| Objective:Diabetic retinopathy(diabetic retinopathy,DR)is one of the most common and serious microvascular complications of diabetes.It is the main cause of the postnatal low vision in adults.Diabetes is mainly divided into non proliferative DR(Nonproliferative diabetic retinopathy,NPDR)and proliferation phase DR(Proliferative diabetic retinopathy,PDR).Non proliferative DR is mainly manifested as fundus microaneurysm,venous bead like change,retinal punctate hemorrhage and hard exudation.If the disease develops further,it will cause vision loss and blindness.Modern medicine has no special treatment for early diabetic retinopathy,while only Calcium dobesilate has certain curative effect.Calcium dobesilate can reduce retinal exudation,hemorrhage and microangioma formation.It can improve the retinal ischemia and hypoxia state,so it is widely used in NPDR.Traditional Chinese medicine requires dialectical treatment and the overall view,in addition to the treatment of local retinal lesions,it also seek the improvement of the systemic symptoms of diabetic patients.In recent years,the traditional medicine treatment for non proliferative DR is quite a lot,and the results are quite abundant,showing some advantages in NPDR treatment.The main thread of this topic is Tangwang I hao,Through two pathways of clinical and animal experiments,the objective is to observe the clinical efficacy of Tangwang I hao in the treatment of non proliferative DR patients with deficiency of Qi and Yin.To observe the effects of Tangwang I hao on the pathological morphology of retina in C57BL/6J mice induced by streptozotocin(STZ).To study the changes in the expression of VEGF and PEDF in the retina.Study on the possible mechanism of Tangwang I hao in the treatment of non proliferative DR.It provides a theoretical basis for the treatment of non proliferative DR with Tangwang I hao,and provides a new idea for the treatment of non proliferative DR with Chinese medicine.The patients signed the informed consent,and those who were treated with calcium dobesilate were divided into western medicine treatment group.The patients who took oral Tangwang I hao were classified as the Chinese medicine treatment group,and the patients who only received the basic treatment of blood glucose control were classified as the control group.Methods:Part one: screen patients with non proliferative diabetic retinopathy from June 2015 to June 2017 in the hospital of Traditional Chinese Medicine in Jimo District,Qingdao,and TCM dialectically conforms to two deficiency syndrome of Qi and Yin.The patients signed the informed consent,and the patients treated with calcium dobesilate were divided into western medicine treatment group.The patients who took oral Tangwang I hao were classified as the Chinese medicine treatment group,and the patients who only received the basic treatment of blood glucose control were classified as the control group.According to the guiding principles of “clinical research on new drugs of traditional Chinese medicine”,the symptoms of all patients in the experiment were quantified.Compare the classification scores of the three groups.To observe and record the best corrected visual acuity of the patients before and after treatment with E international standard visual acuity chart.To observe the change of number of retinal blood vessels in patients with bleeding,exudation range with fundus photography.To observe the changes of retinal vascular leakage,non perfusion area changes and the presence of macular edema with retinal fluorescein angiography(Fluorescein sodium fundus angiography,FFA).foveal thickness were measured in patients with macular edema with optical coherence tomography(Optical correlation tomography,OCT).The difference among each group and the same group for 3 months and 6 months after treatment was compared.Part two: The normal control group of C57BL/6J mice was established,and the diabetic mice were established by intraperitoneal injection of 0.1% STZ.The model group was randomly divided into diabetes control group,Calcium dobesilate group and Tangwang I hao group.The later three groups were treated with Calcium dobesilate,Tangwang I and normal saline orally for 3 months.At the end of the experiment,the cervical dislocations were executed to kill the mice.The mice eyeballs were taken while the right eyes were used for HE and immunohistochemical detection,and the retinal tissue of left eye was taken for cryopreservation at-20 C for PCR quantitative detection.HE staining was used to observe the morphological changes of the retina in mice.Immunohistochemical method was used to observe the expression of VEGF and PEDF in the retina of mice,and the expression of mRNA of VEGF and PEDF were detected by fluorescence RT-PCR.Results:Part one :a total of 150 patients were selected and 18 were lost during the study.132 patients completed the experiment.The control group only received basic diabetes treatment,3 months after and 6 months after the symptom score increased,the difference was statistically significant(P < 0.05),and the difference between 3 months and 6 months was not statistically significant(P < 0.05),suggesting the deterioration of the retina;the western medicine treatment group after the treatment of calcium hydroxybenzosulfonic acid 3 months after the symptom score and The difference was not statistically significant before treatment(P=0.52),but the difference was statistically significant after 6 months treatment(P < 0.05).The symptom score of the Chinese medicine treatment group was decreased 3 months and 6 months after the treatment of Tangwang I hao,and the difference was statistically significant compared with that before treatment(P<0.01).The score of Tangwang I hao group was lower than that of 3 months after 6 months of treatment(P < 0.05).In the control group,the scores increased at 3 and 6 months,and the difference was statistically significant(P < 0.05).The symptom score of the control group increased after 3 months and 6 months later,and the difference was statistically significant(P < 0.05).There was no significant difference between the treatment group and the treatment group for 3 months(P=0.52),but the difference was statistically significant after 6 months of treatment(P < 0.05).The symptom score of the Chinese medicine treatment group