Pharmacokinetics of Sirolimus Eye Drops Following Topical Ocular Administration in Rabbits.

Purpose: To evaluate the pharmacokinetics of sirolimus eye drops following topical instillation in rabbits. Methods: The study included 2 experiments. In single-dose pharmacokinetic study, rabbits received a single bilateral instillation of 0.05% sirolimus eye drops (0.5 mg/mL, 50 µL/eye). In repeat-dose pharmacokinetic study, 0.05% sirolimus eye drops (0.5 mg/mL, 50 µL/eye/time) were instilled into both eyes of rabbits four times a day for 6 consecutive days and one time on day 7. Whole blood, tears, aqueous humor, cornea, and conjunctiva samples were collected. Sirolimus concentration was determined by a validated liquid chromatography-tandem mass spectrometry. Results: Sirolimus was hardly detected in plasma or aqueous humor after either single or repeated dosing. The Cmax of sirolimus in tears, cornea, and conjunctiva after a single instillation was 163.34 ± 69.30 µg/g, 150.56 ± 84.98 ng/g, and 113.22 ± 49.82 ng/g, respectively. As the number of instillation elevated, the Cmax of sirolimus was increased to 486.18 ± 297.93 µg/g, 418.63 ± 41.07 ng/g, and 314.25 ± 63.74 ng/g, respectively. In repeat-dose administration, the steady state of sirolimus concentration was achieved on the third day. Ocular exposure to sirolimus after single and repeated dosing, based on AUC0-t, was highest in tears, followed by cornea and conjunctiva. Compared with single administration, a significant increase in sirolimus exposure as measured by AUC0-t was observed in tears, cornea, and conjunctiva following repeated administration. Conclusions: Topical administration of sirolimus eye drops results in extensive distribution of sirolimus in tears, cornea, and conjunctiva, while aqueous humor and systemic exposure were negligible. Repeat-dose administration increases sirolimus exposure in tears, cornea, and conjunctiva.

Constructing a polygenic risk score for childhood obesity using functional data analysis.

Obesity is a highly heritable condition that affects increasing numbers of adults and, concerningly, of children. However, only a small fraction of its heritability has been attributed to specific genetic variants. These variants are traditionally ascertained from genome-wide association studies (GWAS), which utilize samples with tens or hundreds of thousands of individuals for whom a single summary measurement (e.g., BMI) is collected. An alternative approach is to focus on a smaller, more deeply characterized sample in conjunction with advanced statistical models that leverage longitudinal phenotypes. Novel functional data analysis (FDA) techniques are used to capitalize on longitudinal growth information from a cohort of children between birth and three years of age. In an ultra-high dimensional setting, hundreds of thousands of single nucleotide polymorphisms (SNPs) are screened, and selected SNPs are used to construct two polygenic risk scores (PRS) for childhood obesity using a weighting approach that incorporates the dynamic and joint nature of SNP effects. These scores are significantly higher in children with (vs. without) rapid infant weight gain-a predictor of obesity later in life. Using two independent cohorts, it is shown that the genetic variants identified in very young children are also informative in older children and in adults, consistent with early childhood obesity being predictive of obesity later in life. In contrast, PRSs based on SNPs identified by adult obesity GWAS are not predictive of weight gain in the cohort of young children. This provides an example of a successful application of FDA to GWAS. This application is complemented with simulations establishing that a deeply characterized sample can be just as, if not more, effective than a comparable study with a cross-sectional response. Overall, it is demonstrated that a deep, statistically sophisticated characterization of a longitudinal phenotype can provide increased statistical power to studies with relatively small sample sizes; and shows how FDA approaches can be used as an alternative to the traditional GWAS.

Temporary Upregulation of Nrf2 by Naringenin Alleviates Oxidative Damage in the Retina and ARPE-19 Cells.

