50μL as TE and Control group were injected intradermally into the left and right paws of mice,respectively,the thickness change of the soles within 48 h of mice were measured and recorded,and the percentage of swelling was calculated;The soles of 20 μg and 40μg of TE group as well as Control group at 12 h and 48 h were selected for pathological examination;100 μL Red Blood Suspension（RBS）were incubated with 10 μL of TE（0～78 μg/mL）at 37℃ for 1 h,10 μL of saponin（Saponin,S）（25 μg/mL）as positive control,and the hemolysis was calculated by OD415;30 μL different concentrations of TE（0～200 μg/mL）were added to the 70 μL Raw 264.7 cell suspension and incubated at 37℃ for 2 h,and the cytotoxicity was calculated by OD450;The TE:fibrinogen（Fg）（μg:μg）（0:1～1:0）with different mass ratios was incubated at 37℃ for 16 h and then the protein band and gray value were separated and performed by protein electrophoresis and ImageJ software,respectively,to calculate the degree of degradation of TE to Fg,that is,protease activity;25 μ L of different concentrations of TE（0～86 μ g/mL）and 4-nitro-3-（octanoyloxy）benzoic acid（NOBA）were added to the system and then incubated at 37℃ for 1 h to calculate the degree of degradation of TE to NOB A by OD405,that is,PLA₂ activity.Part Ⅱ:The screening of PLA₂ in TE of jellyfish R.esculentum and the research of its toxicity mechanism3.Construction of TE toxins library:The local Blast tool was used for sequence alignment between UniProtKB database and transcriptome and proteome database of R.esculentum to construct the toxins library and then do the classification and statistics of toxin genes and proteins according to their annotations;Construction of TE-erythrocyte membrane model:According to the method of hemolysis,TE（78 μg/mL）and 1×PBS with RBS were incubated,respectively,and then centrifuged at 4℃,16,000 g for 30 min.The precipitation was centrifuged again after 1× PBS cleaning,which then was performed alignment with the TE toxins library after mass spectrometry analysis to screen the toxin components that interacted with the membrane,especially the PLA₂.4.Expression of PLA₂ proteins:The PLA₂ proteins（PLA₂-Ⅰ and PLA₂-Ⅱ）screened in the erythrocyte membrane model were recombinantly expressed and purified in vitro to obtain pure proteins.Toxicity evaluation of PLA₂ proteins:The detailed method is the same as the toxicity evaluation of TE above,and PLA₂-Ⅰ/Ⅱ instead of TE.Tail vein injection used 3 mg/kg PLA₂-Ⅰ/Ⅱ;Hemolysis assay added different concentrations of PLA₂-Ⅰ/Ⅱ（0～150 μg/mL）;Cytotoxicity assay added PLA₂-Ⅰ/Ⅱ（0～100 μg/mL）;PLA₂ activity assay added PLA₂-Ⅰ/Ⅱ（0～30 μg/mL）.Structural analysis of PLA₂ proteins:Bee venom PLA₂（BVPLA₂）（PDB|1POC|A）was used as a model to perform the sequence alignment,3D structural simulation and evolutionary tree analysis of PLA₂-Ⅰ/Ⅱ proteins.5.Immunofluorescence of PLA₂-Ⅰ/Ⅱ proteins and Raw 264.7 cells:According to the requirements of the dye kit and the above cytotoxicity assay method,PLA₂-Ⅰ/Ⅱ proteins（10 μ g/mL）and 1×PBS were incubated with cells,respectively.The cell membrane,nuclei and PLA₂-Ⅰ/Ⅱproteins were stained and photographed;Transcriptome of Raw 264.7 cells treated by PLA₂-Ⅰ/Ⅱproteins and TE:The detailed method is the same as the method of cytotoxicity assay described above.PLA₂-Ⅰ/Ⅱ proteins（10 μg/mL）,TE（2μg/mL）and 1×PBS were treated with cells,respectively,and then the RNA of cells was detected by transcriptomics technology;Transcriptome of the heart,liver and kidney of mice treated by TE:The detailed method is the same as the above tail vein injection.TE（2 mg/kg）and 1×PBS were used to inject into mice,respectively,and then the RNA of heart,liver and kidney was detected by transcriptomics technology.6.PLA₂ activity determination of PLA₂ inhibitor antagonist PLA₂-Ⅰ/Ⅱ proteins:10 μL Darapladib（0.01 nM～6 μM）or Quinacrine dihydrochloride（QD）（0.01 nM～100 μM）with 20 μ L PLA₂-Ⅰ/Ⅱ（10 μt g/mL）were added to the buffer with 20 μ L NOBA and incubated at 37℃ for 1 h,and then the inhibition degree of DP and QD on PLA₂-Ⅰ/Ⅱ was calculated by OD405.Molecular docking analysis between PLA₂ inhibitors and PLA₂-Ⅰ/Ⅱ proteins:Molecular docking between QD/DP and PAL2-Ⅰ/Ⅱwas performed using AutoDock 