Endogenous Metabolite; Microbial Metabolite

Since Glycoursodeoxycholic acid (GUDCA) has shown to have neuroprotective effects through the prevention of mitochondrial swelling, we have tested the beneficial effects of this bile acid on the UCB-induced alterations on the mitochondrial respiratory chain. GUDCA revealed to be able to completely reverse the inhibition of the cytochrome c oxidase activity (Fig. 1c).[1]
Next, we explored the oxidative status of immature neurons exposed to UCB. We observed that UCB markedly induced the production of reactive oxygen species, namely O2•− (Fig. 2a) and GSSG (Fig. 2b), as well as decreased NADPH concentrations (Fig. 2c). Moreover, pre-incubation of neurons with GUDCA efficiently prevented all these oxidative events caused by UCB. [1]
In view that the mitochondrial respiratory chain thresholds for oxygen consumption (Davey et al. 1998), it could be speculated that the level of cytochrome c oxidase inhibition caused by UCB might not be enough to impair the mitochondrial function. To elucidate this, we first determined the rate of oxygen consumption in the dissociated neurons previously incubated with UCB. As shown in Fig. 3, UCB significantly reduced the rates of oxygen consumption using glucose, succinate, or ascorbate as substrates, effects that were significantly prevented by GUDCA. The effects on O2 consumption are related to mitochondria, since both succinate and ascorbate reproduced the same results as with glucose. In addition, in all cases antimycin or potassium cyanide abolished O2 consumption driven by succinate or ascorbate, respectively (data not shown). The inhibition of oxygen consumption from ascorbate indicates that UCB-inhibition of cytochrome c oxidase, affects cell respiration. To further test this possibility, we assessed the Δψm as an index of the mitochondrial inner membrane integrity; as depicted in Fig. 3b, UCB caused the collapse of Δψm, and GUDCA showed ability to restore mitochondrial integrity. [1]
Curiously, although UCB-induced impairment of mitochondrial respiratory chain function was not accompanied by a reduction of the intracellular ATP levels (data not shown), an increase in the concentrations of extracellular ATP (Fig. 4a) was obtained. In addition, it was also observed an increase in the concentration of intracellular lactate (Fig. 4b), as well as in F1,6P2/F6P ratio (Fig. 4c), suggesting an activation of glycolysis, which was further supported by the concomitant elevated levels of F2,6P2 (Fig. 4d). Noticeably, all these effects were abolished by GUDCA. [1]
Finally, we sought to investigate whether the mitochondrial impairment triggered by UCB was associated with neurotoxicity. As shown in Fig. 5a, UCB enhanced the proportion of annexin V+/7-AAD− neurons, as assessed by flow cytometry; it also triggered an increase in the proportion of condensed or fragmented nuclei, as visualized with DAPI by fluorescence microscopy (Fig. 5b). Cell death by apoptosis was further corroborated by the increase in the activation of caspase 3 (Fig. 5c). Similar increase in the activation of caspase 9 indicates the involvement of mitochondria in this process. Such effects were again completely counteracted by GUDCA. We can then speculate that the increased glycolytic rate (Fig. 4) is a failed attempt to compensate the mitochondrial impairment. These results support the notion that UCB causes nerve cell death by apoptosis, mainly in immature neurons, and confirms that GUDCA efficiently protects cells against this type of neurotoxicity.[1]

