| BackgroundRheumatoid arthritis(RA)is a widespread autoimmune disease characterised by chronic synovial lesions as a key feature.The mechanisms of RA are currently unknown,but inflammatory factors are critical in the pathogenesis of RA.The release of corresponding inflammatory mediators by immune cells,such as synovial macrophages,and synovial fibroblasts is a major contributor to synovial inflammation and bone destruction in RA.There is much evidence that vagus nerve stimulation(VNS)activates the Cholinergic anti-inflammatory pathway(CAP)anti-inflammatory pathway,thereby alleviating synovial inflammation and bone destruction in RA.Since the auricular nerve is the only region where the vagus nerve is distributed on the mammalian body surface,the anti-inflammatory effect of Transcutaneous Auricular Vagus Nerve Stimulation(taVNS)is likely to be mediated by stimulation of the vagus nerve in the auricular region.Given that taVNS and VNS have similar structural and functional bases,and that both animal experiments and clinical studies have suggested that VNS can alleviate synovial inflammation in RA via CAP,the anti-inflammatory effect of taVNS has not yet been demonstrated in RA disease models.Therefore,this paper proposes the scientific hypothesis that taVNS may alleviate synovial inflammation in RA through CAP.Objective1 To clarify the alleviating effect of an auricular nail-vagus nerve stimulation intervention on collagen-induced arthritis(CIA)synovitis in rats.2 To elucidate the relationship between the effect of an auricular nail-vagus nerve stimulation intervention to alleviate experimental RA arthritis and the integrity of the vagus nerve.3 Exploring the intracellular molecular mechanisms underlying the inflammatory effects of auricular nail-vagus nerve stimulation in relieving joints from the perspective of peripheral cholinergic anti-inflammatory pathways.MethodThis experiment is divided into the following four parts:Experiment 1 was to observes the activation of taVNS on brainstem NTS and DMV nuclei;Experiment 2 was to observe the relieving effect of taVNS on synovial inflammation in CIA rats;Experiment 3 was to observe the effect of taVNS on synovial inflammation caused by vagotomy;Experiment 4 was to observe the effect of inhibiting α7AchR(MLA)on the effect of taVNS on regulating synovial inflammation.The experiment 1 was divided into Control group,CIA group and taVNS group;the experiment 2 was divided into CIA group,CIA+vagotomy(VGX)group and CIA+VGX+taVNS group;the experiment 3 was divided into CIA group,CIA+Methyllycaconitine citrate(MLA)group,CIA+MLA+taVNS group.Except for 10 adult healthy male SD(Sprague Dawley)rats(180±20 g),which were not modeled,the rest of the groups were modeled,with 10 rats in each group,and the success rate of modeling was 80%.Four days before the initial immunization,the CIA+VGX group and the CIA+VGX+taVNS group were pretreated by vagotomy before CIA induction,and then recovered for 4 days after the operation.14 days after the initial immunization,after the CIA rat model was successfully established,the rats in the taVNS group were lightly anesthetized with isoflurane by inhalation,and the ear concha vagus nerve was stimulated.On the iron sheet with electromagnets,it was fixed on the bilateral ear conchae area of the rat by means of magnet suction,and the rat auricle kept continuous vibration as the benchmark.Stimulation parameters:continuous wave,frequency 20Hz,intensity 1mA,30min/d,continuous 28d.The treatment time is from 9 to 11 am.The CIA+VGX+taVNS group and CIA+MLA+taVNS group adopted the same intervention method,frequency and time.Rats in the CIA+MLA+taVNS group were pretreated with MLA 3 mg/kg intraperitoneally 15 minutes before taVNS intervention,once a day for 28 consecutive days.Experiments were used to observe the pain behavior,paw swelling,arthritis index,histopathological changes and the expression level of synovial tissue protein in rats.The Vonfrey detector was used to detect the mechanical withdrawal threshold of CIA rats,and the Ugo-37370 bolometer was used to detect the thermal pain latency of the plantar.Paw volume was used to measure paw swelling,and Arthritis Index was assessed by scoring method.Hematoxylin-Eosin Staining was used to observe the pathological changes of synovial inflammation in CIA rats,and MesoScaleDiscovery was used to detect the serum tumor necrosis factor-α,interleukin-6 and interleukin-1βcontent.Western blotting was used to detect α7 Nicotinic Acetylcholine Receptors,phosphorylated nuclear factor kappa B p65/p-p65 in CIA rats,and TNF-α.ResultResults 1:Activation of brainstem NTS and DMV nuclei by taVNSThe number of c-Fos protein-expressing neurons was used as a criterion.Immunohistochemical analysis showed that after taVNS intervention in CIA rats,the number of activation of c-Fos positive