| In recent years,the number of j ellyfish stings has increased explosively and has become a major threat to human life activities.Jellyfish dermatitis is the most common localized symptom of jellyfish stings.Jellyfish venom-releasing nematocysts can rapidly release intense venom into the epidermis and body,resulting in a range of clinical symptoms,including severe pain,necrosis,and scarring,as well as minor localized erythema.which causes great distress to long-term fishermen,tourists and clinical researchers.However,due to the complex and diverse composition of j ellyfish venom,the main molecular mechanisms of venom-mediated skin stings are still unclear,and despite the fact that jellyfish dermatitis treatments have mostly focused on studies to reduce the inflammatory response as well as to neutralize the venom,clinically active and effective therapeutic agents are still lacking.Stomolophus nomurai is the most widely distributed venomous jellyfish in China,concentrating in the North Sea and southeastern coastal areas of China.Therefore,in this study,Therefore,the molecular mechanisms associated with Stomolophus nomurai venominduced dermatitis and the pathophysiologic effects of the major venom-acting proteases inducing jellyfish dermatitis were thoroughly investigated.In the study,we firstly constructed an ex vivo and in vivo sting injury model mediated by Tentacle Extract(TE)of Stomolophus nomurai to reveal the acute cutaneous inflammatory response and oxidative stress injury,Using co-transcriptome sequencing technology,we were able to identify the main differential genes and enriched pathways involved in the Jellyfish toxin-mediated cutaneous damage.In further,we screened the sequences of the major toxin active proteins phospholipase A2(PLA2)and metalloproteinase(MMP)and recombinantly expressed them in vitro by jellyfish toxin proteomics and transcriptomics sequencing technologies,reactivated the skin and explored the skin pathophysiological effects mediated by the major active proteins of Stomolophus nomurai venom.MethodsPart Ⅰ:Construction of an in vitro model of jellyfish dermatitis mediated by Stomolophus nomurai TE and mechanism investigation1.S.meleagris nomurai were collected,To assess the TE-mediated dermatotoxicity,skin HaCaT cells were co-incubated with TE at several doses,and the cell viability was measured using the CCK8 method,TE induced oxidative stress damage in skin,skin cells.HaCaT cells were treated according to2.RT-qPCR was used to determine the expression levels of inflammatory factors TNFα,IL-1β,IL-6,and IL-8 and antioxidant enzyme GSTM2、SOD2、HMOX1 and CAT in the supernatant of HaCaT cells.For different concentrations of TE.Using flow cytometry and reactive oxygen species ROS fluorescence labeling,each group was examined to determine how the concentration-dependent changes in Stomolophus nomurai TE-mediated ROS oxidative stress indicators were occurring.3.Stomolophus nomurai TE-mediated mouse skin transcriptomics construction TE was selected to construct a mouse paw toxicity swelling model,set up a control group,TE was selected as a model concentration to stimulate HaCaT cell toxicity,and total RNA was isolated,which were then kept at-80°,mass spectrometry,and high-throughput sequencing were performed on the tissues,after passing the quality control test to construct a transcriptome database of sand jelly skin stings.The transcriptome database of Stomolophus nomurai skin stings was constructed by mass spectrometry analysis and high-throughput sequencing.Based on the sequencing data,the main differential genes and enrichment pathways were examined.4.Based on the results of cellular level transcriptomic co-sequencing of Stomolophus nomurai TE,the common differential genes will be analyzed together by using the network protein interactions method,and the common enriched pathways will be screened out,including the MAPK and TNF-α signaling pathways;The main expression up-regulated differential genes including Jun,c-Fos,MA2Pk3 and NFkbia,Cebpb were screened out.To further elucidate the accuracy of transcriptome sequencing,RT-qPCR was applied to the aforementioned differential genes;and the expression levels of the pathway nuclear transcription factors Jun and NF-kappaB p65 were measured by Western blotting to detect the phosphorylation of ERK,JNK,P38,and the nuclear transcription factors Jun and NFkappaB p65 at the animal and cellular levels,respectively;the differential expression of phosphorylated pathway proteins was analyzed according to the different temporal gradients of acute inflammatory responses,and