Study On The Role Of Fascia In Signal Conduction Of Acupuncture And Its Relationship With MAPK Pathway.

Since more than half of centuries,widespread research was carried out to explore the essence of the Meridians,including the structure basis of the Meridians, the biological effect of acu-point and the mechanism of acupuncture.Then the biological effect relating to the Meridians was revealed and many kind of theories were proposed.The research of the theory on"connective tissue is related with the Meridians"is becoming hot day by day in this domain.By using the digital anatomic techonology,we constructed the virtual lines and points through the mark and dissection on the fascia converging areas of the trunk and limbs based on the digital dataset of VCH before.When it was reconstructed,the concentrated areas of fascia connective tissue show cord-like and bead-like structures,these structures are similar to that of classical meridians and collaterals. For further verification of the validity of this finding on living human body,image datas of CT and MRI of living human body are rebuild and fascia connective tissue is marked.We get same results from these CT and MRI study.So combinded with developmental biology,Yuan Lin considered that organisms can be divided into both the functional system which composed of differentiated cell as well as supporting and storing system which composed of undifferentiated cell, human body is consist of supporting and storing system which derive from the unspecialized connective tissue and functional system which composed of differentiated cell.The structure and function of the organism are maintained by the incessant supplement and refreshment from the supporting and storing system to the functional system.Meanwhile,under the regulation of nervous system and immune system,the fascia network throughout the body regulates the functional and living status of cells and provides a stable environment for survival of functional cells.Acupuncture regulate human body's functional conditions mainly through stimulating the acupoint,and the cause of"De qi"is that the needle can grasp the fiber of the connective tissue.Therefore,We observed the changes of ERK1/2 and P38 by immunohistochemical and Western-blot and the changes of cell morphology in subcutaneus fascia of SD rats after acupuncture in order to study the biological effect of acupuncture and provide the evidence for the theory of"fascia connective tissue is related with the Meridians".Research Objective1.To explore how acupuncture,as one of mechanical stress,produce the basic biological effect-tissue's morphologic changes.And as one of nociceptive stimulus, how acupunctue affect the medulla spinalis's morphology.2.To explore the mechainsm of acupuncture for organisms from the two anatomic structure and the point of view of cell signal transdunction through observation of the expression,location and translocation of ERK1/2 and P38 in the local subcutaneous fascia and medulla spinalis by immunohistochemistry staining.3.To explore specificity of acupoint from the angle of fascia connective tissue by comparision of the expression of ERK in acupoint and non-acupoint.And to find the best time of drawing the fascia from the rats through observation of the difference among different time-point.4.To explore the effective difference of acupuncture for local fascia and muscle through semi-quantitative observating the expression of ERK1/2 and P38MAPK in local fascia and muscle,and provide the evidence for the theory of"fascia connective tissue is related to the Meridians".Materials and Methods1.Effect of acupuncture on the morphology of local fascia and spinal medulla.25 rats were randomly divided into 5groups,5 rats in each group.Control group: no treatment were given;Hand acupuncture at ST36 group(acu ST36):rats received hand acupuncture at ST36 acupupoint with twist manipulation(120 times per minute) last 4min,Hand acupuncture at non-acupoint group(non-acupoint):rats received acupuncture at the midpoint of inguinal groove with twist manipulation(120 times per minute) last 4min;Electrol-acupuncture at ST36 group(eleacu ST36):rats were treated with eletro-acupuncture at ST36 with the frequency of 2 Hz for 20min;All the treatment were performed once per day.The subcutaneous fascia of the area surrounding the needling-point with diameter 1.5cm,were got from the rats after 10 times treatment,then rinsed with 0.01 mol/L PBS(pH 7.4) and fixed with 4% paraformaldehyde,paraffin sections were respectively made.And then rats were perfused with 4%paraformaldehyde,spinal cord were taken out of rats and stained with hematoxylin-eosine staining.Images were collected with Olympus Image Analysis System.Tension group:Tension was exerted to the back of SD rats'lumbar 1-2 once per day,the control groups did not impose any intervention except anesthesia.After 7 days,stretched preparations were made and were observed by phase contrast microscope。