| BackgroundInflammatory pain is one of the most common clinical pathological pain at present and it is one of serious problems in normal life and work.Capsaicin receptor(TRPV1)belongs to the transient receptor potential capsaicin subfamily members,closely linked with pain transmission membrane ion channel receptor.It plays an important role in inflammatory pain,cancer pain,hyperalgesia and expressed both in the peripheral nervous system and central nervous system.Inflammatory pain induces TRPV1 expression and release other inflammatory mediators such as IL1? and neuropeptides,promotes TrkA and NGF binding,stimulate cell activity,causes nerve inflammation and its autophosphorylation and activation,constitutes pain transmission pathways of energy.Bee venom is a transparent liquid,a specific gravity of 1.1313,bitter,containing a variety of biologically active substances such as active peptides,enzymes,biogenic amines and so on.It can directly or indirectly release inflammatory pain relief,regulate immune function of the whole body.Through bee venom injection Zusanli acupoint to impact the collagen-induced arthritis rats,This study verify the efficacy and explore venom intervention on TRPV1 and TrkA expression inflammatory pain.purposeTo explore venom intervention of collagen-induced arthritis rats and its impact on TrkA and TRPV1 expression.methods1.Materials and MethodsAgents include incomplete Freund's adjuvant(IFA)and bee venom(BV,US Sigma-Aldrich company),type ? bovine collagen(chondrex company),TRPV1 and TrkA kit(abcam company),pentobarbital sodium,and POM,physiological saline and other reagents provided by the Southern Medical University Center agent.Equipment including digital calipers(China Forgester company),life science model 390 thermal pain tester(US IITC company),paraffin slicing machine,electron microscopy and so on.2.modeling and grouping23 healthy Wistar rats,male,8 weeks old,SPF grade,weighing 180-220g.From Southern Medical University Experimental Animal Center of procurement.At Southern Medical University Experimental Animal Center of SPF laboratory.Free laboratory(SPF)reared for 7 days.Maintained the temperature between 18-29?,the humidity at 40%-70%,an indoor wind speed<0.18m/s,ventilation frequency 10-20 times/h,differential pressure 20-50 Pa,day and night(light and dark time)12/12h,noise requirements ? 60 dB.One week before the experiment rats screening samples:heat pain threshold measured value 2?10s were included in the experiment,which meaned rats after the bottom of the heat sources in the plantar base 2?10s those can cause condensation-jerk reaction can be included in the experiment.Pain threshold was less than 2s or more than 10s which prompted the animal may react too sensitive or little sensitive,which rule out the experiment(heat pain threshold measurement method,see below).Excluded three rats that were not suitable,there were 20 rats included in this experiment.They were randomly divided into normal control group,model group,bee venom group and pretreatment groups of five.Preparation of inflammatory pain model:depending on book of Rheumatoid Arthritis,Medical Immunology technology edited by Zhang Jinyu,Ju Songguang,the approach was as below.1 day prior(d0)calf collagen type ? reagent(10mg installed)stored at 4 ? overnight.dl under sterile conditions with an equal volume of IFA in an ice bath,mixed and emulsified sufficiently.Extracted a drop of emulsifier detection through a beaker filled with water,spread evenly prevail.4 ?refrigerator spared.D1 the experimental model group,bee venom group and the pretreatment group rats were modeled of their primary immunization by the base of tail injection of rats pretreated with bovine type ? collagen and IFA emulsifier 0.2ml,the control group by being injected with saline 0.2ml corresponding position in the base of the tail.D8 the three groups were modeled of their second immunization by the base of tail injection with bovine type ? collagen and IFA emulsifier 0.2ml again,control group injected with saline 0.2ml again.d10 modeling rat paw edema appears,swelling gradually increased,activity decreased,walking drag step,d14 they all were swelling and no significant swelling subsided,their swollen joint score(AI)were more than 3 points,and further experiments can be carried out.3.The intervention program3.1 venom Group:d14-d21 to intervene.Interventions:bee venom solution Zusanli point injection.Dose:bee venom solution 0.1ml.Concentration:3mg ˇ ml-1.Injection site:Zusanli.1 day,1 acupoint each time,bilateral Zusanli used interchangeably.Continuous intervention seven days.3.2 Pretreatment Group:d8-d10 to intervene.Interventions:bee venom solution Zusanli point injection.Dose:bee venom solution 0.1ml.Concentration:3mg ˇ ml-1.Injection site:Zusanli.1 day,1 acupoint each time,bilateral Zusanli used interchangeably.Intervention 3 days in a row.3.3 model group:d14-d21 were acupoint injection of saline 0.1ml,injection site:Zusanli.1 day,1 acupoint each time,bilateral Zusanli used interchangeably.7 consecutive days.3.4 normal control group:non-intervention.4.Observation4.1 pairs of lower limb paw thickness:The Chinese company Forgester electronic digital calipers:d15,d16,d17,d18,d19,d20,d21,d22 measuring paw,paws and bottom center of the articular surface of the axis paw thickness,measured three times on each side and averaged,and calculate their sum.4.2 pairs of lower limb pain threshold:US ? TC life science model 390 meter heat pain,setted RI35,the upper limit of 20 s,d21 measuring lower extremity pain threshold,the thermal radiation source heat pain focus instrument aligned at the cross foot rat lower limbs plantar middle of each rat was measured 2 times,each time at least after 10 min,the average value interval.4.3 