Circulating Small Extracellular Vesicles Involved in Systemic Regulation Respond to RGC Degeneration in Glaucoma.

Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by progressive retinal ganglion cell (RGC) degeneration and vision loss. Since irreversible neurodegeneration occurs before diagnosable, early diagnosis and effective neuroprotection are critical for glaucoma management. Small extracellular vesicles (sEVs) are demonstrated to be potential novel biomarkers and therapeutics for a variety of diseases. In this study, it is found that intravitreal injection of circulating plasma-derived sEVs (PDEV) from glaucoma patients ameliorated retinal degeneration in chronic ocular hypertension (COH) mice. Moreover, it is found that PDEV-miR-29s are significantly upregulated in glaucoma patients and are associated with visual field defects in progressed glaucoma. Subsequently, in vivo and in vitro experiments are conducted to investigate the possible function of miR-29s in RGC pathophysiology. It is showed that the overexpression of miR-29b-3p effectively prevents RGC degeneration in COH mice and promotes the neuronal differentiation of human induced pluripotent stem cells (hiPSCs). Interestingly, engineered sEVs with sufficient miR-29b-3p delivery exhibit more effective RGC protection and neuronal differentiation efficiency. Thus, elevated PDEV-miR-29s may imply systemic regulation to prevent RGC degeneration in glaucoma patients. This study provides new insights into PDEV-based glaucoma diagnosis and therapeutic strategies for neurodegenerative diseases.

MicroRNA-18a-5p Administration Suppresses Retinal Neovascularization by Targeting FGF1 and HIF1A.

Pathologic ocular neovascularization commonly results in visual impairment or even blindness in numerous fundus diseases, including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD). MicroRNAs regulate angiogenesis through modulating target genes and disease progression, making them a new class of targets for drug discovery. In this study, we investigated the potential role of miR-18a-5p in retinal neovascularization using a mouse model of oxygen-induced proliferative retinopathy (OIR). We found that miR-18a-5p was highly expressed in the retina of pups as well as retinal endothelial cells, and was consistently down-regulated during retinal development. On the other hand, miR-18a-5p was increased significantly during pathologic neovascularization in the retinas of OIR mice. Moreover, intravitreal administration of miRNA mimic, agomiR-18a-5p, significantly suppressed retinal neovascularization in OIR models. Accordingly, agomir-18a-5p markedly suppressed human retinal microvascular endothelial cell (HRMEC) function including proliferation, migration, and tube formation ability. Additionally, we demonstrated that miR-18a-5p directly down-regulated known vascular growth factors, fibroblast growth factor 1 (FGF1) and hypoxia-inducible factor 1-alpha (HIF1A), as the target genes. In conclusion, miR-18a-5p may be a useful drug target for pathologic ocular neovascularization.

Xiayuxue Decoction ([symbols; see text]) attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4-induced fibrotic liver of mice.

OBJECTIVE: To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice. METHODS: High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively. RESULTS: Amygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 µg/mL, 50 µg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05). CONCLUSIONS: XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.

[Effects of Chinese herbal medicine Xiaopi Pill in preventing rats from dimethylnitrosamine-induced liver fibrosis].

OBJECTIVE: To explore the intervention effects of Xiaopi Pill (XPW), a compound traditional Chinese herbal medicine, on the development progress of dimethylnitrosamine (DMN)-induced liver fibrosis in rats. METHODS: Liver fibrosis model was established by intraperitoneal injection of 0.5% DMN 2 mL/kg thrice a week for 4 weeks. Rats were divided into control group given saline and treatment group given XPW during the 3rd week of DMN injections. Rats were sacrificed at the end of the experiment, and then liver histological changes, liver function and mRNA expression of the liver fibrosis-associated markers were observed. RESULTS: (1) At the end of the 2nd and 4th weeks of DMN injection, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) increased significantly in rats (P<0.01 or P<0.05); content of total bilirubin (TBil) increased significantly compared with the normal group until the end of the 4th week (P<0.05); compared with the model group after 4 weeks of DMN injection, the serum levels of ALT, AST, ALP and TBil were decreased remarkably in the XPW-treated group (P<0.01 or P<0.05). (2) The hepatic inflammation and collagen deposition in hepatic tissues increased by different degrees in experimental rats. Parts of pathological changes in the rat liver were found at the end of the 4th week, including a complete round structure of false flocculus round, meantime, the hydroxyproline content of hepatic tissue was increased significantly at the end of the 2nd and 4th weeks (P<0.05). Compared with the 4-week model group, the hepatic inflammation, collagen deposition and hydroxyproline content in hepatic tissues were alleviated dramatically (P<0.05). (3) Compared with the normal and 2nd week groups, protein expression of alpha-smooth muscle actin (α-SMA) was gradually increased, and that of the 4th week group were aggrandized significantly (P<0.01). Compared with the normal group, the mRNA expression of α-SMA, transforming growth factor-ß1 (TGF-ß1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and heme oxygenase-1 (HO-1) was gradually increased. Further changes in above-mentioned abnormalities were found in the model rats at the end of the 4th week (P<0.01); while compared to the 4th week group, protein and mRNA levels of α-SMA and mRNA levels of TGF-ß1, TIMP-1, and HO-1 were decreased significantly in the XPW group (P<0.01 or P<0.05). CONCLUSION: Progressive DMN-induced liver fibrosis in rats can be suppressed by XPW; the mechanism may be associated with inhibition of the activated hepatic stellate cells.