was decreased 3 months and 6 months after the treatment of Tangwang I hao,and the difference was statistically significant compared with that before treatment(P<0.01).The score of Tangwang I hao was lower than that of 3 and 6 months of treatment(P < 0.05).There was no significant difference between the two groups in scores.Comparison of curative effect: After 3month treatment,western medicine group and Chinese medicine treatment group explicit efficiency were 24.0% and 27.7% respectively;effective rates were 46.0% and 48.9%;Inefficiency were24.0% and 17.0%;the deterioration rate were 6.0% and 6.4%,total effective rate were 70.0% and 76.6% respectively.The total effective rate of the two groups was higher than that of the control group,and the difference was statistically significant(P < 0.05),and there was no statistical difference between the two groups.After 6month treatment,western medicine group and Chinese medicine treatment group explicit efficiency were 26.0% and 29.8% respectively;effective rates were 44.0% and 51.1%;Inefficiency were24.0% and 12.8%;the deterioration rate were 6.0% and 6.3%,total effective rate were 70.0% and 80.9% respectively.The total effective rate of the two groups was higher than that of the control group,and the difference was statistically significant(P < 0.05),and there was no statistical difference between the two groups.The macular fovea thickness of the patients with macular edema in each group was respectively 251.32±14.72 um,249.15±33.22 um,253.72±19.85um;3 months after treatment,the foveal thickness was 272.39±21.52 um,230.89±15.96 um,220.36±19.52um;6 months after treatment the foveal thickness was 259.83±16.71 um,210.79±20.15 um,180.38±18.51 um.Compared with the control group,the macular fovea thickness of the traditional Chinese medicine treatment group decreased in 3 months and 6 months,and the difference was statistically significant(P < 0.05).The foveal thickness had no significant difference in the traditional Chinese medicine treatment group between groups of 3 months and 6 months.There was no significant difference in the foveal thickness of the western medicine treatment group and the control group during the drug use.Part two: among the 50 C57BL/6J mice,30 of them were successful in modeling,the success rate was 71.42%.After 1 weeks,4 weeks,8 weeks and 12 weeks,the body weight and blood sugar of mice in model groups were significantly different from those of the control group(P<0.05).Under light microscope,HE staining showed that all layers of cells in the retina of normal control group were arranged orderly,the core layer is thin,the outer nuclear layer is thick,the inner limiting membrane of the retina complete,no capillary endothelial cells through the inner limiting membrane.While in the diabetic control group,retinal layers loose tissue edema,ganglion cells loose irregular,occasionally capillary endothelial cells projecting from the inner limiting membrane.Compared with the diabetic control group,the retinopathy of the mice in the calcium dobesilate group and Tangwang I hao group was relatively mild.Immunohistochemistry showed that the retinal VEGF in normal control group was mainly distributed in the ganglion cell layer,and also expressed in the nuclear layer,showing a weak positive and pale yellow color.The positive expression of VEGF in the retina of the diabetic mice was almost brown in the retina of the diabetic control group.The expression of VEGF in calcium dobesilate group and Tangwang I hao group was mainly in the inner plexiform layer,the inner nuclear layer and the retinal ganglion cell layer,and the outer nuclear layer also had some positive expression,yellow.The retina PEDF of normal control group showed strong positive reaction,and all levels were expressed in brown and yellow.The expression of PEDF in the diabetic control group was only yellowish in the inner plexiform layer and ganglion cell level of the retina.In the group of Calcium Dobesilate and the group I of sugar net,PEDF was expressed in the inner plexiform layer,the core layer and the ganglion cell layer,and it was yellowish.Determination of optical density score values of the image(Integated optical density,IOD)showed that,compared with the normal control group,the IOD value of VEGF positive particles in Tangwang I hao group,calcium dobesilate group and diabetic control group's mice retina were increased,the difference was statistically significant(P < 0.05).The IOD value of VEGF positive particles in Tangwang I hao group and the calcium dobesilate group was lower than that in the diabetic control group(P < 0.05),while there was no significant difference in the IOD value of VEGF in the Tangwang I hao group and the calcium dobesilate group.Compared with the normal control group,the IOD value of PEDF positive particles in the Tangwang I hao group,calcium dobesilate group and diabetic control group decreased,the difference was statistically significant(P < 0.05).There was no significant difference in IOD among the three groups.The results of fluorescent RT-PCR showed that compared with normal control group,the expression of VEGF mRNA increased in the Tangwang I hao group,calcium dobesilate group and diabetic control group(P<0.05).The VEGF gene expression in theTangwang I hao group and calcium dobesilate group was lower than that of the diabetic control group,and the difference was statistically significant(P<0.05).There was no significant difference in the expression of VEGF gene between the Tangwang I hao group and calcium dobesilate group.Compared with normal control group,the expression of PEDF mRNA in Tangwang I hao group,calcium dobesilate group and diabetic control group were all decreased.The difference was statistically significant(P<0.05),but there was no significant difference in PEDF mRNA among the three groups.Conclusions:Tangwang I hao has a therapeutic effect on non proliferative DR patients with two deficiency of Qi and Yin,including improving symptoms,improving visual acuity and(or)reducing retinal hemangioma and vascular leakage,alleviating macular edema.Tangwang I hao may play its role in treating non proliferative DR by reducing the protein and gene expression of VEGF,and by remolding the dynamic balance of VEGF and PEDF in the retina. |
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