High-Speed Clearing and High-Resolution Staining for Analysis of Various Markers for Neurons and Vessels.

BACKGROUND: Tissue clearing enables deep imaging in various tissues by increasing the transparency of tissues, but there were limitations of immunostaining of the large-volume tissues such as the whole brain. METHODS: Here, we cleared and immune-stained whole mouse brain tissues using a novel clearing technique termed high-speed clearing and high-resolution staining (HCHS). We observed neural structures within the cleared brains using both a confocal microscope and a light-sheet fluorescence microscope (LSFM). The reconstructed 3D images were analyzed using a computational reconstruction algorithm. RESULTS: Various neural structures were well observed in three-dimensional (3D) images of the cleared brains from Gad-green fluorescent protein (GFP) mice and Thy 1-yellow fluorescent protein (YFP) mice. The intrinsic fluorescence signals of both transgenic mice were preserved after HCHS. In addition, large-scale 3D imaging of brains, immune-stained by the HCHS method using a mild detergent-based solution, allowed for the global topological analysis of several neuronal markers such as c-Fos, neuronal nuclear protein (NeuN), Microtubule-associated protein 2 (Map2), Tuj1, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) in various anatomical regions in the whole mouse brain tissues. Finally, through comparisons with various existing tissue clearing methodologies such as CUBIC, Visikol, and 3DISCO, it was confirmed that the HCHS methodology results in relatively less tissue deformation and higher fluorescence retention. CONCLUSION: In conclusion, the development of 3D imaging based on novel tissue-clearing techniques (HCHS) will enable detailed spatial analysis of neural and vascular networks present within the brain.

Three-dimensional visualization of cerebral blood vessels and neural changes in thick ischemic rat brain slices using tissue clearing.

Blood vessels are three-dimensional (3D) in structure and precisely connected. Conventional histological methods are unsuitable for their analysis because of the destruction of functionally important topological 3D vascular structures. Tissue optical clearing techniques enable extensive volume imaging and data analysis without destroying tissue. This study therefore applied a tissue clearing technique to acquire high-resolution 3D images of rat brain vasculature using light-sheet and confocal microscopies. Rats underwent middle cerebral artery occlusion for 45 min followed by 24 h reperfusion with lectin injected directly into the heart for vascular staining. For acquiring 3D images of rat brain vasculature, 3-mm-thick brain slices were reconstructed using tissue clearing and light-sheet microscopy. Subsequently, after 3D rendering, the fitting of blood vessels to a filament model was used for analysis. The results revealed a significant reduction in vessel diameter and density in the ischemic region compared to those in contralesional non-ischemic regions. Immunostaining of 0.5-mm-thick brain slices revealed considerable neuronal loss and increased astrocyte fluorescence intensity in the ipsilateral region. Thus, these methods can provide more accurate data by broadening the scope of the analyzed regions of interest for examining the 3D cerebrovascular system and neuronal changes occurring in various brain disorders.

Effect of Diquafosol on Hyperosmotic Stress-induced Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Corneal Epithelial Cells.

PURPOSE: Diquafosol is a pharmaceutical drug used for dry eye treatment with a novel mechanism of action. It is a purinergic P2Y2 receptor agonist that promotes the secretion of tears and healing of corneal epithelial wounds. However, its inhibitory effect on hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs) remains unclear. METHODS: A hyperosmotic stress model was established by transferring HCECs from isosmotic (312 mOsm/kg to hyperosmotic medium (500 mOsm/kg). HCECs were incubated with 500 mOsm/kg hyperosmotic medium for 30 minutes, and then treated with diquafosol (0.6-6 mg/mL) for 4 or 24 hours. Cells were then harvested and analyzed by western blot, immunocytochemistry, and real-time polymerase chain reaction to evaluate the expression of interleukin-6, tumor necrosis factor-alpha, and the phosphorylation status of nuclear factor-kappa B. RESULTS: Diquafosol significantly decreased the mRNA and protein expression of hyperosmotic stress-induced tumor necrosis factor-alpha and interleukin-6. These results were supported by immunofluorescence staining and quantitative real-time polymerase chain reaction analysis. Furthermore, diquafosol inhibits nuclear factor-kappa B activation by suppressing the phosphorylation and degradation of the inhibitor of кB. CONCLUSIONS: This study shows that diquafosol inhibits nuclear factor-kappa B signaling and inflammatory factors induced by hyperosmotic stress in HCECs. This suggests that using diquafosol for the improvement of dry eye syndrome could be effective in the treatment of inflammation-related corneal and conjunctival diseases.

The physico-chemical alteration of lovastatin and enhanced antioxidant effect of Bacillus subtilis fermented-red yeast rice product.

