Pueraria lobata antioxidant extract ameliorates non-alcoholic fatty liver by altering hepatic fat accumulation and oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata is essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata is widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata has remarkable biological activities in protecting liver, relieving alcoholism, antioxidation, anti-tumor and anti-inflammation in clinic. However, the potential mechanism of ethyl acetate extract of Pueraria lobata after 70% alcohol extraction (APL) ameliorating nonalcoholic fatty liver disease (NAFLD) has not been clarified. AIM OF THE STUDY: This study aimed to investigate the ameliorative effect of P. lobata extract on human hepatoma cells and injury in rats, and to evaluate its therapeutic potential for ameliorating NAFLD. METHODS: Firstly, the effective part of P. lobata extract was determined as APL by measuring its total substances and antioxidant activity. And then the in vitro and in vivo models of NAFLD were adopted., HepG2 cells were incubated with palmitic acid (PA) and hydrogen peroxide (H2O2). In order to evaluate the effect of APL, Simvastatin and Vitamin C (VC) were used as positive control. Various parameters related to lipogenesis and fatty acid ß-oxidation were studied, such as intracellular lipid accumulation, reactive oxygen species (ROS), Western Blot, mitochondrial membrane potential, apoptosis, and the mechanism of APL improving NAFLD. The chemical components of APL were further determined by HPLC and UPLC-MS, and molecular docking was carried out with Keap1/Nrf2/HO-1 pathway related proteins. RESULTS: APL significantly reduced lipid accumulation and levels of oxidative stress-related factors in vitro and in vivo. Immunohistochemical、Western Blot and PCR analysis showed that the expressions of Nrf2 and HO-1 were up-regulated in APL treatment. The Nrf2 inhibitor ML385 can block the rescue by APL of cellular oxidative stress and lipid accumulation induced by H2O2 and PA, demonstrating its dependence on Nrf2. UPLC/MS analysis showed that there were 3'-hydroxyl puerarin, puerarin, 3'-methoxy puerarin, daidzein, genistin, ononin, daidzin and genistein. CONCLUSION: This study further clarified the mechanism of P. lobata extract in improving NAFLD, which provided a scientific basis for developing new drugs to protect liver injury and laid a solid foundation for developing P. lobata Chinese herbal medicine resources.

Observation of Cyclosporin A: Sustained Release Intraocular Lens Implantation in Rabbit Eyes.

PURPOSE: To observe the performance of cyclosporine A (CsA)-loaded intraocular lens (IOLs) implanted into rabbit eyes. METHODS: To prepare a PLGA-based CsA-sustained release IOLs and study the in vitro drug release. Forty-two New Zealand white rabbits were randomly and equally divided into three groups, and all right eyes underwent phacoemulsification. In group A, a common polymethylmethacrylate (PMMA) IOLs was implanted, while polylactide-glycoli acid (PLGA-loaded)-PMMA-IOLs was implanted in group B, and CsA-PLGA-PMMA-IOLs was implanted in group C. All experimental eyes were examined by slit-lamp microscopy. In addition, fundoscopy and the number of corneal endothelial cells, anterior chamber flare grading, and the number of aqueous humor cells were assessed at different time points post-surgery. The wet lens capsule was weighed and histological examination was performed 6 months post-operation. RESULTS: In the early post-operative period, the inflammatory reaction of anterior chamber in groups A and B were more severe than group C. The initial appearance of PCO in group C was much later than the other two groups (F = 68.91; p = 0.000), and PCO grade in group C was much lower than the other two groups (χ2 = 36.07; p = 0.000). The mean weights of wet lens capsules in groups A and B were significantly heavier than group C (F = 134.88; p = 0.00). Histological observation showed no obvious toxic reaction in the intraocular tissues of the CsA-PLGA-PMMA-IOLs group, and the proliferation and accumulation of lens epithelial cells in groups A and B were greater than in group C. CONCLUSION: CsA-sustained release IOLs can effectively prevent PCO in rabbit eyes without defined intraocular toxicity.

Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function.

Etiology and pathogenesis of age-related cataract is not entirely clear till now. Senescence marker protein 30 (SMP30) is a newly discovered anti-aging factor, which plays an important role in preventing apoptosis and reducing oxidative stress damage. Mitochondria are located at the intersection of key cellular pathways, such as energy substrate metabolism, reactive oxygen species (ROS) production and apoptosis. Oxidative stress induced by 4-hydroxynonenal (4-HNE) is closely related to neurodegenerative diseases and aging. Our study focused on the effect of SMP30 on mitochondrial homeostasis of human lens epithelial cells (HLECs) induced by 4-HNE. Western blots and qPCR were used to compare the expression of SMP30 protein in the residual lens epithelial cells in the lens capsule of age-related cataract (ARC) patients and the donated transparent lens capsule. On this basis, SMP30 overexpression plasmid and SMP30 shRNA interference plasmid were introduced to explore the effect of SMP30 on the biological behavior in HLECs under the condition of oxidative stress induced by 4-HNE through immunohistochemistry, ROS evaluation, metabolic spectrum analysis and JC-1 fluorescence measurement. Given that Nuclear Factor erythroid 2-Related Factor 2 (Nrf2)/Kelch Like ECH Associated Protein 1 (KEAP1) signaling pathway is the most important antioxidant stress pathway, we further analyzed the regulatory effect of SMP30 by WB to explore its molecular mechanism. Our study indicated that SMP30 may inhibit ROS accumulation, restore mitochondrial function, activate Nrf2/Keap1 signaling pathway, therefore protecting lens epithelial cells from oxidative stress-induced cell damage.

Free-standing electrochemical biosensor for carcinoembryonic antigen detection based on highly stable and flexible conducting polypyrrole nanocomposite.

A flexible free-standing electrochemical biosensor to detect carcinoembryonic antigen (CEA) is described based on a conducting polypyrrole (PPy) nanocomposite film electrode. The conducting PPy composite was constructed by the sandwiched structure formed by PPy doped with pentaerythritol ethoxylate (PEE) and 2-naphthalene sulfonate (2-NS-PPy) separately via electropolymerization. Gold nanoparticles (AuNPs) were fixed on the PPy composite film by electrodeposition and then connected to CEA aptamer through self-assembly to construct a free-standing electrochemical biosensor breaking away from additional soft substrates and current collector. This PPy composite film-based electrochemical biosensor exhibits satisfying sensing performance for CEA detection, with a linear range from 10-10 g/mL to 10-6 g/mL and a detection limit of 0.033 ng/mL, good specificity and long-term sensing stability (96.8% of the original signal after 15 days). The biosensor also presents acceptable reproducibility with 1.7% relative standard deviation. Moreover, this electrochemical biosensor owns the deformation stability that could bear various deformations (twisting, folding, and knotting) without affecting device's sensing performance. It can even maintain 99.4% of the original signal under 25% strain deformation. Due to the superior sensing performance, high stability (mechanical deformation and long-term storage), and flexibility, this free-standing electrochemical biosensor proves huge potential in application of flexible and wearable electronics.

Glucose Metabolic Characterization of Human Aqueous Humor in Relation to Wet Age-Related Macular Degeneration.

