SIRT1/P53 in retinal pigment epithelial cells in diabetic retinopathy: a gene co-expression analysis and He-Ying-Qing-Re formula treatment.

Objective: Diabetic retinopathy (DR) is a severe diabetic complication that leads to severe visual impairment or blindness. He-Ying-Qing-Re formula (HF), a traditional Chinese medicinal concoction, has been identified as an efficient therapy for DR with retinal vascular dysfunction for decades and has been experimentally reported to ameliorate retinal conditions in diabetic mice. This study endeavors to explore the therapeutic potential of HF with key ingredients in DR and its underlying novel mechanisms. Methods: Co-expression gene modules and hub genes were calculated by weighted gene co-expression network analysis (WGCNA) based on transcriptome sequencing data from high-glucose-treated adult retinal pigment epithelial cell line-19 (ARPE-19). The chromatographic fingerprint of HF was established by ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-Q-TOF-MS). The molecular affinity of the herbal compound was measured by molecular docking. Reactive oxygen species (ROS) was measured by a DCFDA/H2DCFDA assay. Apoptosis was detected using the TUNEL Assay Kit, while ELISA, Western blot, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used for detecting the cytokine, protein, and mRNA expressions, respectively. Results: Key compounds in HF were identified as luteolin, paeoniflorin, and nobiletin. For WGCNA, ME-salmon ("protein deacetylation") was negatively correlated with ME-purple ("oxidative impairment") in high-glucose-treated ARPE-19. Luteolin has a high affinity for SIRT1 and P53, as indicated by molecular docking. Luteolin has a hypoglycemic effect on type I diabetic mice. Moreover, HF and luteolin suppress oxidative stress production (ROS and MDA), inflammatory factor expression (IL-6, TNF-α, IL1-ß, and MCP-1), and apoptosis, as shown in the in vivo and in vitro experiments. Concurrently, treatment with HF and luteolin led to an upregulation of SIRT1 and a corresponding downregulation of P53. Conclusion: Using HF and its active compound luteolin as therapeutic agents offers a promising approach to diabetic retinopathy treatment. It primarily suppressed protein acetylation and oxidative stress via the SIRT1/P53 pathway in retinal pigment epithelial cells.

Hu-Zhang Qing-Mai Formulation anti-oxidative stress alleviates diabetic retinopathy: Network pharmacology analysis and in vitro experiment.

BACKGROUND: In this study, the potential mechanism of the Hu-Zhang Qing-Mai Formulation (HZQMF) on diabetic retinopathy (DR) in inhibiting oxidative stress was explored through network pharmacology analysis and in vitro experiments. METHODS: The Traditional Chinese Medicine Systematic Pharmacology Analysis Platform was used to retrieve the active pharmaceutical ingredients and targets of HZQMF. DR-related genes and oxidative stress-related genes were obtained from PharmGKB, TTD, OMIM, GeneCards, and Drugbank. STRING was used to construct a protein-protein interaction network to screen core targets. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses were performed using R 4.0.3. Network topology analysis was carried out using Cytoscape 3.8.2. Finally, we looked into how well the main API protected human retinal pigment epithelial cells from damage brought on by hydrogen peroxide (H2O2). RESULTS: Quercetin (Que) was identified as the primary API of HZQMF through network pharmacology analysis, while JUN, MAPK1, and STAT3 were identified as the primary hub genes. Kyoto encyclopedia of genes and genomes enrichment analysis showed that the AGE-RAGE signaling pathway may be crucial to the therapeutic process. In vitro experiments confirmed that Que increased cell vitality and inhibited apoptosis. CONCLUSION: Que might significantly reduce H2O2-induced ARPE-19 cell injury by inhibiting apoptosis-related genes of the AGE-RAGE pathway (JUN, MAPK1, STAT3). This study lays the foundation for further research on HZQMF in treating DR.

Application of optical coherence tomography and keratograph in the measurements of lower lid margin thickness.