neurons in the NTS and c-Fos and ChAT+neurons in the DMV area was significantly higher in the taVNS group compared to the CIA group rats(P<0.01,P<0.05).Since the NTS is the vagal sensory centre in the brain,and the DMV is the vagal motor centre,which innervates the activity of vagal efferent nerves,this part of the results suggests that taVNS can activate vagal afferent centres in the brain in addition to vagal reflexes,and thus may activate the complete vagal anti-inflammatory pathway.Results 2:Alleviating effect of taVNS on synovial inflammation in CIA rats1 Effect of taVNS on arthritis index and paw edema in CIA rats1.1 Effect of taVNS on arthritis index in CIA ratsOn day 7 after initial immunization,the difference in arthritis index between the groups was not statistically significant.On day 14 after the initial immunization,the Arthritis Index score of the hind limbs of the successfully modeled CIA rats was>3.On day 14 after initial immunisation,the bipedal arthritis index score of the CIA rats rapidly increased to 6-8,which was significantly higher than that of the Control group(P<0.05).Two weeks after the taVNS intervention(i.e.day 28 after the initial immunisation),the arthritis index of both feet in the CIA rats decreased slowly until the end of the intervention compared to the Control rats,and finally stabilised to 4-6(P>0.05),suggesting that the arthritis index of the CIA rats could still recover mildly without any intervention.After two weeks of taVNS intervention,compared with the CIA rats,the arthritis index of both feet in the taVNS group decreased significantly until the end of the intervention and finally stabilized to 2-4 points(P<0.05),suggesting that taVNS significantly reduced the arthritis index of the CIA rats and significantly reduced the severity of arthritis in the CIA rats.1.2 Effect of taVNS on paw edema in CIA ratsThe difference in paw edema of the hind limbs of the rats was not statistically significant between the groups from the pre-modelling period to day 7 after the initial immunization.On day 14 after the initial immunization,the paw edema of both hind limbs of the rats in the CIA group increased rapidly and was significantly higher than that in the Control group(P<0.05),and reached its peak on day 21 after the initial immunization.On day 28 after the initial immunization,the paw edema of both feet in the CIA rats decreased slowly compared with that in the Control group until the end of the intervention(P<0.05),suggesting that the paw edema of the CIA rats could still be mildly relieved without any intervention.After two weeks of taVNS intervention,the paw edema in the taVNS group decreased significantly compared to the CIA group until the end of the intervention(P<0.05),suggesting that taVNS significantly reduced the paw edema in the CIA rats.2 Effect of taVNS on MWT and TWT in CIA ratsOn day 7 after initial immunization,the differences in MWT and TWT between the pre-modelling groups were not statistically significant.On day 14 after initial immunization,the MWT and TWT of rats in the CIA group decreased significantly compared to those in the Control group(P<0.05).On day 28 after the initial immunization,the MWT and TWT of the CIA group rats slowly increased until the end of the intervention compared to the Control group rats(P<0.05),suggesting that the MWT and TWT of the CIA group rats could still be mildly relieved without any intervention.Two weeks after the taVNS intervention,the MWT and TWT of the rats in the taVNS group increased significantly compared to the CIA group until the end of the intervention(P<0.05),suggesting that taVNS significantly increased the MWT and TWT of the CIA rats and effectively relieved the arthritic pain of the CIA rats.3 Effect of taVNS on toe appearance and joint histomorphology in CIA ratsThe hind limbs of the rats in the Control group were healthy,with no redness or swelling of the ankle joints and toes,and they moved freely,while the hind limbs of the rats in the CIA group had obvious redness and swelling of the ankle joints and toes,and they were even stiff and deformed,in line with the characteristic changes of RA.According to HE staining,the synovial membrane of the ankle joint in Control group rats was structurally intact,the joint surface was smooth and flat,the synovial cells were neatly arranged,and there was no inflammatory infiltration or tissue debris.Compared with rats in the Control group,rats in the CIA group had a narrowed joint cavity,rough and uneven joint surface,and a large number of inflammatory cells infiltrated in the synovial tissue.Compared with rats in the CIA group,the inflammatory response in the synovial membrane of rats in the taVNS group was reduced,and the number of proliferating synovial cells in the thinner synovial layer was reduced,suggesting that taVNS improved