| BackgroundDry eye is a chronic ocular surface disease caused by multiple factors.It is an unstable tear film or an imbalance of the ocular surface microenvironment caused by abnormal tear quality,quantity and dynamics.It may be accompanied by ocular surface inflammatory reactions,tissue damage and neurological abnormalities.,Causing a variety of ocular discomfort symptoms and(or)visual dysfunction.Inflammation has been confirmed as the core mechanism of the pathogenesis of dry eye.Therefore,inhibiting the ocular surface inflammation of dry eye is the key to the treatment of this disease.Clinical studies have found that when acupuncture is used to treat dry eye,selecting acupoints around the eyes can significantly improve the symptoms of patients,which may be related to improving the microcirculation of the tissues around the eyes.Experimental studies have found that acupuncture can effectively repair lacrimal gland cells,promote tear secretion,and inhibit Lacrimal gland tissue cell apoptosis,inhibits the pro-inflammatory activity of inflammatory factors,regulates the level of sex hormones,and promotes the activation of acetylcholine(Ach)neurotransmitter.Therefore,we infer that acupuncture can be used as a conventional treatment for dry eye.Dry eye is closely related to the central nervous system.The brain center transmits inflammatory signals to the efferent vagus nerve,which stimulates the vagus nerve to release ACh and combine with acetylcholine receptor 7(? 7nAChR)to transfer the signal into the cell,regulate the production of cytokines,and exert anti-inflammatory effects.Inflammation,and the anti-inflammatory mechanism in cells is mainly related to the regulation of NF-? B signaling pathway.Therefore,we boldly assume that acupuncture can activate the "cholinergic anti-inflammatory pathway" and analyze the mechanism of acupuncture on dry eye in the dry eye rabbit model.purposeBased on the clinical and experimental research on the treatment of dry eye with acupuncture,as well as the theoretical understanding of the "cholinergcic anti-inflammatory pathway",the inflammatory changes of ocular surface tissues are observed from the microscopic and surface through specific biological indicators,and the acupuncture treatment of dry eye is discussed.Curative effect mechanism of ocular surface inflammation;combined with molecular protein level to observe the pathogenesis of dry eye ocular surface inflammation,expound the influence of acupuncture on cholinergic anti-inflammatory signal pathway and related factors,so as to provide scientific basis for the treatment of dry eye ocular surface inflammation.methodExperiment 1:Healthy New Zealand white rabbits were randomly divided into blank group,model group,sham acupuncture group,acupuncture group,fluorometholone group and acupuncture combined group.Blank group:subcutaneous injection of normal saline 2ml/time 4 times a day for 35 days;model group:subcutaneous injection of scopolamine hydrobromide 2.0 mg/kg/time 4 times a day for 35 days:sham acupuncture group:according to model After the group was successfully modeled,on the 22nd day,the five acupoints Jingming,Sizhukong,Cuanzhu,Tongzilian and Temple were treated with blunt needles.They were pricked separately,but not subcutaneously.At the same time,hydrobromic acid was injected subcutaneously.Scopolamine for 14 consecutive days;Acupuncture group:After successful modeling according to the model group,electro-acupuncture treatment was performed on the 22nd day.The acupoints were the same as those in the sham acupuncture group.Acupoints were selected on both sides,once a day,with a needle retention time of 15 minutes.Scopolamine hydrobromide was injected subcutaneously for 14 consecutive days;fluorometholone group:After successful modeling according to the model group,on the 22nd day,fluorometholone eye drops were given to both eyes 3 times a day,1 drop each time,for 14 consecutive days,at the same time Scopolamine hydrobromide was injected subcutaneously for 14 consecutive days;acupuncture and drug combination group:After successful modeling according to the model group,the 22nd day was treated with fluorometholone eye drops and electroacupuncture at the same time.The treatment plan was the same as the fluorometholone group and Acupuncture group.The amount of tear secretion,tear film rupture time,corneal fluorescence staining,corneal confocal microscopy,light microscopy,and electron microscopy were detected in each group.Enzyme-linked immunoassay was used to detect changes in the levels of ACh and ?7nAChR in the cornea and lacrimal glands.Western blot was used to detect corneal and The expression of NF-kB protein in the lacrimal gland,protein chip technology detects the changes in the content of inflammatory factors in the cornea and lacrimal gland.Experiment 2:Healthy New Zealand white rabbits were randomly divided into blank group,model group,acupuncture group,?7nAChR antagonism group,?7nAChR antagonism plus western medicine group,and ?7nAChR antagonism plus acupuncture group.The modeling and intervention methods of the blank group,model group,and acupuncture group were the same as experiment one;?7nAChR antagonist group:After the successful modeling of the animal,on the 22nd day,intravenous injection of ?-BGT(?-BGT)4.0ug/kg per day,at the same time subcutaneous injection of scopolamine hydrobromide for 14 consecutive days.?7nAChR antagonism plus western medicine group:After the animal model was successfully established,on the 22nd day,?-BGT 4.0ug/kg was injected intravenously into the ear edge,and fluorometholone eye drops were instilled in both eyes at the same time.The method was the same as above.14 days.?7nAChR antagonism plus acupuncture group:After the animal model was successfully established,on the 22nd day,?