| Objective Retinal degeneration(RD)is a group of irreversible eye diseases characterized by progressive apoptotic degeneration of retinal photoreceptors,and microglia activation is involved in photoreceptor degeneration and apoptosis in RD,but the exact effects and mechanisms are unknown.It was found that neuronal degenerative apoptosis and microglia activation are mediated by and closely related to the purinergic receptor P2X7R/CX3CL1/CX3CR1 cascade.Acupuncture can exert neuroprotective effects on RD through multiple targets and mechanisms,but the underlying mechanisms of action need to be further elucidated.In this thesis,based on the previous research,we used the N-methyl-N-nitrosourea(MNU)-induced photoreceptor apoptosis rat model as the entry point,and observed the effect of acupuncture on photoreceptor degeneration and apoptosis in MNU model rats,taking photoreceptor apoptosis as a key pathogenic factor in RD.We investigated the effect of acupuncture on the activation of retinal microglia in the MNU model rats,and explored the association between the effect of acupuncture on microglia activation and the P2X7R/CX3CL1/CX3CR1 pathway,so as to explore the exact effect and possible mechanism of acupuncture on microglia activation,delaying photoreceptor apoptosis and protecting RD.MethodsPart ⅠA classical MNU photoreceptor apoptosis rat model was established using 1intraperitoneal injection of MNU.SD rats were randomly divided into two parts,the first part of rats was randomly divided into control group and model group(divided into 4 subgroups of 3 d,7 d,14 d and 28 d groups);The activation and distribution of microglia were analyzed by immunofluorescence,the structural damage of the rat retina was assessed by HE staining,and the apoptosis of retinal neurons was observed by TUNEL.The SD rats were randomly divided into blank control group,model group,V.A.group,no-acupuncture group and acupuncture group,except for the blank control group,all other groups were injected with MNU intraperitoneally to establish the classical photoreceptor apoptosis rat model.In the acupuncture group,acupuncture points were used to treat the "Jingming","Xinming I","Ganshu","Shenshu"and"Zusanli";in the no-acupuncture group,acupuncture points were used to treat the ingming","Xinming I","Ganshu","Shenshu"and "Zusanli" bilaterally and treated at the same time every day,with a frequency of 1 intervention/d,for 6 d,with an interval of 1 d.A total of 4 cycles of 7 d were used.for a total of 4 cycles of treatment.At the end of each group,the retinal photoreceptor cells,microglia and histopathological damage characteristics were evaluated.The activation and distribution of microglia were analysed by immunofluorescence,the expression of inflammation-related factors TNF-α and IL-1β was verified by ELISA,the structural damage of the rat retina was assessed by HE staining,and the apoptosis of retinal neurons was observed by TUNEL.Part ⅡThe rats were randomly divided into blank control group,model group,V.A.group,no-acupuncture group and acupuncture group,and the acupuncture points "Jingming","Xinming I","Ganshu","Shenshu" and "Zusanli" were used for 6 d of continuous intervention,with an interval of 1 d.7 d was a cycle,and a total of 4 cycles were treated.At the end of each group,CX3CL1 and CX3CR1 distribution markers were analyzed by immunofluorescence,and CX3CL1/CX3CR1 pathway-related factor protein and m RNA expression were observed by Western blot and RT-PCR.Part ⅢThe MNU photoreceptor apoptosis rat model was established and randomly divided into blank control group,model group,BBG(P2X7 receptor antagonist)group,acupuncture group and acupuncture+BBG group.The acupuncture treatment(acupuncture group and acupuncture + BBG group)was started 24 hours after moulding,with 6 d of continuous intervention and 1 d interval,7 d being a cycle,for a total of 4 cycles of treatment.After the intervention,HE staining was performed in each group to assess the structural damage of the retina,TUNEL method was used to observe the apoptosis of retinal photoreceptors,immunofluorescence co-localization was used to observe the expression of P2X7 receptors,activation of retinal microglia and the relationship between the two in each group,and Western blot and RT-PCR were used to investigate the retinal damage at different levels and angles.The expression of P2X7R/CX3CL1/CX3CR1 pathway key factor protein and its m RNA in the retinas of MNU rats after treatment were examined by a series of experiments using Western blot and RT-PCR.Results1.Acupuncture inhibits retinal microglia activity and photoreceptor cell degeneration and apoptosis in MNU model rats(1)A rat model of MNU photoreceptor apoptosis was successfully established.With the increase of time after MNU administration,the arrangement of retinal cells was sparse and disorganized,some of them had deepened staining,and the thickness of the outer nuclear layer was significantly thinned.The thickness of the outer nuclear layer was positively correlated with the modeling time,and there was a significant difference compared with the blank control group(P<0.01);the apoptosis of retinal photoreceptor cells was observed in all groups after MNU modeling by TUNEL,and the apoptosis rate was up-regulated with the prolonged administration time,with the highest apoptosis rate in the model 28 d group;immunofluorescence observation showed that microglia in the blank control group were limited to the inner and outer plexiform cell layers.The expression of IBA-1-positive cells in the retina of rats in the blank control group was limited to the inner and outer plexiform cell layers and RGCs cell layer,and the expression of IBA-1-positive cells was low,and the expression of IBA-1-positive cells increased with the prolonged administration time.