the main interactions between toxin-mediated protein molecules were further investigated.5.TE 30 μg/mL stimulated cutaneous HaCaT cells were incubated for 2h.Immunofluorescence was carried out according to the cell nucleus(DAPI),cell membrane(DIL),FITC and protein nuclear transcription factors Jun and NF-kappaB p65 staining for immunofluorescence assay to analyze the membrane-binding ability of Jun and NF-kappaB p65,respectively,in the TE-mediated MAPK signaling pathway with HaCaT.Part Ⅱ:Construction of an in vivo model of jellyfish dermatitis mediated byStomolophus nomurai TE and the mechanism1.Mice were randomly divided into control and Stomolophus nomurai TE groups:As a control group,mice received an intradermal injection of saline(NS)into their left paw.Different concentrations of TE as different doses of TE groups,the thickness change of the mice’s left paw was measured and recorded,and the percentage of paw swelling was computed.The skin irritation responses were assessed.Subsequently,the paws of mice in the control and 50μg TE groups were selected for pathological examination and analysis.Effectively modeling the in vivo sting pathophysiological manifestations induced by TE2.TE was injected into the paws of mice to construct an in vivo model,n=4,and total RNA was extracted from the tissues;high-throughput sequencing was performed to construct a transcriptome database of stings after passing the quality control test,and the TEmediated jellyfish dermatitis was screened for the main key genes and enriched pathways based on the sequencing.3.Based on the results of co-sequencing,the common enrichment pathway MAPK and Cytokine-cytokine receptor interaction signaling pathway were screened according to the differential genes between TE and control;the expression levels of the pathway nuclear transcription factor Jun,NF-kappaB p65 were detected by immunohistochemistry;and the main key genes of TE-mediated jellyfish dermatitis were verified at the animal level by western blotting.The main pathway proteins ERK,JNK,P38,Jun and NF-kappaB p65 were acvated according to the different temporal gradients of the inflammatory response at the animal level.To further clarify the molecular regulatory mechanism related to jellyfish dermatitis mediated by TE.Part Ⅲ:Screening of PLA2 and MMP components in Stomolophus nomurai TE and their role in the mechanism of jellyfish dermatitis1.Construction of TE toxin library:sequence comparison between proteome and transcriptomics was performed to screen out the main toxin genes and proteins,and statistical evaluation was carried out according to their species,expression amount,and species distribution characteristics;PLA2 and MMP,were selected according to their expression amount:The amino acid sequence structures of PLA2 and MMP were compared,and 3D structure simulation and evolutionary tree analysis were performed to clarify the screening results of PLA2 and MMP sequences.Structural characterization and evolutionary relationships of major toxin proteins.2.For TE major active proteins,in vitro recombinant expression was performed based on the proteins PLA2 and MMP screened in previous proteomics and transcriptomics and purified to obtain single-component purified proteins.Next,PLA2 and MMP proteins will be evaluated for activity and toxicity.PLA2 activity will be determined by adding different concentrations of the PLA2;toxicity will be evaluated by the same specific method as the Stomolophus nomurai TE-mediated cytotoxicity effects were studied by adding different concentrations of PLA2 and MMP,and assayed with PLA2 and MMP equivalent to Stomolophus nomurai TE.3.According to PLA2 and MMP were incubated with skin HaCaT cells,respectively,and reactive oxygen species ROS fluorescence staining and flow cytometry were carried out to detect the effects of PLA2 and MMP on the ROS oxidative stress in skin cells,respectively.4.Proteins PLA2 and MMP were incubated with HaCaT cells for 2 h,and immunofluorescence assay was performed based on nucleus(DAPI),cell membrane(DIL),FITC and protein staining to analyze the membrane-binding ability of PLA2 and MMP with skin HaCaT.For the transcriptome assay of PLA2 and HaCaT cells,the experimental method followed the steps of cytotoxicity assay.PLA2 protein and HaCaT cells were treated with 1×PBS,and transcriptome approaches were utilized to investigate the impact on the key enrichment pathways and differential gene expression in HaCaT cells.In-depth investigation of the regulatory effect of PLA2 protein on TE-mediated gene expression in skin HaCaT cells.Screen the MAPK