Study On The Effect And Mechanism Of Bushen Huoxue Therapyon RGCs Apoptosis Based On NGF/ASK1/FoxO1 And NGF/AKT/CREB Pathways

Background and purpose:The irreversible optic nerve damage of glaucoma poses a great threat to the visual quality of human beings.It is urgent to understand the occurrence and development mechanism of optic nerve damage of glaucoma,so as to exploit the effective neuroprotective drugs for the optic nerve.It was found that progressive apoptosis of retinal ganglion cells(RGCs)caused by high intraocular pressure(HIOP)is the fundamental cause of optic nerve damage in glaucoma.In addition,the expression of pro-survival and pro-death signaling factors play an important role in the apoptosis of RGCs,NGF/ASK1/Fox O1 and NGF/AKT/CREB pathways are strongly linked to cell apoptosis.Therefore,NGF/ASK1/Fox O1 and NGF/AKT/CREB pathways may be involved in the apoptosis of RGCs.In the past,the optic nerve protection of glaucoma mainly used Huoxue Tongluo,with less method of Ziyangganshen,rarely method of combining use of them,and fewer systematic studies.Bujing Yishi tablet withing the effect of Ziyangganshen and Huoxue Tongluo is an experienced prescription of professor Chen dafu,a famous ophthalmologist in traditional Chinese medicine,and it is the representative prescription of Bushen Huoxue method.Our preliminary clinical studies and animal experiments had shown that Bujing Yishi tablet has a good curative effect for protecting optic nerve of glaucoma.Nevertheless,the mechanism of the therapeutic effect still needs further exploration.Firstly,the purpose of this study was to establish the model of RGCs apoptosis induced by high pressure in vitro,so as to explore the effects of different pressure gradients on the apoptosis of RGCs,and the mechanism was studied from the perspective of NGF/ASK1/Fox O1 and NGF/AKT/CREB pathways.Then,the rat model of shareef-sharma chronic HIOP was established for investigating the intervention effect and mechanism of Bushenhuoxue(Bujing Yishi tablet)on the above pathways,for inheriting and exploring the experience of the famous specialists of traditional chinese medicine,so as to provide new ideas and methods for protecting optic nerve in glaucoma.Experiment ?:Study on the effect and mechanism of different pressure gradients on the model of RGCs apoptosis in vitro based on NGF/ASK1/Fox O1 and NGF/AKT/CREB pathwaysObjective:HIOP is an important factor in inducing apoptosis of RGCs.However,the underlying mechanism is still unclear.The study of the pathological characteristics of RGCs in vitro can avoid the limitations of interactions among cells,cells and extracellular matrix,and the complexity of the internal environment in vivo.RGCs apoptosis model(pressurized culture RGCs in vitro)is an ideal model to study the apoptosis mechanism of RGCs in glaucoma.This part of study have investigated the effect and mechanism of different pressure gradients on the model of RGCs apoptosis in vitro based on NGF/ASK1/Fox O1 and NGF/AKT/CREB pathways.Methods:The model of RGCs apoptosis induced by high pressure in vitro was established by open pressure control culture system.Primary cultured SD rat RGCs were exposed to five pressure gradients of 0 mm Hg,20 mm Hg,40 mm Hg,60 mm Hg and 80 mm Hg for 24 h.Morphological changes in RGCs were observed by inverted phase contrast microscope.The viability and apoptosis rate of RGCs were detected by the Cell Counting Kit-8(CCK-8)assay and Annexin V-FITC/PI flow cytometry,respectively.Western blotting and quantitative reverse transcription-polymerase chain reaction(q RT-PCR)were used to detect the expression and m RNA levels of NGF,AKT,ASK1,Fox O1 and CREB.Result:(1)The model of RGCs apoptosis induced by high pressure in vitro was established successfully: The viability rates of RGCsunder 40 mm Hg,60 mm Hg,and 80 mm Hg were significantly lower than0 mm Hgor 20 mm Hg(all P<0.01).The apoptosis rates of RGCs under40 mm Hg,60 mm Hg,and 80 mm Hgwere significantly higherthan 0 mm Hgor 20 mm Hg(all P<0.01).There was no significant difference in activity and apoptosis rate of RGCs between the 20 mm Hg group and the 0 mm Hg group(P> 0.05).In addition,from 40 mm Hg to 80 mm Hg,the viability rates decreased gradually with the increase of pressure(P< 0.05),and the apoptosis rate increased gradually with the increase of pressure(P< 0.05).(2)The effects of different pressure gradients on the morphological changes of RGCs: In the 0 mm Hg group and 20 mm Hg group,there were minute quantity apoptotic morphological changes.In the 40 mm Hg group,parts of the cell were shrunken or disrupted.In the 60 mm Hg group,neurite extension was weakened,and parts of cells were disintegrating or dying.In the 80 mm Hg group,the cells showed severe structural damage,the internal structures of the cells were indistinguishable at all.