Red yeast rice product (RYP) has been used as a food supplement because of its lipid lowering, and in food additives as a natural colorant. Lovastatin of RYP is a hypolipidemic commercial drug. To enhance the beneficial effects of RYP, we performed a bioconversion with Bacillus subtilis. This B. subtilis-fermentation process of RYP increased the ratio of the active open-hydroxyl acid form and the prodrug lactone form of lovastatin, which is a potent cholesterol synthesis inhibitor. 3(2H)-benzofuranone was newly produced in the fermented red yeast rice product (FRYP) as analyzed by GC-MS. FRYP increased the free radical scavenging activity compared with RYP. FRYP blocked xanthine oxidase (XO)-induced oxidative cytotoxicity and inhibited the H2O2-induced intracellular ROS in cells. This is the first study to illustrate that B. subtilis-fermented FRYP is useful for facilitating the alteration in the physico-chemical property of lovastatin and enhancing antioxidant activity, which may have greater pharmacological activity.

Inhibition of Pterygium Fibroblast Migration and Outgrowth by Bevacizumab and Cyclosporine A Involves Down-Regulation of Matrix Metalloproteinases-3 and -13.

We examined the connection between matrix metalloproteinase (MMP) expression/activity and pterygium fibroblast migration, and how these were affected by bevacizumab and/or cyclosporine A (CsA). Fibroblasts were obtained from 20 pterygia and 6 normal conjunctival specimens. Expression levels of MMP-3 and MMP-13 were examined after bevacizumab administration. Immunofluorescence staining was used to examine expression of both MMPs in fibroblasts migrating out from explanted pterygium tissues. Rates of cell migration from explant-cultured pterygia tissues and scratch-wounded confluent pterygium fibroblasts were examined in the presence of MMP-3 or MMP-13 inhibitors, as well as bevacizumab and/or CsA. A scratch wound healing migration assay was performed to determine the effects of bevacizumab and/or CsA. Protein expression of both MMPs in pterygium tissues and in cells migrating from organ-cultured pterygium tissues was greater than that observed in normal cells. Inhibition of the activities of both MMPs decreased their expression levels; these were also significantly reduced in bevacizumab-injected pterygium tissues. Bevacizumab significantly reduced the expression of both MMPs and cell migration. Pretreatment with CsA prior to bevacizumab exposure markedly inhibited cell migration and the expression of both MMPs. CsA enhanced the inhibitory effects of bevacizumab on pterygium fibroblast migration in vitro, possibly by inhibiting expression of both MMPs. These findings suggest that combined CsA and bevacizumab treatment may provide a potential therapeutic strategy for reducing the rate of pterygium recurrence.

Cyclosporine A inhibits TGF-ß2-induced myofibroblasts of primary cultured human pterygium fibroblasts.

Cyclosporine A (CsA), an immunomodulatory drug, and is increasingly used to treat moderate dry eye syndrome and ocular surface inflammation. However, any inhibitory effect on differentiation of fibroblasts to myofibroblasts remains unclear. Here, we show that the inhibitory effect of CsA on transforming growth factor-beta2 (TGF-ß2)-induced myofibroblasts in primary cultured human pterygium fibroblasts. CsA significantly decreased mRNA and protein expression of myofibroblast-related markers including α-SMA, laminin, and fibronectin. These findings were supported by the results from immunofluorescence staining. Taken together, these results indicate the therapeutic potential of CsA against pterygium progression. Further studies are necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of myofibroblast markers in pterygium fibroblasts.

A Distinctive Pattern of Beauveria bassiana-biotransformed Ginsenoside Products Triggers Mitochondria/FasL-mediated Apoptosis in Colon Cancer Cells.

Ginseng is one of the most commonly used adaptogens. Transformation into the minor ginsenosides produces compounds with more effective action. Beauveria bassiana, a teleomorph of Cordyceps bassiana, is a highly efficient producer of mammalian steroids and produces large amounts of sugar-utilizing enzymes. However, the fermentation of steroid glycosides in ginseng with B. bassiana has never been studied. Thus, we evaluated the bioconversion of the major ginsenosides in white ginseng by B. bassiana. Interestingly, B. bassiana increased the total amount of protopanaxadiols and hydrolyzed Rb1 into minor ginsenosides, exhibiting high levels of Rd and Rg3, as well as moderate levels of Rb2 and Rc analyzed by high-performance liquid chromatography coupled with evaporative light-scattering detection. The ß-glucosidase activity was highly increased, which led to the selective elimination of sugar moiety at the 20-C position of Rb1 to Rd, followed by Rg3. Rb2 and Rc accumulated because of the minimal activities of α-L-arabinopyranosidase and α-L-arabinofuranosidase, respectively. The fermentation product exerted dose-dependent cytotoxicity in HCT-15 cells, which are resistant to ginseng. The product, but not white ginseng, exhibited apoptotic effects via the Fas ligand and caspase 8/9. This study demonstrates for the first time that the B. bassiana-fermented metabolites have potent apoptotic activity in colon cancer cells, linking to a therapeutic use.