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GUDCA Prevents Mitochondria Decreased Viability and Apoptosis While Simultaneously Counteracts and Reduces Caspase-9 Activation in NSC-34/hSOD1G93A Cells [2]
Increased apoptosis in MNs from SC was shown to mainly derive from mitochondrial dynamics abnormalities, including abnormal morphology, bioenergetic failure, and degeneration, mediated by mSOD1. In such circumstances, mitochondria-dependent caspase-9 activation was shown to play a crucial role in disease progression of mSOD1 mice. We have previously demonstrated that UDCA and GUDCA were able to inhibit bilirubin-induced apoptosis in neurons and astrocytes and that GUDCA was effective in preserving the activation of caspase-9 in neurons treated with bilirubin. To explore the efficacy of GUDCA in our MN degeneration model of ALS, we assessed the ability of GUDCA in preserving mitochondria dynamic properties, caspase-9 activation and cell death by apoptosis, as well as in recovering the functionality of NSC-34 expressing hSOD1G93A. For this, NSC-34 cells were incubated either alone or with 50 μM of GUDCA at the beginning of cell differentiation (0 DIV), or after 2 days (2 DIV), to distinguish between preventive and restorative capabilities, respectively. Cells were collected at 4 DIV, where increased SOD1 accumulation and apoptosis in NSC-34 expressing hSOD1G93A were manifest. We first observed that cells incubated with GUDCA did not show alterations in the number of PI+ cells (4.2 ± 0.1 in hSOD1wt and 3.8 ± 0.1 in hSOD1G93A at 0 DIV; 4.0 ± 0.5 in hSOD1wt and 3.6 ± 0.1 in hSOD1G93A at 2 DIV) or MTS reduction capacity (102 ± 9.7 % in hSOD1wt and 88 ± 3.3 % in hSOD1G93A at 0 DIV; 99 ± 13.2 % in hSOD1wt and 99 ± 8.7 % in hSOD1G93A at 2 DIV), as compared with nontreated cells, attesting the safety of such bile acid. [2]

Next, we verified that the mitochondria in NSC-34 cells expressing hSOD1G93A were less stained than in the hSOD1wt cells (0.8-fold, p < 0.01), indicating reduced mitochondria viability and energy impairment (Fig. 4a, b). Interestingly, such effect was prevented in mutated cells treated with GUDCA prior to the differentiation process, but not if added later. More importantly, GUDCA was able to prevent caspase-9 activation and to restore basal levels in the NSC-34/hSOD1G93A cells that showed a 1.8-fold increase as compared to NSC-34 cells expressing hSOD1wt (Fig. 4c). In addition, GUDCA was capable of inhibiting nuclei fragmentation in hSOD1G93A cells at 4 DIV that was shown to be 2.1-fold increased (p < 0.05) (Fig. 4d), and in some extent preventing its progression when added at 2 DIV incubation (NS). Collectively, such effects strengthen the stabilizing properties of GUDCA at the level of the mitochondrial membrane and reinforce its indication as an anti-apoptotic compound. [2]

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GUDCA Prevents the Increased Release of NO and MMP-9 by NSC-34/hSOD1G93A Cells While also Effective in Reverting MMP-9 Activation, but not ATP Depletion [2]
Increased extracellular levels of NO and MMP-9 have been associated to ALS pathogenesis and indicated as mediators of oxidative stress and neuroinflammation, respectively. Another pleiotropic cell-to-cell signaling molecule is ATP with diverse and contradictory effects, depending on the dose. ATP depletion was suggested to occur at the time of ALS onset and may derive from mitochondrial-deficient ATP output or calcium dysregulation. In our experimental model of ALS, we observed that NSC-34/hSOD1G93A cells released low levels of ATP (p < 0.01) while producing increased efflux of NO and MMP-9 (p < 0.05) (Fig. 5). Although GUDCA was unable to restore the normal extracellular values of ATP (Fig. 5a), it definitively reduced the production of nitrites when added prior to SOD1 accumulation (p < 0.05), but not thereafter (Fig. 5b). However, GUDCA was helpless in producing significant alterations in SOD1 accumulation in NSC-34 expressing hSOD1G93A when added either at 0 or at 2 DIV. Indeed, the increased density of hSOD1G93A cells with SOD1 inclusions relatively to hSOD1wt ones of 1.7 ± 0.4-fold was maintained unchanged after GUDCA addition (1.4 ± 0.5-fold at 0 DIV; 1.7 ± 0.6-fold at 2 DIV). This finding suggests that the reduction of NO by GUDCA does not derive from a decline in the intracellular accumulation of mSOD1. Considering now the activation of MMPs (Fig. 5c–e), though we did not observe any alteration on MMP-2 activation in the hSOD1G93A cells or modification by GUDCA, interesting inhibitory effects by the bile acid were noticed on the MMP-9 stimulation in hSOD1G93A cells (p < 0.05) (Fig. 5d). Most interesting, GUDCA revealed a window of opportunity relatively to its therapeutic usefulness by attenuating the pathological activation of MMP-9 and associated detrimental outcomes even when administered after the initiation of SOD1 intracellular accumulation at 2 DIV (p < 0.05).

