| BackgroundKnee osteoarthritis(KOA)is the most common disease of joints,mainly clinically characterized by knee pain and limited mobility.Studies have shown that synovitis is an essential factor affecting the course and pain of KOA,and inhibition of synovitis is very important to inhibit the development of pain in patients with KOA.Acupuncture is one of the most common and effective complementary alternative therapies for the clinical treatment of knee OA.High-quality RCT studies at home and abroad have shown that acupuncture is able to reduce knee pain in patients with KOA,improve knee function,reduce pain in patients with KOA,and regulate the levels of related inflammatory factors.Basic research on acupuncture,published in recent years in the international top issue of nature and others,has shown that acupuncture can exert anti-inflammatory effects by activating sympathetic nerves.However,whether acupuncture alleviates KOA by exerting an anti-inflammatory effect through sympathetic immunomodulation remains to be further explored.In this study,we aimed to use sodium monoiodoacetate(MIA)-induced KOA model rats,which were combined with pain behavior testing,immunohistochemistry,immunofluorescence histochemistry,molecular biology,flow cytometry,and Western immunoblotting techniques,including changes in pain behavior,inflammatory factor content of tissues,immune cell populations of tissues,and protein phosphorylation levels of tissue signaling pathways The effects of electroacupuncture at "Zusanli "and "Yanglingquan"acupoints on synovial inflammatory responses in KOA model rats were investigated in terms of local tissues as well as systemic sympathetic noradrenergic pathway activation,and whether sympathetic noradrenergic pathways mediate electroacupuncture exerts anti-inflammatory effects in KOA model synovium.This study will provide a basis for the clinical application of electroacupuncture to alleviate KOA disease.Objective(1)To clarify the effect of electroacupuncture on the pain behavior of the foot Sanli-Yanglingquan acupoint area in KOA model rats and to determine the time point of electroacupuncture intervention intervention.(2)To investigate the anti-inflammatory effect of electroacupuncture on the inflammatory response of the synovial membrane in the KOA model rats..(3)To investigate whether local noradrenergic signals in the synovial membrane are involved in the anti-inflammatory effect of electroacupuncture on the synovial membrane of KOA model rats..MethodsThis experimental study is mainly divided into the following parts:(1)Observation of the effects of electroacupuncture on spontaneous pain and secondary pain behavior in KOA model ratsMale SD rats were randomly divided into control group,model group,early electroacupuncture group and mid-term electroacupuncture group.The rats in the model group,early electroacupuncture group and medium-term electroacupuncture group were injected with 1 mg/50 μL MIA solution in the right knee joint cavity to establish the KOA model.The rats in the control group were injected with the same dose of saline at the same site.After successful modeling,the rats in the electroacupuncture group were given electroacupuncture intervention in the foot Sanli-Yanglingquan acupuncture point area on days 1,2 and 3 after modeling in the early electroacupuncture group,and on days 5,7 and 9 after modeling in the middle electroacupuncture group.Electroacupuncture parameters:1 Hz/15 Hz,1 mA,30 min/day.Baseline pain behavioral indices(weight-bearing rate of the affected limb and plantar mechanical retraction reflex threshold of the affected limb)were measured before and after modeling in each group of rats..(2)Observation of the effects of electroacupuncture on local tissue damage and nerve damage in the joints of KOA model ratsBased on the experimental results in part(1),SD rats with the same conditions were randomly divided into control group,model group and electroacupuncture group.The modeling methods of the model and electroacupuncture groups were the same as above.The rats in the electroacupuncture group were given electroacupuncture intervention on days 5,7 and 9 after modeling.The electroacupuncture parameters were the same as above.The rats in the control and model groups were subjected to the same conditions as those in the electroacupuncture group,except that no electroacupuncture intervention was performed.Synovial histomorphology(HE staining and Masson staining)was observed on day 9 after modeling,and the articular cartilage damage scores on day 14 after modeling were compared.