Experimental Study On The Establishment Of Heat Bi And Cold Bi Syndrome Models In Rheumatoid Arthritis By Sensitization And Desensitization Of TRPV1

BackgroundThe basic pathological changes of rheumatoid arthritis(RA)are synovitis within the joint.It is similar to the paralysis in Chinese medicine.There are two types of manifestations of RA acute active phase,one is manifested as joint redness,swelling,heat and pain are significant,aggravated by heat,which is the "rheumatic fever paralysis" of traditional Chinese medicine,referred to as "Heat Bi";The other is manifested as obvious joint swelling and pain,but the skin color is not red,it is not hot to touch,aggravated by cold,which is the "wind cold and wet paralysis" of traditional Chinese medicine,that is,"Cold Bi".Traditional Chinese medicine plays an important role in the treatment of RA.The animal model of Cold Bi and Heat Bi is the basis of studying the theory of TCM Bi disease prescription.However,the known animal models of Cold Bi and Heat Bi still have limitations.The studies have shown that the collagene-induced arthritis(CIA)model can be used as a heat arthralgia model.In addition,previous studies have shown that Human Tumor Necrosis Factor-α(hTNF-α)transgenic mice(Tgtc)can serve as the basic model of Cold Bi.Neurogenic inflammation refers to the inflammatory response caused by the release of neuromediators from sensory nerve endings after traumatic stimulation,mainly manifested as redness,swelling and heat pain,similar to the joint performance of traditional Chinese medicine Heat Bi.Transient receptor potential vanilloid 1(TRPV1)is the main ion channel involved in neurogenic inflammation.It can be activated by capsaicin(CAP),resiniferatoxin(RTX)and other injurious stimulation.Subtance P(SP)and Calcitonin gene related peptide(CGRP)are two main neuromediators involved in neurogenic inflammation,which can jointly expand blood vessels,promote inflammatory cell infiltration,and stimulate vascular regeneration.After being sensitized by CAP,TRPV1 releases SP and CGRP,causing neurogenic inflammation.RTX is a powerful agonist of TRPV1,which can cause lasting desensitization of TRPV1,decrease or depletion of SP and CGRP release,and reduce the degree of neurogenic inflammation.Therefore,we hope to establish a model of Heat Bi and Cold Bi on the basis of hTNF-a transgenic mouse through TRPV1 sensitization or desensitization,and to explore the influence of TRPV1 desensitization on TCM syndromes of CIA mouse model,so as to better apply it to the research of the basic theory of TCM paralysis.Objective:1)To observe the effect of TRPV1 sensitization on the TCM syndrome of arthritis in Tgtc mice,and to explore its pathogenic mechanism2)To observe the effect of TRPV1 desensitization on the TCM syndrome of arthritis in Tgtc mice,in order to verify the relevant role of TRPV1 sensitization in the establishment of TCM Heat Bi animal model,and to explore its pathogenic mechanism3)To investigate the effect of TRPV1 desensitization on the TCM syndrome and pathogenic parenchyma in mice with collagen-induced arthritisMethods:1)Ten SPF-level female FVB background mice and thirty SPF-level female Tgtc mice were divided into negative control group(NC),blank control group(BC),model group(MD),experimental group(CAP),ten animals in each group,the drug intervention was carried out on CAP group,absolute ethanol intervention was given to NC group and BC group,and no treatment was done in MD group.After the intervention,the weight,ankle diameter,joint deformation index,skin red value,ankle skin surface temperature,mechanical pain threshold,thermal pain threshold and cold pain threshold of each group were observed and recorded2)Eight SPF-level female FVB background mice and twenty four SPF-level female Tgtc mice were divided into NC group,BC group,MD group,RTX group,animals in each group,the drug intervention was carried out on RTX group,absolute ethanol intervention was given to NC and BC,and no treatment was done in MD.After the intervention,the weight,ankle diameter,joint deformation index skin red value,ankle skin surface temperature,mechanical pain threshold,thermal pain threshold and cold pain threshold of each group were observed and recorded.Two weeks later,the laser speckle sprankle flow imaging test of double ankle joint,serum SP and CGRP ELISA detection,ankle histopathology and immunohistochemistry were detected3)Twenty four female DBA mice with SPF level were divided into NC group,BC group,MD group,RTX,six mice in each group,among which the NC and BC groups were given subcutaneous injection of absolute ethanol neck as the control,the MD group was not treated,and the RTX group was given RTX pretreatment.Two weeks after desensitization,CIA modeling was performed in the BC group,MD group and RTX group,and the weight,ankle diameter,joint deformation index,skin red value,ankle skin surface temperature,mechanical pain threshold and thermal pain threshold of each group were observed and recorded after the first modeling.After Three weeks of booster immunization,laser speckle blood flow imaging detection of double ankle joint,serum P substance(SP)and calcitonin gene related peptide(CGRP)ELISA detection,ankle histopathology and other tests were performedResults:1)Tgtc mice died after subcutaneous injection of CAP2)After RTX pre-stimulation of Tgtc mice,they showed poor mental status,decreased dietary water intake,messy and dull hair,reduced activity,soft stool,and heavy urine.RTX reduces joint swelling,lowers joint surface temperature,and ultimately lowers the red color of its skin,but does not cause hyperalgesia and an increase in joint SFI(P>0.05).Compared with BC group and MD group,there were no significant differences in inflammatory infiltration and bone destruction scores(P>0.05),but pannus was significantly reduced(P<0.05),and TRPV1 content in synovial tissue was significantly down-regulated(P<0.05).The contents of SP and CGRP in serum and CGRP in synovium were not significantly different among the four groups.Compared with NC group,SP content in synovial tissue of Tgtc mice was significantly increased(P<0.05)3)After RTX intervention,CIA mice showed poor mental status,reduced dietary water intake,messy and dull hair,reduced activity,soft stool texture,and slightly more urine.There were no significant differences in ankle diameter,arthritis index score,joint color change,joint surface temperature,mechanical pain threshold,thermal pain threshold,serum CGRP content and joint pathological manifestations compared with BC and MD groups(P>0.05).After RTX intervention,ankle SFI values and serum SP levels were significantly reduced(P<0.05,P<0.01)Conclusion:1)The experiment of Tgtc mice sensitized by TRPV1 to establish TCM Heat bi syndrome model has not been successful,but it is still a meaningful attempt for establishing TCM Heat Bi syndrome model in the future2)Tgtc mice can be used as a model of TCM Cold Bi through TRPV1 desensitization3)TRPV1 desensitization can cause cold syndrome performance in CIA mice,which can reduce joint blood flow,suggesting that it is related to the decrease of serum SP content.