Purpose: Energy compromise underpins wet age-related macular degeneration (wAMD) pathogenesis, but the relationship between glucose metabolism and the disease remains unclear. Here, we characterized aqueous humor (AH) to elucidate glucose-related metabolic signatures in patients with wAMD. Methods: In total, 25 eyes of 25 patients with wAMD were divided into phakic (15 eyes), pseudophakic (10 eyes), and intravitreal injections of ranibizumab (13 eyes) wAMD groups. Twenty patients with cataract (21 eyes) served as controls. Ultrahigh-performance liquid chromatography tandem mass spectrometry was used to quantitatively characterize AH. Results: Twenty-one metabolites related to glucose metabolism were identified in AH from 45 patients. Tricarboxylic acid (TCA)-related metabolic substrates, including citrate, were detected in AH and were significantly increased in AMD (P < 0.01) and AMD pseudophakic groups (P < 0.05). In contrast, α-ketoglutarate levels were decreased in the AMD group (P < 0.05). The α-ketoglutarate/citrate ratio was significantly decreased, corresponding to 71.71% and 93.6% decreases in the AMD (phakic and pseudophakic) groups as compared with controls (P < 0.001), revealing a significant positive correlation with glutamine. A lower mean glutamine and higher glutamate level were detected in AMD cases compared with controls. No significant differences were observed for lactic acid or other Krebs cycle metabolites. Intravitreal injection significantly alleviated mean central foveal thickness but did not significantly alter metabolites. Conclusions: Compromised glucose TCA cycle and altered glutamine metabolism are implicated in the AH metabolism in wAMD. These findings highlight potential treatments for alleviating wAMD from a metabolic perspective.

Metabolomic Profiling of the Aqueous Humor in Patients with Wet Age-Related Macular Degeneration Using UHPLC-MS/MS.

Assessing metabolomic alterations in age-related macular degeneration (AMD) can provide insights into its pathogenesis. We compared the metabolomic profiles of the aqueous humor between wet AMD patients (n = 26) and age- and sex-matched patients undergoing cataract surgery without AMD as controls (n = 20). A global untargeted metabolomics study was performed using ultra-high-performance liquid chromatography tandem mass spectrometry. Univariate analysis after the false discovery correction showed 18 significantly altered metabolites among the 291 metabolites measured. These differential metabolomic profiles pointed to three interconnected metabolic pathways: a compromised carnitine-associated mitochondrial oxidation pathway (carnitine, deoxycarnitine, N6-trimethyl-l-lysine), an altered carbohydrate metabolism pathway (cis-aconitic acid, itaconatic acid, and mesaconic acid), which plays a role in senescence and immunity, and an activated osmoprotection pathway (glycine betaine, creatine), which potentially contributes to the pathogenesis of the disease. These results suggested that metabolic dysfunction in AMD is mitochondrial-centered and may provide new insights into the pathophysiology of wet AMD and novel therapeutic strategies.

KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis.

BACKGROUND: Many studies have confirmed that high myopia is related to the high prevalence of cataracts, which results from apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress. Krüppel-like factor 6 (KLF6) is a tumor suppressor that is involved in the regulation of cell proliferation and apoptosis. PURPOSE: In this study, our purpose was to find the relationship between KLF6-induced apoptosis in LECs and ATF4 (activating transcription factor 4)-ATF3 (activating transcription factor 3)-CHOP (C/EBP homologous protein) signaling pathway. METHODS: KLF6, ATF4, ATF3, and CHOP were ectopically expressed using cDNAs subcloned into the pCDNA3.1+ vector. ATF4, ATF3, and CHOP knockdown were performed by small interfering RNA (siRNA). Expression of relative gene was tested using QT-PCR and western-blot. Then, accompanied by UVB stimulation, cell viability was measured by CCK-8 assay; The cell damage was examined by live & dead staining; The apoptotic markers Bax and Bcl-2 were detected by immunoblotting; Quantitative apoptotic levels were measured with the Apoptosis Detection Kit; The expression level of reactive oxygen-free radical (ROS) was analyzed by DCFH-DA` probe. RESULTS: Ectopically expressed ATF4, ATF3, and CHOP-induced apoptosis in cells, whereas ATF4, ATF3, and CHOP knockdown by small interfering RNA (siRNA) blocked KLF6-induced apoptosis. In addition, we determined that ATF4 regulates ATF3 and CHOP expression and that ATF3 silencing reduces CHOP upregulation without changing ATF4 levels; however, ATF4 and ATF3 expression was unaffected by blockade of CHOP, suggesting that KLF6 triggers endoplasmic reticulum stress in LECs by mediating the ATF4-ATF3/CHOP axis. Besides, KLF6 overexpression significantly induced LEC apoptosis under UV radiation, as demonstrated by the elevated Bax/Bcl-2 ratio. CONCLUSION: The ATF4-ATF3-CHOP pathway plays an important role in KLF6-induced apoptosis in HLECs. Our results increase our understanding of the mechanisms that regulate LEC apoptosis and contribute to the development of a new preventative strategy for cataract.