After staining with phalloidin(10mg/L) and PI(0.01%),images were acquired by fluorescence microscope and confocal laser scanning microscopy. 2.Observation on the expression of ERK1/2 and P38 in fascia and medulla spinalis by immnohistochemistry staining.Experimental animals were ut supra,and control group,hand acupuncture at ST36 group,eletrol-acupuncture at ST36 group and hand acupuncture at non-acupoint group were chosen,and the treatment methods were ut spura.After subcutaneous fascia and medulla spinalis were got,two anti-shed paraffin sections were made and stained with S-P immunohistochemistry staining.Images were collected by Olympus Image Acquisition System and analyzed with Imagepro-Plus 6.0.Mean Density were used as comparison index.The results dates were analyzed with SPSS 13.0 software package.3.The expression of ERK1/2 in subcutaneous and the relationship between the expression of ERK1/2 and time point after acupuncture by Western-blot technology. 18 SD rats were randomly divided into 6 groups.After rats were received eletro-acupuncture at the regions of inguinal groove,the superfacial fascia of the area surrounding the acupuncture-point,whose diameters was about 1.5cm,were got from the rats of acpuncture 1,2,3,4 and 5 groups at 0h,1h,6h,12h and 36h respectively.While the same fascia were got from the Zusanli(ST36) and the same area corresponding to the acupuncture-ponit from the rats of control group.The exprssion of ERK1/2 and phosphorylated ERK1/2(p-ERK1/2) were detected by Western-blotting.And colouration were got using enhanced chemiluminescence kit. Images were got with Kodak Image Station 2000 MM imaging system.Gray scale was detected and analyzed by Image Tool 3.0.The dates were analyzed with SPSS 13.0 software package.4,Observation on the expression of ERK1/2 and P38 in fascia and medulla spinalis by Western-blot method.Materials:KeyGene extraction of protein kit and Keygene protein assay kit were from Guangzhou Jetway Biotechnology A mouse monoclonal antibody recognizing phosphorylated ERK 1/2(Tyr204;1:400) and phosphorylated P38(Tyr182;1:400),rabbit polyclonal antibodies recognazing total ERK1/2(1:400) and rabbit polyclonal antibody recognizing P38,PVDF Immobilon membranes and ECL chemiluminescence detection kit were from Santa Cruz Biotechnology(Santa Cruz,CA,USA).Goat anti-rabbit IgG and anti-mouse peroxidase-conjugated secondary antibodies were from Zhoushan Goldenbridge Biotechnology(Beijing, China).SABC(rabbit IgG) -AP kit and SABC(mouse IgG)- AP kit were provided by Wuhan Boster.Tissue homogenizer were from Fluko Company,Germany,and Trans-blot SD semi-dry electrophoretic transfer cell were from BIO-RAD. Experiment animals and grouping were as the same as Materials and Methods 1. After the rats in control group,hand acupuncture at ST36 group,eletro-acupuncture at ST36 group and hand acupuncture at non-acupoint were treated,local fascia and muscle of needling-point were got from the rats.Then the expression of ERK1/2 and P38 were detected by Western-blot.Results1.Fibers in fascia of rats in hand acupuncture at ST36 group and hand acupuncture at non-acupoint group distributed afferensly around the center of stretch point and their tensile force increased.The morphous of histiocyte nucelus become"flat spindle"and"sliver"shape after acupuncture while it of control group was"ellipse"or"round"shape.Fluorescence double staining indicated that in the streched group:cells arranged in the same direction with the force,the cell density were increased and the cystoskeleton reconstributed as"flat spindle"shape.The longitudinal axis of cytoplasm was long and compact,boundaries of cell processes were unclear and nucleus is small with fusiform shape(fig.2A).While in control group,many cells filled in fiber network disorderly and cell density was little in unit area with clear boundaries among them.Cytoskeleton were stretched as"flat"shape with many processes touching with each other and nucleus located at the center of cytoplasm in"approximately to round"shape.However,the morphous of fascia changed not significantly after receving eletro-acupuncture and the morphous of medulla spinalis hadn't changed after receiving acupuncture.2.ERK1/2 immunohistochemistry positive staining coloration in cornu dorsale medullae spinalis of rats was Buffy color.Positive cells distributed in gray nucleus that nerve cell concentrated,and deep staining region localized at the nucelus of neurocyte.Positive staining area of ERK1/2 