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UDCA and GUDCA protect HBMEC from UCB-induced apoptosis, but only GUDCA is effective in reducing caspase-3 activation [3]
UCB-induced apoptosis in the HBMEC line includes the presence of apoptotic features that increased with the time of exposure and reached maximal levels at 48 h (Palmela et al., 2011). Thus, this time was chosen to evaluate the ability of UDCA and GUDCA to protect HBMEC from UCB-induced apoptosis. The bile acids were added at three different time points, evaluating their potential when added before and after the injury. Addition of UDCA and GUDCA reduced UCB injury, regardless of the time of addition (Figure 1). This protective effect was maximal in the treatments with GUDCA, especially when added at 1 h prior to UCB addition (54% reduction from UCB values, P < 0.001, vs. 42% for UDCA at the same time point, P < 0.01). Importantly, bile acids partially reverted UCB injury with a nearly 30% protection rate reduction compared to UCB damage (Figure 1), when added 8 h after UCB addition.

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Ultrastructural changes induced by UCB in HBMEC are abrogated by UDCA and GUDCA [3]
Based on the UCB-induced effects on apoptosis after 48 h incubation and the rescue ability of both UDCA and GUDCA to partially restore cell functionality, we decided to further assess whether changes at HBMEC ultrastructural level were produced by UCB and prevented by bile acid treatment. The transmission electron microscopy analysis revealed a marked reduction in the amount of ribosomes in UCB-treated cells, with an evident recovery in the presence of both bile acids (Figure 3). The same occurred relatively to cellular fragments detaching from the cultured HBMEC and mitochondrial cristae disruption observed, showing the damaging effects of UCB, once again markedly reduced in the presence of each of the bile acids.

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UCB-induced increase of interleukin-6 mRNA and cytokine expression in HBMEC is more effectively reduced by UDCA than by GUDCA [3]
One of the important effects of UCB on HBMEC previously observed by us was the upregulation of interleukin-6 mRNA levels and protein secretion (Palmela et al., 2011). This previous work indicated that UCB induced the maximum cytokine secretion at 4 h, while the highest mRNA expression was at 1 h following UCB exposure. These time points were then selected and the bile acids were added 1 h prior to UCB incubation. As seen in Figure 4, both bile acids abrogated interleukin-6 mRNA upregulation (Figure 4A), with reductions from UCB values of 27% for GUDCA (P < 0.05) and 46% for UDCA (P < 0.001). On the other hand, only UDCA showed preventive effects on UCB-induced release of interleukin-6 (Figure 4B), decreasing UCB-induced cytokine secretion by 35% (P < 0.001).

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UDCA and GUDCA prevent and rescue disruption of HBMEC integrity by UCB [3]
The ability of the tested bile acids to counteract UCB-induced upregulation of interleukin-6, led us to hypothesize that UDCA and GUDCA would protect against the consequent endothelial hyperpermeability. Thus, we measured the paracellular permeability to the low molecular weight compound, sodium fluorescein. This is a widely used indicator of the barrier properties, with several studies showing increased values in conditions associated with hyperpermeability (Hülper et al., 2013; Labus et al., 2014). In our previous study we showed that this parameter is significantly enhanced upon prolonged UCB exposure (Palmela et al., 2012), as also observed in the present study (Figure 5). Here, we also observed that UDCA and GUDCA alone do not affect the HBMEC integrity, since we did not observe any changes in permeability values. However, analysis of the bile acids effect on the permeability to sodium fluorescein revealed that only UDCA prevented UCB injury, and if added before (22% reduction from UCB values, P < 0.01) or at 4 h (18% protection from UCB values, P < 0.05). In fact, while UCB induced an increased passage of sodium fluorescein molecules from 1.42 × 10−5 cm/s in controls to 2.48 × 10−5 cm/s in UCB-treated samples, incubation with UDCA reduced such value to 1.95 × 10−5 cm/s or to 1.99 × 10−5cm/s in cells pre-treated or treated 4 h after UCB addition. In contrast, the values obtained for GUDCA were 2.19 × 10−5 and 2.24 × 10−5 cm/s (pre- and 4 h after UCB addition treatments, respectively), thus not different from UCB values.

