| Inflammatory pain is caused by a variety of stimulating factors,such as mast cells,macrophages,neutrophils and nerve endings to release inflammatory factors,causing local tissue inflammatory response.The current drugs for the treatment of inflammatory pain are often greatly affected by drug resistance and side effects.According to previous studies,Bufo bufo gargarizans,as an amphibian,contains a variety of active substances in its skin's secretion,which has a variety of pharmacological activities,such as anti-inflammatory,analgesic,anti-cancer and so on.However,the specific factors related to this function are rarely known,so this study was carried out.In this study,the skin glands(retroauricular glands)of Bufo bufo gargarizans were observed by transmission electron microscope((TEM))and scanning electron microscope((SEM)).The ultrastructure of the glands was obtained,and it was speculated that the secretion mode was whole plasma secretion.After that,the main functions of Chan Su polypeptide were predicted by glandular transcriptional group sequencing and secretory proteome sequencing and functional annotation,and the inflammatory pain inhibitory factor-neuropeptide B(Bg-NPB)was identified.In order to verify the reliability of this function prediction,the anticancer activity identified in Chan Su was tested in this study.The results showed that the water extract of Chan Su polypeptide could effectively inhibit the proliferation of cancer cells.At the same time,the effect of Chan Su polypeptide on angiogenesis was detected.It was found that Chan Su polypeptide inhibited the proliferation of human umbilical vein endothelial cell(HUVEC)in a dose-dependent manner,and played an anti-angiogenic effect by inhibiting VEGF165-VEGFR2-RAS signal pathway,which further achieved the anticancer effect.Therefore,the above results verify the reliability of the functional prediction and identification of Chan Su polypeptide including analgesic factor-neuropeptide B(Bg-NPB).Furthermore,the inflammatory pain regulation activity of the factor was studied: using the method of recombinant expression in vitro,the recombinant plasmid(Bg-NPB-p ET-32a)was constructed,and the prokaryotic expression was induced by IPTG,and the best induction conditions were selected(0.8 m M IPTG induction for 2 h),to prepare its active polypeptide.The inflammatory pain regulation activity of Bg-NPB peptide was studied by constructing inflammatory pain mouse model and pain ion channel cell line.The results of mouse writhing test and rat foot licking test showed that Bg-NPB peptide had anti-inflammatory pain activity in a concentration-dependent manner.The experimental results of related pain ion channels show that the percentage blocking rate of Bg-NPB peptides to most ion channels is less than 10%.It is speculated that the inhibition of pain signals by the peptide may not play a role through these ion channels.Finally,the inflammatory pain regulation mechanism of this factor was studied,and the recombinant plasmid(Bg-NPB-p GBKT7)was constructed by yeast two-hybrid technique.The new receptor protein of Bg-NPB was obtained from the peripheral nerve of mice,and the changes of the expression of related pain proteins in the L4-L6 segment of spinal cord of rats after corresponding treatment were measured to study the changes of the expression of related pain proteins in the signal pathway.The molecular mechanism of anti-inflammatory and analgesic effects of Bg-NPB was summarized from the above two aspects.This study finally contributes to the development of new analgesics derived from natural drugs. |
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