(3)Observation of the effect of electroacupuncture on the cytokine content of synovial tissue in KOA model ratsMale SD rats were randomly divided into control group,model group,early electro-acupuncture group and mid-term electro-acupuncture group.The rats in the model group,early electroacupuncture group and medium-term electroacupuncture group were injected with 1 mg/50 μL MIA solution in the right knee joint cavity to establish the KOA model.The rats in the control group were injected with the same dose of saline at the same site.After successful modeling,the rats in the electroacupuncture group were given electroacupuncture intervention in the foot Sanli-Yanglingquan acupuncture point area on days 1,2 and 3 after modeling in the early electroacupuncture group,and on days 5,7 and 9 after modeling in the middle electroacupuncture group.Electroacupuncture parameters:1 Hz/15 Hz,1 mA,30 min/day.Synovial inflammatory factor content was measured on day 1 after modeling in the early electroacupuncture intervention experiment and on day 9 after modeling in the medium-term electroacupuncture intervention experiment.Multi-spot detection(MSD)kits were used to detect inflammatory factors,including pro-inflammatory cytokines:tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β;anti-inflammatory cytokines:IL-4(IL-10,IL-13,Interferon gamma,IFN-y);chemokines:chemokine(C-X-C motif)ligand 1(CXCL1).(4)Observation of the effects of electroacupuncture on local adrenergic sympathetic activation and its transmitter content and receptor expression in the synovial membrane of KOA model ratsSD rats with the same condition were randomly divided into control group,model group and electroacupuncture group,and the modeling method was the same as above.The intervention method for the electroacupuncture group was the same as(2).After two hours of electroacupuncture on the 9th day after modeling,the rats were executed and plasma and synovial membrane of the affected limbs were collected for the following tests.(1)Norepinephrine(NE)was measured in the protein supernatant and plasma of synovial tissues by ELISA;(2)The expression level of beta 2 adrenergic receptor(beta2-AR)in synovial tissues was measured by protein immunoblotting;(3)Immunohistochemical staining was used to observe the expression level of dopamine in synovial tissues.The expression of dopamine βhydroxylase(DBH)markers in synovial tissues was observed by immunohistochemical staining..(5)Observation of the effect of blocking sympathetic norepinephrinergic signaling on the anti-inflammatory effect of electroacupunctureSD rats with the same conditions were randomly divided into control group,model group,electroacupuncture group,6-OHDA+electroacupuncture group and ICI118,551+electroacupuncture group.The modeling method was the same as above.Sympathetic noradrenergic signaling was blocked:6-Hydroxydopamine hydrobromide(6-OHDA)(150μg/50μl,dissolved in 0.02%ascorbic acid saline)was injected into the knee joint cavity the day before modeling);or ICI 118,551(1.5μg/50μl,dissolved in sterile saline);or ICI 118,551(1.5μg/50μl,dissolved in sterile saline).50μl,dissolved in sterile saline)was injected into the joint cavity half an hour before each electroacupuncture session.The electroacupuncture intervention was performed as in(2).Two hours after electroacupuncture on day 9,the weight-bearing rate of the affected limb was measured in each group of rats.Another group of rats was executed two hours after electroacupuncture on the 9th day after modeling,and the levels of pro-inflammatory cytokines TNF-α,IL-6,and chemokine CXCL1 in the synovial tissue protein supernatant were measured.(6)Observation of the effect of electroacupuncture on the activity of CXXL1/CXCR2 axis in the synovial membrane of KOA model ratsSD rats with the same conditions were randomly divided into control group,model group and electroacupuncture group,and the modeling method was the same as above.The intervention method for the electroacupuncture group was the same as(2).After two hours of electroacupuncture on the 9th day after modeling,the rats were executed,and serum and synovial membrane of the affected limbs were collected for the following tests.(i)the expression level of chemokine(C-X-C motif)receptor 2(CXCR2)in synovial tissue wasdetected by protein immunoblotting assay;(ii)the expression of CD11b+immune cell markers in synovial tissue was observed by immunohistochemical staining technique,and the expression of CXCR2 on CD11b+ expression of CXCR2 on macrophages.(7)Observation of the effects of electroacupuncture on synovial inflammatory response and related signaling pathways in KOA model ratsSD rats with the same conditions were randomly divided into control group,model group and electroacupuncture group,and the modeling method was the same as above.The intervention method for the electroacupuncture group was the same as(2).After two hours of electroacupuncture on the 9th day after modeling,the rats were executed,and the serum and synovial membrane of the affected limbs were collected for the following tests.