Quantitative analysis of metabolites in glucose metabolism in the aqueous humor of patients with central retinal vein occlusion.

Quantitative analysis of aqueous humor (AH) was performed to investigate glucose metabolism in patients with central retinal vein occlusion (CRVO), and to explore metabolic changes after anti-vascular endothelial growth factor (VEGF) treatment. AH samples were collected from 35 patients. Participants diagnosed with CRVO (n = 15) were compared to participants who underwent cataract surgery (n = 20). Thirteen of the participants with CRVO received second-round anti-VEGF treatments. Ultra-high performance liquid chromatography tandem-mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites of the AH. Central macular thickness (CMT) and retinal ganglion cell layer (RGC) thickness were measured using spectral-domain optical coherence tomography. Thirteen metabolites involved in glucose metabolism were identified. Among these metabolites, succinate, glutamate, and glutamine were significantly decreased for the CRVO group (p = 0.028, 0.009, and 0.017, respectively). The α-ketoglutarate/citrate (K/C) ratio had a significant positive correlation with glutamine levels for both control (r = 0.922, p < 0.001) and CRVO groups (r = 0.674, p = 0.006). A significant increase in lactate was observed after intravitreal anti-VEGF administration (t = 2.273, p = 0.045); the change in CMT was negatively correlated with this increase (r = -0.745, p = 0.003). The alteration of RGC thickness was negatively correlated with increases in both glutamine (r = -0.619, p = 0.024) and glucose (r = -0.754, p = 0.003). These results indicate that, compared to glucose metabolism, glutamine was significantly decreased in the AH of patients with CRVO, and may therefore serve as a potential target for CRVO therapy. The glycolytic pathway might be enhanced after intravitreal anti-VEGF injection, which is an important insight into CRVO pathophysiology.

Biodegradable nanoprobe based on MnO2 nanoflowers and graphene quantum dots for near infrared fluorescence imaging of glutathione in living cells.

Near infrared (NIR) emitting semiconductor quantum dots can be excellent fluorescent nanoprobes, but the poor biodegradability and potential toxicity limits their application. The authors describe a fluorescent system composed of graphene quantum dots (GQDs) as NIR emitters, and novel MnO2 nanoflowers as the fluorescence quenchers. The system is shown to be an activatable and biodegradable fluorescent nanoprobe for the "turn-on" detection of intracellular glutathione (GSH). The MnO2-GQDs nanoprobe is obtained by adsorbing GQDs onto the surface of MnO2 nanoflowers through electrostatic interaction. This results in the quenching of the NIR fluorescence of the GQDs. In the presence of GSH, the MnO2-GQDs nanoprobe is degraded and releases Mn2+ and free GQDs, respectively. This gives rise to increased fluorescence. The nanoprobe displays high sensitivity to GSH and with a 2.8 µM detection limit. It integrates the advantages of NIR fluorescence and biodegradability, selectivity, biocompatibility and membrane permeability. All this makes it a promising fluorescent nanoprobe for GSH and for cellular imaging of GSH as shown here for the case of MCF-7 cancer cells. Graphical abstract A biodegradable NIR fluorescence nanoprobe (MnO2-GQDs) for the "turn-on" detection of GSH in living cell was established, with the NIR GQD as the fluorescence reporter and the MnO2 nanoflower as the fluorescence quencher.

Combination of Toric and multifocal intraocular lens implantation in bilateral cataract patients with unilateral astigmatism.