Dry age-related macular degeneration (dAMD) is a chronic degenerative ophthalmopathy that leads to serious burden of visual impairment. Antioxidation in retinal pigment epithelium (RPE) cells is considered as a potential treatment for dAMD. Our previous studies have showed that naringenin (NAR) protects RPE cells from oxidative damage partly through SIRT1-mediated antioxidation. In this study, we tested the hypothesis that the Nrf2 signaling is another protective mechanism of NAR on dAMD. NaIO3-induced mouse retinopathy and ARPE-19 cell injury models were established. Immunochemical staining, immunofluorescence, and western blotting were performed to detect the protein expressions of Nrf2 and HO-1. In addition, ML385 (activity inhibitor of Nrf2) and zinc protoporphyrin (ZnPP, activity inhibitor of HO-1) were applied to explore the effect of NaIO3 or NAR. The results showed that NAR increased the protein expressions of Nrf2 and HO-1 in the retinas in mice exposed to NaIO3 at the early stage. NAR treatment also resulted in a stronger activation of Nrf2 at the early stage in NaIO3-treated ARPE-19 cells. Moreover, inhibition of HO-1 by ZnPP weakened the cytoprotective effect of NAR. The constitutive accumulation and activation of Nrf2 induced by NaIO3 led to the death of RPE cells. However, NAR decreased the protein expressions of Nrf2 and HO-1 towards normal level in the mouse retinas and ARPE-19 cells exposed to NaIO3 at the late stage. Our findings indicate that NAR protects RPE cells from oxidative damage via activating the Nrf2 signaling pathway.

Naringenin protects RPE cells from NaIO3-induced oxidative damage in vivo and in vitro through up-regulation of SIRT1.

BACKGROUND: Dry age-related macular degeneration (dAMD) leads to serious burden of visual impairment and there is no definitive treatment. Previous studies have showed that naringenin (NAR) significantly increased electroretinography (ERG) c-wave in sodium iodate (NaIO3)-treated rats and viability of NaIO3-treated ARPE-19 cells. But the underlying mechanism is still unknown. PURPOSE: We tested the hypothesis that anti-oxidation mediated by Sirtuin 1 (SIRT1) was important to the protective effect of NAR on dAMD. STUDY DESIGN/METHODS: NaIO3-induced mice retinopathy and ARPE-19 cells injury models were established. In vivo, the protective effect of NAR eye drops on retina was evaluated by flash ERG (FERG) recording and histopathological examination. In vitro, viability of ARPE-19 cells, and the levels of lactic dehydrogenase (LDH), reactive oxygen species (ROS) and carbonyl protein were detected. Protein expression of SIRT1 was analyzed by immunochemical staining, immunofluorescence and western blotting. RESULTS: NAR eye drops improved retinal function and morphology and normalized the protein expression of SIRT1 in mice exposed to NaIO3. NAR promoted the survival of ARPE-19 cells in a concentration-dependent manner. NAR up-regulated SIRT1 protein expression, and decreased levels of ROS and carbonyl protein. Moreover, EX527, a selective inhibitor of SIRT1, abolished the effects of NAR on the cell viability and ROS. In addition, SRT1720, a selective agonist of SIRT1, improved the viability of cells and suppressed the production of ROS. CONCLUSION: Our findings indicate that SIRT1-mediated anti-oxidation contributes to the protective effect of NAR eye drops on dAMD.

Nuclear HMGB1 promotes the phagocytic ability of macrophages.

Phagocytosis is a basic immune response to the invasion of pathogens. High mobility group protein B1 (HMGB1) is a DNA chaperone that is associated with phagocytosis. However, its influence on phagocytosis is debated. In the present study, HMGB1-mutant, HMGB1-overexpressing and HMGB1-silenced RAW264.7 cells were constructed. In addition, HMGB1 conditional knockout mice were constructed to determine the influence of HMGB1 on phagocytosis. Lipopolysaccharide (LPS) was used to stimulate the translocation of HMGB1 from the nucleus to the cytoplasm. Zymosan particles were used to test the phagocytic function of the macrophages. Our results showed that the accumulation of HMGB1 in the nucleus enhances the phagocytic function of the macrophages. By interacting with P53, nuclear HMGB1 may remain in the nucleus and decrease the negative influence of P53 on the phosphorylation of focal adhesion kinase (FAK). The increase in phosphorylated FAK promotes the formation of pseudopods and enhances the phagocytic ability of macrophages.