PURPOSE: This study aims to investigate the applicability of lower lid margin thickness (LLMT) measurements in adults with and without meibomian gland dysfunction (MGD) by optical coherence tomography (OCT) and keratograph. METHODS: This is a cross-sectional, observational study. A hundred and eight volunteers aged 20 to 79, including 68 MGD patients and 40 normal subjects, were recruited. Using OCT and keratograph to measure the LLMT from the posterior lash line to anterior edge or outer edge of the tear meniscus was separately performed two times by the same person. RESULTS: The mean age of normal and MGD subjects was 50.5 ± 14.2 years and 55.8 ± 15.5 years, respectively. The LLMT with OCT and keratograph in MGD patients was significantly greater than that in normal subjects (1.06 ± 0.27 and 1.03 ± 0.25 mm vs. 0.90 ± 0.20 and 0.86 ± 0.16 mm, respectively). In both normal and MGD subjects, the tear meniscus height and LLMT with OCT were both greater than that with keratograph (P < 0.05), and intraclass correlation coefficient (ICC) demonstrated a good agreement in the LLMT measurements between two devices (ICC = 0.83 and 0.79, respectively). Additionally, the LLMT in MGD patients was appeared to be positively correlated with meiboscore (rs = 0.37, P = 0.002). CONCLUSIONS: The OCT and keratograph were two reliable tools in the LLMT measurements, which may have potential applications for diagnosis and evaluation of MGD. Furthermore, we found that the LLMT measured by OCT was greater than that measured by keratograph.

Mechanisms of Qing-Gan Li-Shui Formulation in Ameliorating Primary Open Angle Glaucoma: An Analysis Based on Network Pharmacology.

Objective: In this study, we investigated the mechanism of Qing-Gan Li-Shui formulation (QGLSF) in treating primary open glaucoma (POAG) by network pharmacology and in vitro experiments. Methods: The active pharmaceutical ingredients (APIs) of GLQSF (prepared with Prunella vulgaris, Kudzu root, Plantago asiatica, and Lycium barbarum) were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Yet Another Traditional Chinese Medicine database (YATCM). The targets of POAG were screened out with GeneCards, OMIM, PharmGKB, Therapeutic Target Database (TTD), and DrugBank databases. The Venny platform was used to summarize the core targets. Topological analysis was performed using Cytoscape3.8.0. A protein-protein interaction network was plotted by STRING online. The key targets were subjected to GO and KEGG enrichment analyses. Finally, the effects of APIs were verified by a model of chloride hexahydrate (CoCl2)-induced retinal ganglion cells-5 (RGC-5). Results: The main APIs were selected as quercetin (Que) by network pharmacology. Nine clusters of QGLSF targets were obtained by the PPI network analysis, including AKT-1, TP53, and JUN. KEGG enrichment analysis showed that these targets were mainly involved in the AGE-RAGE signaling pathway. By in vitro experiments, Que promoted cell proliferation. The secretion of AKT-1, TP53, JUN, AGE, and RAGE in the cell culture supernatant decreased, as shown by ELISA. The mRNA levels of AKT-1, TP53, JUN, and RAGE decreased, as shown by RT-PCR. QGLSF may employ the AGE-RAGE signaling pathway to counter POAG. Conclusion: This study preliminarily elucidates the efficacy and mechanism of QGLSF in the treatment of POAG.

Bie-Jia-Ruan-Mai-Tang, a Chinese Medicine Formula, Inhibits Retinal Neovascularization in Diabetic Mice Through Inducing the Apoptosis of Retinal Vascular Endothelial Cells.

Proliferative diabetic retinopathy (PDR) is one of the main complications of diabetes, mainly caused by the aberrant proliferation of retinal vascular endothelial cells and the formation of new blood vessels. Traditional Chinese medicines possess great potential in the prevention and treatment of PDR. Bie-Jia-Ruan-Mai-Tang (BJ), a Chinese medicine formula, has a good therapeutic effect on PDR clinically; however, the mechanism of action involved remains unclear. Therefore, we investigated the effect of BJ on PDR through in vitro and in vivo experiments. A diabetic mouse model with PDR was established by feeding a high-fat-high-glucose diet combined with an intraperitoneal injection of streptozotocin (STZ), while high-glucose-exposed human retinal capillary endothelial cells (HRCECs) were employed to mimic PDR in vitro. The in vivo experiments indicated that BJ inhibited the formation of acellular capillaries, decreased the expression of VEGF, and increased the level of ZO-1 in diabetic mice retina. In vitro experiments showed that high glucose significantly promoted cell viability and proliferation. However, BJ inhibited cell proliferation by cycle arrest in the S phase, thus leading to apoptosis; it also increased the production of ROS, decreased the mitochondrial membrane potential, reduced the ATP production, and also reduced the expressions of p-PI3K, p-AKT, and Bcl-xL, but increased the expressions of Bax and p-NF-κB. These results suggest that BJ induces the apoptosis of HRCECs exposed to high glucose through activating the mitochondrial death pathway by decreasing the PI3K/AKT signaling and increasing the NF-κB signaling to inhibit the formation of acellular capillaries in the retina, thus impeding the development of PDR.

Abnormal Levels of Serum Ferroptosis-Related Biomarkers in Diabetic Retinopathy.

