Effect Of Acupotomology On L₃Transverse Process Syndrome Rat About P38MAPK/CREB Signal Pathway

Purpose：This topic will investigate the effect of acupotomology on L3transverse processsyndrome of rat model’s pain threshold and tissue morphology, the expression levels of NGFand BNDF in the organization, the changes of SP,5-HT and β-Ep in the serum; the changes ofp-p38MAPK, in p-CREB, p38MAPKmRNA, CREBmRNA, NGFmRNA, BDNFmRNA inthe lumbar spinal cord and dorsal root ganglia. Through these indicators, we can explore thecontact within pain threshold-pain substance-nerve growth factor-signaling pathways,which aims to provide the experimental evidence for the mechanism of analgesia withacupotomy.Material and method：1Animal grouping and modelingThirty-six SD rats were divided into four groups, including normal group, modelgroup, electro acupuncture(EA) group, and acupotomy group, there were9rats in each group.The third lumbar transverse process syndrome rats model were used which was established byWang Jianrui. Modeling method: under sterile conditions in the lumbar portion of SD ratsvertical incision3cm, peel erector both sides, reveal L3~L4spinous process, and completelyseparate the lumbar spinal nerves piercing the deep fascia parts. The0.5×0.5cm size gelatinsponge is implanted to the latter half of the third lumbar transverse process under the deepfascia, keep fascia intactly and stop bleeding, with No.0catgut sutures equidistant sacralspine fascia and skin of animals, use Gentamicin to flush cut. Twenty-seven rats weremodeled, fifty days after modeling, a rat was killed in each group, observed pathological tissue morphology and verified the success of modeling.2Intervention measuresAcupotomy group: After the successful model validation, in sections fifteenth andtwenty-first days performed the acupotomy operation under anesthesia. Procedure can bereferred to Professor Zhu Hanzhang’s “four-step” process in acupotomology textbook editoracupotomy action, fixing, directing, pressure separation and piercing. Pierce approximately1.5-2.0cm until arrive to the tip of the third lumbar transverse process, first parallel with thelongitudinal spine2knives, and then rotate the handle90°cross a knife, press the pinholebleeding, and band-aid sticking.EA group: On the fifteenth, seventeenth, nineteenth, twenty-first, twenty-third,twenty-fifth days after modeling, conduct electro-acupuncture treatment, totally six times.Bundled with limb joint Budou law fixed rat, choose rat left "shenshu" and "Yaoyangguan"two points referring to "experimental acupuncture", with mm needle into approximately0.5-1cm, then electro-acupuncture device is powered EA frequency density wave2/100Hz,the current intensity is no more than2mA, with dorsal tremble and animals do not bray as thedegree,20minutes each time.Model group and normal group were given normal bundle process, do not give othertherapeutic measures.3Pain threshold detectionUsed TF2light and heat pain measurement instrument to measure the pain threshold inrats, on the modeling days and the third, fifth, ninth, fourteenth, sixteenth, twentieth,twenty-second, twenty-seventh after modeling, totally nine times. In irradiated rats to radiantheat tail flick reflex time for the pain threshold, each rat was repeated measured three times toget the pain threshold, at intervals of five minutes or more, averaged for statistical analysis.4HE stainingTwenty-eight days after modeling, the remaining rats in each group were sacrificed.Visually observed the third lumbar transverse process syndrome rat model of local tissuechanges, took partial skeletal muscle tissue (approximately2cm×2cm size) into neutralformalin solution fixed for a week, HE staining,then observed the morphological changes.5To detect the content of SP,5-HT, β Ep in serum On the28th day after modeling, post-anesthesia took the blood through heart about3ml,puted it in pink vascular coagulation (PT tube) to stand for one hour, in the low temperaturecentrifuge (4℃,3500rev/min) for15min, then separated the serum, packed-20℃preservation, numbered censorship. Detected SP,5-serotonin, β-Ep content in serum byELISA, the steps were carried out in accordance with the kit instructions.6NGF, BDNF were detected by immunohistochemistryOn the28th days after modeling, the rats were killed by heart exsanguination, took thethird lumbar transverse sections of skeletal muscle tissue, neutral formalin solution andfixed,then NGFand BDNF were detected by immunohistochemistry, obtained the averageoptical density values to be statistically analyzed.7Using RT-PCR to dectect the expression of NGFmRNA, BDNFmRNA in the lumbar spinalcord and dorsal root gangliaOn the28th day after modeling, the rats were sacrificed by the heart exsanguination,thenlumbar spinal cord and dorsal root ganglia were quickly took, foil wrap,-70℃preservation.To detect the expression of NGFmRNA and BDNFmRNA in the spinal cord and the dorsalroot ganglia of the model rats through RT-PCR method, use2-△△ctmethod for amplification ofsamples analyzed.8Using RT-PCR to dectect the expression of p38MAPKmRNA and CREBmRNA in thelumbar spinal cord and dorsal root gangliaOn the28th day after modeling, the rats were sacrificed by the heart exsanguination andlumbar spinal cord and dorsal root ganglia were quickly took, foil