signaling pathway proteins ERK,JNK,P38,Jun,NF-kappaB p65 phosphorylation expression level for validation analysis.Part Ⅳ:Inhibition of TE-mediated jellyfish dermatitis toxicity by PLA2 and MMP inhibitors1.The left paw of mice was selected for intradermal injection of the PLA2 inhibitor Darapladib(DPD)and the MMP inhibitor Ethylenediaminetetraaceticacid(EDTA),and randomly divided into the NS group,TE,TE-DPD group,and TE-EDTA group.DPD,and TE-EDTA groups;the pathology tests performed on the paws of the mice in the NS,TE,TEDPD,and TE-EDTA groups.The cytotoxicity assays of the Study of the toxicoprotective effects of DPD and EDTA on TE:At the same time,cytotoxicity assays were performed to study the toxic antagonistic effects of DPD and EDTA on TE.TE and different concentrations of DPD as well as EDTA were added to HaCaT cell suspension for mixing and incubation.The expression levels of inflammatory factors were detected using RT-qPCR to investigate their effects on the inflammatory response.The impact of DPD and EDTA on the key phosphorylated proteins in the MAPK signaling pathway that are mediated by TE is examined using the WB technique.Results1.Stomolophus nomurai TE mediated significant in vivo and vitro dermal toxicity.Compared with the control group,mice in the TE group showed obvious swelling of the paw and the inner side of the lower leg,and some paws were necrotic;skin irritation scores were significantly elevated in a toxin time-and dose-dependent manner;pathology showed that the paw of mice in the TE group showed separation of the true epidermis with oedema and separation of the collagen fibers,and mixed inflammatory cell infiltration of neutrophils and lymphocytes in the dermis and subcutaneous skin.TE showed dose-dependent cytotoxicity to HaCaT cells in vitro,and cell viability was inversely proportional to TE Concentration,LC50(Lethal Concentration 50%)=29.75 μg/mL.2.Stomolophus nomurai TE mediates cellular inflammatory responses and oxidative damage:The TE group exhibited considerably greater mRNA expressions of inflammatory factors,namely IL-1β,IL-6,IL-8,and TNF-α,than the control group,as demonstrated by quantitative PCR data.Furthermore,the inflammatory effects of IL-6 and TNF-α were more pronounced.The average fluorescence intensity grew progressively at various TE concentrations in a dose-dependent manner,as demonstrated by the oxidative stress index ROS flow cytometry and fluorescence.While the mRNA expression levels of catalase(CAT)were not significantly decreased by fluorescence quantitative PCR,the antioxidant enzymes glutathione transferase 2(GSTM2),heme oxygenase-1(HMOX1),and superoxide dismutase 2(SOD2)were significantly lower in the TE group than in the control group.A statistically significant difference was present.3.Investigation of the molecular mechanism of jellyfish dermatitis mediated by Stomolophus nomurai TE through MAPK pathway:TE-mediated in vivo and ex vivo transcriptomic data showed that the common differential genes of HaCaT cells in the paw and skin of toxin-treated mice included Jun,NFkbia,FOS,IL6,and IL-1β,and that the enrichment of the differential genes was mainly concentrated in the inflammation-and apoptosis-related pathways,including the TNF signaling pathway,MAPK signaling pathway,IL-17 signaling pathway,NF-kappaB signaling pathway,Cytokine-cytokine receptor interaction;TE similarly elicited overall animal and cellular level RT-qPCR showed that mRNA expression of differential genes,comparing to the control group,there was a substantial increase in Jun,c-Fos,MA2Pk3,and NFkbia.which further clarified the accuracy of the transcriptome sequencing results;immunohistochemistry of Jun and NF-kappaB p65 proteins in skin tissues showed that,compared with the control group,the TE group mediated a significant increase in the phosphorylation level of MAPK signaling pathway proteins ERK,JNK,P38 and the main nuclear transcription factors Jun and NF-kappaB p65,and peaked in one hour,then saw a steady decline in phosphorylation over the next six to twelve hours,TE cytokine assay showed that the phosphorylation levels of proteins ERK,JNK,P38,Jun,and NF-kappaBp65.The immunofluorescence showed that the main proteins of the TEmediated MAPK signaling pathway,Jun and NF-kappaB p65,all bind to the skin HaCaT cells,and the protein Jun binds to skin cells in a statistically significant manner.Jun had a stronger binding ability to skin cells.4.Successful sequence screening of PLA2 and MMP proteins in Stomolophus nomurai TE:proteomic and transcriptomic sequencing results screened the main toxin proteins and genes,and