| Our lab has committed to the bioactivities and mechanisms of the active substances from the jellyfish Cyanea capillata(C.capillata)for many years.We found that jellyfish tentacles exhibit a strong self-repair capacity and rapid pro-proliferation ability of sting cells,suggesting that some highly active growth factors may exsit in the tentacles.Furthermore,during the study of cell activities of the tentacle extract(TE)of C.capillata,we found an interesting phenomenon that the lower concentration of TE could induce cell proliferation.Together with relevant literature reports,we speculated that similar proproliferative components may also exist in the TE of C.capillata.In our previous studies,we have constructed a c DNA library of the tentacle from C.capillata to fully understand its biological activities.The multiple sequence alignment indicated that there are four vascular endothelial growth factor(VEGF)-like sequences in the library,providing an important theoretical principle for extracting pro-proliferative components from TE.Combined application of various separation techniques,we have successfully extracted the polypeptide Cyanea capillata granulin-1(Cc GRN-1),with a possessed growth-promoting activity.Therefore,this article would focus on the pro-proliferative ability and the possible mechanisms of TE and Cc GRN-1.Through this study,it is expected to provide new ideas to solve the problem of wound healing in the injuries caused by some specific diseases or environments.MethodsPart I: Bioactivities of TE from C.capillataFirstly,we determined the viability of HUEVCs by Cell Count Kit-8(CCK-8).Then,we detected the effects of TE on cell cycle progression of HUVECs by flow cytometry and determined whether TE would affect the expression of cell cycle-related proteins,including Cyclin B1 and Cyclin D1.Subsequently,using western blotting,we investigated the effects of TE on proliferation-related signaling pathways,including PI3K/Akt,ERK1/2 MAPK,JNK MAPK,p38 MAPK and NF-κB pathways,as well as downstream proteins,including caspase-3,8,9 and Cyto C.Simultaneously,immunofluorescence technique was selected to verify the cascades of signaling pathways.Afterwards,we selected the TE-activated signal pathway inhibitors to determine their effects on cyclins.Finally,the wound healing assay was employed to detect wether TE could induced cell migration in HUVECs.Part II: Bioactivities of Cc GRN-1 from C.capillataFirstly,we investigated the viability of HUEVCs by CCK-8.Next,we detected the effect of Cc GRN-1 on cell cycle progression of HUVECs by flow cytometry and determined the effects of Cc GRN-1 on the expression of cell cycle-related proteins,including Cyclin B1 and Cyclin D1.Subsequently,using western blotting,we investigated the effects of Cc GRN-1 on proliferation-related signaling pathways,including PI3K/Akt,ERK1/2 MAPK,JNK MAPK and NF-κB pathways.Simultaneously,immunofluorescence technique was selected to verify the cascades of signaling pathways.Afterwards,we selected the signal pathway inhibitors to determine their effects on cyclins.Finally,the wound healing assay was employed to detect whether Cc GRN-1 could induced cell migration in HUVECs.Meantime,using the same methods,Cc GRN-1 was compared with commercial product-VEGF to explore its possible bioactivities and mechanisms.ResultsPart I: Bioactivities of TE from C.capillataThe results by CCK-8 showed that the higher doses of TE exerted a dose-dependent cytostatic effect,while the lower doses of TE significantly enhanced in the viability of HUVECs.In addition,the results by flow cytometry showed that TE could promote cell transition from the G1 phase to the active phase of cell division(S and G2 phases)to accelerate cell cycle progression.Furthermore,TE could also increase the expression of cell cycle-related proteins.In signaling pathways,TE could activate PI3K/Akt,ERK1/2 MAPK and JNK MAPK pathways,but not affect the phosphorylations of p38 MAPK and NF-κB pathways.In the downstream of signaling pathways,TE had no effect on the expression of caspase-3,8,9 and Cyto C proteins,either.Immunofluorescence results vertified that TE could activate PI3K/Akt,ERK1/2 MAPK and JNK MAPK pathways.On the other hand,the results of signaling pathway intervention showed that PD98058 blocked the expression of cyclins,while LY294002 and SP600125 were ineffective,which indicated that TE mainly upregulate the expression of cyclins through ERK1/2 MAPK pathway.Finally,the results of wound healing assay showed that TE could induce the migration of HUVECs.Part II: Bioactivities of Cc GRN-1 from C.capillataThe results by CCK-8 showed that both Cc GRN-1 and VEGF enhanced the HUVECs’ viability in the time-and dose-dependent manners.In addition,the results by flow cytometry showed that both Cc GRN-1 and VEGF could promote cell transition from the G1 phase to S and G2 phases to accelerate cell cycle progression.Furthermore,both Cc GRN-1 and VEGF can also increase the expression of cell cycle-related proteins.As for signaling pathways,both Cc GRN-1 and VEGF could activate PI3K/Akt and ERK 1/2 MAPK pathways,but they had no effect on JNK MAPK and NF-κB pathways.And the immunofluorescence results vertified that both Cc GRN-1 and VEGF could activate PI3K/Akt and ERK1/2 MAPK pathways.On the other hand,the results of signaling pathway intervention showed that PD98059 blocked the expression of cyclins,while LY294002 was ineffective,indicating that Cc GRN-1 mainly upregulate the expression of cyclins through activating ERK1/2 MAPK pathway.Finally,the results of wound healing assay showed that Cc GRN-1 and VEGF could induce the migration of HUVECs.ConclusionLower concentration of TE could increase cell viability,accelerate cell cycle progression and improve the expression of Cyclin B1 and Cyclin D1,suggesting that TE could promote cell proliferation.In terms of the possible mechanism,we found that TE could phosphorylate PI3K/Akt,ERK1/2 MAPK and JNK MAPK pathways.Among them,ERK1/2 MAPK pathway may be closely correlated with the expression of cell cycle proteins.Lastly,the wound healing assay suggested that TE could induce cell migration.On the other hand,Cc GN-1 and VEGF have the similar biological activities,for example to improve cell viability,accelerate cell cycle progression and promote the expression of cyclins,suggesting Cc GRN-1 is consistent with VEGF on cells proliferation.In terms of the possible mechanism,both Cc GRN-1 and VEGF could phosphorylate PI3K/Akt and ERK1/2 MAPK pathways.Among them,ERK1/2 MAPK pathway may be closely correlated with the expression of cell cycle proteins.Lastly,the results of wound healing assay indicated that both Cc GN-1 and VEGF could promote cell migration.Based on the similar characteristics of Cc GRN-1 and VEGF in promoting cell proliferation,we believe that Cc GRN-1,as a special peptide derived from marine organisms,may have a promising prospects in solving the problem of wound healing in the injuries caused by some specific diseases or environments. |
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