Purpose: This study aimed to measure the concentrations of ferroptosis-related biomarkers, namely, iron (Fe), lipid peroxide (LPO), reactive oxygen species (ROS), glutathione peroxidase-4 (GPX4), and glutathione (GSH) in DR in the attempt to evaluate the diagnostic performance of these biomarkers. Methods: This study included 30 NPDR patients, 30 PDR patients, and 30 healthy subjects matched in age and sex. The concentrations of Fe, LPO, ROS, GPX4, and GSH in serum of the subjects were measured. Results: Compared with the normal group, GPX4 and GSH concentrations were significantly lower, and LPO, Fe, and ROS concentrations were significantly higher in DR patients. Compared with the PDR group, the NPDR group had higher concentrations of LPO, Fe, and ROS and lower concentrations of GPX4 and GSH, but there was no statistical difference in Fe, GPX4, and GSH. ROC curve shows that ferroptosis-related biomarkers have accumulated accuracy in NPDR and PDR. Conclusion: This study shows that ferroptosis-related biomarkers may be involved in the pathological process of DR and can be used as one of the biomarkers of DR.

Effect of the Meibomian Gland Squeezer for Treatment of Meibomian Gland Dysfunction.

PURPOSE: To investigate the effect of the meibomian gland squeezer for treatment of meibomian gland dysfunction (MGD). METHODS: Seventy patients (140 eyes) with MGD were randomly divided into 2 groups: 36 patients who were treated by the meibomian gland squeezer as the treatment group and 34 patients were selected as the control group. Patients were evaluated at baseline, and 2-week and 1-month visits for subjective symptoms, objective signs and pain assessments, including ocular symptom scores, Ocular Surface Disease Index, tear breakup time, corneal fluorescein staining, Schirmer scores with no anesthetic (Schirmer I test), meibum quality, meibum expressibility, and Numeric Rating Scale-11. RESULTS: Sixty-five patients were followed in the study, and mean (±SD) age was 57.0 (±12.6) years. Compared with baseline, the 2 groups had varying degrees of improvement in ocular symptom scores and Ocular Surface Disease Index at the 2-week and 1-month visits; there was a statistically significant difference between groups (P < 0.001). At the 1-month visit, the treatment group showed a greater improvement in the breakup time (3.8 ± 1.6 vs. 1.8 ± 1.0 seconds, P < 0.001), corneal fluorescein staining (-2.1 ± 2.13 vs. -0.9 ± 1.3, P = 0.03), Schirmer I test (5.3 ± 2.9 vs. 2.3 ± 2.8 mm, P < 0.001), meibum quality (-7.5 ± 2.9 vs. -5.3 ± 2.4, P = 0.004), and meibum expressibility (-1.2 ± 0.8 vs. -0.7 ± 0.4, P = 0.007). In the treatment group, the mean (±SD) of total pain scores was 2.4 ± 1.0, which indicated that mild pain was still predominant under topical anesthesia. CONCLUSIONS: The meibomian gland squeezer may be safe, effective, and helpful for treatment of MGD and may offer an attractive treatment option for some patients with MGD, although it can cause mild pain or discomfort.

Neuroprotective effect of Qinggan Lishui formula on retinal ganglion cell apoptosis in a microbead-induced rat chronic glaucoma model.

OBJECTIVE: To investigate a possible mechanism for protective effects of a decoction of the Qinggan Lishui formula (QF) on retinal ganglion cells (RGCs) in a rat model of microbead-induced chronic intraocular hypertension (COH). METHODS: The COH model was generated by injecting microbeads (superparamagnetic iron oxide) into the anterior chamber of rat eyes. QF was given by intragastric administration (gavage) once daily at a dose of 6.2 g/kg until day 28, following microbead injection. Cholera toxin B subunit (CTB) retrograde labeling and immunohistochemistry were used to evaluate changes in the number of RGCs in the retina. Terminal dUTP nick end labeling (TUNEL) staining was used to assess apoptotic changes in RGCs. RESULTS: Microbead injection induced a steady increase in intraocular pressure (IOP) of rats. Elevated IOP resulted in a progressive reduction in the number of CTB-labeled RGCs, 2-4 weeks after microbead injection. QF administration may moderately reduce IOP in the rat COH model and attenuate reduction of the number of CTB-labeled RGCs in COH rats. Furthermore, elevated IOP resulted in a progressive increase in the number of TUNEL-positive RGCs, 2-4 weeks after microbead injection, suggestive of an increase in the extent of RGC apoptosis. There was a significant reduction in the number of TUNEL-positive signals in QF-treated COH retinas, compared with untreated COH retinas. CONCLUSION: QF decoction may provide a protective effect for RGCs in COH retinas by reducing RGC loss; these effects may be mediated by inhibition of RGC apoptosis.

