| Background: Diabetic retinopathy(DR)is one of the serious microvascular complications of diabetes,and is still the main cause of blindness among working-age people in developed countries.The prevention and development of DR has become one of the important goals to improve the quality of life of diabetic patients.The pathogenesis of DR is complex and has not been fully elucidated.At present,it is considered as a neurovascular complication.Inflammation plays an important role in the pathogenesis of neurovascular unit injury and may be a key therapeutic target in the early stage of DR.Epigenetics,as the mediating role of environment and genes,has gradually attracted people's attention in the pathogenesis of DR.Changes in micro RNA(mi RNA)level play an important role in the changes of retinal vascular endothelial function,apoptosis,inflammation and other pathological changes of DR,which has become a research hotspot.Qi-huang-mingmu capsule is a prescription formed in repeated clinical practice according to the theory of collateral diseases in Traditional Chinese medicine,which can improve the fundus microvascular lesions in early DR patients.Previous studies have found that QiHuang-Mingmu capsule can play a role by simultaneously participating in the regulation of multiple signal pathways in the pathogenesis of DR,but the specific mechanism of action needs to be further studied.Objective: Under the guidance of the theory of collateral-disease in Traditional Chinese medicine(TCM)and combined with the hot research on the regulation of micro RNA in epigenetics,the relevant mechanism of qi Huang Mingmu capsule in the prevention and treatment of DR was explored at the animal level: 1.To investigate whether Qi-Huang-Mingmu capsule could inhibit the NF-?B signaling pathway mediated by IL-1R/TLR and GRCP and inhibit retinal inflammation by up-regulating mi R-146 a.Targeted IL-6 regulates STAT3/VEGF signal transduction,reduces cell apoptosis and improves retinal endothelial function.The expression of extracellular matrix protein FN was down-regulated and the retinal microvascular fibrosis was reduced.2.To investigate whether Qi-Huang-Mingmu capsule can reduce retinal vascular permeability and protect the blood-retina barrier by reducing the amount of Evans Blue(EB)leakage and up-regulating the expression of tight junction protein.3.To investigate whether QiHuang-Mingmu capsule can improve retinal nerve function in diabetic rats.Methods: Male SD rats were fed a high-glucose and high-fat diet for 2 weeks +50mg/kg STZ intraperitoneal injection to induce type II diabetes model.According to the administration cycle,the experiment was randomly divided into three batches of animals according to their body weight.The disease course of the first group of animals was 1 week: blank control group,model group and model antagonist group.The model antagonist group was injected with 5?g mi R-146 a antagomir in single eye vitreous cavity,and the blank control group and model group were injected with the same amount of NC.One week later,the samples were taken for Western Blot to detect the protein expressions of mi R-146 a target genes IRAK1,TRAF6 and CRAD10.The disease course of the second group of animals was 6 weeks: blank control group,model group,positive drug group,qi-Huang-Mingmu capsule low-dose,medium-dose and high-dose control group and corresponding antagonistic group.Rats in the low,medium and high dose antagonistic groups were injected with 5?g mi R-146 a Antagomir in the vitreous cavity of single eye,and the other groups were injected with the same amount of NC in the control eye,once a week,for 6 consecutive times.The next day after the first injection of antagonist,animals in each group were given intragastric administration.Positive group,low,medium and high dose control group and antagonistic group were given calcium hydroxybenzoate(0.15g/kg)and low,medium and high dose Qi Huang Mingmu capsule(0.27g/kg,0.54g/kg and 1.08g/kg,respectively.Blank control group and model group were given pure water intragastric administration,once a day.After 6 weeks,IRAK1,TRAF6,CARD10,NF-?B,ICAM1,IL-6,STAT3(phosphorylation),VEGF,FN protein or m RNA expression were detected by Western Blot and real-time PCR.The course of the third group of animals was 12 weeks: blank control group,model group,positive group,low dose group,medium dose group and high dose group.Positive drug group and low,medium and high dose