Paeonol attenuates inflammation by targeting HMGB1 through upregulating miR-339-5p.

Sepsis is a life-threatening disease caused by infection. Inflammation is a key pathogenic process in sepsis. Paeonol, an active ingredient in moutan cortex (a Chinese herb), has many pharmacological activities, such as anti-inflammatory and antitumour actions. Previous studies have indicated that paeonol inhibits the expression of HMGB1 and the transcriptional activity of NF-κB. However, its underlying mechanism is still unknown. In this study, microarray assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results confirmed that paeonol could significantly up-regulate the expression of miR-339-5p in RAW264.7 cells stimulated by LPS. Dual-luciferase assays indicated that miR-339-5p interacted with the 3' untranslated region (3'-UTR) of HMGB1. Western blot, immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analyses indicated that miR-339-5p mimic and siHMGB1 both negatively regulated the expression and secretion of inflammatory cytokines (e.g., HMGB1, IL-1ß and TNF-α) in LPS-induced RAW264.7 cells. Studies have confirmed that IKK-ß is targeted by miR-339-5p, and we further found that paeonol could inhibit IKK-ß expression. Positive mutual feedback between HMGB1 and IKK-ß was observed when we silenced HMGB1 or IKK-ß. These results indicated that paeonol could attenuate the inflammation mediated by HMGB1 and IKK-ß by upregulating miR-339-5p expression. In addition, we constructed CLP model mice by cecal ligation and puncture. Paeonol was used to intervene to investigate its anti-inflammatory effect in vivo. The results showed that paeonol could improve the survival rate of sepsis mice and protect the kidney of sepsis mice.

Consumption of Bifidobacterium animalis subsp. lactis BB-12 in yogurt reduced expression of TLR-2 on peripheral blood-derived monocytes and pro-inflammatory cytokine secretion in young adults.

PURPOSE: Probiotic bacteria modulate immune parameters and inflammatory outcomes. Emerging evidence demonstrates that the matrix used to deliver probiotics may influence the efficacy of probiotic interventions in vivo. The aims of the current study were to evaluate (1) the effect of one species, Bifidobacterium animalis subsp. lactis BB-12 at a dose of log10 ± 0.5 CFUs/day on immune responses in a randomized, partially blinded, 4-period crossover, free-living study, and (2) whether the immune response to BB-12 differed depending on the delivery matrix. METHODS: Healthy adults (n = 30) aged 18-40 years were recruited and received four treatments in a random order: (A) yogurt smoothie alone; smoothie with BB-12 added (B) before or (C) after yogurt fermentation, or (D) BB-12 given in capsule form. At baseline and after each 4-week treatment, peripheral blood mononuclear cells (PBMCs) were isolated, and functional and phenotypic marker expression was assessed. RESULTS: BB-12 interacted with peripheral myeloid cells via Toll-like receptor 2 (TLR-2). The percentage of CD14+HLA-DR+ cells in peripheral blood was increased in male participants by all yogurt-containing treatments compared to baseline (p = 0.0356). Participants who consumed yogurt smoothie with BB-12 added post-fermentation had significantly lower expression of TLR-2 on CD14+HLA-DR+ cells (p = 0.0186) and reduction in TNF-α secretion from BB-12- (p = 0.0490) or LPS-stimulated (p = 0.0387) PBMCs compared to baseline. CONCLUSIONS: These findings not only demonstrate a potential anti-inflammatory effect of BB-12 in healthy adults, but also indicate that the delivery matrix influences the immunomodulatory properties of BB-12.

Consumption of Bifidobacterium animalis subsp. lactis BB-12 impacts upper respiratory tract infection and the function of NK and T cells in healthy adults.