(1)After the synovial tissue was processed into single cell suspension,the number of synovial CD11b+immune cell population and CD11b+CD86+M1 macrophage population were detected by using the corresponding antibody staining and flow cytometric fluorescence sorting technique;(2)The expression of CD68+macrophage markers in synovial tissue and IL-6 on CD68+macrophages were observed by using immunohistochemical staining technique.expression in synovial tissues;(iii)using MSD kits,the levels of inflammatory factors in serum were detected;(iv)the phosphorylation levels of ERK1/2 and MEK,representative molecules of mitogen-activated protein kinase(MAPK)pathway,were detected in synovial tissues by protein immunoblotting assay.(8)Effects of electroacupuncture on late saphenous nerve structure in KOA model ratsSD rats with the same conditions were randomly divided into control group,model group and electroacupuncture group,and the modeling method was the same as above.The intervention method for the electroacupuncture group was the same as(2).The rats were executed on the 14th day after modeling,and the ultrastructure of the saphenous nerve was observed by transmission electron microscopy.Results(1)Determination of the time point of electroacupuncture interventionOn the first day after modeling,the rats showed lameness symptoms and progressive aggravation,suggesting successful modeling.Compared with the model group,no improvement was observed in the weight-bearing and plantar mechanical pain thresholds of the affected limb in the early electroacupuncture group for 14 days,and no difference was observed in the synovial content of inflammatory factors on day 1 after modeling.In contrast,the weight-bearing of the affected limb in the medium-term electroacupuncture group improved on days 7,9,and 11 after modeling compared with the model group,suggesting that medium-term electroacupuncture improved spontaneous pain in the KOA model rats.At the same time,the plantar mechanical pain threshold of the affected limb on day 14 after modeling improved in the medium-term electroacupuncture group compared with that of the model group,suggesting that medium-term electroacupuncture improved the secondary touch stimulation-induced pain in the KOA model rats.(2)Electroacupuncture improved the local pathological damage of the joint in KOA model ratsThe results of synovial tissue morphological examination(HE staining and Masson staining)on the 9th day after modeling showed that the model group rats had obvious synovial tissue destruction,a large number of superficial synovial cells migrated to the subendothelial layer and showed obvious hyperplasia and inflammatory cell infiltration,while a large number of collagen fibers were seen,suggesting synovial fibrosis.Compared with the model group,the synovial surface of the rats in the electroacupuncture group was relatively smooth and more orderly,with less cell proliferation,no obvious mucus-like hyperplasia,less inflammatory cell infiltration and less synovial fibrosis.The injury score of knee cartilage showed that on the 14th day after modeling,the injury score of rats in the model group increased compared with that of the control group(P<0.001);compared with that of the model group,the injury score of rats in the electroacupuncture group decreased(P<0.05)..(3)Medium-term electroacupuncture reduces local synovial pro-inflammatory cytokine content in KOA model ratsThe results of MSD assay showed that,compared with the control group,the TNF-α,IL-6,IL-1β and CXCL1 contents in the synovial tissues of rats in the model group increased on day 1 after modeling(P<0.01).Compared with the control group,the levels of TNF-α,IL-6,IL-1β and CXCL1 in the synovial tissues of rats in the model group increased on day 9 after modeling(P<0.01);compared with the model group,the levels of TNF-α,IL-6,IL-1βand CXCL1 in the synovial tissues of rats in the mid-term electroacupuncture group decreased(P<0.05),while no significant differences were observed in the levels of various inflammatory factors in the serum of rats in each group.It is suggested that the modeling did not induce a systemic inflammatory response.(4)Electroacupuncture activates local sympathetic noradrenergic signals in synovial membraneOn the 9th day after modeling,the NE content in the synovial membrane of rats in the model group decreased compared with that in the control group(P<0.01);compared with that in the model group,the NE content in the synovial membrane of rats in the electroacupuncture group increased(P<0.01).And there was no significant difference in NE content in the plasma of rats in each group.Meanwhile,the results of protein immunoblotting experiments showed that the expression level of β2-AR in the synovial tissues of rats in the model group was decreased compared with that in the control group(P<0.001),and that the expression level of β2-AR in the synovial tissues of rats in the electroacupuncture group was increased compared with that in the model group(P<0.01).Immunohistochemical staining showed that the expression of DBH in the synovial membrane of rats in the electroacupuncture group was increased compared with that in the model group.It was suggested that electroacupuncture activated local adrenergic sympathetic nerves in synovial membrane,increased local NE content and activated β2-AR on synovial immune cells..