PLA2 and MMP protein sequences were screened according to toxin expression,combined with the structural analysis of PLA2 and MMP showed that the PLA2 screened by histology compared with the simulated sequences had more obvious sequence homology;MMP sequences compared with the template sequences MMP sequence has certain structural homology with the template sequence.5.Recombinant expression of PLA2 and MMP in vitro:The effective in vitro expression and purification of PLA2 and MMP proteins were demonstrated by protein gel electrophoresis and protein blotting;the assessment of proteotoxicity revealed that the concentrations of PLA2 and MMP did not result in discernible toxic effects.Additionally,it was discovered that PLA2 and MMP activities exhibited protein dose-dependent features,with PLA2’s activity being noticeably higher than MMP’s.6.PLA2 and MMP bind to HaCaT cell membrane:Cellular immunofluorescence results showed that the proteins PLA2 and MMP mainly acted on the membrane of skin HaCaT cells,and the binding force of PLA2 to the cell membrane was stronger than that of MMP;the measurement of reactive oxygen species(ROS)indicated that In addition to having the ability to cause oxidative damage between cells,PLA2 and MMP also had higher average fluorescence intensities.7.PLA and MMP are major active proteases in the molecular mechanism of jellyfish dermatitis:Transcriptome sequencing analysis showed that recombinant protein PLA2 stimulated skin HaCaT cells to enrich the concentration of pathways including MAPKsignaling pathway,PI3K-Akt signaling pathway,JAK-STAT signaling pathway,ECM-receptor interaction,AGE-RRS signaling pathway,ECM-receptor interaction,and ECM-receptor interaction,and the average fluorescence intensity of PLA2 was higher than MMP.interaction,AGE-RAGE signaling pathway;protein blotting showed that recombinant protein PLA2 and MMP-mediated MAPK signaling pathway increased the phosphorylation level of the main proteins ERK,JNK,P38,Jun,and NF-kappaB p65,and the results were statistically different.8.PLA2 and MMP inhibitors antagonize TE-mediated jellyfish dermatitis:The PLA2 and MMP inhibitors DPD and EDTA antagonized Stomolophus nomurai TE-mediated paw swelling and inhibited toxin-induced acute inflammatory response in the paw of mice;the antagonistic effect of DPD and EDTA on skin HaCaT cytotoxicity showed that both DPD and EDTA increased cell survival;RT-qPCR showed that DPD and EDTA reduced the expression level of TE-mediated inflammatory factors in TE.RT-qPCR showed that DPD and EDTA may lower the degree of inflammatory factor expression in TE-mediated cells;protein blotting showed that DPD and EDTA had a significant antagonistic effect level of phosphorylated proteins of the TE-mediated MAPK signaling pathway.Conclusion:1.By upregulating the generation of reactive oxygen species(ROS)and cellular inflammatory components,TE can cause skin inflammation and oxidative stress damage.It also has evident skin toxicity at the animal and cellular levels.2.According to combined transcriptomic sequencing,TE can activate the nuclear transcription factors and major proteins of the MAPK and NF-kappaB signaling pathways.This suggests that the primary molecular mechanism underlying the pathophysiology of medusa dermatitis is the molecular interaction between pathway proteins and nuclear transcription factors.Induce oxidative stress and skin inflammation by activating nuclear transcription factor transduction and downstream inflammatory impact molecules.3.Jellyfish TE through the MAPK signaling pathway protein ERK,JNK,P38,Jun,NFkappaB p65 phosphorylation regulatory interactions,together involved in mediating the pathogenesis of jellyfish dermatitis.Its main molecular mechanism through the toxin directly stimulate the cell membrane surface receptor activation activate MAPK signaling pathway upstream protein ERK,JNK,P38,phosphorylation,and further activation of intracellular nuclear transcription factors Jun,NF-kappaB signaling,regulated by the transcription factor signaling to initiate the downstream inflammatory effect of molecules released rapidly,ultimately leading to jellyfish dermatitis inflammation and oxidative stress effects.4.Both PLA2,the MMP inhibitors DPD and EDTA all antagonized Stomolophus nomurai TE-mediated paw swelling and cutaneous HaCaT cytotoxicity in mice,and inhibited the level of phosphorylation of MAPK signaling pathway,which attenuated TEmediated inflammatory responses.This study provides new ideas for the treatment of jellyfish dermatitis. |
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