| ObjectiveThe main purpose of this study was to investigate the gene expression changes and potential biological marker associated with diabetic retinopathy in rats using transcriptional sequencing and metabonomic technolog. And clarify the mechanism of QiDengMingMu capsule in diabetic retinopathy treatment by combination of the anti-inflammatory and anti-oxidative effects.Methods1. The animal model of diabetic retinopathy was induced by a high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ; 40 mg·kg-1). The blood glucose was determined 72 h after STZ injection and the value greater than 16.7 mmol·L-1 was considered to be the model of diabetic rats. The retinal vascular digest preparations, HE staining and transmission electron microscopy methods were used to evaluate the model of diabetic retinopathy at the end of 16 and 20 weeks after STZ.2. The model rats were divided into 6 groups, including calcium dobesilate group (150 mg·kg-1), QiDengMingMu capsule group (600 mg·kg-1), total puerariae flavones group (421 mg·kg-1), erigeron breviscapus flavonoids group (136 mg·kg-1), astragalus saponins group (43 mg·kg-1), and model control group. Other ten healthy rats were made as a normal control group. After treatment for 10 weeks, level of vascular endothelial growth factor ?VEGF?, angiotensin ? ?ANG ??, intercellular adhesion molecule-1 ?ICAM-1?, tumor necrosis factor-a ?TNF-a?, nitric oxide ?NO?, hypoxia inducible factor 1 alpha ?HIF-la?, Malondialdehyde ?MDA? and superoxide dismutase ?SOD? were tested.3. Total RNA of retinal was extracted by Trirol method. The quantitative and quality of RNA samples were evaluated by 1.5% agarose gel electrophoresis and Agilent 22 TapeStation. A high-throughput transcriptome sequencing ?RNA-seq? technology using Illumina GA?x was used to characterize the entire retinal transcriptome from nondiabetic and the diabetic retinopathy model rat. The differentially expressed genes were screened, and the bioinformatics analysis was done by GO and KEGG.4. Metabolic profiling changes were analyzed by ultra-performance liquid chromatography-quadrupole-Exactive Orbitrap-mass spectrometry ?UPLC-Q-Exactive Orbitrap-MS?. The principal component discriminant analysis ?PCA-DA?, partial least-squares discriminant analysis ?PLS-DA? and orthogonal partial least-squares discriminant analysis ?OPLS-DA? were used for multivariate analysis. The information of potential biomarkers was obtained by searching databases of KEGG, Lipidmaps and Metlin based on the accurate molecular weights and isotope shapes.Results1. Compared with the normal group, the body weight of model rats were lost, and the blood glucose were significantly increased. Retina HE staining showed that there were no significantly changes in normal group. But in DR model group, the inner retina structure was lost; cells were arranged in irregularly, tissue edema. Retinal ganglion cells were swelling, arranged in irregularly, the number were decreased, inner plexiform layer were swelling, Inner and outer nuclear layer cells had a disordered arrangement, density decreased, capillary diffusion, congestion, endothelia cell were proliferated, vascular wall was thickening. The outer plexiform layer the capillaries appeared. The retinal pigment epithelial cells were proliferated, and the choroidal capillary expanded obviously.2. QiDengMingMu capsule and each treatment group had no significant effect on body weight and blood glucose of rats. Compared with the normal group, the content of HIF-la, VEGF, Ang II, ICAM-land MDA was increased obviously, SOD1 was decreased significantly. Compared with the model group, the content of VEGF, Ang II and MDA can be obviously decreased by QiDengMingMu capsule and the Pueraria extract. Besides QiDengMingMu capsule can also decrease the content of HIF-la. The extract of Erigeron breviscapus can decrease the content of Ang II and MDA markedly. The content of SOD1 can be markedly increased by extract of Astragalus. Principal component analysis showed that there was considerable difference between the model and normal group. Calcium dobesilate and QiDengMingMu capsule have significant effects on DR rats. Among the three components, the Pueraria extract is the most effective material.3. RNA-seq technique provided comprehensive informations of transcripts that are altered in diabetic retinopathy. There were a total of 785 genes differentially expressed between DR and normal rats. Most of the differentially expressed genes were up-regulated. These transcripts encoded proteins involved in diabetes disease-associated pathways such as inflammation, oxidative stress, microvasculature formation, PI3K-Akt signaling pathway. JAK-STAT signaling pathway, cAMP signaling pathway, amino acid metabolism, and lipid metabolism. The differentially expressed genes in QiDengMingMu capsule group are enriched in the pathway of oxidative phosphorylation, glutathione metabolism, and PI3K-Akt signaling pathway, the cAMP signaling pathway, HIF-1 signaling pathway, peroxisomes, amino acid metabolism, and lipid metabolism. The difference expression genes are mainly enriched in glycolytic pathway, amino acid, and lipid metabolism pathway with the treatment of Pueraria extract. The difference expression genes are mainly enriched in retinol metabolism, vascular smooth muscle contraction, p53 signaling pathway, tight junction, and complement and coagulation cascades with Erigeron breviscapus treatment.4.Eleven potential metabolite biomarkers, including PC?2O:4?5Z,8Z,11Z,14Z? /18:1?11Z??, PC?22:0/12:0?, PC?21:0/0:0?[U], Leu Ser Lys Lys, Phylloquinone, Gly His Arg Gly, Lys Lys Ser,5'-Methylthioadenosine,2-Hexylglycerol,7-keto-n-caprylic acid,7-Oxoheptanoic acid have been identified, which are mainly related to the coagulation, lipid metabolism and amino acid metabolism. PCA-DA scores plots indicated that biochemical changes in diabetic rats were gradually restored to normal after administration of QiDengMingMu capsule and the Pueraria extract.ConclusionThe QiDengMingMu capsule has protective effects on diabetic retinopathy rats, and its mechanisms are may be associated with inhibiting oxidative damage, preventing angiogenesis, and regulating the disturbed amino acid and lipid metabolism. The Pueraria extract is the main effective components, and other ingredients have synergistic effect through different ways. |
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