concentrated surrounding the nucleus of fibroblasts and macrophages,and the staining level enhanced afer acupuncture, especially in fascia of hand acupuncture at non-acupoint group enhanced significantly(P＜0.05).Positive staining cells of P38 in fascial were a few and only in fascia of non-acupoint the staining level enhanced signifcantly(P＜0.05).However,the changes of ERK1/2 and P38 in medulla spinalis were not completely corresponding to the changes of them in fascia.3.ERK1/2 and p-ERK1/2 expressed in the area both of acupoint and of non-acupoint,but there were no significant difference between them(P＞0.05).The expressions of ERK1/2 were higher in the sides of acupuncture than the own control sides after theatment in the rats of each acupuncture group.The expression level of ERK1 achieved the maximum at 12h after acupuncture,but the expression of ERK2 and the expression of ERK1/2 were achived the peak at 6h after acupuncture.4.Western blotting examination showed that the expressions of ERK1/2, p-ERK1/2 and P38 were enhanced markedly after acupuncture in ench group, especially in the fascia and muscle of acupuncture at non-acupoint group,and followed by eletro-acupuncture at ST group and hand acupuncture at ST36.Conclusions1.That the morphous of fascia changed not significantly after receving eletro-acupuncture showed that acupuncture can make local fascial morphous change through manupulation of twist,extract and interpolation.On the basis of extensive evidence in cultured cells,these morphological changes are likely to be accompanied by important modification of cell signaling,gene expression,and matrix adhesion and may also produce variable connective tissue tension.These cellular events may also be key to the therapeutic mechanism of a wide variety of treatments using mechanical stimulation of connective tissue including physical therapy,massage, andacupuncture.2.Immunohistochemistry showed that acupuncture can up-regulate the expression of ERK1/2 and P38 of local fascia of acupoint and non-acupoint and especially in fascia of acupuncture at non-acupoint enhanced significantly.It perhaps because that subcutaneous fascia of non-acupoint is rich andand twisting needle can spur wide-region connective tissue and need more"pull-out"force,however,the subcutaneus fascia below the ST36 region was relatively less,therefor,twisting needle can spur little fascia connective tissue.The translocation of ERK1/2 and P38 positive staining area in medulla spinalis showed that acupuncture can activate these signal proteins.That the changes of ERK1/2 and P38 in medulla spinalis were not completely corresponding to the changes of them in fascia indicated that that fascia connctive tissue network all over the body is another initial regulation system besides"nerve-humoral regulation"for acupunture to regulate the human body.3.ERK and phosphorylated ERK are expressed in normal loose connective tissue.And the signal transduction protein is related with increment and differentiation.It indicated that in the developmental biologic point of view,very archaic anatomical structure-loose connective tissue play an athletic and energetic role in organisms' metabolic process.As cell signal transduction is concerned, subcutaneous fascia of acupoint is unspecialized. 4.That acupuncture can up-regulate the expression of ERK1/2 and P38 of local fascia of acupoint and non-acupoint was further confirmed by Western-blot.ERK1/2, phosphorylated ERK1/2 and P38 expressed in local muscle of needle-point and enhance after acupuncture.In summary,the research initiated to study the stretch of fascia and stimulating of muscle by the needle,which is the beginning step of acupuncture for the body, through HE staining,immunohistochemistry and Western-blot technology.And the reserch pattern differed from the traditional acupuncture study pattern"stimulating acupoint-reaction of target organ".That to explore the general biologic mechanism of acupuncture through observation of ERK1/2 and P38 is a new try of studying the mechanism of acupuncture in the fascial point of view.There is evidence that soft tissue fibroblasts form an extensively interconnected cellularnetwork and in it intercellar communication is through close cell-to-cell connnections.We can hypothesized that fascia connective tissue affect the immunity, vessels and nerve cell in the different way as the nerve system,and MAPK signal pathway reside in almost all eucaryotic cells and is one archaic communication mode in organic evolution process.Therefor,acupuncture perhaps affect the cellular mophology of fascia connective tissue,then affect the expression of MAPKs signal protein and organism's biochemical effect and finally regulate the organism.