The small GTPases regulate HMC05-induced NQO-1 expression with an antioxidant effect in smooth muscle cells.

Recently, Banhabackchulchunmatang (HMC05) has been implicated as a preventive and/or therapeutic candidate for cardiovascular diseases due to its inhibition of atherosclerosis lesions and its reduction of neointima formation. Knowledge of the mechanism of HMC05 in smooth muscle cells (SMC) is limited. However, SMC may be a potential target for HMC05 therapy because they are supported by the HMC05-mediated preservation of medial smooth muscle cell layers in pathogenic progression. Therefore, in the present study, we hypothesized that the effect of HMC05 is associated with reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H):quinone oxidoreductase-1 (NQO-1) gene regulation, which precipitates an antioxidant effect in SMC. HMC05 significantly increased NQO-1 gene expression in a dose- and time-dependent manner. The reactive oxygen species-mediated toxicity that was generated by xanthine/xanthine oxidase was suppressed by HMC05. The knockdown of the NQO-1 gene abrogated the HMC05-mediated cytoprotection. Interestingly, pretreatment with a chemical inhibitor of geranylgeranyl transferase 1 or farnesyl transferase abolished the NQO-1 gene induction and cytoprotection by HMC05. The transfection of dominant negative RhoA or Ras suppressed HMC05-induced gene expression. Berberine and hesperidin, which are found in large quantities in HMC05, also induced NQO-1 gene expression. Taken together, this is the first study to demonstrate that HMC05 is efficacious in protection against oxidative stress through NOQ-1 gene induction via the regulation of RhoA and/or Ras, and that berberine and hesperidin are major components of NQO-1 gene induction. This study provides mechanistic targets of HMC05 in reducing atherosclerotic lesions in atherosclerosis.

Differential hepatic GSTA2 expression of arylalkyl isothiocyanates in vivo and in vitro: the molecular mechanism of gene induction by phenethyl isothiocyanate.

SCOPE: Naturally occurring isothiocyanates (ITCs) have been investigated for their chemopreventive actions. However, limited studies have determined the potential for regulation of hepatic glutathione S-transferase A2 (GSTA2) by arylalkyl or alkyl ITCs. METHODS AND RESULTS: The present study was designed to investigate the effects of ITCs on GSTA2 expression and upstream signaling analyzed by Northern blotting, RT-PCR, immunoblotting, and promoter-luciferase assay. A single dose of ethyl isothiocyanate and phenethyl isothiocyanate (PEITC) enhanced GSTA2 mRNA levels in rats. The three consecutive injections of arylalkyl ITCs resulted in the gene induction. PEITC was the most potent and sustained inducer of GSTA2 in vivo. PEITC induced GSTA2 mRNA and protein expression in H4IIE cells. PEITC increased the levels of nuclear factor erythroid 2 related factor 2 and CCAAT/enhancer binding protein ß (C/EBPß). The cells transfected with a pGL-ΔARE or pGL-ΔC/EBP-deleted promoter construct failed to increase GSTA2 promoter activity by PEITC. These effects of PEITC were downstream of multiple cellular signaling, including phosphoinositide 3-kinase (PI3K), c-jun N-terminal kinase (JNK), and/or protein kinase A (PKA). CONCLUSION: These results demonstrated that PEITC is the most efficacious of the arylalkyl ITCs for the induction of GSTA2 in vivo and in vitro, and that transactivation of C/EBP and nuclear factor erythroid 2 related factor 2 downstream of multiple signaling pathways is involved in this induction.

The amelioration of N-acetyl-p-benzoquinone imine toxicity by ginsenoside Rg3: the role of Nrf2-mediated detoxification and Mrp1/Mrp3 transports.

Previously, we found that Korean red ginseng suppressed acetaminophen (APAP)-induced hepatotoxicity via alteration of its metabolic profile involving GSTA2 induction and that ginsenoside Rg3 was a major component of this gene induction. In the present study, therefore, we assessed the protective effect of Rg3 against N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolic intermediate of APAP. Excess NAPQI resulted in GSH depletion with increases in the ALT and AST activities in H4IIE cells. Rg3 pretreatment reversed GSH depletion by NAPQI. Rg3 resulted in increased mRNA levels of the catalytic and modulatory subunit of glutamate cysteine ligase (GCL), the rate-limiting steps in GSH synthesis and subsequently increased GSH content. Rg3 increased levels of nuclear Nrf2, an essential transcriptional factor of these genes. The knockdown or knockout of the Nrf2 gene abrogated the inductions of mRNA and protein by Rg3. Abolishment of the reversal of GSH depletion by Rg3 against NAPQI was observed in Nrf2-deficient cells. Rg3 induced multidrug resistance-associated protein (Mrp) 1 and Mrp3 mRNA levels, but not in Nrf2-deficient cells. Taken together, these results demonstrate that Rg3 is efficacious in protecting hepatocytes against NAPQI insult, due to GSH repletion and coordinated gene regulations of GSH synthesis and Mrp family genes by Nrf2.