SCOPE: Probiotics can modulate immunity and reduce upper respiratory tract infections (URTI) in humans; however few studies have examined both outcomes in the same trial. The goal of the current study was to investigate the effect of Bifidobacterium animalis subsp. lactis BB-12, on natural killer (NK) and T-cell function in conjunction with self-reported cold/flu outcomes in healthy adults. METHODS AND RESULTS: In a randomized, partially blinded, four-period crossover study, healthy adults (n = 30) were recruited, and received four treatments for 4 weeks in a random order: (i) yogurt smoothies alone (YS); smoothies with BB-12 added (ii) before (PRE) or (iii) after (POST) yogurt fermentation, or (iv) BB-12 capsule (CAP). NK- and T-cell function was assessed at baseline and after each treatment. Incidence and severity of cold/flu infection was quantified using self-reported URTI questionnaires. Participants on YS, PRE, or CAP treatments had elevated IL-2 secretion and NK-cell cytotoxicity, concurrently with fewer days with URTI. However, the POST treatment did not change immune outcomes or the severity of URTI. CONCLUSION: The timing of BB-12 addition to yogurt smoothies in relation to the fermentation process influenced the impact of BB-12 on immune function and cold/flu severity in young healthy adults.

Danhong Huayu Koufuye combined with metformin attenuated diabetic retinopathy in Zucker diabetic fatty rats.

AIM: To evaluate effects of Danhong Huayu Koufuye (DHK, a Chinese medicinal formulae) alone or combined with metformin on diabetic retinopathy (DR) in Zucker diabetic fatty (ZDF) rats, an animal model of obese type-2 diabetes, and then to investigate the mechanisms. METHODS: ZDF (fa/fa) rats were administered with vehicle (distilled water), metformin, DHK, and DHK plus metformin. Electrophysiological and histological analysis were applied to evaluated effects of DHK alone or combined with metformin on DR. The levels of fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) in blood were measured to evaluate the antihyperglycemic activity of DHK. Furthermore, levels of nitric oxide (NO), malondialdehyde (MDA) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in serum were measured to study effects of DHK on oxidative stress in ZDF rats. In addition, body weight, lipidic indexes and insulin level were also assessed. RESULTS: DHK combined with metformin significantly reversed the prolongation of latency times of flash electroretinogram (FERG) and oscillatory potentials (OPs) in diabetic rats. Furthermore, DHK alone or combined with metformin showed a remarkable suppression of retinal neovascularization and amelioration of retinal internal limiting membrane morphology. Moreover, DHK alone or plus metformin reduced FBG (P<0.05), HbA1c (P<0.01) and MDA (P<0.01) levels in diabetic rats. In addition, reductions in levels of triglycerides (TG) (P<0.01) and low density lipoprotein cholesterol (LDL-c) (P<0.01 and P<0.05, respectively) were also observed in diabetic rats treated with DHK alone or plus metformin. CONCLUSION: DHK in combination with metformin had a preventive and therapeutic effect on DR in type-2 diabetic rats, and the possible mechanisms may be alleviating hyperglycemia, reducing oxidative stress and improving lipid metabolism.

Ocular pharmacokinetics of naringenin eye drops following topical administration to rabbits.

PURPOSE: To investigate the ocular pharmacokinetics of 1% naringenin eye drops following topical administration to rabbits. METHODS: One drop (50 µL) of 1% naringenin eye drops was instilled into both eyes of each rabbit. The animals were sacrificed at predetermined intervals after dosing, and ocular tissues and plasma were then collected. Concentrations of naringenin were analyzed using specific electrospray ionization liquid chromatography-tandem mass spectrometry method, which is proved to be sensitive, specific, precise, and suitable for determination of naringenin in ocular tissues and plasma of rabbits. RESULTS: Ocular exposure to naringenin, based on AUC(0-t), was highest in cornea, followed by aqueous humor, retina, and vitreous body. The Cmax of naringenin in cornea, aqueous humor, vitreous body, and retina were 67945.30 ± 4109.34 ng/g, 1325.69 ± 239.34, 160.52 ± 38.78 ng/mL, and 1927.08 ± 660.77 ng/g at 0.083, 0.75, 0.083, and 0.083 h after topical administration, respectively. The half-lives for these tissues were 9.37, 0.65, 1.17, and 4.62 h, respectively. There was no significant difference between free naringenin and total naringenin in plasma based on Cmax and Tmax. Cmax of total naringenin in plasma at 0.083 h was 35.12 ± 0.54 ng/mL. CONCLUSIONS: Measurable concentrations of naringenin were achieved in ocular tissues after topical application in rabbits. Topical instillation of naringenin may be an effective approach in the treatment of posterior section diseases.