groups were given calcium hydroxybenzoate(0.15g/kg)and low,medium and high dose Qi Huang Mingmu capsules(0.27g/kg,0.54g/kg,1.08g/kg)intragastric administration,blank control group and model group were given pure water intragastric administration,once a day,after 12 weeks,ERG and fundus photography were performed.Samples were stained with HE,EB retinal perfusion,Western Blot and real-time PCR were used to detect the protein or m RNA expressions of IRAK1,TRAF6,CARD10,NF-?B,ICAM1,IL-6,STAT3(phosphorylation),VEGF,FN,Occludin and Claudin-5.The expression of ZO-1,Occludin and Claudin-5 was detected by immunohistochemistry.Results: 1.Effects of Qi-Huang-Mingmu Capsule on general state,body weight,blood glucose and retina morphology in diabetic rats:(1)The body weight of rats in blank control group increased gradually with the age of weeks,while the body weight of rats in other groups increased slowly or decreased.After 2 weeks of modeling,the body weight of rats in each group was significantly decreased compared with that in blank control group(P < 0.05).There was no significant difference in fasting blood glucose in each group before modeling,and the blood glucose in blank control group was always normal.After modeling,fasting blood glucose of diabetic rats was significantly higher than that of blank control group(P < 0.05).There were no significant differences in body weight and fasting blood glucose in model group,positive drug group,low,medium and high dose Chinese medicine groups.(2)After 6 weeks of model formation,some of the diabetic rats began to have unilateral or binocular lens opacity,and the lens opacity rate reached 97% at 12 weeks of disease.No obvious lens opacity was observed in blank control group,while the other groups showed different degrees of lens opacity.(3)The course of the disease was 12 weeks.Fundus photography of rats in the blank control group showed that optic disc was located in the central posterior part of the eyeball,and several straight thin arteries were issued from optic disc,accompanied by thick veins.No macula,exudation,bleeding,etc.There was no significant difference between the groups.(4)Retinal HE staining at 12 weeks showed that the structure of all retinal layers was clear and complete in blank control group.In the model group,the retinal structure was slightly damaged,the lamination was not clear,the nerve fiber layer was edema,and the ganglion cells were disordered and the number was reduced.In the positive group,the retinal structure was intact and the stratification was obvious,and part of the nerve fiber layer was edema.In the low-dose group,the retinal structure was intact,the number of ganglion cells in the local area was reduced,a few ganglion cells were degeneration and necrosis,blood vessels were hyperplasia and dilated,and the nerve fiber layer was edema to varying degrees.The structure of the retina in the medium and high dose groups was relatively intact,and the ganglion cells were reduced to varying degrees,and their arrangement was more scattered.In general,the overall lesion degree was relatively heaviest in the model group,followed by the low-dose group,and slightly lighter in the medium-dose and high-dose groups than in the low-dose group and the model group,but there was no difference between groups.The overall lesion degree was lighter in the positive drug group than in the medium-dose and high-dose groups.2.Effects of Qi-Huang-Mingmu capsule on retinal function in diabetic rats:(1)Scotopic0.01 ERG B wave: Compared with blank control group,the amplitude of B wave in model group was significantly decreased(P < 0.05);Compared with model group,b wave amplitude increased in positive drug group,low dose and high dose groups(P < 0.05).(2)Scotopic 3.0 ERG B wave:Compared with blank control group,b wave amplitude in model group was significantly decreased(P < 0.05);Compared with model group,the amplitude of B wave in high dose group was increased(P < 0.05).(3)OS2 wave of Scotopic 3.0 OPS: Compared with blank control group,THE amplitude of OS2 in model group was significantly lower(P < 0.05);Compared with model group,the amplitude of OS2 in high dose group was increased(P < 0.05).In addition,the number of retinal ganglion cells in the 12-week model group was significantly lower than that in the blank control group(P < 0.05).Compared with model group,the number of retinal ganglion cells in positive drug group,medium and high dose Chinese medicine groups was significantly increased(P < 0.05).3.Effects of qi-huang-mingmu capsule on blood-retina barrier in diabetic rats :(1)Compared with blank control group,EB osmotic volume in model group was significantly increased(P <0.05).EB osmotic volume in positive drug group,medium and high dose groups was lower than that in model group(P < 0.05).