Effect And Molecular Mechanism Of Thyroxine On Lens-Induced Myopia

Objective1.To study the effect of excess thyroxine on the spherical equivalent,axial length,anal temperature and bodyweight of negative lens-induced myopia in guinea pigs,laying the foundation for exploring the relationship between hyperthyroidism and myopia and providing new ideas for the prevention and control of myopia.2.The serum levels of estradiol（E2）,testosterone（T）,free triiodothyronine（FT3）,and free thyroxine（FT4）,cyclic adenosine monophosphate（c AMP）and cyclic guanosine monophosphate（cGMP）were analyzed in guinea pigs by enzyme-linked immunosorbent assay（ELISA）.To explore the changes in serum hormone levels and cyclic nucleotide levels in hyperthyroid guinea pigs and to preliminarily elucidate the biological basis and molecular mechanism of myopia exacerbated by excess thyroxine.3.To investigate the changes of m RNA and protein expression levels of insulin-like growth factor（IGF-1）in the retina,matrix metalloproteinase-2（MMP-2）and signal transducer and activator of transcription 3（STAT3）in the sclera of negative lens-induced myopia in guinea pigs by Real-Time quantitative PCR（qPCR）and Western blot,and to further clarify the molecular mechanism of excess thyroxine aggravating the course of myopia.Methods1.45 two-week-old British male tricolor short-hair guinea pigs were randomly divided into 3 groups,including normal control（NC）group,lens-induced myopia group（LIM group）and thyroxine+lens-induced group（TH+LIM group）.During experiment,the environment temperature was controlled at about 24℃,and the circadian rhythm of 12h/12h was maintained.TH+LIM group was intraperitoneally injected levothyroxine sodium solution(0.05mg·kg^-1)between 8:30 and 10:30 am.NC group and LIM group were intraperitoneally injected with equivalent volumes of saline at the same time.From the 4th day of intraperitoneal injection,the right eyes of LIM group and TH+LIM group were all wearing-6.00D lens for 4 weeks.The left eyes of them as the self-control eyes were not wearing glasses.The eyes of NC group as the blank control eyes without any intervention.General behavior was observed.The body weight of guinea pigs（accurate to 0.01g）was recorded at0 weeks,1 week,2 weeks,3 weeks and 4 weeks after combined treatment.The anal temperature of guinea pigs at 0 weeks,1 week,2 weeks,3 weeks and 4 weeks after combined treatment was measured by Vicks thermometer.Spherical equivalent by streak retinoscopy and axial length by Ophthalmology A ultrasonic instrument were measured and recorded respectively at 0 weeks,2 weeks and 4 weeks.2.The levels of hormones and cyclic nucleotides in guinea pig serum were detected by ELISA.After treatment for 4 weeks,blood was collected by cardiac puncture,and serum was obtained after centrifugation,which was placed in the-80°C refrigerator.Levels of free FT3,FT4,E2,T,c AMP and cGMP were detected by ELISA.3.Posterior scleral thickness was measured.After treatment for 4 weeks,the guinea pigs were anesthetized by intraperitoneal injection of pentobarbital.The eyes were quickly removed,fixed with eye fixing solution,dehydrated,embedded in paraffin,and sliced parallel to the optic nerve on a paraffin slicer.Sections about 4μm thick were collected when approaching the optic disk.Paraffin sections obtained by hematoxylin-eosin（HE）.The thickness of sclera at the posterior pole of each group was measured by the 200×magnification light microscope.The thickness of the central sclera was measured for each section.4.After 4 weeks of joint modeling,the guinea pigs were sacrificed by anesthesia and their right eyes were removed and preserved.The m RNA and protein expression levels of IGF-1 in retina,MMP-2 and STAT3 in sclera were determined by qPCR and Western blot.Results1.The guinea pig model of hyperthyroidism and lens-induced myopia was successfully constructed using intraperitoneal injection of thyroxine combined with negative lens induction.After 4 weeks of joint modeling,the guinea pigs in the TH+LIM group showed clinical symptoms of hyperthyroidism,such as dry hair,hair shedding,eating too much but losing weight,black and dry stools,irritability,aggression,difficulty grasping,increased activity frequency and so on.The NC group and LIM group performed normally.After 1weeks of intraperitoneal injection of thyroxine,the anal temperature was significantly increased in the TH+LIM group than those of NC group and LIM group.After 2 weeks of intraperitoneal injection of thyroxine,the bodyweight of guinea pigs in the TH+LIM group was significantly decreased,which continued until the end of the 4-week modeling.After 4weeks of joint modeling,compared with the NC group,the spherical equivalent and the spherical equivalent difference between the left and right eyes（right eye minus left eye）were significantly decreased in the LIM group.Compared with the LIM group,the TH+LIM group’s spherical equivalent and the spherical equivalent difference of two eyes were significantly decreased.The axial length and the difference of the axial length between the left and right eyes of LIM group were significantly longer than that of NC group.The axial length of TH+LIM group was shorter than that of LIM group,but the difference between left and right eyes was obviously longer than that of LIM group.2.After 4 weeks of treatment,compared with NC group,the serum levels of FT3,FT4,E2,T,c AMP,cGMP and c AMP/cGMP ratio of LIM group were unchanged.The serum levels of FT3,FT4,E2,c AMP and the c AMP/cGMP ratio in TH+LIM group were significantly increased,the T hormone level was significantly decreased,and the cGMP hormone level was unchanged.Compared with LIM group,the serum levels of FT3,FT4,E2,c AMP and the c AMP/cGMP ratio in TH+LIM group were increased significantly,and the hormone levels of T was decreased significantly.There was no significant difference in cGMP hormone levels.3.The HE staining results showed that after 4 weeks of joint modeling,the sclera thickness of posterior pole of NC group,LIM group and TH+LIM group became thinner in order.The sclera thickness of the posterior pole of the guinea pigs in the NC group was normal,the collagen fibers were thicker,and the fibers were arranged regularly and tightly without breakage.The sclera of the guinea pigs in the LIM group became thinner,the collagen fibers became looser and thinner,and their gap became larger,still no fiber fracture phenomenon.The sclera of posterior pole of TH+LIM group was the thinnest,collagen fibers were the loosest,and some fibers were broken.4.The results of molecular experiment showed that,after 4 weeks of modeling,compared with NC group,the m RNA and protein expression levels of IGF-1 in retina,MMP-2 and STAT3 in sclera of LIM group and TH+LIM group were increased significantly.Compared with LIM group,the m RNA and protein levels of IGF-1 in retina,MMP-2 and STAT3 in sclera of TH+LIM group were increased significantly.ConclusionThyroxine deepens the degree of myopia in negative lens-induced guinea pigs,which may be related to the increased expression levels of IGF-1 in retina and MMP-2 and STAT3in sclera.