AIM: To assess the binocular visual function in bilateral cataract patients with unilateral astigmatism after combined implantations of Toric with multifocal intraocular lens (IOL), and to compare with that of Toric and monofocal IOL implantation. METHODS: All the 30 patients with unilateral astigmatism suffered bilateral cataract were randomly divided into two groups: Toric plus multifocal IOL group and Toric plus monofocal IOL group. Uncorrected and corrected visual acuity at distance (5.0 m), intermediate distance (0.6 m), and near (0.33 m), contrast sensitivity, and stereopsis were assessed 6mo after surgery. Patients were also surveyed for visual disturbances and spectacle dependence. RESULTS: Binocular uncorrected visual acuity (LogMAR) of Toric/multifocal IOL eyes at distance, intermediate, near were 0.05±0.05, 0.24±0.10, and 0.14±0.06 respectively. The values of Toric plus monofocal IOL eyes were 0.06±0.07, 0.26±0.08, and 0.37±0.10 respectively. These values did not indicate significant differences between two groups with exception of near visual acuity. In the photopic condition (with or without glare), the contrast sensitivity of multifocal IOL eyes was significant lower than the monofocal IOL eyes in 18 cpd. In the mesopic condition, the contrast sensitivity of multifocal group was significant lower than monofocal group in 12 cpd, and in mesopic glare condition, this significant difference was found both in 6 cpd and 12 cpd. The stereopsis of Toric/multifocal IOL eyes decreased slightly (100±80 seconds of arc, t=2.222, P=0.136). Mean near vision for patient satisfaction was statistically significantly higher in Toric/multifocal IOL group patients versus than that in Toric/monofocal IOL group (80% vs 25.5%, P=0.000). Visual disturbance was not noticed in either group. CONCLUSION: Although the combination of Toric and multifocal IOL implantation results in compromising stereoacuity, it can still provide patients with high levels of spectacle freedom and good overall binocular visual acuity.

Comparison of Artisan iris-claw intraocular lens implantation and posterior chamber intraocular lens sulcus fixation for aphakic eyes.

AIM: To compare the efficacy and complications of Artisan iris-claw intraocular lens (IOL) implantation and posterior chamber IOL sulcus fixation for the treatment of aphakic eyes without capsular support after vitrectomy. METHODS: A prospective study of 45 cases was conducted. Forty-five eyes without sufficient lens capsule support following pars plana vitrectomy (PPV) combined lens extraction were divided into two groups. Group A: 25 eyes received Artisan iris-claw IOL implantation. Group B: 20 eyes received posterior chamber IOL sulcus fixation. The corrected distance visual acuity (CDVA) and intraocular pressure (IOP), corneal endothelial cell loss rate, surgical time and complications were compared between the two groups. Pigment changes of trabecular meshwork and anterior chamber depths were measured at each time point in Artisan group. RESULTS: The mean surgical time of Artisan group was significantly shorter (P<0.05). No statistically significant difference in endothelial cell loss rate was noted between two groups at any time point (P>0.05). CDVA of Artian group was better than that of the sulcus fixation group 1d after surgery (P<0.05) and there was no statistically significant difference 1 and 3mo after surgery (P>0.05). Mean IOP showed no significant differences between groups before and after surgery. The postoperative complications of Artisan group were anterior uveitis, iris depigmentation, pupillary distortion and spontaneous lens dislocation. The complications of sulcus fixation group include choroidal detachment, intraocular haemorrhage, tilt of IOL optic part and retinal detachment. CONCLUSION: Secondary Artisan IOL implantation can be performed less invasively and in a shorter surgical time period with earlier visual recovery after surgery compared to transscleral suturing fixation of an IOL. This technique is an effective and safe procedure. It is a promising option for the treatment of aphakic eyes without capsular support after vitrectomy.

[Artisan iris claw intraocular lens implantation for the correction of aphakia after pars plana vitrectomy].

