| Neoneurogenesis has become an emerging hallmark of cancer.Studies have shown that the activated sympathetic nerve through specifically gene technology can promote the progress of breast cancer.while the activated parasympathetic nerve can inhibit the growth of breast cancer.The molecular mechanism of neoneurogenesis in regulating tumor has become a focus in the tumor research.It may provide an important target for anti-tumor therapy by blocking neurogenesis.However,the research on inhibitors of nerve growth factors and related receptors is in the initial stage.Peripheral needling is a traditional Chinese acupuncture method,which can regulate qi and activate blood circulation,remove blood-stasis and dredge collaterals through connecting meridians,channels,superficial vessels,and skin in the local parts.It is a technique of acupuncture treatment along the edge of the tumor.Previous studies have shown that the expression of p75-neurotrophin receptor(p75NTR)in tumor tissues increased significantly after peritumoral electroacupuncture(EA),which was consistent with the change trend of tumor inhibition rate.Meanwhile,it was involved in regulating the expression of semaphoring-3A(Sema3A)in tumor tissues,suggesting that peripheral needling may affect tumor nerves.However,whether peripheral needling can regulate nerve and the specific mechanism remain to be further studiedThe first part is literature review in this study,mainly including two aspects:On the one hand,this research introduced the clinical application and curative effect of peripheral needling in tumor from the perspective of clinical practice of traditional Chinese medicine;On the other hand,the research progress of neurogenesis and p75NTR in breast cancer was summarized from the perspective of modern science.The second part is the experimental study in this studyObjective:1 To compare the effects of peritumoral EA on tumor growth and apoptosis in breast cancer xenografts;2 To observe the effects of peritumoral EA on tumor nerves,neurotransmitters,neurotrophins(NTs)and related receptors.3 To explore the candidate targets and related molecular mechanism of peritumoral EA in regulating tumor neurogenesis through high-throughput detection.4 Taking p75NTR as a breakthrough point,to observe the efficacy of p75NTR on regulating nerve,cell apoptosis and tumor growth by peritumoral EA.Methods:In this study,the 4T1 breast cancer model was established by using animal experimental method.Experiment 1 was divided into TG group,ETG group,and ATG group.The mice in the TG group are grasped for 3 minutes a day,the mice in the ETG group were treated with peritumoral EA for 3 minutes per day,and the mice in the ATG group were treated with peritumoral acupuncture for 3 minutes per day.Samples were taken at 1 week,2 weeks,and 3 weeks after the intervention.Tumor growth curve was drawn,tumor inhibition rate was calculated,and tumor cell apoptosis was detected by TUNEL.The effects of peritumoral EA and peritumoral acupuncture on tumor growth and cancer cell apoptosis were compared.Experiment 2 was divided into TG group,ETG group,NG group,and ENG group.The intervention of TG group and ETG group was the same as experiment 1.The NG group and ENG group were normal mice.The mice in the NG group were grasped for 3 minutes a day.The mice in the ENG group were treated with peripheral EA in which the sites and methods were the same as those in the ETG group.HE,IHC,and ELISA were tested to clarify the effects of peritumoral EA on tumor nerves,neurotransmitters,NTs and receptors.The samples of experiment 3 were frozen tumor tissues from Experiment 2 after 1 week of intervention.There were 3 samples in the TG group and ETG group,respectively.A total of 6 samples are used for parametric transcription sequencing and bioinformatics analysis to explore the effects of peritumoral EA on tumor nerves.Based on the previous study on the up-regulation of p75NTR on peritumoral EA,the experiment 4 were divided into TG group,ETG group,ETTG group.The intervention of the TG group and ETG group was the same as experiment 1.The peritumoral EA of the ETTG group was the same as that of the ETG group.Meanwhile,the p75NTR receptor inhibitor TAT-Pep5(75μg/kg·d)was intraperitoneally injected at 15min after peritumoral EA in the ETTG group to determine the effect of inhibiting p75NTR signal transduction