(2)The positive expression of retinal tight junction protein Claudin-5 and Occludin was decreased in model group compared with blank control group(P <0.05).Compared with model group,positive drug group and TCM high-dose group were more widely expressed(P < 0.05).(3)After 12 weeks of disease,compared with blank control group,the m RNA relative expression levels of Claudin-5 and Occludin in model group decreased significantly(P < 0.05);Compared with model group,m RNA relative expression levels of Claudin-5 and Occludin in positive drug group and high dose group were significantly increased(P < 0.05).4.Effects of Qi-Huang-Mingmu Capsule on NF-?B inflammatory signaling pathway in diabetic rats:(1)Western Blot results of vitreous injection of Mir-146 a antagonist for 1week: Compared with blank control group,the protein expression levels of CARD10,IRAK1 and TRAF6 in retinal tissues of model group were increased(P<0.05);The protein expression levels of CARD10,IRAK1 and TRAF6 in retinal tissues of model antagonist group were increased compared with blank control group and model group(P<0.05).(2)After 6 weeks of disease,the expression of CARD10,TRAF6,IRAK1,NF-?B and ICAM1 protein in retina of model group was significantly increased compared with blank control group(P<0.05).Compared with model group,the protein expression levels of CARD10,TRAF6,NF-?B and ICAM1 in low,medium and high dose groups were decreased(P<0.05).The expression level of IRAK1 protein was significantly decreased in the medium and high dose groups(P<0.05).The expression of TRAF6 protein in the retina of the antagonistic groups with low and medium doses of Traditional Chinese medicine was higher than that of the control groups with low and medium doses of Traditional Chinese medicine(P<0.05).The expression levels of TRAF6 and ICAM1 protein in the high-dose antagonist group were higher than those in the control group(P<0.05).The expression of retinal CARD10 protein in medium-dose antagonistic group was higher than that in control group(P<0.05).(3)Compared with blank control group,the m RNA expression levels of CARD10,IRAK1,NF-?B and ICAM1 in model group were significantly increased(P<0.05).Compared with model group,m RNA expression levels of CARD10,IRAK1,NF-?B and ICAM1 in low,medium and high dose groups were significantly decreased(P<0.05).The m RNA expression levels of NF-?B and ICAM1 in the retina of the low-dose and high-dose antagonistic groups were higher than those of the low-dose and high-dose control groups(P<0.05).The m RNA expression levels of CARD10,NF-?B and ICAM1 in the retina of medium-dose antagonistic group were higher than those in the control group(P<0.05).(4)Compared with blank control group,CARD10,IRAK1,TRAF6,NF-?B and ICAM1 protein expressions in model group were significantly increased during 12 weeks(P<0.05).Compared with model group,protein expression levels of TRAF6 and ICAM1 were significantly decreased in medium dose group(P<0.05),and protein expression levels of CARD10,IRAK1,TRAF6,NF-?B and ICAM1 were significantly decreased in high dose group(P<0.05).(5)After 12 weeks of disease,the m RNA levels of CARD10,IRAK1,NF-?B and ICAM1 in retinal tissues of model group were increased compared with blank control group(P<0.05).Compared with model group,the m RNA levels of NF-?B in low dose group were decreased,and the m RNA levels of CARD10,IRAK1,NF-?B and ICAM1 were significantly decreased in medium and high dose groups(P<0.05).5.Effects of qi-huang-mingmu capsule on il-6 /STAT3/VEGF signaling pathway in retina of diabetic rats :(1)compared with blank control group,the expression of il-6,p-stat3 and VEGF protein in retina of model group was significantly increased at 6 weeks(P<0.05);Compared with model group,the protein expression levels of RETINAL IL-6,P-STAT3 and VEGF were significantly decreased in low-dose,medium-dose and high-dose groups(P<0.05).The protein expression levels of IL-6,P-STAT3 and VEGF in retina of low-dose antagonist group were higher than those of control group(P<0.05).The protein expression levels of p-STAT3 and VEGF in retina of mediumdose antagonistic group were higher than those of control group(P<0.05).