the appearance of the toe and the pathological morphology of the joint tissue in the CIA rats.4 Effect of taVNS on changes in serum levels of relevant inflammatory factors in CIA ratsCompared with the Control group,the levels of TNF-α,IL-6 and IL-1β in the serum of rats in the CIA group were significantly higher(P<0.01,P<0.01,P<0.05),suggesting that the inflammatory cytokine content in the serum of rats in the CIA group increased substantially after modeling.Compared with the CIA group,the serum expression of TNF-α and IL-6 in the taVNS group was significantly lower(P<0.01,P<0.01),but the difference in the expression of IL-1β was not statistically significant(P>0.05),demonstrating that taVNS significantly reduced the content of inflammatory factors in the serum of CIA rats.5 Effect of taVNS on the expression of CAP regulatory-related proteins in the synovial tissue of CIA ratsCompared with Control rats,the expression levels of α7AchR in the synovial tissues of CIA rats were lightly up-regulated(P>0.05)and the expression levels of NF-κB p-p65/NF-κB p65,and TNF-α were significantly up-regulated(P<0.05),suggesting that the expression levels of α7AchR in the synovial tissues of CIA rats were reduced after moulding,and the expression levels of NF-κB p-p65/NF-κB p65 and TNF-α expression levels were increased,and the inflammatory response of synovium was aggravated.After four weeks of taVNS intervention,the expression levels of α7AchR were significantly up-regulated(P<0.05)and the expression levels of NF-κB p-p65/NF-κB p65 and TNF-α were significantly down-regulated(P<0.05,P<0.05)in the synovial tissues of rats in the tavNS group compared with those in the CIA group,suggesting that taVNS significantly increased the expression levels of α7AchR in the synovial tissues of CIA rats.The expression levels of the key CAP regulatory receptor α7AchR were significantly increased in the synovial tissues of CIA rats,thereby inhibiting the protein expression of NF-κB signaling and downstream TNF-α inflammatory factors and reducing the inflammatory response in the synovial membrane.Result 3:The effect of taVNS to alleviate synovial inflammation requires an intact vagus nerve1.Effect of vagal nerve dissection on taVNS regulation of arthritis index and paw edema in CIA rats1.1 Effect of vagal neurotomy on taVNS regulation of arthritis index in CIA ratsFrom day 0 to day 21 after the initial immunisation,the arthritis index of both feet was slightly higher in the CIA+VGX group compared to the CIA group.The arthritis index peaked on day 21 after initial immunization in both the CIA and CIA+VGX groups,after which the arthritis index in both groups decreased slowly and steadily and finally stabilized between 4-6 points(P>0.05),suggesting that vagotomy did not have a significant effect on the appearance and behavioural phenotype of arthritis symptoms per se in the CIA rat model.Compared with the CIA+VGX group,the CIA+VGX+taVNS group showed a similar slow decrease in the arthritis index of both feet and the arthritis index of both feet eventually stabilized between 4-6 points(P>0.05).The above experimental results indicate that the alleviating effect of taVNS on the arthritis index of CIA rats disappeared after vagotomy.The alleviating effect of taVNS on the arthritis index requires the involvement of the intact vagus nerve.1.2 Effect of vagotomy on taVNS-regulated paw edema in CIA ratsWithin 7 days of initial immunization,the paw edema of the hind limbs of the rats in the CIA+VGX group showed a slow decrease compared to that of the rats in the CIA group.On day 7 after the initial immunization,the paw edema of the hind limbs in both the CIA+VGX and CIA groups increased significantly and reached a peak on day 21 after the initial immunization,after which the paw edema of the hind limbs in both groups decreased slowly until the end of the intervention(P>0.05),suggesting that vagotomy had no effect on the paw edema of the hind limbs in the CIA rats.On the 28th day after the initial immunization,compared with the CIA+VGX group,the trend of paw edema of the hind limbs in the CIA+VGX+taVNS group was similar to that of the CIA+VGX+taVNS group,with both groups slowly decreasing and the paw edema of the hind limbs in both groups eventually being about 3-4 mL(P>0.05).The effect of taVNS on the paw edema of the hind limbs of CIA rats disappeared after vagotomy,which means that the intact vagus nerve is required for the relief effect of taVNS on the paw edema of the hinds of CIA rats.2 Effect of vagotomy on taVNS modulation on MWT and TWT in CIA ratsOn day 28 after initial immunization,the MWT and TWT of rats in the CIA+VGX group showed a similar trend compared to those in the CIA group,both slowly increasing until the end of the intervention(P>0.05),suggesting that vagotomy had no effect on the MWT and TWT of CIA