| Objectives:Amphibians,as a transitional group from aquatic fish to terrestrial vertebrates,play an important role in biological evolution.In order to adapt to the environment,species evolved in a wide variety of directions,venomous animal was one of them.There are the distributions of venomous animals in both aquatic and terrestrial vertebrates,but not amphibians.Amphibians were generally considered as poisonous animals rather than venomous animals due to the absence of venom delivery pipeline mechanism,which seems to be an evolutionary loss phenomenon.In addition,amphibians have been extensively concerned and studied because of their abundant glandular secretory system in the skin.However,most of these studies focus on the exploitation and exploration of natural bioactive peptide resources in their skin secretions.The biological roles of skin secretions in the process of amphibian adaptation to the environment remained systematic studies and elaborations.The purpose of this study are to to explore whether Odorana andersonii can be redefined as a venomous animal by systematically studying the biological functions of skin secretions and maxillary teeth of O.andersonii,and to systematically explore and explain the biological role of skin secretions of O.andersonii in its environmental adaptation,and to further isolate,purify and study the functions of wound-healing promoting peptides in the secretions.Methods:Adults of O.andersonii were collected in the field,and enough skin secretions were collected by electrical stimulation.The skin structure and gland distribution of O.andersonii were analyzed by scanning electron microscopy(SEM)and paraffin section staining.At the same time,the maxillary teeth of O.andersonii were analyzed by paraffin section staining.In addition,the composition of the secretion was analyzed by SDS-PAGE and RP-HPLC,and then the alkaline protease(AKP)activity of the skin secretions was detected.Then,the biological effects of skin secretion in the survival activities of O.andersonii were analyzed by toxicity test,anti-predatory ability test,antioxidant activity test,bacteriostasis test,and overall animal level restorative activity test.After confirming that the skin secretion of O andersonii could promote the healing of skin wounds,the secretion were separated and purified by a RP-HPLC.Cell scratch test was used to track the ability of wound-healing promoting of the peaks,and the primary structure of the active peptide was determined.At the same time,the skin cDNA library was constructed to commercialize the peptide sequence.After synthesis of this sample,the acute toxicity test,hemolytic test,free radical scavenging activity test in vitro,antimicrobial test and stability analysis were carried out.In addition,wound-healing promoting ability of the samples was tested by cell scratch test,cell proliferation test in vitro and full-thickness skin wound repairing test in mice at animal level.As well,the skin samples of animal model were stained with H&E to observe and analyze the formation of neoepidermis and granulation in the process of skin wound-healing.Results:(1)There are granular glands and mucous glands in dermal spongy layer of O.andersonii skin,and the distributions of glands in different skin sections are different:the larger granular glands are majorly located in the head skin and back skin;(2)There are two kinds of gland openings on the skin surface of O.andersonii:type-1 openings have valvular structure,and type-2 openings are irregular circular;(3)There are a large number of proteins and peptides in skin secretions,and the proteins showed AKP activity,and the skin secretion showed lethal toxicity;(4)The maxillary teeth of O.andersonii are directly connected with the premaxilla and maxilla,and can be divided into type-a and type-b,type-a shaped in hollow cone,and type-b shaped in parenchymal bony spurs;(5)The skin secretion has cytotoxicity,hemolytic activity,and can cause acute pain and edma responses after subcutaneous injection;(6)Skin secretion has anti-predatory effect;(6)Skin secretion has antioxidant capacity,antimicrobial activity and wound-healing promoting activity;(7)A novel and highly active natural wound-healing promoting peptide was successfully identified from the skin secretion of 0.andersonii:OA-GL21,with the primary structure of"GLLSGHYGRVSTQSGHYGRG",has a relative molecular weight of 2188.0 Da;(8)OA-GL21 showed no acute toxicity to mice,no hemolytic activity,and no antimicrobial activity,but weak free radical scavenging activity in vitro,as well with certain stability;(9)OA-GL21 promoted the scar healing of cutaneous keratinocytes and fibroblasts,but had no significant effect on cell proliferation;(10)OA-GL21 promoted skin wound healing on mice,and showed the potential to inhibit the hyper-proliferation and reduce the scar formation.Conclusions:(1)The odorous frog O.anersonii could be regarded as a venomous animal,its skin secretion could be redefined as animal venom and maxillary teeth could be used as a venom delivery tool.It is inferred that other poisonous frogs with similar structures such as maxillary teeth could also be regarded as venomous animals.(2)Skin secretions of O.anersonii could be used as a chemical weapon to help O.anersonii effectively predation and defense,which participated in several survival behaviors,including predatory&anti-predatory behaviors,preventing from the disoperation of other species,protecting skin from oxidative damage,preventing the microorganisms invasion and promoting the skin wound healing.Skin secretion is an effective survival strategy of O.anersonii.(3)The skin secretions of O.anersonii contained some wound-healing promoting peptides,which could provide a large number of molecular templates for the development of new wound-healing promoting drugs. |
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