Active Peptide OM-LV20 Promotes The Repair Of Cerebral Ischemia Injury Mediated By Tryptophan Hydroxylase-1 And Its Mechanism

Objective:Stroke leads to serious nerve injury and severe social and economic burden.Because of the high complexity of repair mechanisms after injury and the extremely scarce drugs approved for use,it is of great significance to discover new anti-apoplexy drugs,new mechanism analysis,and new drug target discovery.Peptide molecules have become a powerful tool for the discovery and confirmation of new drug targets and the important source of leading compounds in new drug creation,which plays a unique and irreplaceable role in basic scientific research,new biological industry,and innovative drug research and development.Tryptophan hydroxylase 1(TPH1)is involved in many related diseases,such as mental and nervous diseases,but its effect on stroke has not been reported.The aim of this study is to clarify the neuroprotective effect and mechanism of exogenous OM-LV20 on rat cerebral ischemia-reperfusion model and the injury of astrocytes under oxidative stress.The possibility of endogenous TPH1 as a target of anti-apoplexy drugs was explored with OM-LV20 as a molecular probe.It provides a molecular biological basis for the treatment and prevention of the following neurological diseases,especially cerebral ischemia-reperfusion.Methods:1)The half life of active peptide OM-LV20 at 4?,37? and in plasma were detected by the HPLC system.2)Sprague Dawley rats were used as the research object,after 7 days of continuous intraperitoneal injection(OM-LV20),the model of cerebral ischemia/reperfusion(I/R)was established,i.e.,middle cerebral artery embolization for 2 h/reperfusion for 24 h.The neuroprotective effect of OM-LV20 on cerebral I/R was evaluated by using mNSS behavioral score,TTC staining,and quantification of cerebral infarction area;the protective effect of OM-LV20 on morphology and survival status of brain cells and neurons was evaluated by H&E staining and Nissl staining.3)The mechanism of OM-LV20 on I/R rats(In vivo):the neuroprotective mechanism of OM-LV20 to I/R rats was studied by a molecular docking simulation experiment,high-throughput glucagon-like peptide-1 receptor(GLP-1R),and transcriptional sequencing analysis to explore the neuroprotective mechanism of OM-LV20 to I/R rats;western blotting,real-time quantitative PCR Time quantitative poly chain reaction(RT-qPCR)and cyclic adenosine monophosphate(cAMP)were used to study whether OM-LV20 can directly activate GLP-1R,activate mitogen-activated protein kinase(MAPKs)signal pathway,BDNF/AKT signal pathway and regulate the levels of pituitary adenylate cyclase activated peptide receptor type 1(PAC1R),cAMP and tryptophan hydroxylase 1(TPH1)for protecting the nerve damage of I/R rats.4)The expression of TPH1 on rat astrocytes:the expression of TPH1 in rat brain tissue was studied by the primary culture of rat hippocampal astrocytes,frozen sections of rat brain tissue,immunofluorescence three-standard,RT-qPCR,sequence comparison,and western blotting.5)The neuroprotective effect of OM-LV20 on rat astrocytes under oxidative stress(In vitro):CTX-TNA2 cell line was used as the research object,and hydrogen peroxide(H₂O₂)(300 ?M)was used at different time points(2 h,4 h,6 h,12 h,and 24 h),to explore whether the neuroprotective mechanism of the active peptide on CTX-TNA2 cells stimulated by H₂O₂ is through the "PAC1R-MAPKs-TPH1" axis by western blotting and RT-qPCR.6)Neuroprotective effects of TPH1:CTX-TNA2 cells were transfected with lentivirus to construct and screen cell lines stably overexpressing and interfering with TPH1;oxidative stress model was established by stimulating with H₂O₂ for 2 h;the changes of cell viability,and CAT and LDH levels were detected to explore the neuroprotective effects of TPH1.Results:(1)The stability of OM-LV20 was pretty goodat 4? and 37?.The half-life of the peptide in plasma was 2.834 h.(2)OM-LV20 effectively reversed the nerve function damage and reduced the formation of cerebral infarction area caused by I/R and improved the abnormal neurobehavioral of rats.OM-LV20(20 ng/kg)effectively improved the survival rate of neurons in the hippocampus(p<0.05)and cortex(p<0.0001)of rats,and improved the chances of cell and tissue state caused by I/R,such as the deepening of cell cytoplasm staining,cell shrinkage,increase of cell gap and vacuoles formation and structural disorder of CA1 area in the hippocampus.