on tumor nerves,tumor cell apoptosis and tumor growth.Results:1 Tumor growth inhibition in 4T1 breast cancer mice by peritumoral EA and peritumoral acupuncture(1)The tumor growth curve showed that the ETG group had the slowest tumor growth.the TG group had the fastest tumor growth,and the ATG group was between ETG group and TG group.On the 15th day of the experimental intervention,the tumor growth curves in the three groups began to converge.The comparison between the groups showed that the tumor volume in the ETG group was significantly smaller than that in the TG group at 5-18 days of the intervention(p<0.05),and the tumor volume in the ATG group was significantly smaller than that of TG group at 6-14 days(p<0.05).(2)Comparison of tumor weight at three sampling points showed that the ETG group had the smallest tumor weight,the TG group had the largest tumor weight,and the ATG group was between the ETG group and the TG group.(3)At three sampling points,the tumor inhibition rates were 36.93%,46.49%and 27.89%in the ETG group,respectively,and were 30.79%,26.61%and 21.67%in the ATG group.(4)TUNEL staining showed that the apoptosis index in the ETG group was significantly higher than that of the ATG group and TG group(p<0.05)and was the highest after 2 weeks of experimental intervention There was similar apoptosis index between ATG group and TG group2 The effect of peritumoral EA on the nerve in 4T1 breast cancer mice(1)The results of HE staining and IHC labeled by PGP 9.5 showed that there were innervations in cancer tissues and adjacent tissues in the 4T1 breast cancer.(2)The results of IHC showed that the β 3-tubulin positive nerve fiber in the ETG group were less than that in the TG group at 1 week and 2 weeks of intervention,while there was no difference between the two groups at 3 weeks of intervention.With regard to nerve filament,it was more in the ETG group than that in the TG group at three sampling time points.The positive expression area ofβ 3-tubulin in the ETG group was significantly less than that in the TG group at three sampling time points(p<0.05).(3)IHC labeled TH to determine the effect of peritumoral EA on the sympathetic nerve.The results showed that TH positive nerve were in the form of filaments,distributed in tumor stroma and mostly around blood vessels.The number of TH positive nerve filaments in the ETG group was critically less than that in the TG group at three sampling points.(4)Serum neurotransmitters:The NE,ACH and AD content in the ENG group was transitorily higher than that in the NG group,in which the difference of AD content at 3 weeks of intervention showed statistically significant.Meanwhile,the NE,ACH and SP content in the ETG group was transitorily higher than that in the TG group,in which the difference of NE content at 3 weeks of intervention showed statistically significant.(5)Tumor tissue neurotransmitters:NE content in the ETG group was lower than that in the TG group at three sampling points,and the difference was statistically significant at 2 and 3 weeks after intervention.ACh level in the ETG group was also lower than that in the TG group,and the difference was statistically significant at 2 weeks of intervention.(6)Serum NTs:The contents of NGF,BDNF,NT-3 and NT-4 showed no difference among the three sampling points.(7)Tumor tissue NTs:NGF content in the ETG group was higher than that in the the TG group at 1 week and 2 weeks after intervention(p<0.05).The level of NT-3 in the ETG group was significantly higher than that in the TG group at 3 weeks after intervention(p<0.05).Both BDNF and NT-4 had no difference in the ETG and TG groups,respectively.Besides,the relative expression of pro-NGF in the ETG group was higher than that in the TG group at three sampling time points.