The Studies On Low Hydraulic Resistance Channels Along Meridians In Rats And Its Morphology And Differential Proteome

BACKGROUNDIn more than half a century,the research on the biological connotation of the meridians and collaterals was one of the core components of scientific researches on traditional Chinese medicine(TCM).Many researchers have performed meridians research using biological,physical and imaging technology to confirm the existence of meridians and to explore its essence.Visualizing the paths and structures of meridians was an important part.It proved the existence of meridians.Even though,there were many defects in the bio-safety,marking and repeating for the most visualization methods of meridians.So,it limited the further researches on the structure and function of the displayed tracks along meridians.It was impossible to explain the biological essence of the tracks.Therefore,it is necessary to do some further researches on developing the new technologies of meridian visualization.Now,at home and abroad,many scholars have proposed the hypothesis that the interstitial flow(IF)and tissue interstitial space were related to the structure and function of meridians.Zhang established the method for visualizing the interstitial channels of Gephyrocharax Melanocheir fish and the pathological model of mini pig's meridians blockage to provide a lot of data.Zhang analysed the mechanics of IF flow and direction in the interstitium,and he proposed that meridians would be continuous and porous channels with low hydraulic resistance(LHRC).Some researchers recognized these results.Although,it is difficult to perform a large number of LHRC studies in mini pigs,and there is not a perfect visualization method of LHRC along meridians(LHRCM)right now.Further investigations and verifications of LHRCM are difficult too.Therefore,to explore an ideal animal model and experimental method,and to establish the display technology of LHRCM,it is the key to promote the LHRCM development.It will provide new data for revealing the biological connotation of meridians and collaterals.OBJECTIVEBased on the works of LHRC,in this study,a technique for measuring interstitial hydraulic resistance(Rh)in rats was established to detect the Rh along the meridians in rats.In order to explore a new experimental model and method for the study of LHRCM,a special dye was injected into the low Rh point(LHRP)along meridians to display the LHRC.The relationship between the track's location and meridians' paths was investigated.The relationship between of the tracks' histomorphology and meridians'function was analysed.The expression of the differential protein between the tracks along conception vessel(CV)and non-meridians' tissue were analysed to explore the material basis and biological function of LHRCM.It aimed to provide more data to explain the biological nature of the LHRCM,and to develop a new direction for the study of biological connotation of meridians.METHODSExperiment 1:The measurement of the LHRP along meridians1.The low impedance points(LIP)of the skin along the governor vessel(GV)and CV path were determined using a 57-6F30 meridian position indicator instrument.2.A method consisting of continuous differential pressure measurements was used to detect the Rh of subcutaneous tissues along GV and CV path,to establish the new method of Rh determination for the rat.The differences of the Rh between the subcutaneous tissues along meridian path and the non-meridians were analysed.Experiment 2:The observation of migration characteristics after AB injection into LHRP along meridians1.The Alcian Blue(AB)solution was injected into the LHRP along the GV and CV(n=23).The injection parameters were screened through the comparison of AB migration characteristics with different injection time,injection dose and observation time.2.The interstitial fluid pressure(IFP)of LHRP before and after AB injection were measured in rats(n=5).The changes of IFP were analysed.3.The LHRPs of GV(n=10)and CV(n=27)were injected with AB.The distribution characteristics of AB were observed.The relationships of AB tracks(ABT)and TCM meridian paths was analysed.The direction,length,width and occurrence frequency of ABT were recorded.Then it was compared with AB injection into high hydraulic resistance points(GV&CV with 8 rats each group),ink injection into LHRP(HLRP)(GV&CV with 5 rats each group).The migration of ABTs was analysed.Experiment 3:The histomorphology of the ABT along meridians in ratsThe histomorphology of the ABT was investigated by compound staining with frozen section of fresh tissue.The histological difference between ABT and the tissues nearby ABT was compared.The correlations of ABT and vessels,nerves and mast cells(MC)were analysed.Explore the relationship between morphological characteristics of ABT along meridians and meridian function.Experiment 4:The proteomic analysis of differentially expressed proteins of subcutaneous ABT along CV and peripherally non-meridian tissues connective tissue in ratsProteomics based on LC-MS was introduced into subcutaneous ABT along CV and peripherally non-meridian tissues connective tissue in rats.Total proteins were identified from sclerotia and hyphae at intital,developmental and mature phases based on data dependent acquisition(DDA)model.The proteome of subcutaneous ABT and peripherally non-meridian tissues connective tissue(0.5-1 cm from the low resistance line along CV)were quantified from intital sclerotia and hyphae based on SWATH(Sequential Window Acquistion of all theoretical fragment ions)label-free model.The differential proteome between them were analyzed by non-standard and quantitative method of MSALL.Then the GO,KEGG,PPI,BP link KEGG analysis were performed to understand the function of differentially expressed proteins and the biological process involved.The data of differential proteins and TCM meridian nature researches were analysed to screen the proteins which were the highest correlation