[Dynamic change of lipid peroxidation-related protein expression and the intervention effects of Yiguanjian decoction in a rat model of CCl4-induced liver fibrosis].

To investigate the dynamic change of lipid peroxidation-related protein expression and the intervention effects of Yiguanjian (YGJ) Decoction on liver fibrosis induced by CCl4 in rat. Fifty-seven male Wistar rats were randomly divided into a liver fibrosis group (n = 39) and a normal group (n = 18). The liver fibrosis was treated with peritoneal injection of 50% CCl4 for nine weeks. At the end of weeks 3 and 6 of CCl4 treatment, six rats were sacrificed to assess the status of liver fibrosis. At the end of week 7, the remaining -fibrotic rats were randomly divided into an untreated model group (M, n=15) and a YGJ-treated group (n = 12). The YGJ group was administered daily, oral YGJ Decoction for three weeks, concomitant with continued CCl4 treatment. The M group and normal group received the same treatment oral regimen and volume of distilled water. At the end of week 8, four rats in group M were sacrificed to observed the fibrosis status. At the end of week 9, the fibrotic rats were sacrificed for sampling. Liver function, histological changes, contents of hydroxyproline (Hyp) and malondialdehyde (MDA), activity of super oxidase dismutase (SOD) and L-glutathione (GSH), protein expression of heat shock protein (HSP)70, heme oxygenase (HO)-1, transferrin, peroxiredoxin (Prxd) 6 and liver fatty acid binding protein (L-FABP) were detected. Compared with normal group-, the MDA content was increased significantly in M group at week 6 (M: 4.23+/-0.45 nmol/mg vs. normal: 2.22+/-0.59 nmol/mg, F = 60.13, P less than 0.01) and week 9 (M: 6.29+/-1.23 nmol/mg vs. normal: 2.22+/-0.59 nmol/mg, F = 66.99, P less than 0.01), but the SOD activity was decreased significantly at the same time points [week 6: (M: 196.94+/-39.20 U/mg vs. normal: 264.50+/-30.44 U/mg, F = 11.12, P less than 0.01]); [week 9: (M: 152.2+/-51.65 U/mg vs. normal: 264.50+/-30.44 U/mg, F = 23.11, P less than 0.01)], as were the GSH content [week 6: (M: 48.47+/-7.27 nmol/mg vs. 60.74+/-9.04 nmol/mg, F = 6.71, P less than 0.05]]; [week 9: (M: 37.89+/-9.01 nmol/mg vs. 60.74+/-9.04 nmol/mg, F = 24.06, P less than 0.01]]. Compared with group M at week 9, the YGH-treated model group had markedly decreased MDA (YGJ: 4.25+/-0.86 nmol/mg vs. M: 6.29+/-1.23 nmol/mg, F = 19.52, P less than 0.01], but significantly increased SOD (YGJ: 198.35+/-46.48 U/mg vs. 152.21+/-51.65 U/mg, F = 4.65, P less than 0.05] and GSH (YGJ: 53.73+/-7.54 nmol/mg vs. M: 37.89+/-9.01 nmol/mg, F = 19.23, P less than 0.01). Compared to normal rats at week 9, group M had significantly higher protein levels of HSP70 (normal: 1.21+/-0.06 vs. M: 0.58+/-0.07, F = 166.87, P less than 0.01) and HO-1 (normal: 1.11+/-0.06 vs. M: 0.58+/-0.06, F = 123.96, P less than 0.01), but significantly decreased levels of Prxd6 (normal: 0.04+/-0.05 vs. M: 1.49+/-0.05, F = 1215.85, P less than 0.01), transferrin (normal: 0.67+/-0.03 vs. M: 1.67+/-0.04, F = 301.35, P less than 0.01), and L-FABP (normal: 0.24+/-0.02 vs. M: 1.44+/-0.14, F = 219.05, P less than 0.01). Compared to group M at week 9, the YGJ treatment group showed significantly reduced HSP70 (YGJ: 0.82+/-0.04 vs. M: 1.21+/-0.06, F = 92.31, P less than 0.01) and HO-1 (YGJ: 0.90+/-0.04 vs. 1.11+/-0.06, F = 26.89, P less than 0.01), but significantly increased Prxd6 (YGJ: 0.88+/-0.11 vs. 0.04+/-0.05, F = 150.17, P less than 0.01), transferrin (YGJ: 1.36+/-0.13 vs. 0.24+/-0.02, F = 237.19, P less than 0.01), and L-FABP (YGJ: 1.04+/-0.12 vs. 0.67+/-0.03, F = 27.53, P less than 0.01). YGJ treatment of fibrotic liver rats reduces lipid peroxidation damage by preventing generation of oxidizing substances.