4.2.6,AutoDockTools 1.5.6 and Python 2.5,and the results were demonstrated using PyMol 2.4.1 software,respectively.Part Ⅲ:The antagonistic effects of PLA₂ inhibitors and molecular hydrogen on TE toxicity of jellyfish R.esculentum7.Toxicity evaluation of PLA₂ inhibitor antagonism TE:The detailed method is the same as the toxicity evaluation of TE above.In the injection of tail vein and the determination of hematologic index,the mice were randomly divided into TE,DP-TE,QD-TE（the final concentrations of TE,DP and QD were 2 mg/kg,0.5 mg/kg and 0.3 mg/kg,respectively）and Control group（1×PBS）;In the intradermal injection model of the soles,mice were randomly divided into TE,DP-TE and QD-TE group,in which the final concentration of TE was 20 μg,the final concentration of DP or QD was 100 μM,250 μM and 1,000 μM;the soles of TE,1,000 μM DP-TE,100 μM QD-TE and Control group at 12 h and 48 h were selected for pathological examination,respectively;In the hemolysis assay,10 μL of TE（40 μg/mL）and 10 μL of DP（0.1 nM～15 μM）or QD（0.1 nM～15 μM）were added to 90 μL RBS;In the cytotoxicity assay,30 μL TE（10 μg/mL）and 10 μL QD（0.1 nM～15 μM）or QD（0.1 nM～15 μM）were added to 60 μL Raw 264.7 cell suspension;In the protease activity assay,DP and QD were incubated after mixing with TE,respectively,in which the final concentrations of DP and QD were 0.5 mg/mL and 0.3 mg/mL,respectively;The PLA₂ activity determination is the same as the above.The final concentration of DP and QD was 0.01 nM～6 μM and 0.01～100 μM,respectively.8.Toxicity evaluation of antioxidant antagonism TE:The detailed method is the same as the toxicity evaluation of PLA₂ inhibitor antagonism TE above.In the intradermal injection model,mice were randomly divided into TE,salvianolic acid B-TE（Salvianolic acid B-TE,SAB-TE）and molecular hydrogen-TE group,in which the final concentration of TE was 20 μg,the final concentration of SAB including 100 μM,250 μM and 1,000μM,the hydrogen final concentration of intraperitoneal injection of molecular hydrogen（H2+IP）and mixed paw（H2+Mix）was 1.5 ppm and 0.75 ppm,respectively.In cytotoxicity assay,the method is the same as that of TE,one group is put in a normal incubator and another group in a hydrogen incubator.Results 1.Electrophoretic gel and concentration detection displayed that the total RNA and protein of R.esculentum tentacles were successfully extracted,in which the OD260/OD280 of RNA was around 2.0,and the RNA bands were complete and bright.The protein was qualified and its bands were clear and not degraded;A total of 29,401 unigenes and 3,383 proteins were obtained,and their length distribution,expression and GC or molecular weight distribution were concentrated,which met the standard of building library;The morphological structure of R.esculentum is consistent with the reported R.esculentum,a TRINITY_DN9273_c0_g2 sequence annotated COI was screened from the constructed database,which has the highest similarity with R.esculentum through sequence alignment and evolutionary tree analysis,so the jellyfish was identified as R.esculentum;2.The results of tail vein injection showed that TE on the survival of mice showed a dose-dependent effect in the range of 0～8.6 mg/kg,its LD50 was 2.0 mg/kg;The results of hematological indicators showed that TE significantly increased or decreased the indexes of heart,liver and kidney in the blood of mice;Pathology showed that TE caused congestion,edema and bleeding of heart,liver and kidney;Intradermal injection of TE of mice showed time-and concentration-dependent edema,which caused edema inside and outside the prickle cells,telangiectasia at superficial dermal and infiltration of inflammatory cells;The results of hemolysis and cytotoxicity were indicated that TE showed dose-dependent toxicity to erythrocytes and Raw 264.7 cells;Enzyme viability assay indicated that TE also has dose-dependent protease activity and PLA₂ activity;3.TE toxins library showed that 370 toxin genes were screened from R.esculentum transcriptome,and 182 corresponding proteins were identified in the proteome,of which 12 and 5 PLA₂ were identified at the gene and protein level,respectively;the model of TE-erythrocyte membrane showed that there are 62 