Effects Of Astragaloside IV On Retinal Microvascular Pericytes And Ganglion Cells In Rats Under Hypoxi

Obstract:Diabetes retinopathy（DR）is one of the most common neurovascular complications of diabetes and one of the main causes of blindness.With the increasing incidence rate of diabetes in China.DR blindness is also on the rise.Research shows that 30%～50%of diabetes patients have retinopathy,and 1/4 of them have obvious visual impairment,with a blindness rate of 8%～12%.The pathogenesis of DR is complex.In recent years,it is generally believed that the early lesions of DR are mainly retinal microvascular injuries.The persistent hyperglycemic environment in the blood vessels of diabetes patients causes ischemia and hypoxia in the local microenvironment of the retina,promotes the release of a large number of angiogenic factors,and generates new blood vessels.The immature new blood vessels are very fragile,easy to leak and bleed,resulting in vitreous cavity hemorrhage,traction retinal detachment and other complications,Seriously endangering the patient’s vision.Pericyte（RMPs）are the main parietal cell of microvessels.Selective apoptosis of pericyte is the first observed pathological change of DR retina.However,after in-depth research on DR,scholars found that neurodegenerative changes had occurred before the retinal microvascular pathological changes in diabetes,so they believed that DR was not only a microvascular disease,but also a neurodegenerative disease.The study also found that the destruction of the blood retinal barrier caused by retinal hypoxia,the increase of glutamate excitotoxicity,the damage of oxygen free radicals and inflammatory reactions can all cause damage or even death of retinal ganglion cell,leading to irreversible optic nerve damage.Diabetes retinopathy is one of the leading blinding eye diseases in the world,which seriously affects the quality of life of patients.Research shows that neovascularization is one of the main causes of visual loss in diabetes patients,and ischemia and hypoxia in the retina is the key factor of neovascularization.Since most patients in the early stage of DR have no conscious symptoms in the eyes,once the vision declines,it means that the retina has irreversible pathological changes.Laser photocoagulation and comprehensive control of blood sugar level can slow down the progress of DR,but it cannot reverse the clinical symptoms of diabetes retinopathy,and can not restore the blindness caused by diabetes.Therefore,early diagnosis and treatment are the key to preventing and treating DR.Astragalus membranaceus is a traditional Chinese medicine that uses its roots as a medicine.It has the functions of tonifying qi and elevating yang,consolidating the surface,stopping sweat,promoting diuresis and reducing swelling.Historical records have shown that Astragalus membranaceus was a commonly used medicine for treating thirst before the Ming Dynasty.Astragaloside Ⅳ（AS-Ⅳ）has the highest content in Astragalus membranaceus and is also the main active ingredient of Astragalus membranaceus.Modern research shows that AS-Ⅳ can significantly reduce the levels of blood sugar,triglyceride and insulin in T2DM mice,and can reduce liver gluconeogenesis.It can improve the damaged endothelium dependent relaxation in vivo and in vitro,reduce the apoptosis of venous endothelial cells and reduce inflammatory reaction,and has a good protective effect on the retina of diabetes patients.Therefore,the main research objective of this experiment is to explore whether AS-Ⅳ can protect retinal blood vessels and nerve cells,improve the local ischemic and hypoxic state of DR,prevent and delay the progression of DR,achieve early prevention and treatment,and reduce the blindness rate of patients.Direction:To explore the effect and mechanism of AS-Ⅳ,the main effective ingredient of Astragalus membranaceus,on the injury of retinal vascular pericyte（RMPs）and retinal ganglioncells（RGCs）in rats under hypoxia environment,and to study the effect of AS-Ⅳ at different doses on the experimental results,so as to provide an experimental basis for inhibiting the damage of RMPs and RGCs in early diabetes to delay the progression of retinopathy and clinical medication.Methods:Cells in the first generation of logarithmic growth phase cultured in vitro with good growth status were cultured with DMEM（low sugar type,containing 5.5mmol/l glucose,20%FBS,without green chain double antibody）for 24 hours before modeling.During the modeling process,peripheral and ganglion cells were cultured separately.The experimental group cells were rinsed twice with an oxygen free and sugar free equilibrium salt solution（D-Hank solution）,and then added to a sugar free and serum free DMEM culture medium.The cells were placed in a three gas incubator and treated in a mixture of 95%N2 and 5%CO₂ at 37℃ for the corresponding time according to their respective groups,establishing an oxygen deficient cell model.The blank group was cultured in low sugar DMEM medium containing 20%FBS under normal conditions of 37℃,95%O₂,and 5%CO₂.After 1,6,and 12 hours of cultivation,the experimental group was taken out and then cultured in DMEM（low sugar type,containing 5.5 mmol/L glucose,without FBS,and without green chain double antibody）for 48 hours.The survival rate of the two types of cells was measured relative to the blank group using the CCK8 method.Subsequent experiments were conducted on the number of hours when both cell survival rates were greater than and closest to 50%.After modeling,the original culture solution was aspirated and cleaned with PBS for three times.The drug added DMEM without serum and double antibodies was used for 48 hours,which was divided into five groups（including 15 groups）.Each group was provided with three double holes:a.blank group（5.5 mmol/L glucose low glucose DMEM+pericyte,ganglion cells,pericyte+ganglion cells）,b.control group（5.5 mmol/L glucose low glucose DMEM+pericyte,ganglion cells,pericyte+ganglion cells）,c.Pericyte+AS-Ⅳ low,medium and high dose group（1,10,100 mg/l+low glucose DMEM）,d.Ganglion cells+AS-Ⅳ low,medium and high dose group（1,10,100 mg/l+low glucose DMEM）,e.pericyte+Ganglion cells+AS-Ⅳ low,medium and high dose group（1,10,100 mg/l+low glucose DMEM）.The ratio of pericyte to ganglion cells in co culture was 1:1.After 48 hours of cultivation,the proliferation of RMPs and RGCs in each group was detected using the CCK8 method,and the proliferated cells were identified using immunofluorescence assay.Results:Compared with the control group,different concentrations of AS-Ⅳ groups（1,10,and 100mg/l）can promote the proliferation of RMPs cultured under hypoxia in vitro,and there is a dose-effect relationship within a certain concentration,with significant differences;Different concentrations of AS-Ⅳ groups（1,10,and 100mg/l）can promote the proliferation of RGCs cultured under hypoxia in vitro,and there is a dose-effect relationship within a certain concentration.The proliferation promoting effect of the AS-Ⅳ 100mg/l group was better than that of the AS-Ⅳ 1mg/l and 10mg/l groups,and the results were applicable to both individual and co cultured cells,with significant differences.The intervention of AS-Ⅳ on the combined cultivation of RMPs and RGCs in hypoxic environments is not as effective in promoting growth as solely cultivating RMPs or RGCs.After drug added culture,all pericyte alone expressed pericyte markers α-SMA,no expression of glial cell marker GFAP,single ganglion cell group all expressed ganglion cell markers Brn3a and Map2,no expression of GFAP,pericyte+ganglion cell mixed group all expressed their respective markers α-SMA and Brn3a do not express GFAP.Conclusions:AS-Ⅳ has a promoting effect on the growth of rat retinal microvascular RMPs and RGCs cultured in an in vitro hypoxic environment,and exhibits a dose-response relationship within a certain concentration.AS-Ⅳ intervenes in the co cultivation of RMPs and RGCs in hypoxic environments,but its growth promoting effect is not as good as that of RMPs or RGCs cultured alone.This suggests that under co culture conditions,there may be some interactive relationship between the two types of cells,which can avoid excessive cell growth.The specific mechanism needs further research.