Platycoside O, a new triterpenoid saponin from the roots of Platycodon grandiflorum.

A new unusual minor triterpenoid saponin, platycoside O (1), was isolated from the 75% EtOH extract obtained from the roots of Platycodon grandiflorum, together with four known saponins: platycoside M-3 (2), platycoside J (3), platycoside F (4) and platycoside B (5). The structure of 1 was determined as 3-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl-2ß,3ß,16α,23-tetrahydroxyolean-12-en-24-methoxyl, 24-oxo-28-oic acid 28-O-ß-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside on the basis of spectral analysis and chemical evidence.

Simultaneous determination of five nucleosides and nucleobases of Rehmannia glutinosa Libosch. by high performance liquid chromatography.

This study is to establish a method for simultaneously determination of five nucleosides and nucleobases, including hypoxanthine, uridine, adenine, guanosine and adenosine in Rehmannia glutinosa Libosch. which was collected from different regions in China. A Diamonsil C18 column (250 mm x 4.6 mm, 5 microm) was used. Acetonitrile and 0.04 mol L(-1) potassium dihydrogen phosphate solution were adopted as mobile phase with gradient elution. The flow rate was 1 mL min(-1) and column temperature was 30 degrees C. The detection wavelength was at 254 nm. The method had good linearity over the range of 1.0 - 16.0 microg mL(-1) (r2 = 0.999 8), 5.0 - 80.0 microg mL(-1) (r2 = 0.999 8), 1.0 - 16.0 microg mL(-1) (r2 = 0.999 5), 1.25 - 20.0 microg mL(-1) (r2 = 0.999 8) and 1.0 - 16.0 microg mL(-1) (r2 = 0.999 8) for hypoxanthine, uridine, adenine, guanosine and adenosine, respectively. The average recoveries were between 98.8% and 100.7%. The content of hypoxanthine, uridine, adenine, guanosine and adenosine in Rehmannia glutinosa Libosch. from different regions was significantly different. This established method was sensitive and reliable for the quantification of five chemical constituents in Rehmannia glutinosa Libosch.

Pharmacokinetic behavior of 16-dehydropregnenolone after intramuscular administration in rats.

The pharmacokinetics of 16-dehydropregnenolone (16-DHP), a sterols compound isolated from Solanum lyratum Thunb., was investigated in rats following a Single intramuscular administration (40 mg/kg). The concentration of 16-DHP in rat plasma was determined by a high Performance liquid chromatography (HPLC) method with UV detection. Levonorgestrel was used as the internal Standard (IS). The pharmacokinetic parameters of 16-DHP were derived by non-compartmental method. After a Single intramuscular administration, the maximum plasma concentration (Cmax) was (289 ± 25) ng/mL, time to reach Cmax(tmax) was (0.38 ± 0.14) h, the elimination half-life (t1/2) was (2.5 ± 1.1) h, the area under the plasma concentration-time curve from time zero to the time of the last measurable concentration (AUC(0-t)) was (544 ± 73)ng · h/mL. The results indicated that 16-DHP was absorbed quickly and eliminated rapidly in rats after the intramuscular injection.