(2)After the establishment of MNU photoreceptor apoptosis rat model,treatment interventions such as acupuncture were given,and HE staining showed that the retinal tissue damage was improved compared with the model after V.A.,no-acupuncture and acupuncture treatment,and certain ONL thickness could be preserved(P<0.01),and cell loss was reduced.The TUNEL method observed that compared with the model group,the V.A.,no-acupuncture group and acupuncture group all reduced photoreceptor cell apoptosis after treatment(P<0.05),and the effect of acupuncture treatment was better than no-acupuncture treatment;IBA-1 positive microglia migrated from the inner retina to the photoreceptor layer of the retina in the model group,resulting in a significant increase in the expression of microglia in the outer retina(P<0.01),and IBA-1(+)expression was significantly lower than that in the model group after acupuncture treatment and the V.A.intervention(P<0.01),while IBA-1(+)expression in the s no-acupuncture group was slightly weaker than that in the model group but the difference was not significant(P>0.05);Elisa results showed that retinal TNF-α and IL-1β levels were significantly increased in the model group compared with the blank control group(P<0.01),and compared with the model group,the acupuncture group intervention was significantly decreased the level of IL-1β and TNF-α in the retina(P<0.01),and was better than that in the no-acupuncture group(P<0.05).2.Acupuncture may regulate retinal photoreceptor-microglia interactions via CX3CL1/CX3CR1 signalling in MNU model ratsImmunofluorescence showed that the expression of CX3CL1 in the retina of the model group increased significantly after MNU injury(P<0.01),and the expression of CX3CL1 in the V.A.and acupuncture groups was significantly downregulated compared with that of the model group(P<0.01),while no-acupuncture could reduce its expression but the effect was not significant compared with that of the model group(P>0.05).The expression of CX3CR1 was significantly increased in the retina of the model group(P<0.01),and the increase was located in the photoreceptor layer;compared with the model group,the expression of CX3CR1 was significantly down-regulated in the V.A.,no-acupuncture group and acupuncture group(P<0.01);Western blot and RT-PCR results showed that the expression of CX3CL1,CX3CR1 protein and m RNA in the retina of the model group was significantly higher than that of the blank control group(P<0.01).CX3CL1 and CX3CR1 protein and m RNA expression in the retina of the model group were significantly lower than those in the model group(P< 0.05);p-P38 and p-SAPK/JNK protein levels were increased in the model group(P< 0.05)and p-ERK protein levels were decreased in the model group(P<0.05);compared with the model group,the p-P38 protein expression level was reduced in the V.A.group and acupuncture treatment(P<0.05).P38 protein expression levels(P<0.05)and up-regulated p-ERK protein expression(P<0.05),compared with the model group;the no-acupuncture group regulated neither p-P38 nor p-ERK expression significantly(P>0.05).3.Acupuncture may inhibit microglia activity and photoreceptor apoptosis through modulation of P2X7 receptor-mediated CX3CL1/CX3CR1 signaling(1)P2X7 receptor expression was inhibited by intraperitoneal injection of the antagonist BBG into MNU model mice.The level of apoptosis was significantly lower in the BBG,acupuncture and acupuncture+BBG groups than in the model group(P<0.01);immunofluorescence double-labeling showed that IBA-1 and P2X7 receptors were co-localized in microglia,and the expression of IBA-1 and P2X7 receptors were significantly up-regulated after MNU induction(P<0.01),while the positive expression of IBA-1 and P2X7 receptors was significantly down-regulated in the BBG,acupuncture and acupuncture+BBG groups(P<0.01).Compared with the model group,the BBG,acupuncture and acupuncture + BBG treatment interventions significantly reduced the expression levels of IL-6,TNF-α and IL-1β in the retina(P<0.01),and the acupuncture + BBG group had a better effect than the BBG group in reducing IL-1β content(P<0.05).(2)The protein expression of P2X7 R,CX3CL1,CX3CR1 and p-P38 in the MNU-induced photoreceptor apoptosis rat model group was significantly higher than that in the blank control group(P<0.01),while the expression level of P38 did not show any significant change(P>0.05),compared with the model group,the retinal P2X7 R,CX3CL1,CX3CR1 and p-P38 protein expression was inhibited in the BBG group and the retina in the acupuncture group(P<0.05),and the protein expression of CX3CL1 and CX3CR1 was significantly downregulated after acupuncture + BBG intervention(P<0.01),and the downregulation effect of CX3CR1 protein expression was better than that in the BBG group(P<0.05);the m RNA expression of P2X7 R,CX3CL1 and CX3CR1 in the retina of the model group were significantly higher than that of the The m RNA expression of P2X7 R,CX3CL1 and CX3CR1 in the model group was significantly lower than that in the blank control group(P<0.01),and the m RNA expression of P2X7 R,CX3CL1 and CX3CR1 in the acupuncture group,BBG group and acupuncture+BBG group were all significantly lower(P<0.05),and the effect of down-regulating P2X7 R and CX3CR1 m RNA expression in the acupuncture+BBG group was better than that in the BBG group(P<0.05).Conclusion1.Retinal microglia activation is involved in MNU-induced photoreceptor apoptosis,and acupuncture can improve the structural damage of retinal tissue in MNU model rats,inhibit microglia activation and migration,reduce the concentration of inflammatory factors,and decrease photoreceptor apoptosis.2.Acupuncture may interfere with retinal photoreceptor-microglia interactions through CX3CL1/CX3CR1 signaling,thereby inhibiting retinal damage in the MNU model mouse.3.Acupuncture may inhibit retinal microglia activity and proliferation,downregulate their mediated inflammatory factor expression,suppress inflammatory responses and slow retinal photoreceptor apoptosis by regulating the expression of P2X7 receptors and its mediated CX3CL1/CX3CR1 signaling pathway-related factors.P2X7R/CX3CL1/CX3CR1 is a possible mechanism of action of acupuncture in the treatment of RD. |
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