Research Of The Mechanism Of A Small Analgesic Peptide Derived From Naja Atra Neurotoxin

Pain is one of the most frequent signals in different diseases,bring great misery to patients and causing seriously affect their daily life and work.At present,most effective clinic analgesic drugs(such as morphine,codeine,fentanyl,pethidine,and so on)for the treatment of pain are addictive,dose-dependently,toxic.It is urgent and valuable for seeking analgesic drugs with low or non-addictivity,and longer effective time.Snake venom showed some advantages.Lots of evidences have demonstrated that the snake venoms have an obvious analgesic effect,has long time of analgesic effect,however,the analgesic mechanism of snake venom is largely unknown.Previous studies thought that the analgesic effect of snake venom was caused by its toxicity,and binding activity to AchRs.However,tens of studies suggested that some snake weak neurotoxins and cardiotoxins showed stronger analgesic effects,and might be related to the central nervous system,which is quite different to the previous views.Furthermore,the analgesic effects by oral administration can not be explained by current views.Thus,we hypothesized that the activity centers of analgesic and toxic were independent,at least not totally overlapped.In this study,we first collected the LD50 and the dose of analgesic of snake venoms with analgesic activity in literature,and comparison and analyzed results suggested that they arenot positively or negatively correlated,indicating that two activities and the activity centers are independent in TFTs.Our previous results showed that the short neurotoxin C/coborotoxin c(CBT c)from Naja atra(Chinese Cobra)had relatively weak neurotoxicity,but significantly analgesic activity by oral administration.There should be a segment in CBT c.which is digested from CBT c by pepsin and resistant to pepsin for further digestion,showing analgesic effects.Hence,we analyzed that the amino acid sequence and structure of CBT c.and designed a ten peptide(KDHRGTRIER),according to the selective csite of pepsin.The analgesic activity of ten peptides was determined by hot plate method,acetic acid writhing test and formalin test.The results indicated that the peptide showed significant analgesic activity;the LD50 and the confidence limit of 95%were 8.4 g/kg and 7.3-9.7 g/kg,respectively,which indicated that the toxicity of ten peptides was very low or non-toxic.The results are also consistent to our hypothesis that the centers of the analgesic activity and the toxicity were two different domains in three finger toxins(TFTs).Referred to long chain neurotoxins with analgesic activity,and based on the 10 peptide,13,15 and 16 peptide were designed and tested with hot plate and acetic acid writhing experiments.The results showed that the analgesic activity of the 16 peptide was significantly higher than that 13 peptide and 15 peptide.and the original 10 peptide,indicating that the structure of peptide is more stable and the analgesic activity of peptide is stronger.According to the imaging results of 5-TAMRA labeled 10 peptide in the normal model,the right leg pain model and the left leg pain model,we concluded that there are differences analgesic mechanism of 10 peptide in the two kinds of administration mode of intragastric administration and intraperitoneal injection.It may be associated with the specific receptor or ion channel of gastrointestinal tract for ten peptides by intragastric administration,and may be closely related to the dorsal root ganglion by intraperitoneal administration.The fluorescence of the labeled peptide with 5-TAMRA was enriched at the pain site,and it was speculated that 10 peptide might be active analgesia.