Korean red ginseng extract prevents APAP-induced hepatotoxicity through metabolic enzyme regulation: the role of ginsenoside Rg3, a protopanaxadiol.

BACKGROUND: Inappropriate use of acetaminophen (APAP) can lead to morbidity and mortality secondary to hepatic necrosis. AIMS: We evaluated the beneficial effect and molecular mechanism of Korean red ginseng (KRG) on the APAP-mediated hepatotoxicity and identified a major component of KRG for hepatoprotection. METHODS: Survival test, liver function test, histopathological study, APAP-metabolic profiling and gene expression were examined in mice. We determined the enzyme expression and upstream signalling in H4IIE cells analysed by RT-PCR, immunoblotting, siRNA gene knockdown and promoter-luciferase assay. RESULTS: High doses of KRG reduced mortality at the LD50 of APAP. APAP increased AST and ALT activities, which were abrogated by low doses of KRG. These protective effects were consistent with the results from histopathological examinations. KRG altered APAP metabolic profiles through inhibition of cytochrome P450 2E1 and induction of glutathione S-transferase A2 (GSTA2). Knockdown of GSTA2 catalyses the conjugation of glutathione reversed KRG-mediated protection against N-acetyl-p-benzoquinone imine in H4IIE cells. The nuclear Nrf2 and C/EBPß, which are essential transcriptional factors for GSTA2 were increased by KRG. These effects were downstream of multiple signalling, including PI3K, JNK or PKA. Ginsenoside Rg3 but not Rb1, Rc and Rg1 significantly increased GSTA2 protein expression. Rg3 resulted in the transcriptional activation of GSTA2 downstream of the multiple cellular signalling. CONCLUSIONS: These results demonstrate that KRG is efficacious in protection against APAP-induced hepatotoxicity and mortality through metabolic regulation and that Rg3 is a major component of KRG for the GST induction, implying that Rg3 should be considered to be a potential hepatoprotective agent.

Recent updates on acetaminophen hepatotoxicity: the role of nrf2 in hepatoprotection.

Acetaminophen (APAP) known as paracetamol is the main ingredient in Tylenol, which has analgesic and anti-pyretic properties. Inappropriate use of APAP causes major morbidity and mortality secondary to hepatic failure. Overdose of APAP depletes the hepatic glutathione (GSH) rapidly, and the metabolic intermediate leads to hepatocellular death. This article reviews the mechanisms of hepatotoxicity and provides an overview of current research studies. Pharmacokinetics including metabolism (activation and detoxification), subsequent transport (efflux)-facilitating excretion, and some other aspects related to toxicity are discussed. Nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated gene battery plays a critical role in the multiple steps associated with the mitigation of APAP toxicity. The role of Nrf2 as a protective target is described, and potential natural products inhibiting APAP toxicity are outlined. This review provides an update on the mechanism of APAP toxicity and highlights the beneficial role of Nrf2 and specific natural products in hepatoprotection.

The potent protective effect of wild ginseng (Panax ginseng C.A. Meyer) against benzo[alpha]pyrene-induced toxicity through metabolic regulation of CYP1A1 and GSTs.

Wild Panax ginseng C.A. Meyer (WG) is a well-known medicinal herb. In this study, the protective effects of a water extract from the root of WG on benzo[alpha]pyrene (BP)-induced hepatotoxicity and the mechanism of these effects were investigated for the first time. The effects of WG on liver toxicities induced by BP were assessed by blood biochemical and histopathological analyses. BP caused severe liver injury in rats, as indicated by elevated plasma ALT, AST and LPO levels. Pretreatment with WG for 4 weeks completely abrogated increases in the ALT, AST and LPO levels when challenged with BP. Reductions in GSH content and GST activity by BP were reversed by WG. These protective effects of WG against BP-induced toxicity were consistent with the results of histopathological examinations. We next examined the effects of WG on the gene expression of the enzymes that metabolize BP in H4IIE cells. CYP1A1 mRNA and protein expression were increased by BP. WG moderately inhibited BP-induced CYP1A1 gene expression. Moreover, GSTA2, GSTA3 and GSTM2 gene expressions were significantly increased by WG through the Nrf2/antioxidant responsive element pathway for enzyme induction. In summary, WG is efficacious in protecting against BP-induced hepatotoxicity as results of metabolic regulations through both the inhibition of metabolic enzyme activation and the enhancement of electrophilic detoxification, implying that WG should be considered a potential chemopreventive agent.