with meridians.The expression levels of the screened proteins were measured by western-blot analysis.RESULTSExperiment 1:The paraments of Rh measurement for rats:four points of the abdominal skin were fastened to the animal operating table using surgical sutures such that the abdominal wall formed a horizontal plane in vivo,the 5#needle with a side hole was used,the saline velocity was 10 drop/min.The case detection rate of GV was 72%,and CV with 73.3%was similar.The subcutaneous Rh value along GV(7.99 ± 1.88,×106 dyne·s·cm-5)was significantly lower than that the non-meridian points alongside GV(10.63± 1.51,×106 dyne·s·cm-5),the subcutaneous interstitial space 0.5-1 cm from the low resistance line along the meridian.The Rh value along the CV(10.37±1.26,×106 dyne·s·cm-5)was significantly lower than that the non-meridian points alongside CV(19.13 ± 1.37,×106 dyne·s·cm-5)(p<0.01).Experiment 2:1.The paraments of AB injection:injection volume 0,2 mL,injection time 10 min,observation time 48 h after AB injection.At the end of AB injection,the IFP of LHRP was 2.52 mmHg higher than before AB injection.The local IFP of LHRP increased slightly before and after injection.2.At 48 h after injecting AB solution into the LHRP of the meridian,AB solution diffused in the subcutaneous tissue to gather in the centre where the needle tip entered,and some linear tracks parallel to the long axis of the body with different lengths and widths extended beyond the border of the AB solution gather area.After AB injection into subcutaneous LHRP along GV,ABTs distributed along GV,or bladder meridian in rats.The occurrence rate was 80%.The ABTs migrated towards the head at most.The longest ABT was along the GV path.Starting at the injection point,the length of ABTs migrating along GV ranged from 1.1 to 4.7 cm,with a mean of 2.74 ± 0.36 cm.Starting at the border of AB diffusion,the length of the linear tracks ranged from 0.3 to 1.4 cm,with a mean of 0.78± 0.12 cm and an average width of 0.125± 0.02 cm.3.After AB injection into subcutaneous LHRP along CV,ABTs distributed along CV,or stomach meridian(ST),or spleen meridian(SP),or kidney meridian(KI)in rats.The occurrence rate was 81%.These ABTs migrated towards the tail at most.The longest ABT was along the SP path.Starting at the injection point,the length of ABTs migrating along SP ranged from 0.4 to 4.9 cm,with a mean of 2.12 ± 1.96 cm.Starting at the border of AB diffusion,the length of the linear tracks ranged from 0.3 to 4.1 cm,with a mean of 1.12± 0.42 cm and an average width of 0.15± 0.07 cm.The migration characteristics of ABTs are consistent with the distributions of TCM meridian paths.The ABTs appeared in one or two directions,with a single path or multiple paths.No tracks appeared in those rats after injecting ink into the LHRP or AB into the HLRP.The migration of the AB solution indicated that interstitial fluid flowed through the LHRCM in the tissue.Experiment 3:1.Each ABT was a reticular space channel with large gaps in the loose subcutaneous connective tissue consisting of a mass of collagen fibres,elastic fibres,other fibrous components and acid mucopolysaccharides attached to the fibres.The interstitial spaces of these ABTs were larger than those around ABTs in SCT.2.There are a large number of MCs around the fibers of the ABT,and there are abundant blood vessels,cholinergic nerves and MCs in the ABT.Experiment 4:There were 468 proteins expressed differentially in the ABT along CV compared with peripherally connective tissue of non-meridian.The Go analysis:indicated that the cell components of differential proteins were the cytoplasm,intracellular organelle,vesicle,intracellular vesicle,extracellular exosome and extracellular membrane-bounded organelle,etc.Their molecular functions were homologous protein binding,coenzyme binding,enzyme binding,nucleoside binding,cell adhesion molecule binding,protein complex binding,oxidoreductase activity,glutathione peroxidase activity.The biological processes of the differential proteins mainly were metabolic process,response to hormone,and response to wounding.KEGG analysis indicated the metabolic process of difference proteins were mainly metabolic,celluar process,human disease.It indeed that Glyceraldehyde-3-phosphate dehydrogenase(GAPDH),ATP synthase subunit epsilon(ATP5E),Voltage-dependent calcium channel subunit alpha-2/delta-1(CACNA2D1),Glutathione peroxidase 3(Gpx-3)in the ABT along CV path expressed differentially compared with peripherally connective tissue of non-meridian.Western-blot results indicated the expression levels of GAPDH,ATP5E and CACNA2D1 of ABT were significantly higher than that peripherally connective tissue of non-meridian(P<0.05).The expression level of Gpx-3 was higher than that peripherally connective tissue of non-meridian(P=0.08).CONCLUSIONS1.In this study,a noval measurement method of tissue Rh in the rats was established successfully.The Rh value of the subcutaneous tissue along GV or CV path was lower than that aeras along non-meridians in rats.It was suggested that there was the LHRCM in rats.2.The display method of LHRCM with AB injection at LHRP in rats was established.The liner ABTs appeared in the subcutaneous connective tissue layer,which were similar to the characteristics of the TCM meridians and collaterals paths on the body surface.3.In the ABT,there were some large gaps,numerous mast cells,blood vessels and cholinergic nerves.It provided morphological evidence for explaining the biophysical properties of the LHRCM.4.There were 468 proteins expressed differentially in the ABT along CV compared with peripherally connective tissue of non-meridian.The biological processes of ATP5E,CACNA2D1,GAPDH and Gpx-3 which were up-regulated expression in ABT were closely related to meridian phenomenon and acupuncture effect.Those proteins would be the characteristic substances of LHRC along CV in the rats.These results provided new evidences for explaining the biological mechanism of LHRCM.