OBJECTIVE: To compare the efficacy and complications of Artisan iris claw intraocular lens implantation with those of posterior chamber intraocular lens implantation with ciliary sulcus suture fixation for correction of aphakia in vitrectomized eyes without capsular support. METHODS: This was a prospective interventional case series. From Jan 2009 to Feb 2012, 53 aphakic vitrectomized eyes without sufficient capsular support were divided into two groups according to the condition of residual capsule. Group one:28 eyes received Artisan iris claw intraocular lens implantation. Group two: 25 eyes received posterior chamber intraocular lens implantation with ciliary sulcus suture fixation. The best corrected visual acuity and intraocular pressure, corneal endothelial cell loss rate, duration of surgery and complications were compared between two groups. Two-independent t-test,Mann-Whitney test and χ² test were used to analyze measurement data of normal distribution, non-normal distribution and count data respectively. RESULTS: The mean duration of surgery in group one [(11.23 ± 1.54) min] was much shorter (t = -26.60, P < 0.05) than that in group two [(31.68 ± 3.15) min]. The endothelial cell loss rate of group one were (4.39 ± 1.85)%, (4.76 ± 2.06)% and (6.30 ± 2.71)% at 3 months, 6 months and 1 year after surgery, and the data of group two were (3.92 ± 1.85)%, (4.33 ± 1.80)% and (5.73 ± 2.12)% . No statistically significant difference was noted between two groups at any time point (t = 0.77,0.66, 0.69; P > 0.05). The best corrected visual acuity of group one (0.40, 0.12-0.80) was better than that of group two (0.30, 0.08-0.60) at 1 day after surgery(t = -2.16, P < 0.05).However, there was no statistically significant difference at 1 month and 3 months after surgery (P > 0.05). The intraocular pressure of group two [ (11.63 ± 2.29) mmHg, 1 mmHg = 0.133 kPa] was much lower (t = 2.34, P < 0.05) than that of group one [(13.61 ± 3.37) mmHg] at 1 day after surgery. There was no statistical significant difference at 1 month and 3 months after surgery between two groups(P > 0.05). The postoperative complications of group one were anterior uveitis, iris depigmentation, pupillary distortion and spontaneous lens dislocation. Those of group two were choroidal detachment, intraocular haemorrhage, intraocular lens decentration and retinal detachment. CONCLUSIONS: Iris claw intraocular lens implantation is a simple, fast, less complications and minimally invasive method for the correction of aphakic eyes after vitrectomy. There were on difference in stability and safety between these two implantation methods. Due to the difference of indication choice, either of them can be a viable complement for the other.

Morphological changes in the lingual papillae and their connective tissue cores on the 7,12-dimethylbenz[alpha]anthracene (DMBA) stimulated rat experimental model.

The aim of the study was to analyze morphological changes of the epithelial surface and underlying connective tissue cores (CTCs) of the lingual mucosa in the rat using a DMBA induced pre-cancerous experimental model. Lightmicroscopically, initially DMBA treated sections exhibited infiltration of chronic inflammatory cells. At 16 weeks, aldehyde-fuchsin (AF) positive elastic fibers decreased and were scanty in the juxtaepithelium. On the other hand, rather densely packed thick bundles of AF positive fibers were observable in the deep layers of lamina propria. Carcinomas were not found at any stage, however, epithelial dysplasia was observed at 24 weeks post-treatment with DMBA. Scanning electron microscopy revealed an irregular arrangement of filiform papillae 4-12 weeks following DMBA stimulation. Patchy degenerated areas were observed especially at 16-24 weeks post-treatment and filiform papillae were totally attenuated on the central part of the degenerated areas. After removal of the epithelium, attenuated CTCs were observed from 4-8 weeks. Morphology of CTCs seemed to be gradually remodeled and severely altered in the later stage. The CTCs were however attenuated and exhibited a patchy distribution. The animal experimental model in this study revealed degenerative morphological changes of CTCs of the lingual papillae in the precancerous stage induced by DMBA.