Neuroprotective effect of He-Ying-Qing-Re formula on retinal ganglion cell in diabetic retinopathy.

ETHNOPHARMACOLOGY RELEVANCE: He-Ying-Qing-Re Formula (HF) was empirically modified from Si-Miao-Yong-An Decoction (SD), which was recorded in the literature of Divine Doctor's Secret Transmission, and has been utilized for centuries to treat vasculopathy through clearing heat and accelerating bloodstream. HF has been used as an effective holistic treatment of diabetic retinopathy (DR) for decades and experimentally reported to ameliorate retinal condition in diabetic mice. AIM OF THE STUDY: Our study aims to investigate the effect of HF in preventing sustained hyperglycemia and hyperlipidemia-associated retinal ganglion cell (RGC) cell death and its possible mechanism. MATERIALS AND METHODS: Chromatographic fingerprint of HF was obtained upon the UPLC-based analytic system; Diabetic retinopathy was established in streptozotocin (STZ) injection-induced hyperglycemic mice; Alterations of retinal structure was assayed by H&E staining. Expression of PSD-95 and CHOP in retinae was assessed by immunofluorescence; RGC cell line (mRGC) was used for in vitro study. Cell death was analyzed by flow cytometry; Intracellular reactive oxygen species (ROS) was measured by 2',7'-dichlorofluorescin diacetate (DCFDA); Apoptosis-related proteins and signaling were monitored with immunoblotting and colorimetric assay. RESULTS: Chlorogenic acid, ferulic acid, and rutin were identified in HF. HF attenuates the loss of RGCs, thinning of inner retinal layers in diabetic mice. Furthermore, expressions of Brn3a and PSD-95 were restored while CHOP level was downregulated upon HF treatment. In vitro study, HF alleviates H2O2-induced apoptosis of mRGCs and loss of postsynaptic protein via scavenging ROS and suppressing ATF4/CHOP-mediated endoplasmic reticulum stress and mitochondria-related pro-apoptotic factors, probably as cleaved-caspase-3, and phospho-p38 mitogen-activated protein kinase (MARK). Meanwhile, both pro-survival protein levels like Bcl-2/Bcl-xL and postsynaptic protein of PSD-95 were upregulated upon HF treatment. CONCLUSION: HF administration was a valid therapeutic approach for DR treatment, oriented at the blockade of endoplasmic reticulum- and mitochondria-dependent oxidative stress-induced retinal neurodegeneration including RGC apoptosis.

Protective effect of a Chinese Medicine formula He-Ying-Qing-Re Formula on diabetic retinopathy.

ETHNOPHARMACOLOGY RELEVANCE: He-Ying-Qing-Re Formula (HF) is a formula modified from "Si-Miao-Yong-An Decoction", a traditional Chinese medical classic emerged in the Qing dynasty and has been reported for treatment of vascular diseases. HF, containing 8 herbs, has been used in local hospital for decades as a complementary method for diabetic retinopathy (DR) with retinal vascular dysfunction. Clinical reports revealed HF could ameliorate vision defects, microaneurysms, hemorrhages and macular edema. The aim of this study is to investigate the anti-DR action of HF and its underlying mechanism experimentally. METHODS: Chromatographic fingerprinting of HF and rodent model of DR were established; hypoglycemic effect of HF was measured by fasting, random blood glucose and glucose tolerance test; vascular degeneration was measured by retinal digestion; blood-retina-barrier (BRB) permeability was assessed with Evans Blue leakage assay. Advanced glycation end products (AGEs) were measured in vitro and in vivo level; Migration of retinal vascular endothelial cells were determined by wound healing and transwell chamber assays; permeability of endothelial monolayer was monitored with dextran transport. AGEs-related proteins and signaling were measured with immunoblotting and immunohistochemistry. RESULTS: Chlorogenic acid, ferulic acid and arctin were identified as major components in HF; HF suppresses retinal vasculature degeneration and BRB permeability damage without significant inhibition on hyperglycemia; HF reduces in vitro and in vivo formation of AGEs and AGEs-induced migration as well as permeability of retinal vascular endothelial cells. Expression of tight junction proteins Zo-1 and Claudin-1 was increased while activation of AGEs receptor and downstream signaling Akt were suppressed upon HF treatment. CONCLUSIONS: HF exhibits protective effect against diabetic retinopathy, which may be associated with inhibition on AGEs and recovery on endothelial dysfunction via modulation of tight junction and AGEs downstream signaling.