rats.On day 28 after initial immunization,the CIA+VGX+taVN S group showed a similar trend compared to the CIA+VGX+taVNS group,both slowly increasing to a final MWT and TWT of approximately 40-60g(P>0.05),suggesting that vagotomy significantly reversed the pain-relieving effect of taVNS on CIA rats,i.e.the pain-relieving effect of taVNS on arthritis pain in CIA rats The relieving effect requires the involvement of the intact vagus nerve.3 Effect of vagal nerve dissection on the appearance and joint histomorphology of the toe in taVNS-modulated CIA ratsThe rats in the CIA group,CIA+VGX group and CIA+VGX+taVNS group all showed purple skin colour,sparse hair and lack of lustre on the hind limbs,and all three groups showed moderate to severe swelling of the ankle joint and toes.According to the HE staining,the three groups of rats showed similar HE staining results,and all of them showed massive hyperplasia of synovial tissue,massive inflammatory cell infiltration and tissue debris in the synovial space.The above experimental results suggest that the effect of vagotomy on the appearance and joint morphology of the ankle joint in the CIA rats was not significant,while the alleviating effect of taVNS on the appearance and joint morphology of the ankle joint in the hind limbs of CIA rats disappeared after vagotomy,suggesting that the alleviating effect of taVNS on the appearance and joint morphology of the toe in CIA rats requires the involvement of the intact vagus nerve.4 Effect of vagotomy on changes in serum levels of relevant inflammatory factors in taVNS-regulated CIA ratsCompared with rats in the CIA group,there was no significant difference in the levels of inflammatory factors TNF-α,IL-6 and IL-1β in the serum of rats in the CIA+VGX group(P>0.05,P>0.05,P>0.05);compared with rats in the CIA+VGX group,there was no significant difference in the expression of TNF-α,IL-6 and IL-1βin the serum of rats in the CIA+VGX+taVNS group(P>0.05,P>0.05,P>0.05).The above experimental results indicated that vagotomy had no significant effect on the inflammatory response of CIA rats,while vagotomy significantly reversed the inhibitory effect of taVNS on the serum levels of inflammatory factors in CIA rats,suggesting that taVNS had a The mitigating effect of taVNS on inflammatory factors in serum of CIA rats requires the involvement of the intact vagus nerve.5 Effect of vagotomy on the expression of CAP-regulated proteins in the synovial tissue of taVNS-regulated CIA ratsThe expression levels of α7AchR,NF-κB p-p65/NF-κB p65 and TNF-α in the synovial tissues of rats in the CIA+VGX group were not significantly different compared to those in the CIA group(P>0.05,P>0.05 P>0.05);compared to rats in the CIA+VGX group,rats in the CIA+VGX+taVNS group The expression levels ofα7AchR,NF-κB p-p65/NF-κB p65 and TNF-α in synovial tissues were also not significantly different(P>0.05,P>0.05,P>0.05).The above experimental results indicated that vagotomy had no significant effect on the expression of α7AchR and the expression of the above three inflammation-related proteins in synovial tissues of CIA rats.Vagotomy significantly reversed the upregulation of α7AchR expression and the downregulation of NF-κB signaling and downstream TNF-α protein expression in the synovial tissue of CIA rats,suggesting that the regulatory effect of taVNS on CAP-related proteins in the synovial tissue of CIA rats requires the involvement of the intact vagus nerve.Results Ⅳ:Effect of α7AchR(MLA)inhibition on the inflammatory effects of taVNS on the synovial membrane1 Effect of MLA on taVNS regulation of arthritis index and toe swelling in CIA rats1.1 Effect of MLA on taVNS regulation of arthritis index in CIA ratsOn day 28 after initial immunization,the arthritis index of both feet was slightly higher in the CIA+MLA group compared to the CIA group,and then the trend was similar,with both rats slowly decreasing until the end of the intervention(P>0.05),suggesting that MLA injection did not have a significant effect on the appearance and behavioural phenotype of arthritis symptoms in the CIA rat model itself.On day 28 after the initial immunization,the arthritis index of rats in the CIA+MLA+taVNS group showed a similar trend compared to rats in the CIA+MLA+taVNS group,both slowly decreasing until the end of the intervention(P>0.05),indicating that the alleviating effect of taVNS on AI in CIA rats disappeared significantly after MLA injection,and since α7AchR is a key receptor for CAP,the above the experimental results suggest that the alleviating effect of taVNS on the severity of arthritis in CIA rats is related to the activation of CAP.1.2 Effect of MLA on paw edema in taVNS-regulated CIA ratsOn the 28th day after the initial immunization,the paw edema of the hind limbs was slightly higher in the CIA+MLA group compared with the