(3)Molecular docking simulation experiment suggested that OM-LV20 and GLP-1R may bind through Ile-19,Asp-12,Lys-7,Lys-4 and Leu-1;GLP-1R activation experiment suggested that OM-LV20 was not a direct agonist of GLP-1R.(4)The results of transcriptome sequencing showed that there were 3089 significantly different genes between the OM-LV20 and I/R groups.KEGG indicates that these differential genes are mainly enriched in the MAPKs signaling pathway,cAMP signaling pathway,and endoplasmic reticulum processing pathway.After validated by experiments,the neuroprotective mechanism of OM-LV20 on cerebral I/R injury in rats may be related to inhibition of phosphorylation of MAPK signaling pathway,activation of BDNF/AKT signaling pathway,promotion of TPH1 and PAC1R levels,and regulation of the cAMP level.(5)Distribution of TPH1 in rat brain:TPH1 was significantly distributed in the hippocampus,thalamus,and raphe,especially in the CA1 area of the hippocampus.TPH1 was expressed in CA1 astrocytes in hippocampus of rat and DI-TNC1 and CTX-TNA2 cell lines.(6)The levels of TPH1 at different times with H₂O₂ treated were changed:the mRNA and protein levels of TPH1 decreased first(at 2 h)and then increased(reaching the peak at 12 h).(7)OM-LV20 increased the mRNA and protein levels of TPH1 in CTX-TNA2 cells with concentration dependence(10 pM,100 pM,1nM).(8)The neuroprotective effects of OM-LV20 on CTX-TNA2 cells in rats stimulated by H₂O₂:OM-LV20 can improve cell damage under oxidative stress by improving cell viability(p<0.01),inhibiting phosphorylation of MAPK signaling pathway(p<0.05)and increasing PAC1R(p<0.001),TPH1(p<0.05)and CAT(p<0.05)levels,and reducing the LDH(p<0.0001)level.(9)OM-LV20 increased the TPH1 level by inhibiting JNK phosphorylation:OM-LV20 decreased JNK phosphorylation and increased TPH1(p<0.05).After OM-LV20 and Anisomycin were used at the same time,TPH1 level decreased(p<0.01);used SP600125 alone increased TPH1 level(p<0.05).These results indicate that OM-LV20 regulates the TPH1 level by regulating the phosphorylation of the JNK signaling pathway,and there is a negative correlation between the p-JNK level and TPH1 levels.(10)OM-LV20 may regulate the level of TPH1 by partly combining PAC1R:The docking mode experiment indicated that OM-LV20 had the potential to bind PAC1R.The application of OM-LV20 increased the level of TPH1 in CTX-TNA2 cells(p<0.01),while PACAP6-38 decreased the level of TPH1(p<0.05).(11)Overexpression of TPH1 had no protective effect on the activity damage of CTX-TNA2 cells stimulated by H₂O₂.The overexpression of TPH1 had an antioxidative stress effect on CTX-TNA2 cells stimulated by H₂O₂:Compared with the H₂O₂ group,overexpression of TPH1 could reverse the decrease of CAT content(25.01%±12.62%)and the abnormal increase of LDH level(313.53%±65.39%),Conclusion:The vivo results suggest that intraperitoneal injection of active peptide OM-LV20 can significantly reduce the infarct volume,improve the neurobehavioral abnormalities,and protect the survival of neurons in the cortex and hippocampus of the infarct area caused by I/R.The potential molecular mechanism mediated by OM-LV20 involves the activation of MAPKs and BDNF/Akt signaling pathway,and the changes of TPH1,cAMP,and PAC1R levels.The results of in vitro experiments suggest that the neuroprotective mechanism of OM--LV20 on rat astrocytes under oxidative stress may be mediated by "PAC1R-MAPKs-TPH1"molecular axis.In addition,this study first reported the expression of TPH1 in rat astrocytes,explored the expression of TPH1 in CTX-TNA2 cells stimulated by H₂O₂ at different time points,and found that TPH1 may protect astrocytes against oxidative stress by reducing the release of LDH and increasing the level of CAT.The neuroprotective effect of amphibian skin-derived peptide OM-LV20 on I/R rats was reported for the first time.It provides a candidate template for leading molecules of new anti-stroke drugs,a possible mechanism for reducing brain I/R injury by targeting TPH1 and antioxidant stress pathway,and a potential target for neuroprotective drug research and development of brain I/R.