Study On The Effect And Mechanism Of Fushiming Capsule In Ameliorating Diabetic Retina Damage

BackgroundDiabetes retinopathy(DR)is a neurovascular complication of diabetes and is one of the most common causes of blindness in working-age adults.The number of people with diabetes worldwide reached 400 million in 2017 and is expected to double by 2030.Among them,more than one third of diabetic patients have clinical symptoms of DR.At present,effective methods of DR treatment mainly include laser photocoagulation,vitrectomy and intravitreal injection of corticosteroids or anti-VEGF drugs.Although the above methods have certain clinical efficacy,they cannot completely inhibit the clinical progress of DR or reverse retinal injury.In addition,frequent intraocular injections can cause risks such as neurodegeneration and choroidal capillary atrophy,not to mention the high medical cost burden of frequent eye clinics.The available treatment options for patients are limited,which makes finding new drugs an urgent task.Traditional Chinese medicine is the most commonly used medicine for the prevention and treatment of diseases in clinical practice.The application of Chinese herbal medicine in the treatment of DR is one of the important clinical means of traditional Chinese medicine,which plays an important role in overcoming the limitations of chemotherapy in the treatment of DR.Fushiming capsule(FSM),a compound traditional Chinese medicine,is composed of 12 kinds of Chinese herbal medicine,such as angelica sinensis,American ginseng,astragalus root,prepared rhizome of rehmannia,cassia seed,safflower,pearl,whitmania pigra.In 2004,it was applied as a military hospital medical [LanLian system(2004),NO.FP68095] for clinical purposes.It is confirmed that the prescription has the function of tonifying Qi,promoting blood circulation,nourishing Yin and kidney,clearing collaterals and brightening eyes,and is applicable to treat diabetic retinopathy caused by Qi-deficiency and blood stasis,liver and kidney deficiency,and blood vessel congestion.However,the systematic pharmacological mechanism of FSM on DR remains obscure.ObjectivesThe purpose of this study was to investigate the effect of FSM on diabetic retinal pathological changes and its effects on oxidative stress and inflammatory response.By exploring the protective effect and mechanism of acetaldehyde dehydrogenase 2(ALDH2)on DR,it was further studied whether FSM could exert its anti-oxidative stress and anti-inflammation effects via ALDH2 pathway.Methods1.The antioxidant and anti-inflammatory effects of Fushiming capsule on diabetic rat retina damageDiabetic rat model was induced by 6-week high-fat and high-sugar diet combined with 35 mg/kg streptozotocin(STZ).30 days after successful establishment of diabetic rat model,full field electroretinograms(ffERG)and optical coherence tomography(OCT)were performed to detect retinal pathological alterations.According to the amplitude of dark adaptation OPs2,diabetic rats were randomly divided into 5 groups: model group(DM),Calcium Dobesilate positive group(CaD),FSM high-dose group(High),FSM medium-dose group(Medium)and FSM low-dose group(Low).Age-matched normal rats were selected as normal group(N).Rats in each group were given corresponding drug intervention for 42 days by gavage,and rats in DM group and N group were given normal saline of equal volume.By using methods such as ffERG,OCT,PCR,Western Blot and ELISA,the retinal function and structure were examined,retinal vascular endothelial growth factor-α(VEGF-α),glial fibrillary acidic(GFAP)and vascular cell adhesion protein 1(VCAM-1)expressions were measured both on mRNA and protein levels,and a series of blood metabolic indicators characterizing oxidative stress,inflammatory reaction and lipid metabolism of the body were also assessed,so as to evaluate the effect of Fushiming capsule on attenuating diabetic retina damage.2.The effect of ALDH2 on early-stage STZ-induced diabetic rat retina damage via Sirt1/Nrf2 pathway24 aged male diabetic Sprague-Dawley(SD)rats induced by a single intraperitoneal injection of STZ(60 mg/kg)were randomly divided into Alda1-treated group and dimethylsulfoxide(DMSO)group.Rats were intraperitoneally injected with 10 mg/kg ALDH2 activator Alda1(or DMSO)3 days before STZ injection and 30 days afterwards.A series of detections on retinal structural,functional and molecular levels