toxins in TE interacted with the membrane,including two PLA₂（PLA₂-Ⅰ and PLA₂-Ⅱ）;4.Protein gel showed that PLA₂ proteins were successfully expressed and purified in vitro and PLA₂（0～30 μg/mL）were no obvious toxic effect at animal and cell level;The activity of PLA₂ was dose-dependent,of which PLA₂-Ⅱ was stronger than that of PLA₂-Ⅰ;Structural analysis indicated that PLA₂-Ⅰ and PLA₂-Ⅱ have high sequence and structural homology with Bee venom PLA₂（BVPLA₂）（PDB|1POC|A）;5.Immunofluorescence showed that PLA₂ proteins are acted on the cell membrane,rather than passing through the membrane into the cell interior to play a role;Transcriptome data indicated that the most enriched pathways of cells treated with PLA₂-Ⅰ/Ⅱwere related to inflammation,including TNF signaling pathway,IL-17 signaling pathway,NF kappa B signaling pathway,Toll-like receptor signaling pathway,Jak-STAT signaling pathway and MAPK signaling pathway;TE also induced inflammatory responses at the cellular and animal levels;6.The activity antagonism of PLA₂ inhibitors to PLA₂-Ⅰ/Ⅱ proteins showed that DP can effectively inhibit the PLA₂ activity of PLA₂-Ⅰ and PLA₂-Ⅱ,while QD had no significant inhibitory effect;Molecular docking analysis of PLA₂ inhibitors and PLA₂-Ⅰ/Ⅱ proteins showed there were different degrees and forms of forces between PLA₂-Ⅰ/Ⅱ and ligands DP and QD,in which one hydrogen bond was formed between PLA₂-Ⅰ and QD and three between PLA₂-Ⅱ and DP;7.Tail vein injection of PLA₂ inhibitor antagonism against TE showed both DP and QD can significantly improve the survival of mice;Hematological indicator indicated both DP and QD can significantly inhibit the index abnormalities caused by TE,and even return to the normal level;Pathology showed that both DP and QD can improve the organ damage caused by TE,and the effect of DP was better;Intradermal injection showed DP can significantly reduce the swelling of mice soles with a dose-dependent manner,while QD had no significant effect;At the same time pathology showed both DP and QD can significantly improve the edema and inflammation caused by TE;PLA₂ inhibitors for hemolysis and cytotoxicity of TE showed that both DP and QD can effectively antagonize the hemolysis of erythrocyte and cytotoxicity of Raw 264.7 cell induced by TE and improve their survival;The results of enzyme activity inhibition showed that both DP and QD can effectively inhibit the protease and PLA₂ activity of TE;8.The toxicity evaluation of antioxidant antagonism TE were proved that SAB can significantly reduce the swelling of mice soles;Intraperitoneal injection of hydrogen-rich water（TE+H2 IP）and mixture of hydrogen-rich water with TE（TE+H2 Mix）effectively reduced the swelling of mice soles;Pathology showed SAB and molecular hydrogen effectively can improve the edema of prickle and basal layer,telangiectasia of dermis,and separation and fracture of collagen caused by TE;Cell viability showed the proliferation rate of Raw 264.7 cell and the LD50 of TE for Raw 264.7 cell in the hydrogen incubator were significantly higher than that of the cell in the ordinary incubator.Conclusion:R.esculentum TE has obvious toxic effects and strong enzyme activities at the animal and cellular level;TE toxic components are complex and diverse,of which PLA₂ is the potential active component acting on cell membranes in R.esculentum,and the PLA₂ proteins expressed recombinantly in vitro has activity and structural characteristics of the PLA₂.And like R.esculentum TE,it can also stimulate cells to produce inflammatory factors and induce synchronized inflammatory responses in vivo;PLA₂ inhibitors（DP and QD）can effectively inhibit the toxicity of TE in vivo and in vitro,which possibly achieved by inhibiting the activity of PLA₂ toxins;Antioxidants（SAB and molecular hydrogen）may play a protective role by mitigating TE-induced in vivo and intracellular damage.In short,PLA₂ plays an important role during the toxic effect of R.esculentum,it may be one of the important toxins of TE to induce multi-organ and intracellular inflammatory responses.Inhibitors of specific toxins,antioxidants and "medical gases" may be effective strategies for treating R.esculentum and even jellyfish stings.