(5)Local sympathetic noradrenergic signaling in synovium mediates the anti-inflammatory effect of electroacupunctureOn day 9 after modeling,compared with the control group,the weight-bearing rate of the affected limb in the model group decreased(P<0.001),and the contents of TNF-α,IL-6 and CXCL1 in synovial tissue increased(P<0.01);compared with the model group,the weight-bearing rate of the affected limb in the electroacupuncture group increased(P<0.01),and the contents of TNF-α,IL-6 and CXCL1 in synovial tissue decreased(P<0.05)..Compared with the electroacupuncture group,6-OHDA+electroacupuncture group and ICI118,551+electroacupuncture group rats had a lower weight-bearing rate of the affected limb(P<0.01)and higher TNF-α,IL-6 and CXCL1 contents in synovial tissues(P<0.05),and the pain relief and anti-inflammatory effects of electroacupuncture were affected in the KOA model rats.It is suggested that local sympathetic norepinephrinergic signaling in synovial membrane mediates the anti-inflammatory effect of electroacupuncture.(6)Electroacupuncture regulates CXCL1/CXCR2 axis activity,activates MAPK signaling pathway activity,regulates synovial macrophage function,and alleviates synovial inflammatory response in KOA model ratsThe results of the correlation analysis of double quantitative variable data suggested that CXCL1 content and IL-6 content in synovial membrane were highly correlated(n=90,r=0.6457,P<0.0001).The results of protein immunoblotting experiments showed that the CXCR2 expression level in the synovial tissue of rats in the model group was increased compared with the control group(P<0.01),and the CXCR2 expression level in the synovial tissue of rats in the electroacupuncture group was decreased compared with the model group(P<0.05).Immunohistochemical staining showed that the expression of immune cells CD11b and CXCR2 increased in the synovial tissues after modeling,and the expression of CD11b and CXCR2 decreased in the rats of the electroacupuncture group compared with that of the model group.It was suggested that the activity of CXCR2 on synovial immune cells in KOA rats was reduced after electroacupuncture.On day·9 after modeling,the number of CD 11 b+immune cell population and CD11b+CD86+M1 macrophage population in the synovial tissues of rats in the model group was increased compared with that in the control group(P<0.001);the number of CD11b+immune cell population and CD11b+CD86+M1 macrophage population in the synovial tissues of rats in the electroacupuncture group was decreased compared with that in the model group(P<0.001).Immunohistochemical staining showed that the expression of macrophage markers CD68 and IL-6 secreted by M1-type macrophages was increased in the synovial tissues of rats in the model group compared with the control group;the expression of CD68 and IL-6 in the synovial tissues of rats in the electroacupuncture group was decreased compared with the model group.The synovial macrophage population and its functional activities(M1-type polarization and pro-inflammatory cytokine secretion)were reduced in KOA rats after electroacupuncture.The results of protein immunoblotting showed that the phosphorylation levels of ERK1/2(P<0.001)and MEK(P<0.001),representative molecules of MAPK signaling pathway,were increased in the synovial tissue of rats in the model group compared with the control group,and the phosphorylation levels of ERK1/2(P<0.05)and MEK(P<0.001)were decreased in the synovial tissue of rats in the model group compared with the electroacupuncture group.The above results suggest that electroacupuncture alleviates the inflammatory response in the synovial membrane of KOA model rats by regulating CXCR2 expression and MAPK signaling pathway activity.(7)Electroacupuncture improved nerve demyelination in KOA rats at the later stage of the diseaseThe saphenous nerve in the model group showed obvious destruction of myelin structure,disintegration and loosening of myelin laminae,extrusion of axons or thickening of laminae and the appearance of fissures;compared with the model group,the damage of saphenous nerve in the electroacupuncture group was significantly reduced.It was suggested that electroacupuncture alleviated the tissue damage and late nerve injury in KOA model rats.Conclusion(1)Electroacupuncture alleviated spontaneous pain and touch-induced pain in KOA model rats and reduced local pathological joint damage.(2)Electroacupuncture activated local sympathetic nerve release of norepinephrine in the knee joint of KOA model rats,which acted on β2 receptors and alleviated local inflammatory response in the synovial membrane.(3)Electroacupuncture inhibits synovial CXCL1-CXCR2 expression and MAPK signaling pathway,inhibits the release of inflammatory factors from Ml-type macrophages in the synovial membrane of KOA model rats,and alleviates the synovial inflammatory response. |
PDF Full Text Request