(2)After 6 weeks of disease,compared with blank control group,the m RNA expressions of IL-6,STAT3 and VEGF in model group were significantly increased(P<0.05);Compared with model group,the m RNA expression levels of IL-6,STAT3 and VEGF in low,medium and high dose groups were significantly decreased(P<0.05).The m RNA relative expressions of RETINAL STAT3 and VEGF in low dose antagonistic group were higher than those in control group(P<0.05).The m RNA relative expressions of IL-6,STAT3 and VEGF in the retina of medium-dose antagonist group were higher than those of control group(P<0.05).The relative expression of RETINAL STAT3 m RNA in the high-dose antagonist group was higher than that in the control group(P<0.05).(3)After 12 weeks of disease,compared with blank control group,the expression of IL-6,P-STAT3 and VEGF protein in model group was significantly increased(P<0.05);Compared with model group,the expression levels of RETINAL IL-6 and VEGF in TCM medium dose group were decreased(P<0.05).The protein expression levels of IL-6,P-STAT3 and VEGF in retina were decreased in high dose group(P<0.05).(4)After 12 weeks of disease,compared with blank control group,the m RNA levels of IL-6,STAT3 and VEGF in model group were increased(P<0.05).Compared with the model group,the m RNA expression levels of STAT3 and VEGF were decreased in the lowdose group(P<0.05),and the m RNA expression levels of IL-6,STAT3 and VEGF were decreased in the medium-dose and high-dose groups(P<0.05).6.Effects of Qi-Huang-Mingmu capsule on retinal extracellular matrix protein FN in diabetic rats:(1)The course of disease was 6 weeks: compared with blank control group,the expression level of FN protein in model group was increased(P<0.05);Compared with model group,FN protein expression in low,medium and high dose groups was significantly decreased(P<0.05);The expression of retinal FN protein in the antagonistic groups with low and medium dose of TCM was higher than that in the control groups with low and medium dose of TCM(P<0.05).(2)Duration of 12 weeks: Compared with blank control group,FN protein expression in model group was increased(P<0.05);Compared with model group,FN protein expression in medium and high dose groups was significantly decreased(P<0.05).(3)6 weeks: Compared with blank control group,FN m RNA expression in model group was increased(P<0.05);Compared with model group,FN m RNA expression in low,medium and high dose groups was significantly decreased(P<0.05);The relative expression of FN m RNA in the low,medium and high dose antagonistic groups was higher than that in the corresponding low,medium and high dose control groups(P<0.05).(4)Duration of 12 weeks: Compared with blank control group,FN m RNA expression in model group was increased(P<0.05);Compared with model group,FN m RNA expression in low,medium and high dose groups was significantly decreased(P<0.05).(5)Course of disease 12 weeks: Compared with blank control group,the expression of Mir-146 a in retina of model group was significantly decreased(P<0.05);Compared with model group,Mir-146 a m RNA levels in medium and high dose groups were significantly increased(P<0.05).Conclusions:1.Type II diabetic rats fed with high sugar and high fat +STZ induced early retinal pathological changes of DR after 12 weeks of disease.Qi-huang-mingmu capsule can alleviate the retinal pathological manifestations of diabetic rats.2.Qi-huang-mingmu capsule can reduce the loss of ganglion cells,increase the amplitude of ELECTROretinogram B wave and OS2 wave,and improve the inner retinal function and retinal microcirculation in diabetic rats.3.Qi-huang-mingmu capsule can reduce the retinal EB permeability of diabetic rats and upregulate the expression of tight junction proteins claudin-5 and Occludin,thus reducing the permeability of retinal blood vessels and protecting the blood-retina barrier.4.Qi-huang Mingmu capsule plays an anti-inflammatory role in diabetic rat retina by upregulating mi R-146 a,inhibiting THE NF-?B signaling pathway mediated by IL-1R/TLR and GPCR,and targeting down-regulating the IL-6/STAT3/VEGF signaling pathway.5.Qi-huang-mingmu capsule can regulate the expression of retinal extracellular matrix protein FN by up-regulating mi R-146 a and reduce retinal microvascular fibrosis in diabetic rats. |
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