-BGT 4.0ug/kg was injected into the marginal ear vein and acupuncture treatment was performed at the same time.The acupuncture method was the same as above,and scopolamine hydrobromide was injected subcutaneously for 14 consecutive days.The detection method is the same as Experiment 1.Result(1)Tear flow test results:In experiment 1,on the 21st day of the experiment,the tear flow of each group decreased significantly compared with before the experiment,and there were significant differences,and the tear flow of the model group continued to decrease on the 27th and 35th days.And compared with the 21-day tear flow in the same group,there is a significant difference.The tear flow test results on the 35th day showed that the tear flow of the acupuncture group and the western medicine group was higher than that of the model group,and there was a significant difference.After using the ?7nAChR antagonist ?-BGT in experiment 2,the tear flow test results on the 35th day showed that there was no significant difference in tear flow between the model group and the antagonist group;the tear flow of the acupuncture group was higher than that of the model group,the antagonist group and the antagonist group There are significant differences in the acupuncture group;the tear flow of the antagonistic western medicine group is significantly higher than that in the model group,and there are significant differences.(2)Corneal fluorescence staining test results:Experiment 1 started on the 21st day of the experiment.The corneal fluorescence staining scores of each group were higher than those before the experiment.There were significant differences,and the corneal fluorescence staining scores of the model group were higher than those of the experiment on the 27th and 35th days.The former significant difference is more obvious.The corneal fluorescence staining score results on the 35th day showed that there was no significant difference between the model group and the sham acupuncture group.The corneal fluorescence staining scores of the acupuncture group and the western medicine group were lower than the model group,and there was a significant difference.Experimental results of the second experiment found that after using the?7nAChR antagonist ?-BGT,there was no significant difference in corneal fluorescence staining scores between the model group and the antagonist group on day 35;the corneal fluorescence staining scores of the acupuncture group were lower than those of the model group,antagonist group and antagonistic needle Acupuncture group;the corneal fluorescence staining score of the antagonistic western medicine group is lower than that of the model group,and there are significant differences in the above results.(3)The tear film rupture time comparison test results:the tear film rupture time was significantly decreased on the 7th day compared with the pre-experiment,and there was a significant difference,and each group was compared with the pre-experiment from the 21st day of the model building The significance of the difference is more obvious.The model group continued to decrease on the 27th and 35th day compared to the pre-experimental period.There was no significant difference between the model group and the sham acupuncture group on the 35th day.The tear film rupture time of the acupuncture group and the western medicine group was higher than that of the model group.Significant differences.In experiment two,after using the ?7nAChR antagonist ?-BGT,there was no significant difference in tear film rupture time between the model group and the antagonist group on day 35;the tear film rupture time of the acupuncture group and the antagonistic western medicine group was higher than that of the model group and the antagonist group;simultaneous acupuncture The tear film rupture time of the acupuncture group was higher than that of the antagonistic acupuncture group,and there were significant differences in the above results.(4)The results of the corneal stromal layer confocal microscope:the stromal layer cells of the blank group were in a quiet state,the nerve fiber branch reflections were uniform and coherent,and there were no abnormal nerve fiber numbers and walking shapes;a large number of globular immune cells were seen in the model group and the sham acupuncture group And the activated stromal layer cells with unclear borders and irregular sizes can be seen in areas with irregular intercellular gaps.The width of the nerve changes,and the reflection rate of the nerve branches is reduced.The branches are intermittent,all of which indicate the existence of inflammation;the stromal layer cell morphology of the acupuncture group The cells were slightly activated,nerve reflexes were occasionally intermittent,and nerve thickness was uneven;the shape and size of the stromal layer cells in the western medicine group increased regularly,the cells were slightly activated,nerve reflexes were occasionally intermittent,and nerve reflexes were not See obvious exception.(5)The results of light microscopy of the cornea and lacrimal gland:The corneal light microscope results showed that the corneal epithelium of the blank group showed 4-6 layers of epithelial cells,the cell layer was clear,the matrix was uniform,the polar orientation was normal,and the blood vessels and lymphocytes were not infiltrated;the surface of the corneal tissue of the model group and the sham acupuncture group Excessive keratinized squamous epithelial cells,lymphocyte infiltration,increased focal epithelial cell layers,shedding of surface epithelial cells,and polar disorder.In the acupuncture group and western medicine group,the number of corneal epithelial layers was slightly increased,a small amount of lymphocyte infiltration was seen,and corneal epithelial cells were slightly shedding.Evaluate the number of corneal epithelial cells:Compared with the blank group,the number of corneal epithelial cells in the model group and the sham acupuncture