(3)The effects of different pressure gradients on the related proteins expression of NGF,AKT,ASK1,Fox O1 and CREB in NGF relevant pathway: Compared with 0 mm Hg group,there was no significant expression change of NGF,AKT,ASK1,Fox O1 and CREB in the 20 mm Hg group(P> 0.05).However,in the 40 mm Hg group,the 60 mm Hg group,andthe 80 mm Hg group,there were significant differences of NGF,AKT,ASK1,Fox O1 and CREBcompared the 0 mm Hg group and 20 mm Hg group(P < 0.01).The protein expression levels of NGF,AKT and CREB were decreased gradually with the increase of pressure(P< 0.05),while the protein expression levels of ASK1 and Fox O1 were increased gradually with the increase of pressure(P< 0.05).(4)The effects of different pressure gradients on the m RNArelative expression of NGF,AKT,ASK1,Fox O1 and CREB in NGF pathway: Compared with 0 mm Hg group,there was no significant of m RNArelative expression of NGF,AKT,ASK1,Fox O1 and CREB in the 20 mm Hg group(P> 0.05).However,in the 40 mm Hg group,the 60 mm Hg group,and the 80 mm Hg group,there were significant differences of m RNArelative expression of NGF,AKT,ASK1,Fox O1 and CREB compared the 0 mm Hg group and 20 mm Hg group(P <0.01).The m RNArelative expression levels of NGF,AKT and CREB were decreased gradually with the increase of pressure(P <0.05),while the m RNArelative expression levels of ASK1 and Fox O1 were increased gradually with the increase of pressure(P <0.05).(5)Comparison of NGF/ASK1/Fox O1 pathway and NGF/AKT/CREB pathway.There was no significant difference between the ASK1,Fox O1 protein expression,m RNA relative expression of NGF/ASK1/Fox O1 pathway and the CREB protein and m RNA relative expression of NGF/AKT/CREB pathway in the 20 mm Hg group(P >0.05).However,in the 40 mm Hg group,the 60 mm Hg group,the protein and m RNA relative expression of CREB of NGF/AKT/CREB pathway were significant higher than the ASK1,Fox O1 protein expression,m RNA relative expression of NGF/ASK1/Fox O1 pathway.Conclusion:(1)The model of RGCs apoptosis induced by high pressure in vitro can be successfully produced by adopting an open pressure control system.(2)The pressure of 40 mm Hg,60 mm Hg and 80 mm Hg can induce apoptosis morphological changes of cultured RGCs in vitro,reduce the activity of RGCs and increase the apoptosis rate of RGCs.However,20 mm Hg is similar to 0mm Hg of failing to cause corresponding changes.(3)The regulation of NGF/ASK1/Fox O1 and NGF/AKT/CREB signal pathways may be the molecular biological mechanism of RGCs apoptosis induced by pressure.(4)The NGF/AKT/CREB pathway may play a major role in the apoptosis of RGCs.Experiment ? : Study on the effect and mechanism of Bushen Huoxue(Bujing Yishi tablet)on retinal injury and RGCs apoptosis of the rat model of chronic HIOP based on NGF/AKT/CREB pathwayObjective:Our previous clinical study and animal experiments have also found that the Bushen Huoxue(Bujing Yishi tablet)has a good protective effect on the optic nerve of glaucoma.In addition,Experiment I " To study the effect and mechanism of different pressure gradients on the model of RGCs apoptosis in vitro based on NGF/ASK1/Fox O1 and NGF/AKT/CREB pathways " had found that NGF/AKT/CREB pathway may play a more important role in the apoptosis of RGCs.In order to further explore whether NGF/AKT/CREB signal pathway is involved in the optic nerve protection mechanism by Bushen Huoxue(Bujing Yishi tablet).Shareef-Sharma rats model of chronic HIOP was established to explore theeffect and mechanism on retinal injury and RGCs apoptosis of Bushen Huoxue(Bujing Yishi tablet).Method:The rat model of shareef-sharma chronic HIOP induced by cauterizing the superior scleral vein was established.Sixty rats were randomly divided into the normal group(20 rats),the treatment group(20 rats),and the model group(20 rats).The IOP of rats was monitored,and the correlation detections was implemented at 3 days and 2 monthsafter modeling.Retinal morphological structure was observed under high magnification of the microscope,retinal thickness and the number of RGCs were measured,the apoptosis rate of RGCs in rats was detected by Tunel method,and the expression of NGF,AKT and CREB proteins in RGCs were detected by immunohistochemistry.Result :(1)The rat model of shareef-sharma chronic HIOPwas established successfully: 3 days after modeling,IOP in the model groupwas significantly higher than that before modeling(P< 0.01).The IOP of the model group at 2 months after modeling was higher than the IOP of the model group before modeling and the normal group at 2 months after modeling.