Experimental Study Of Electroacupuncture Intervention On Ciliary Muscles From Lens-induced Myopia Guinea Pigs

ObjectiveTo establish a lens-induced myopia(LIM)guinea pig animal model and to analyze the pathological changes of ciliary muscle tissues during the development of myopia,and further to explore the molecular mechanisms that electroacupuncture at bilateral Taiyang and Hegu points delays the development of myopia of LIM guinea pigs,to provide theoretical basis for treating myopia using electroacupuncture in clinical practice.MethodsThe experiment included three parts.Part one:The role of electroacupuncture in the expression of epidermal growth factor and its receptor in the ciliary muscle from LIM guinea pigs.In this part,2-week-old healthy pigment guinea pigs(n=72)were randomly divided into normal control(NC)group,lens-induced myopia(LIM)group,lens-induced myopia+electroacupuncture(LIM+EA)group,and lens-induced myopia+electroacupuncture at sham acupoint(Sham)group,each group was divided into two time points of 2-week(n=9)and 4-week(n=9)according to the modeling time.Guinea pigs in the NC group were raised normally and received no treatment.Right eyes of the guinea pigs in the LIM group,LIM+EA group and Sham group were all wore-6 D lenses to induce myopia,while the fellow eyes treated nothing and were as self-control.Guinea pigs in LIM+EA group received electroacupuncture stimulation at bilateral acupoints(Hegu and Taiyang)simultaneously,while guinea pigs in Sham group were given electroacupuncture to stimulate the hips.The diopter and axial length of guinea pigs of each group were detected before enrollment and after 2-or 4-week treatment with myopia induction/electroacupuncture intervention.At 2-or 4-week treatment after modeling,histopathological section was prepared to observe the change of morphology in ciliary muscles,the expression of epidermal growth factor(EGF)and epidermal growth factor receptor(EGFR)at m RNA and protein levels were detected by quantitative polymerase chain reaction(q PCR)and enzyme-linked immunosorbent assay(ELISA).Part two:The effect of electroacupuncture on the homeostasis of potassium ions in the ciliary muscle tissue of lens-induced myopia guinea pigs.Guinea pigs(n=132)were randomly divided into four groups on average,and each group was divided into two time points of 2-week(n=15)and 4-week(n=18)according to the treatment duration.Others were identical to the part 1.The diopter and axial length of guinea pigs in each group were detected before enrollment and after 2-or 4-weektreatment with myopic induction/electroacupuncture intervention.At 2 and 4 weeks after modeling,the contents of adenosine triphosphate(ATP)and lactic acid(LA)in ciliary muscles were detected,the expression and activity of Na~+/K~+-ATPase in ciliary muscle tissues were determined.Moreover,the changes in potassium ion(K~+)flux in the ciliary muscles in each group were monitored at 4-week time point after modeling.Part three:Transcriptome analysis of ciliary muscle of lens-induced myopia guinea pigs.Twenty-seven guinea pigs were randomly divided into three groups,including NC group,LIM group and LIM+EA group.Others were identical to the contents in the first part,and the modeling time in each group maintained for 2 weeks.The diopter and axial length of guinea pigs in each group were detected before enrollment and after treatment for 2 weeks with myopic induction/electroacupuncture intervention.Ciliary muscle tissues of right eyes from 3 guinea pigs were randomly selected for transcriptome gene sequencing.Differentially expressed genes(DEGs)among groups were performed Gene Ontology(GO)biological process and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses,and the another ciliary muscle tissues in each group were used to verify the significant up-regulated expression factors in the complement and coagulation cascades pathway in KEGG analysis involved by DEGs in the LIM group including guinea pig complement factor H(CFH),guinea pig complement factor I(CFI)and guinea pig complement C4,and to investigate the effect of electroacupuncture on the expression of CFH,CFI and C4 in LIM guinea pig ciliary muscles.ResultsAfter modeling for 2 or 4 weeks,the myopic diopter and axial length of myopic eyes increased in each group compared with self-control eyes and the right eyes of the NC group,and the degree of myopia deepened as modeling time prolonged.The myopic diopter and axial length of myopic eyes in LIM+EA group were statistically lower than those in LIM group.There was no statistically significant difference in myopic diopter and axial length between Sham and LIM groups.Pathological results showed that the ciliary muscle fibers of the guinea pigs in the NC group were intact and well-ordered.The ciliary muscle fibers in LIM and Sham groups were broken,dissolved,and disordered,while those in the LIM+EA group were relatively complete and orderly arranged.Compared with NC group,EGF and EGFR expression,ATP content,Na~+/K~+-ATPase expression and activity in ciliary muscles of guinea pig in LIM group were significantly reduced,and LA content was significantly increased,while those in the LIM+EA group were not statistically different from the NC group;compared with LIM group,the EGF and EGFR expression,ATP content,Na~+/K~+-ATPase expression and activity in ciliary muscles of guinea pig in LIM+EA group were significantly increased,and the LA content was significantly reduced,while those in the Sham group were not statistically different from the LIM group.After modeling for 4 weeks,the K~+flux in ciliary muscles of guinea pig in both NC and LIM+EA groups showed fluctuations around the baseline,and the flow values were small;the K~+flux in ciliary muscles of guinea pig in both LIM and Sham groups showed an absorption state,and the flow values were large.After modeling for 2 weeks,there were 404 DEGs in the ciliary muscles of guinea pig in LIM group compared with those of NC group,including 131 up-regulated genes and 273 down-regulated genes.Compared with the LIM group,there were10 DEGs in the ciliary muscles of guinea pig in LIM+EA group,including 7 up-regulated genes and 3 down-regulated genes.The GO enrichment analysis of DEGs in each group mainly focuses on the aspects of cells,biological regulation,metabolism and immune system in biological processes.Further analysis revealed that DEGs in LIM group had 9significantly enriched KEGG pathways,mainly including S.aureus infection,complement and coagulation cascade pathways,rheumatoid arthritis,systemic lupus erythematosus.The upregulated molecules such as CFH,CFI and C4 in the complement and coagulation cascades pathway of the KEGG pathway enriched by DEGs in LIM group were verified.Compared with the NC group,the protein expression of the three molecules in LIM group were significantly up-regulated,and the results were consistent with those in transcriptomics analysis.The expression of the three molecular proteins in LIM+EA group was significantly down-regulated by contrast with those in NC group,and there was no statistically significant difference between LIM+EA group and NC group.ConclusionsLIM guinea pigs can induce ciliary muscle morphological damage,energy metabolism disorders and immune dysfunction,resulting in the disruption of K~+balance.Electroacupuncture stimulation at bilateral Taiyang and Hegu acupoints of the myopic guinea pig can maintain K~+homeostasis by increasing the expression and activity of Na~+/K~+-ATPase,thereby improving the physiological function and accommodative function of ciliary muscle and delaying the development of myopia.Moreover,electroacupuncture stimulation at bilateral Taiyang and Hegu acupoints can reduce the expression of related complement factors in the ciliary muscle tissues from LIM guinea pigs.