Protective Effect of Tongluo Mingmu Formula on Retinal Ganglion Cell and Optic Nerve in Rat Model of Glaucoma Based on HSP72 Regulation
OBJECTIVE To investigate the protective effect of Tongluo Mingmu Formula on retinal ganglion cell(RGC)and optic nerve in rat models of glaucoma by regulating heat shock protein 72(HSP72).METHODS A total of 40 rats were used to establish the glaucoma model using the Shareef-Sharma method and were randomly divided into the model group(MG)and the Tongluo Mingmu Formula group(TM),with 20 rats in each group.An additional 24 rats underwent sham surgery and served as the control group(CG).Rats in the TM group were orally administered Tongluo Mingmu Formula at a dose of 8.61 g/kg daily,while rats in CG and MG were orally administered an equivalent volume of 0.9%sodium chloride injection daily for 21 days.On the first,seventh,14th,and 21st day after modeling,intraocular pressure was measured,and 0.5 mL of blood was collected from the retro-orbital sinus of the opposite eye of the rats.The enzyme-linked immunosorbent assay(ELISA)was used to detect serum HSP72 levels.After completion of treatment,hematoxylin-eosin(HE)staining was performed to observe the morphological structure of the rat retina,and TdT-mediated dUTP nick-end labeling(TUNEL)assay was used to detect the number and apoptosis rate of RGC in rats.Retinal thickness was also measured.Immunofluorescence and immunohistochemical methods were used to detect the expression of HSP72 in RGC.RESULTS(1)Intraocular pressure:In the MG group,intraocular pressure was higher than that in the CG group at first,seventh,14th,and 21st day after modeling(t1 d=7.758,t7 d= 10.913,t14 d=17.353,t21 d=7.955,all P=0.000).In the TM group,intraocular pressure was lower than that in the MG group at first,seventh,14th,and 21st dayafter modeling(t7 d=-2.604,P=0.021;t14 d=-8.815,P=0.000;t21 d=-4.163,P=0.001),with statistically significant differences.There were no statistically significant differences at other time points(P>0.05).(2)Serum HSP72:Serum HSP72 levels in the MG group were higher than those in the CG group at first,seventh,14th,and 21st day after modeling(t1 d=16.450,t7 d=23.576,t14 d=13.206,t21 d=8.934,all P=0.000).Serum HSP72 levels in the TM group were higher than those in the MG group at day 21 after modeling(t= 5.936,P=0.000),with statistically significant differences.There were no statistically significant differences at other time points(P>0.05).(3)Retinal morphology and thickness:RGC in the MG group showed reduced numbers and vacuolization,disordered arrangement and looseness of inner nuclear layer cells,edema of nerve fiber layer,and thinner inner nuclear layer compared to the CG group.In contrast,RGC in the TM group showed mild reduction in number,vacuolization,and less severe changes in inner nuclear layer cells compared to the MG group,with greater retinal thickness than the MG group.(4)RGC number and apoptosis rate:The number of RGC in the retina of rats in the MG group was lower than that in the CG group at 14th and 21st day after modeling(t14 d=-5.886,P=0.000;t21 d=-4.424,P=0.001),and the apoptosis rate was higher than that in the CG group at first,seventh,14th,and 21st dayGC in the retina of rats in the TM group was higher than that in the MG group at 14th and 21st day after modeling(t14 d=3.014,P=0.010;t21 d= 3.444,P=0.004),and the apoptosis rate was lower than that in the MG group at first,seventh,14th,and 21st dayafter modeling(t7 d=-9.762,t14 d=-8.639,t21 d=-9.674,all P=0.000),with statistically significant differences.There were no statistically significant differences at other time points(P>0.05).(5)Retinal HSP72:HSP72 expression in the retina of rats in the CG group was almost absent,while HSP72 expression in the MG and TM groups increased with time.HSP72 expression in the TM group was higher than that in the MG group at the same time points.CONCLUSIONS HSP72 has a protective effect on RGC in rat models of glaucoma,and Tongluo Mingmu Formula may enhance and maintain HSP72 expression to increase RGC survival,thereby protecting against glaucomatous optic neuropathy.
heat shock protein 72glaucomaTongluo Mingmu Formulaoptic nerveintra-ocular pressure
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