limb joint swelling score:0,no swelling;1,little toe joint mild swelling;2 points,the little toe joints and paw swelling;3,paw swelling following ankle;4,including ankle including all feet,joint swelling.Add all the scores of joints as its AI.4.4 Ankle HE section pathology observed:rapidly remove the ankle,about 0.7 x 0.5 × 0.5mm3,placed in fixative containing 4%paraformaldehyde fixed,decalcified,gradient dehydrated,embedded in paraffin.HE staining observed damage synovial,cartilage and the like under an electron microscope.4.5 dorsal root ganglia TrkA content:The gray values of TrkA in the western blotting detection in DRG.The left ventricle,aortic cannulation for cardiac perfusion fixation after lumbar spine taken along the sciatic nerve track DRG(L4-6),gently DRG removed from the intervertebral foramen.The quality of post-crush,cleavage,centrifuged and the supernatant,protein concentration determination and so on.Cleaning slide,glue,loading,run gel electrophoresis,transferred to a membrane,closed,1:5000 dilution TrkA antibody(abcam),followed by secondary antibodies were incubated overnight and washed three times,the last color,exposure,scan picture,using Quantity One software for Western blotting strips gray analysis,recording its gray value.The ratio of the relative content of protein with the target protein and the optical density of the internal control band said,according to measurement data for statistical analysis.4.6 dorsal root ganglion TRPV1-positive cells:The number in the immunohistochemical DRG TRPV1-positive neurons.The left ventricle,aortic cannulation for cardiac perfusion fixation after lumbar spine taken along the sciatic nerve track DRG(L4-6),gently DRG removed from the intervertebral foramen.Conventional specimen dehydration,embedded in paraffin,sectioned,slice thickness 4?m.Conventional specimen dehydration,embedded in paraffin,sectioned slice thickness 4?m.Conventional dewaxing to water,incubated at room temperature,PBS wash,hot fixes,closed,dropping 1:100 dilution TRPV1 primary antibody(abcam),4? overnight.Washing,secondary antibodies,incubation,washing,etc.,color,wash,stained,dehydration,positive cells was observed under the sealing,and placing the eyepiece(x 200)micrometer.5.Statistical analysisAll datas are used spss13.0 statistical software for statistical analysis.Comparison between multiple way are used analysis of variance,variance in one-way ANOVA,two further compares the LSD,variance are used when not neat Welch,two further compares the Dunnett's T3;Different time points compared with repetitive measure analysis of variance,and comparing two further use of LSD.All data are expressed with X ąSD P<0.05 for the difference was statistically significant.Results1.The model group than the control group significantly increased paw thickness,bee venom venom intervention group decreased by 7 days paw thickness,a significant difference compared with the model group.Pretreated group at 7 days after the bee venom intervention paw thickness lower than the model group,there are significant differences.2.AI model group increased significantly compared with normal control group,the bee venom group AI group was significantly lower than the model,pretreatment group was significantly lower than the model group.3.The pain threshold of the model group were significantly lower than normal,significantly increased the pain threshold of bee venom group compared with model group,pretreatment group was significantly higher than the model group.4.HE staining pathological model group observed visible near the joint infiltration of inflammatory cells,cartilage destruction and pannus dark;venom were observed in the joint of inflammatory cell infiltration,cartilage damage lighter than the model group;pretreatment group showed a small amount or cell infiltration,articular cartilage damage or no light,the overall situation compared with the model group light.5.Western blotting detection DRG expression level of TrkA,TrkA expression seen in the model group was significantly higher than the normal control group,low venom group compared with model group,low-pretreated group compared with the model group.6.IHC-P detection DRG TRPV1 expression levels,we can see the model group TRPV1-positive cells compared with normal control group was significantly higher than the low-venom group model group,low-pretreated group compared with the model group.Conclusions1.Zusanli Point Injection of Bee Venom rats with inflammatory pain intervention can reduce the level of expression of TRPV1 in DRG,which affects the regulation of inflammatory pain of TRPV1.2.Zusanli Point Injection of Bee Venom rats with inflammatory pain intervention can reduce the level of expression of TrkA in DRG3.Zusanli Point Injection of Bee Venom rats with inflammatory pain interventions can improve pain threshold,reducing the thickness of the paw,joint swelling score.Zusanli Point Injection of Bee Venom rats with inflammatory pain intervention can effectively reduce swelling ankle inflammatory cell infiltration,cartilage destruction and pannus formation.4.Pretreatment venom can effectively reduce the thickness of the paw of rats with inflammatory pain,joint swelling score,effectively increase the pain threshold,effectively reduce the swelling of the ankle joint inflammatory cell infiltration,pannus formation and cartilage damage and reduce DRG the TRPV1,TrkA expression levels,and thus indirectly affect regulation of TRPV1 in inflammatory pain.5.TrkA and TRPV1 pain is an important signal transduction molecule.One of the analgesic effect of bee venom molecules are simultaneously regulate TRPV1,TrkA expression levels,thus affecting the regulation of inflammatory pain of TRPV1. |
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