(8)Nerve growth factor receptors in the tumor tissues:The relative expression of p75NTR in the ETG group was significantly higher than that in the TG group at three sampling points(p<0.05),while there was no difference in the relative expression of TrkA and TrkB between ETG and TG group.3 Transcriptome sequencing to explore the pathway of peritumoral EA.Compared with the TG group,the ETG group obtained 116 differential expressed genes,including 66 up-regulated genes and 50 down-regulated genes.Go functional annotation analysis showed that the differential expressed genes related to peritumoral EA were mainly relevant to several biological processes,such as immune response,cell death and homeostasis,oxidative stress,substance and energy metabolism.KEGG pathway enrichment analysis showed that the differential expressed genes related to peritumoral EA were mainly relevant to nerve,immune response,substance and energy metabolism.With regard to biological process of nerve,there are four neurotransmitter excitatory synaptic pathways and one axon guidance pathway,involving 11 differential genes.Among these genes,5-HT2,VGCC,COX,TRPC1 and Ablim were down-regulated,while Gi/o,PLA2,GLS,CREB,Boc,and Netin-G2 were up-regulated.These genes involve in exocytosis,calcium influx,neuronal excitability,neuroprotection,axon guidance,feedback inhibition regulation of neurotransmitter release,and synaptic plasticity.4 The mechanism of peritumoral EA improving cancer cell apoptosis by p75NTR regulating tumor nerveTAT-Pep5 was injected intraperitoneally to inhibit p75NTR signal transduction,and the effect of electroacupuncture on apoptosis of tumor nerve and cancer cells was observed.(1)β3-tubulin positive nerve:There was a decrease in the number of(33-tubulin positive total nerves and nerve filaments in the ETTG group compared with those in the ETG group(p>0.05).While the number of β 3-tubulin positive nerve fiber in the ETTG group were increased compared with those in the ETG group(p>0.05).The area of β 3-tubulin positive expression in the ETTG group was higher than that in the ETG group at 1 week and 2 weeks after intervention.(2)TH positive nerve:TH positive nerve filaments in the ETTG group were significantly more than those in ETG group at three sampling time points(p<0.05).While,the TH positive nerve filaments in the ETTG group were equivalent to these in the TG group at 1 and 2 weeks after intervention,and were significantly lower than those in the TG group at 3 weeks after intervention(p<0.05).(3)Neurotransmitters(NE and ACH)in tumor tissues:NE content in the ETTG group was between the TG group and the ETG group.ACH content in the ETTG group was higher than that of the ETG group at the three sampling time points,and the difference of the experimental intervention for 2 weeks was statistically significant.While ACH content in the ETTG group was not compared with that in the TG group.(4)NGF and proNGF in tumor tissues:NGF content in ETTG group was significantly higher than that in TG group at three sampling points(p<0.05).It was equivalent to that in the ETG group at 1 week,significantly lower than that in the ETG group at 2 weeks,and higher than that in the ETG group at 3 weeks.The expression of proNGF in ETTG group was equivalent to that in ETG group at three sampling points,while significantly higher than that in TG group(p<0.05).(5)Tumor growth:In the ETTG group both tumor volume and weight were between ETG group and TG group.The tumor inhibition rate in ETG group was significantly lower than that in ETG group at three sampling points.(6)Apoptosis:The apoptosis index in ETTG group was significantly lower than those in ETG group at three sampling points(p<0.05),while was comparable to that observed in the TG group.Conclusions:1.Both peritumoral EA and peritumoral acupuncture can inhibit tumor growth,respectively.The anti-tumor effect of peritumoral EA shows stronger and more lasting effect than that of peritumoral acupuncture.One of anti-tumor mechanisms on the peritumoral EA is involved in the increase of cancer cell apoptosis.2.Peritumoral EA can regulate tumor nerve,promote neurogenesis,inhibite nerve infiltration and sympathetic nerve formation,regulate tumor tissue neurotransmitters(ACh,AD and NE)and NTs(NGF,proNGF and NT-3),and increases the expression of p75NTR,which may be the potential molecular mechanism of peritumoral EA on tumor inhibition.3.Peritumoral EA may inhibit the release of neurotransmitters in the tumor microenvironment by weakening exocytosis,inhibiting calcium influx,regulating nerve excitability,and promoting neuroprotection and axonogenesis.4.Peritumoral EA induced cell apoptosis by upregulating the expression of p75NTR to directly inhibit tumor growth.Meanwhlie,it regulated tumor microenvironment by inhibiting nerve infiltration and sympathetic innervation and down-regulating the content of NE and ACh in tumor tissue to play indirect effect in inhibiting tumor growth. |