(2)The detection of retinal morphology,RGCs apoptosis rate,retinal thickness and the number of RGCs of shareef-sharma chronic HIOP rats: 3 days after modeling,in the model group and treatment group,the retinal structure was significantly damaged,interstitial edema,the number of RGCs was significantly lower than that of the normal group(P< 0.05),the apoptosis rate of RGCs in model group on 3 days after modeling was higher than that of the normal group(P< 0.05),and the retinal thickness was thicker than that of the normal group(P< 0.05),some RGCs presented irregular shape,become pale and vacuolate,and disorderly arrangement,and the inner and outer nuclear layers presented loosely arrangement.At 2 months after modeling,the retinal thickness was thinner than that of the normal group(P< 0.05),and the arrangement of inner and outer nuclear layers was significantly loose,the apoptosis rate of RGCs in model group at 2 months after modeling was higher than that of the normal group(P< 0.05),the number of RGCs was significantly lower than that of the normal group(P < 0.05),the RGCs presented an irregular arrangement and most RGCs had become vacuoles.In addition,compared with the model group of 3 days after modeling,the apoptosis rate of RGCs in model group at 2 months after modeling was increaser(P< 0.05),the number of RGCs was decreased(P< 0.05).(3)The influence of the traditional Chinese medicine of Bushen Huoxue(Bujing Yishi tablet)on the IOP,retinal morphology,RGCs apoptosis rate,number and retinal thickness of the rat model of shareef-sharma chronic HIOP: At 3 days after modeling,there were no significant differences in IOP,retinal morphology,RGCs apoptosis rate,number and retinal thickness between the treatment group and the model group(P >0.05).At 2 months after modeling,the IOP of the treatment group was lower than model group and the IOP of the treatment group at 3days after modeling(P< 0.05).Compared with the model group at 2 months after modeling,the retinal layers of the treatment group at 2 months after modeling were thicker(P< 0.05),the apoptosis rate was less(P< 0.05),the number of RGCs was increaser(P< 0.05),the RGCs morphology was generally normal,arrangement rule,the inner and outer nuclear layers were slightly disordered.In addition,compared with the treatment group at 3 days after modeling,the apoptosis rate of RGCs was lower than that of the treatment group at 2 months after modeling(P < 0.05).(4)The effect of Bushen Huoxue(Bujing Yishi tablet)on the expression of NGF,AKT and CREB of NGF/AKT/CREB pathway in RGCs of shareef-sharma rats model of chronic HIOP: At 3 days after modeling,the expression of NGF,AKT,CREB in the model group were markedly less compared with the normal group(P <0.05),there were no significant differences in NGF,AKT and CREB expression between the model group and the normal group.At 2 months after modeling,the expression of NGF,AKT and CREB in the model group was less than the model group at 3 days after modeling(P <0.05),the expression of NGF,AKT and CREB in the treatment group at 2 months after modeling were significantly increaser than the model group(P <0.05).In addition,the expression levels of NGF,AKT and CREB in the treatment group at 2 months after modeling were higher than the treatment group at 3 days after modeling(P< 0.05).Conclusion:(1)The rat model of shareef-sharma chronic HIOP induced by cauterizing the superior scleral vein can obtain stable and lasting HIOP.It can also simulate the progressive retinal pathology damage caused by HIOP in glaucoma: Progressive damage of retinal morphological structure,progressive thinning of retinal thickness,gradual increase of RGCs apoptosis rate and gradual decrease of the number of RGCs.(2)Bushen Huoxue(Bujing Yishi tablet)can inhibit the apoptosis of RGCs,prevent the reduction of RGCs,improve the pathological structural damage of retina,prevent the thinning of retinal thickness,and help to slightly reduce IOP in shareef-sharma chronic HIOP rat model,thus playing a role in protecting the optic nerve.(3)Bushen Huoxue(Bujing Yishi tablet)can promote expression of survival genes NGF,AKT and CREB of NGF/AKT/CREB signal transduction pathway in RGCs of chronic HIOP rats model of shareef-sharma.Furthermore,the expression of NGF,AKT and CREB were increasing with the prolongation of treatment time.It may play an important role in the mechanism for protecting glaucomatous optic nerve of Bushen Huoxue(Bujing Yishi tablet).