Study On The Mechanism Of M(?)ller Cell Regulation Of Optic Nerve Protection By Chinese Medicine Compound VIS Based On ERK1/2 Pathway

Objective:To explore the regulatory effect of the traditional Chinese medicine compound VIS on the Müller cells of the glaucoma visual model and its possible mechanism.Methods:1.Study on the effect of traditional Chinese medicine compound VIS on the activation of Müller cells in the glaucoma model(1)Establish a rat model of chronic ocular hypertension by the method of suprascleral vein cauterization.Sixty SD rats were randomly divided into blank group(n=15),model group(n=17)and VIS group(n=18)after modeling.Three days after modeling,the model group and the VIS group were given normal saline and traditional Chinese medicine compound VIS solution by gavage treatment respectively.After 6 weeks,the materials were taken,and HE staining was used to evaluate the pathological changes such as retinal morphology,structure and thickness.The TUNEL method was used to detect the apoptosis of RGCs.Immunofluorescence,Western blot and qRT-PCR were used to detect the protein and mRNA expression levels of GFAP,p-ERK 1/2,and ERK 1/2,respectively.(2)Purify and culture RGCs and retinal Müller cells in vitro,establish a co-culture model,and divide them into a blank group,a pressurized group,and a pressurized dosing group(the pressurized dosing components are 4 VIS concentration groups:based on the LBP concentration in the compound Conversions are 20,40,80,and 160 mg/L respectively).An open pressure-controlled culture system was used to expose the RGCs and Müller co-cultured cells to a hydrostatic pressure of 60 mmHg for 24 h.The CCK-8 method was used to determine the cell survival rate to screen the optimal concentration of the drug for subsequent experiments.The TUNEL method is used to detect cell apoptosis,and immunofluorescence and Western blot are used to detect the protein expression of pERK 1/2 and ERK 1/2,respectively.2.Study on the mechanism of traditional Chinese medicine compound VIS to protect RGCs by regulating the metabolic function of Müller cells(1)Establish a rat model of chronic ocular hypertension through the suprascleral vein burning method,detect the level of ROS in the retina by flow cytometry,and detect the expression levels of GS,GLAST protein and mRNAby immunofluorescence,Western blot and qRT-PCR,respectively,and observe the retina Changes in the metabolic function of Müller cells.(2)Purify and culture RGCs and retinal Müller cells in vitro and establish a co-culture model.The mixed cells are divided into a blank group,a pressure group,and a pressure dosing group.Flow cytometry was used to detect the level of ROS in cells,and immunofluorescence and Western blot were used to detect the expression of GS and GLAST proteins,respectively.Results:1.The effect of traditional Chinese medicine compound VIS on the activation of Müller cells in the glaucoma model:(1)Intraocular pressure:In the 6th week,the intraocular pressure of the rats in the VIS group and the model group were maintained at 23.04 ±0.79 and 24.35± 1.02 mmHg,respectively,which were significantly higher than the blank group(P<0.05);the intraocular pressure after the intervention of the traditional Chinese medicine compound VIS It was slightly lower than the model group(P<0.05).HE staining:The retina in the model group showed obvious pathological changes.The thickness of the whole retina,ganglion cell layer(GCL),and inner plexiform layer(IPL)were significantly thinner than those in the blank group and the VIS group(P<0.05),and the GCL layer in the model group.The number of RGCs(17.67±2.08)was significantly reduced compared with the blank group(30.33±2.08)and the VIS group(23.00±1.73)(P<0.05),which further verified the success of the rat model of chronic ocular hypertension.Apoptosis rate of RGCs:Compared with the blank group,the apoptosis rate of RGCs in the model group and the VIS group was significantly higher(P<0.001),and the apoptosis of the VIS group was reduced compared with the model group after treatment.The apparent increase in apoptosis of RGCs in this model further confirmed the success of the rat chronic glaucoma animal model.Immunofluorescence:The blank group only expressed green fluorescently labeled GFAP at the end of Müller cells.The GFAP protein of the model group was significantly higher than that of the blank group(P<0.001);the increase in the VIS group was significantly better than that of the model group(P<0.001);The blank group expressed very little P-ERK 1/2,compared with the model group and the VIS group,the expression of P-ERK 1/2 increased significantly(P<0.05),while the expression of P-ERK 1/2 in the VIS group decreased compared with the model group(P<0.05).Western Blot:For GFAP and p-ERK 1/2 protein expression,the model group and VIS group increased significantly compared with the normal group(P<0.05),and the VIS group compared with the model group showed a decrease in the expression of both proteins(P<0.05).The p-ERK 1/2:ERK 1/2 ratio of the model group was significantly higher than that of the blank group and the VIS group(P<0.05).The qRT-PCR test results showed that the expression of GFAP mRNA was significantly reduced in the model group and the VIS group compared with the blank group(P<0.001).There was no significant change in ERK 1/2 mRNA in different groups.