CIA group,after which the paw edema of the hind limbs in both groups decreased slowly until the end of the intervention(P>0.05),suggesting that the injection of MLA slightly increased the paw edema of the hind limbs in the CIA rats.On the 28th day after the initial immunization,the paw edema of the hind limbs in the CIA+MLA+taVNS group showed a similar trend compared to that in the CIA+MLA+taVNS group,with both groups slowly decreasing and finally stabilizing at 3-4 mL(P>0.05).The above experimental results indicated that the effect of taVNS on the paw edema of hind limbs of CIA rats disappeared after MLA injection,suggesting that the effect of tavNS on the paw edema of hind limbs of CIA rats was related to the activation of CAP.2 Effect of MLA on taVNS regulation of MWT and TWT in CIA ratsOn day 28 after the initial immunization,the MWT and TWT of rats in the CIA+MLA group showed similar trends compared to those in the CIA group,both slowly increasing until the end of the intervention(P>0.05),suggesting that the injection of MLA did not affect the MWT and TWT of CIA rats.Compared with rats in the CIA+MLA group,rats in the CIA+MLA+taVNS group showed a similar trend,with both slowly increasing until the end of the intervention(P>0.05),indicating that the pain-relieving effect of taVNS on CIA rats disappeared significantly after MLA injection,suggesting that the pain-relieving effect of taVNS on arthritis pain in CIA rats was related to the activation of CAP.3 Effect of MLA on toe appearance and joint histomorphology in taVNS-conditioned CIA ratsThe rats in the CIA group,CIA+MLA group and CIA+MLA+taVNS group all showed purple skin and sparse hair on the hind limbs as well as moderate to severe swelling of the ankle joint and toes of the hind limbs in all groups,as seen in the external view.According to HE staining,the above three groups of rats showed similar pathological manifestations of synovial tissues,including massive hyperplasia of synovial tissues and massive infiltration of inflammatory cells.The above experimental results suggest that the effect of MLA on the appearance and joint tissue morphology of the hind limb ankle joint of the CIA rats was not significant,while the alleviating effect of taVNS on the appearance and joint tissue morphology of the hind limb ankle joint of the rats disappeared after MLA injection,suggesting that the alleviating effect of taVNS on the appearance and joint tissue morphology of the toe of the CIA rats was related to the activation of CAP.4 Effect of MLA on changes in serum levels of relevant inflammatory factors in taVNS-regulated CIA ratsCompared with rats in the CIA group,the expression of inflammatory factors TNF-α,IL-6 and IL-1β in the serum of rats in the CIA+MLA group were not significantly different(P>0.05,P>0.05,P>0.05);compared with rats in the CIA+MLA group,the expression of TNF-α,IL-6 and IL-1β in rats in the CIA+MLA+taVNS group were not significantly different(P>0.05,P>0.05,P>0.05).β expressions were not significantly different(P>0.05,P>0.05,P>0.05).The above experimental results indicated that ML A injection had no significant effect on the inflammatory response of CIA rats,while the inhibitory effect of taVNS on inflammatory factor content in serum of CIA rats disappeared after MLA injection,suggesting that the inhibitory effect of taVNS on inflammatory factor content in serum of CIA rats The inhibitory effect was related to the activation of CAP.5 Effect of MLA on the expression of CAP regulatory-related proteins in synovial tissue of taVNS-regulated CIA ratsThe expression levels of α7AchR,NF-κB p-p65/NF-κB p65,and TNF-α in the synovial tissues of rats in the CIA+MLA group were not significantly different compared to those in the CIA+MMLA group(P>0.05,P>0.05,P>0.05);the expression levels of α7AchR,NF-κB p-p65/NF-κB p65,and TNF-α in the synovial tissues of rats in the CIA+MLA group were not significantly different compared to those in the CIA+MLA group(P>0.05,P>0.05).There was no significant difference in the expression levels of α7AchR,NF-κB p-p65/NF-κB p65 and TNF-α in synovial tissue(P>0.05,P>0.05).The above experimental results suggest that MLA injection had no significant effect on the expression of α7AchR and the expression of the above three inflammation-related proteins in the synovial tissue of CIA rats However,the upregulation of α7AchR expression and the inhibition of its downstream NF-κB signal and TNF-α inflammatory factor protein expression by taVNS were significantly lost after MLA injection,which proved that the alleviating effect of taVNS on arthritis symptoms in CIA rats was related to the activation of CAP.ConclusiontaVNS was effective in alleviating the symptoms of synovitis in CIA rats,and this effect was dependent on the activation of intact vagal and cholinergic anti-inflammatory pathways. |