wrap,-70℃preservation.To detecte the expression of p38MAPKmRNA, CREBmRNA in spinal cord and dorsal rootganglia of the model rats, use RT-PCR method, use2-△△ctmethod for amplification ofsamples analyzed.9Using Western Blot to dectect the expression of p-p38MAPK and p-CREB in the lumbarspinal cord and dorsal root gangliaTo detecte the L3transverse process syndrome model rats expression of p-p38MAPK andp-CREB in spinal cord and dorsal root ganglia by Western Blot method, obtain P-P38MAPK,P-CREB and action grayscale ratio for statistical analysis. Results：1The tissue repair of the third lumbar transverse process syndrome ratsTwenty-eight days after modeling showed that there was no significant fibroblastproliferation about the structure of the normal group; at the third lumbar transverse surfaceprojection of the model group could see local capillary congestion, darker connective tissue,fibroblasts, fiber fracture, scattered red blood cell distribution with capillary proliferation;electro-acupuncture group and acupotomy group were saw relatively bright colorssurrounding the tissue, compared with the model group significantly reduced inflammation,visible fibrous tissue neatly arranged in rows, occasionally inflammatory cells, acupotome setlower than other groups to form collagen fibers, scar area is small, a higher degree of tissuerepair.2Effect of acupotomy on the third lumbar transverse process syndrome model rats about painthresholdBy watching the pain threshold changes of rats,discovered acupotomy and EAinterventions can increase the pain threshold on the third lumbar transverse process syndromemodel rats.3Effect of Acupotomy on the third lumbar transverse process syndrome model rats about thecontent of SP,5-HT, β-Ep in serumOn the28th day after modeling, detected the content of SP,5-HT, β-Ep in serum byELISA from the third lumbar transverse process syndrome model rats, there was a significantdifference (P <0.01) between acupotomy group and model group, there was a significantdifference (P <0.01) between EA group and model group, the content of SP,5-HT in serumfrom acupotomy group and EA group,the levels tended to decrease compared with the modelgroup, β-Ep content showed an increasing trend.4The expression of NGF and BDNF in the skeletal muscle about acupotomy on the thirdlumbar transverse process syndrome model ratsOn the28th day after modeling, the left side of the third lumbar transverse processsyndrome model rats of skeletal muscle around the third lumbar transverse process, there wasa significant difference (P <0.01) in the mean optical density of NGF and BDNF between acupotomy group and model group, there was a significant difference (P <0.01) betweenelectro-acupuncture group and model group, compared with model group, acupotomy groupand EA group showed a decreasing trend.5The expression of NGFmRNA and BDNFmRNA in the spinal cord and dorsal root gangliaabout acupotomy on the third lumbar transverse process syndrome model ratsOn the28th day after modeling, the expression of NGFmRNA and BDNFmRNA in thespinal cord and dorsal root ganglia, there was a significant difference (P <0.01) between theacupotomy group and model group,and there was a significant difference between theelectro-acupuncture group and model group (P <0.01), compared with the model group, theacupotomy group and EA group showed a decreasing trend.6The expression of38MAPKmRNA and CREBmRNA in the spinal cord and dorsal rootganglia about acupotomy on the third lumbar transverse process syndrome model ratsThe expression of p38MAPKmRNA, CREBmRNA in the spinal cord and dorsal rootganglia, there were significant differences between acupotomy and model groups (P <0.01),there was a significant difference between electro-acupuncture group and model group(P<0.01). Compared with model group, acupotomy group and EA group showed a decreasingtrend.7The expression of P-P38MAPK and P-CREB in the spinal cord and dorsal root gangliaabout acupotomy on the third lumbar transverse process syndrome model ratsOn the28th day, drawn from the third lumbar transverse process syndrome,then to detectthe expression of P-P38MAPK and P-CREB in the spinal cord and dorsal root ganglia, therewas a significant difference between acupotomy and model groups (P <0.01), there was asignificant difference between electro-acupuncture group and model group(P <0.01), bothgroups showed a decreasing trend compared with the model group.Conclusion：1Acupotomology therapy may inhibit the third lumbar transverse process syndrome modelrats SP、5-HT levels and improve the content of β-EP in serum, so played a role in analgesiato increased pain threshold;2Acupotomology therapy can regulate the synthesis and release of pain of the third lumbar transverse model rats, probably by inhibiting the expression of NGF、BDNF in muscle tissueand the expression of NGFmRNA、BDNFmRNA in spinal cord and dorsal root ganglia.3Acupotomology therapy can regulate the expression of NGF、BDNF in skeletal muscletissue, expression of NGFmRNA、BDNFmRNA in the spinal cord and dorsal root ganglia，probably by inhibiting the expression of p38MAPKmRNA、CREBmRNA、P-P38MAPK、P-CREB in the pinal cord and dorsal root ganglia of the third lumbar transverse processsyndrome model rats, adjusted the levels of the SP,5-HT, β-EP, so then played the analgesiceffect.