group was abnormally increased,p<0.05,which had a significant difference;compared with the model group,the cornea of the acupuncture group and the western medicine group The number of epithelial cells decreased,p<0.05,with a significant difference.In experiment two,after using the ?7nAChR antagonist,hyperkeratinized epithelial cells and lymphocyte infiltration were seen in the corneal tissue of the antagonistic acupuncture group,the number of focal epithelial cells increased,the surface epithelial cells fell off,and the polar direction was disordered.At the same time,the number of corneal epithelial cells in the antagonistic western medicine group was reduced compared with the model group,which has a significant difference;the number of corneal epithelial cells in the antagonistic acupuncture group was increased compared with the acupuncture group,with a significant difference.The results of lacrimal gland light microscopy showed that the blank group had no obvious abnormality in the columnar epithelial cells of the lacrimal gland tissue,with uniform size and rich cytoplasm;the epithelial cells of the lacrimal gland tissue in the model group and the sham acupuncture group showed obvious atrophy,the glandular cavity expanded,the secretion decreased,and the focal lymph nodes were visible.Cell infiltration;part of the epithelial cells of the lacrimal gland tissue in the acupuncture group and fluorometholone group atrophy,part of the gland cavity expands,and lymphocyte infiltration can be seen.Assess the area of lacrimal gland atrophy:Compared with the blank group,the area of lacrimal gland atrophy in the model group,sham acupuncture group,acupuncture group and western medicine group increased,with significant difference;compared with the model group,the acupuncture group and western medicine group had a significant difference in the area of lacrimal gland atrophy Decrease,there is a significant difference.In experiment two,after using the ?7nAChR antagonist ?-BGT,the corneal tissue of the antagonistic acupuncture group showed hyperkeratinized epithelial cells,lymphocyte infiltration,increased focal epithelial cell layers,shedding of surface epithelial cells,and polar disorder.At the same time,the number of corneal epithelial cells in the antagonistic western medicine group was reduced compared with the model group,which has a significant difference;the number of corneal epithelial cells in the antagonistic acupuncture group was increased compared with the acupuncture group,with a significant difference.(6)The content of ACh and ?7nAChR in cornea and lacrimal gland tissue:Experiment 1 The content of ACh and ?7nAChR in the model group in cornea and lacrimal gland tissue was significantly reduced,lower than that of the blank group,the difference was statistically significant;acupuncture and fluorometholone treatment Later,the content of ACh and ?7nAChR was significantly higher than that of the model group,and the difference was statistically significant.In Experiment 2,after using the ?7nAChR antagonist,compared with the acupuncture group,the content of ?7nAChR in the antagonistic acupuncture group was significantly reduced,and the difference was statistically significant.(7)The protein expression level of NF-?B in cornea and lacrimal gland tissue:Experiment 1 showed that compared with the blank group,the p-NF-?B/total NF-?B of the model group increased,indicating that the relative expression of NF-?B protein increased.The difference was statistically significant;compared with the model group,the p-NF-?B/total NF-?B decreased in the acupuncture group and the western medicine group,indicating that the relative expression of NF-?B protein was decreased,which was statistically significant;the second experiment was antagonized by ?7nAChR Compared with the acupuncture group after administration,the p-NF-?B/total NF-?B increased in the antagonistic acupuncture group,and the relative expression of NF-?B protein increased.The difference was statistically significant.(8)Detection results of inflammatory factors in cornea and lacrimal gland tissue:IL-1a,IL-1b,IL-8,TNF-?,IL-17A,MMP-9,IL-21,Leptin,The concentration of MIP-1b and NCAM-1 was higher than that of the blank group;the concentration of IL-1b and TNF-? in the lacrimal gland model group was higher than that of the blank group,the difference was statistically significant;compared with the model group,acupuncture improved dry cornea The expression of IL-1b,IL-21,IL-17A,MIP-1b and the expression of IL-1a,IL-1b,MIP-1b,TNF-?,and IL-8 in the lacrimal gland tissue showed statistically significant differences.In experiment two,after using ?7nAChR antagonist,the corneal antagonism acupuncture group compared with the acupuncture group IL-1b,IL-17A,MMP-9,TNF-? expression and lacrimal gland antagonism acupuncture group IL-1a,IL-1b,The expression of IL-8 and TNF-? both increased,and the difference was statistically significant.Conclusion(1)Acupuncture can reduce corneal fluorescence staining,increase tear secretion and tear film rupture time,improve corneal and lacrimal gland tissue morphology;increase the expression of Ach and ?7nAChR receptors,and down-regulate the expression of NF-?B protein and related pro-inflammatory cytokines These beneficial effects are related to the up-regulation of ?7nAChR and the down-regulation of NF-?B.(2)After using the ?7nAChR antagonist,the beneficial effects of acupuncture are inhibited,which indicates that the treatment of dry eye by acupuncture is through the anti-inflammatory pathway mediated by ?7nAChR:that is,acupuncture can inhibit the activation of the NF-?B signal pathway by regulating ?7nAChR And reduce the expression of inflammatory factors to play an anti-inflammatory effect,so the ?7nAChR-mediated NF-?B signaling pathway may serve as a potential therapeutic target for acupuncture treatment of dry eye. |
PDF Full Text Request