(2)Cell viability:When co-cultured cells were exposed to 60 mmHg hydrostatic pressure,the cell viability decreased significantly(P<0.05).Different concentrations of traditional Chinese medicine compound VIS(LBP concentrations were 20,40,80,160 mg/L)Can increase cell activity to varying degrees,and when the LBP concentration is 40 mg/L,the cell activity is the highest(P<0.05).Apoptosis rate of RGCs:The apoptosis rate of RGCs under hydrostatic pressure was significantly higher than that of the blank group(P<0.001).After the intervention of the traditional Chinese medicine compound VIS,cell apoptosis was inhibited,and the apoptosis rate was lower than that of the model group(P<0.001).Immunofluorescence:The ERK 1/2 signal pathway of mixed cells is activated under high pressure in vitro,which is manifested by increased expression of p-ERK 1/2 protein.The expression of p-ERK 1/2 protein in the pressurized group was significantly higher than that in the blank group(P<0.001).Western Blot:The p-ERK 1/2 in the compression group and the compression group were significantly increased compared to the blank group(P<0.001),and the expression of p-ERK 1/2 in the compression group was reduced compared with the compression group.(P<0.05),the expression of ERK 1/2 protein did not change.The p-ERK 1/2:ERK 1/2 ratio in the compression group was significantly higher than that in the blank group(P<0.001),and the compression group was reduced compared with the compression group(P>0.05).2.Study on the mechanism of traditional Chinese medicine compound VIS to protect RGCs by regulating the metabolic function of Muller cells:(1)Retina ROS level:The chronic HIOP model damages the rat retina and causes excessive ROS production in the cells.The positive rates of ROS in the blank group,model group and VIS group were 29.93±3.54%,42.66±2.67%and 35.97±2.71%,respectively.Compared with the blank group,ROS in the model group was significantly increased(P<0.05),and the positive expression of ROS in the VIS group was improved compared with the model group,and the difference was statistically significant(P<0.05).Immunofluorescence:GS and GLAST were specifically expressed in Muller cells.The expression of model group and VIS group was significantly lower than that of normal group(P<0.05).The expression of GS in VIS group had a tendency to increase compared with model group(P>0.05).GLAST protein The expression was significantly higher than that in the model group(P<0.05).Western Blot:The expression of GS protein and VIS group in the model group was significantly lower than that in the blank group(P<0.05),and the expression of GS in the VIS group and the model group was increased(P>0.05).Compared with the blank group,the expression of GLAST was significantly reduced in the model group and the VIS group(P<0.05),and the VIS group increased after intervention compared with the model group(P<0.05).qRT-PCR results:Regarding the expression of GS mRNA,the GS expression level of the model group and the VIS group was significantly lower than that of the blank group(P<0.05);the expression of GLAST mRNA in the three groups had the same changing trend,and the model and VIS groups were similar to the blank group.Compared with,the expression level of GLAST mRNA decreased(P>0.05).(2)ROS levels in mixed cells:In vitro high pressure induced an increase in intracellular ROS levels.The positive rates of ROS in the blank group,pressurized group,and pressurized group were 30.17 ± 3.25%,44.45 ± 2.53%and 36.06 ± 2.86%,respectively.Compared with the blank group,it was significantly increased(P<0.05).The pressurized drug group inhibited the production of ROS in mixed cells,which was statistically significant compared with the pressurized group(P<0.05).Immunofluorescence:GS and GLAST in the Müller cells of the mixed cell pressurization group under high pressure in vitro were significantly reduced compared with the blank group and the pressurization plus medicine group(P<0.001).Western Blot:Regarding the expression of GS and GLAST protein,the expression of the pressurized group and the pressurized drug group was lower than that of the blank group(P<0.05).The expression of GS and GLAST in the pressurized drug group was higher than that of the pressurized group.Trend(P>0.05).Conclusion:1.The traditional Chinese medicine compound VIS has the effect of slightly lowering IOP,and at the same time,it improves the morphological and structural damage of rat retina caused by high intraocular pressure,reduces the thinning of retinal thickness,reduces the apoptosis of RGCs in vivo and in vitro,and has certain optic nerve protection.2.Chronic HIOP rat model and in vitro high-pressure cell model showed that the expression of GFAP and p-ERK 1/2 increased,which marked the glial activation of Müller cells;the traditional Chinese medicine compound VIS could inhibit the expression of GFAP and p-ERK 1/2.It is suggested that preventing glial activation of Müller cells may be one of the mechanisms of VIS's glaucoma optic neuroprotection.3.Both the chronic HIOP rat model and the in vitro high-pressure cell model resulted in a decrease in the expression of GS and GLAST,marking the abnormal glutamate metabolism of Müller cells;the traditional Chinese medicine compound VIS has the effect of up-regulating the expression of GS and GLAST proteins,suggesting that it can improve Müller cell valleys Amino acid metabolism may be one of the mechanisms of VIS's glaucoma optic neuroprotection.4.Traditional Chinese medicine compound VIS can improve the oxidative stress response of chronic HIOP rat model and high-pressure cell model in vitro,so oxidative stress may be one of the targets of VIS.