were applied at 1 d,7 d and 30 d after aged diabetic rats model established.3.The mechanism of Fushiming capsule on STZ-induced diabetic rat retina damage via ALDH2 pathwayHealthy male SD rats were selected for a single intraperitoneal injection of STZ(60 mg/kg)to establish a diabetic rat model.Diabetic rats were randomly divided into diabetes model group(DM),calcium dobesilate positive group(DM+CaD),Fushiming group(DM+FSM),Fushiming+Alda1 group(DM+FSM+Alda1)and Fushiming+daidzin group(DM+FSM+daidzin)at 60 days post diabetic rat model established.Age-matched normal rats were selected as blank control group(Control).Alda1 is an agonist of ALDH2,and daidzin is an inhibitor of ALDH2.Rats in each group were given corresponding drugs for intervention by gavage,and normal saline and DMSO of the same volume were given to DM group and Control group.After 30 days of drug intervention,the effects of different drugs on DR were evaluated by detecting retinal function and structure,as well as related molecules of oxidative stress and inflammatory response.Results1.The antioxidant and anti-inflammatory effects of Fushiming capsule on diabetic rat retina damageIn DR rats,FSM(1.0g/kg and 0.5g/kg)treatment group significantly restored retinal function(b-wave in dark-adaptation 3.0 response and OPs2 wave)(P < 0.01)and prevented the decrease of retinal thickness including inner nuclear layer(INL),outer nuclear layer(ONL)and entire retina(P < 0.01).Additionally,FSM(1.0g/kg and 0.5g/kg)dramatically decreased VEGF-α,GFAP and VCAM-1 expressions in retinal tissues(P < 0.05).Moreover,FSM(1.0g/kg and 0.5g/kg)notably improved serum antioxidative enzymes glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)and catalase activities(CAT),whereas it reduced serum advanced glycation end products(AGEs),methane dicarboxylic aldehyde(MDA),nitric oxide(NO),total cholesterol(TC)and triglycerides(TG)levels(P < 0.05).2.The effect of ALDH2 on early-stage STZ-induced diabetic rat retina damage via Sirt1/Nrf2 pathwayOptical coherence tomography(OCT)revealed that the thickness of outer nuclear layer(ONL)and whole retinas in Alda1-treated group were thicker than DMSO group(ONL: 7 d and 30 d,P < 0.05;the whole retina: 30 d,P < 0.05).Full field electroretinograms(ffERG)showed a higher amplitude wave(b-wave in dark-adaptation 3.0 response and OPs wave)in Alda1-treated group(P < 0.05).In addition,the levels of retinal tumor necrosis factor(TNF-α)and interleukin-6(IL-6)from Alda1-treated group were lower(TNF-α: P < 0.05;IL-6: 1 d and 7 d,P < 0.05)whereas superoxide dismutase(SOD)activity was notably higher(P < 0.05).Moreover,the expressions of ALDH2,silence information regulation factor 2 related enzyme I(Sirt1)and nuclear factor erythroid 2-related factor 2(Nrf2)in Alda1-treated group retinas were significantly increased(P < 0.05),while the expression of vascular endothelial growth factor(VEGF-α)was dramatically decreased(7 d and 30 d,P < 0.05).3.The mechanism of Fushiming capsule on STZ-induced diabetic rat retina damage via ALDH2 pathwayFfERG showed that compared with the DM group,the amplitude of b-wave in dark-adaptation 3.0 response,b-wave in dark-adaptation 0.01 response,and OPs2 wave in DM+FSM+Alda1 group was significantly increased(P < 0.01,P < 0.01,P < 0.05),and the peek time of b-wave in dark-adaptation 3.0 response was notably shortened(P < 0.05).HE staining showed that compared with the DM group,the retinal ONL,NFL and the entire retinal thickness in the DM+FSM group and the DM+FSM+Alda1 group were thicker(P < 0.05).Elisa results showed that the levels of retinal TNF-α and IL-6 in DM+CaD group,DM+FSM group and DM+FSM+Alda1 group were lower than those of DM group(P < 0.05,P < 0.01),while SOD activity was significantly higher than that of DM group(P < 0.01).Meanwhile,immunohistochemistry showed that compared with the DM group,the expression levels of ALDH2 and SOD in the retinas of DM+CaD group,DM+FSM group and DM+FSM+Alda1 group were significantly increased,while the expression level of VEGF-α was significantly decreased.Conclusions1.FSM could ameliorate diabetic rat retina damage possibly via inhibiting inflammation and improving anti-oxidation.2.ALDH2 could ameliorate early-stage STZ-induced aged diabetic rat retina damage possibly via increasing Sirt1 and Nrf2 expression.3.FSM can exert its anti-oxidative stress and anti-inflammation effects through ALDH2 pathway.