Protective effects of naringenin eye drops on N-methyl-N-nitrosourea-induced photoreceptor cell death in rats.

AIM: To investigate the effects of naringenin eye drops on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell death in rats. METHODS: Photoreceptor cell death was induced by single intraperitoneal injection of MNU (60 mg/kg) in rats. Both eyes of all animals were instilled with one drop of vehicle, 0.5% or 1.0% naringenin eye drops three times per day from 7d before to 17d after MNU injection. Effects of naringenin on MNU-induced photoreceptor cell death were evaluated by electrophysiological and histological analysis. RESULTS: Flash electroretinography (FERG) and oscillatory potentials (OPs) recordings showed that the vehicle control group had remarkable reduction of amplitudes and prolongation of latency times. FERG and OPs responses were significantly reversed in MNU-induced rats treated with 0.5% or 1.0% naringenin eye drops compared with the vehicle control. The retinal morphological results showed that naringenin dose-dependently preserved the outer nuclear layer, outer retina and total retina. CONCLUSION: These results indicate that topical treatment with naringenin eye drops prevented retinal neurons from MNU-induced structural and functional damages.

[Effect of danhong huayu oral liquid combined with insulin on prevention and progression of early diabetic cardiomyopathy in rats].

OBJECTIVE: To investigate effects of Danhong Huayu Koufuye (DHK), insulin and their combination on diabetic cardiomyopathy (DC) in streptozotocin (STZ, 50 mg/kg, ip)-induced diabetic rats. METHODS: Rats were divided into five groups: normal control, diabetic treated with vehicle, insulin, DHK, and DHK plus insulin. The animals were treated once daily for 15 weeks starting one week after STZ injection. RESULTS: The combination of DHK with insulin significantly reduced cardiac index (P < 0.05), serum LDH (P < 0.05), AST(P < 0.05), ALT(P < 0.05) and HDL-C (P < 0.05) level, and promoted pancreatic and cardiac morphological changes as compared with the model group. CONCLUSION: It is suggested that DHK may be a valuable adjuvant therapy for DC.

Preventive effect of danhong huayu koufuye on diabetic retinopathy in rats.

AIM: To study the effects of danhong huayu koufuye (DHK) on fasting blood glucose (FBG) and diabetic retinopathy (DR) in streptozotocin (STZ)-induced type 1 diabetic rats to facilitate the rational usage of this drug. METHODS: Diabetic rats were induced by injection of a single dose of STZ intraperitoneally at 50mg/kg. Flash electroretinogram (FERG) and oscillatory potentials (OPs) were used to measure retinal function. The microvascular perfusion of ears was performed to study the microcirculation in rats. FBG, body-weight, and 24-h urine volume, water intake and diet intake were also assessed. RESULTS: DHK had no effect on FBG in normal rats. However, STZ + DHK group were significantly different from those of Model and moved toward those of normal control. It reversed the increase in diet intake (P≤0.05 vs model control) and the loss in body-weight (P≤0.05 vs model control) in diabetic rats. DHK decreased the FBG of diabetic rats by 25.6% (P≤0.05) and 37.9% (P≤0.01) after 14 and 21 days administration as compared with the model control, respectively. Moreover, DHK significantly increased the FERG b-wave amplitude by 80% (P≤0.05 vs model control) and decreased the FERG b-wave latency by 15.3% (P≤0.01 vs model control) after 24 days administration. The OP(1) and OP(2) amplitudes in DHK group were 2.6 (P≤0.01) and 2.0 (P≤0.01) times of model group after 24 days of DHK treatment, respectively. At the same time, OP1 and OP2 latencies in DHK group reduced by 16.0% (P≤0.001) and 14.7% (P≤0.001) as compared with the model control, respectively. Furthermore, the microvascular perfusion of DHK group was 2.4 times of model group (P≤0.001) after 21 days administration. CONCLUSION: DHK had no effect on normal FBG. But it had antihyperglycemic activity, and had a preventive and therapeutic effect on DR in diabetic rats.