| Purpose:The purpose of this study was to explore the efficacy of the self-made Chinese herbal compound "Huanxietang" on non-proliferative and proliferative diabetic retinopathy rat model.The main mechanisms of diabetic retinopathy including insulin resistance,degree of fundus hemorrhage and number of choroidal neovascularization were analyzed based on the method of promoting blood circulation and removing blood stasis.To provide a new method for the prevention and treatment of diabetic retinopathy from the perspective of traditional Chinese medicine.Material and method:1.Drug preparation: According to the equivalent dose ratio calculated by animal and human surface area in Pharmacological Experimental Methodology edited by Professor Xu Shuyun,self-designed blood decoction low-dose,medium-dose and high-dose subjects were 0.272 g,0.544 g and 1.088g/ kg,respectively;For positive control group,take twice the adult daily dosage,add distilled water to 50 ml and stir evenly,1ml each time,twice a day.2.Modeling and drug administration: Adaptability of SD rats fed 7 d,watching animal general status,then randomly divided into 6 groups,namely blank group,model group,positive medicine group,the blood soup smart particles of low,medium and high dose group,respectively,according to the animal body surface area to the dose conversion,each group 5,positive-controlled(hydroxyl benzene sulfonic acid calcium)for 1 times that of the adult dosage,The dosage of Huaxue Decoction intelligent granules in low,medium and high dose groups was 0.5,1 and 2 times of adult daily dosage,respectively.Rats in the blank group were placed in cages and fed with food and water twice a day,with normal light circulation.Rats in model group,positive administration group,low-dose,medium-dose and high-dose administration groups were placed in cages and fed with high-fat diet and sugar water twice a day,with normal light circulation.Color fundus in vivo examination was performed on all rats one day before intragastric administration,and fundus photos were collected before treatment.The successful modeling of nonproliferative diabetic retinopathy(NPDR)rats was determined by fasting blood glucose concentration(FBG > 13.9 mmol/ L(250 mg/ d L))and changes of hemangioma and microvascular abnormalities in fundus photographs.After successful modeling(DAY0),the first-stage drug intervention(non-proliferative sugar net efficacy study)was conducted,that is,low-dose,medium-dose and high-dose groups were fed different doses of self-designed Blood decoction TCM intelligent granules.Twice a day(9am,2pm)for 28 days;Positive administration group was given equal proportion of adult daily dose of calcium hydroxybenzenesulfonic acid mixture of 1 times;The model group and the normal control group were not given drugs.Twenty-eight days after administration,PDR rat model was established after the in vivo index detection of all rats,and the success of PDR rat model was confirmed according to whether the blood glucose and fundus images of rats showed the symptoms of proliferative diabetic retinopathy such as choroidal neovascularization(CNV).After all the rats in the experimental group were successfully modeled,the second stage of drug intervention(sugar net effect study in the proliferative stage)was carried out,and the intervention method was the same as that in the first stage.On the last day of drug intervention,the eyeballs of rats were collected for subsequent histopathological section detection.Histopathological section staining included HE staining,immunohistochemistry and immunofluorescence staining.3.Detection indexes: Before modeling,14 days after modeling,28 days after modeling,42 days after modeling,and 56 days after modeling,the physical and chemical indexes of experimental animals such as blood glucose,intraocular pressure,and body weight were measured.The pathological tissues were stained with HE,TUNEL,VEGF and TNF-α.4.Statistical analysis: Graphpad PRISM 8 software was used for statistical processing to compare the differences between each group and the model group.Statistical data were expressed as Mean± standard deviation(Mean±SD)and analyzed by one-way ANOVA.P <0.05 was statistically significant.Results:1.In the first stage(NPDR stage)experiment,compared with the blank control group,the body weight of other groups was significantly decreased on the 28 th day after treatment(P <0.05);Compared with model group,the body weight of self-designed blood decoction medium and high dose groups was significantly increased(P < 0.01);Compared with the positive drug group,the body weight of the self-designed blood decoction high-dose group was significantly increased(P < 0.01).During the second stage(PDR stage),the weight of rats in the blank control group still increased uniformly.The body weight of rats in low-concentration dose group decreased gradually;The weight of the other groups still increased in different degrees.On day 56 after treatment,compared with the model group,the body weight of the self-designed blood decoction high-dose group increased(P < 0.05);Compared with the positive drug group,the body weight of the self-designed blood decoction high-dose group was increased(P < 0.05).2.In the first stage(NPDR stage),on the 28 th day after treatment,compared with the model group,the fasting blood glucose concentration(FBG)of the self-designed blood decoction high-dose group was decreased(P < 0.05);Compared with the positive drug group,FBG in the self-designed blood decoction high-dose group was decreased(P < 0.05).In the second stage(PDR stage),FBG in the self-designed blood decoction medium-dose and high-dose groups decreased compared with the model group on day 56 after treatment(P < 0.05).3.On the 28 th day of drug intervention,compared with the model group,fundus color images showed that the number of hemangiomas in the self-designed blood decoction high-dose group was reduced;Compared with the positive drug group,the number of hemangiomas in the self-designed blood decoction high-dose group also decreased.On the 56 th day of drug intervention,compared with the model group,the number of new blood vessels in the self-designed blood decoction high-dose group was reduced by fundus color imaging.4.The results of dark field flash electroretinogram(ERG)showed that,compared with the blank control group,the amplitude intensity of A-wave in positive drug group and self-designed blood soup low-dose group was significantly decreased(P <0.01),while the amplitude intensity of A-wave in model group and self-designed blood soup medium-dose and high-dose groups was decreased(P <0.05);Compared with the model group,there was no significant difference in a-wave amplitude intensity and no statistical significance.Compared with blank control group,b-wave amplitude intensity of positive drug group and self-designed blood decoction low-dose group decreased significantly(P <0.0001),b-wave amplitude intensity of model group decreased significantly(P <0.001),b-wave amplitude intensity of high-dose group decreased somewhat(P <0.001),and b-wave amplitude intensity of medium-dose group decreased slightly(P<0.05).Compared with the model group,there was no significant difference in b-wave amplitude intensity among all groups,showing no statistical significance.5.HE staining results showed that after 56 days of intragastric administration of self-designed blood decoction,the inner core layer,outer core layer and total retinal thickness of rats were significantly increased compared with the model group,and the effect of self-designed blood decoction in high concentration group was significantly better than that in other treatment groups,with statistical significance(P <0.05).6.The results of TUNEL ganglion cell apoptosis showed that compared with the modeling group,the self-designed blood decoction high-dose group had improved retinal injury,and the number of retinal ganglion cells per unit length was also significantly increased(P < 0.05).Conclusion:1.The self-made Chinese herbal compound Huaxue Decoction has therapeutic effects on st Z-induced insulin deficient diabetic retinopathy rats in all stages,and its therapeutic effects may be related to inhibiting weight loss,increasing blood glucose tolerance,controlling progressive blood glucose rise,inhibiting neovascularization and retinal cell apoptosis.2.The effect of high dose of TCM compound self-designed Blood decoction on non-proliferative diabetic retinopathy was better,that is,after 28 days of treatment,body weight,fasting blood glucose and ERG in the self-designed blood decoction high dose group were significantly improved compared with the model group,with statistical significance(P <0.01);Compared with self-designed blood decoction low-dose and medium-dose groups,all indexes were improved,and the difference was statistically significant(P < 0.05).Compared with before intervention,body weight,fasting blood glucose and other basic values were improved(P < 0.05).3.Compared with non-proliferative diabetic retinopathy,self-designed blood decoction has no obvious therapeutic effect on proliferative diabetic retinopathy. |
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