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UDCA and GUDCA cross the HBMEC monolayer in a time-dependent manner [3]
The addition of 50 μM of each of the studied bile acids to the upper (“blood”) compartment of an insert culture system was performed to evaluate if they were able to cross the HBMEC monolayer and thus hypothetically achieve the brain parenchyma. After a short period of incubation (4 h) the bile acids were barely detectable in the lower chamber of the culture plate. However, when longer periods of treatment were applied (48 h) a significant increase in the bile acid passage through the monolayer was obtained (18.8 ± 4.8 and 16.2 ± 3.9 μM, for UDCA and GUDCA, respectively).

Gelatin Zymography [2]
MMP-2 and MMP-9 extracellular levels were determined by performing a SDS-PAGE zymography in 0.1 % gelatin-10 % acrylamide gels, under nonreducing conditions as described. Afterwards, the gels were washed for 1 h with 2.5 % Triton X-100 (in 50 mM Tris pH 7.4; 5 mM CaCl2; 1 μM ZnCl2) to remove SDS and to renature the MMP species in the gel. To induce gelatin lysis, the gels were incubated in the same buffer without Triton X-100 overnight at 37 °C. For enzyme activity analysis, the gels were stained with 0.5 % Coomassie Brilliant Blue R-250 and distained in 30 % ethanol/10 % acetic acid/H2O. Gelatin activity was detected as a white band on a blue background and measured by using computerized image analysis and normalized for total cellular protein.

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Nitrite Assay [2]
The production of NO was estimated by measuring the accumulated level of nitrites in the extracellular media of NSC-34 cells with Griess reagent, as previously described by us. Briefly, culture supernatant was mixed with Griess reagent [1 % (w/v) sulfanilamide in 5 % H3PO4 and 0.1 % (w/v) N-1 naphtylethylenediamine, in a proportion of 1:1 (v/v)] in a 96-well tissue culture plate for 10 min in the dark, at RT. The absorbance at 540 nm was determined using a microplate reader. A calibration curve was used for each assay. All samples were measured in duplicate and the mean value was used.

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ATP Assay [2]
Samples were treated on ice to avoid degradation of ATP. For the determination of extracellular ATP levels, the incubation media was collected and treated with 2 M perchloric acid. Next, neutralization was achieved with 4 M KOH solution. To remove cellular debris, the samples were centrifuged for 5 min at 10,000 × g at 4 °C. ATP levels were determined by an enzymatic assay, and the corresponding fluorescence intensity was quantified using a fluorimeter at λem = 410–460 nm and λex = 365 nm. An ATP standard curve was generated each time the assay was run.

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Mitochondria Viability Assay [2]
To stain viable mitochondria, cells were incubated for 30 min at 37 °C with 500 nM of MitoTracker Red® solution as usual in our lab, and then fixed with 4 % (w/v) paraformaldehyde. Cell nuclei were stained with Hoechst 33258 dye. Red fluorescence and UV images of at least ten random microscopic fields (original magnification = ×400) were acquired per sample using a fluorescence microscope. Fluorescence intensity was quantified by ImageJ software and normalized to the total number of cells.

Treatment of neurons [1]
Neurons were incubated in Neurobasal medium without (control) or with 50 μM UCB (from a 10 mM stock solution) in the presence of 100 μM HSA (UCB/HSA molar ratio of 0.5) for 1 h at 37°C. Stock UCB solutions were extemporarily prepared in 0.1 M NaOH under the dark and the pH adjusted to 7.4 using 0.1 M HCl. When appropriate, neurons were pre-incubated with Glycoursodeoxycholic acid (GUDCA) (50 μM) 1 h prior to UCB addition.

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Cell Differentiation and Treatment [2]
To slow the proliferation rate and enhance cell maturation, we cultured low concentrations of NSC-34 cells in low FBS-supplemented media, an established protocol to investigate the mechanisms of cellular toxicity in NSC-34 cells. Therefore, after 48 h, the proliferation medium was replaced by a fresh medium comprising DMEM-F12 plus FBS (1 %), nonessential amino acids (1 %), penicillin/streptomycin (1 %), and G148 (0.1 %), as previously described. Evaluations were performed at 0, 2, and 4 DIV to assess transfection efficiency, accumulation of SOD1, and cell viability. In another set of experiments, cells were incubated with 50 μM Glycoursodeoxycholic acid (GUDCA) at 0 DIV and at 2 DIV to assess the ability of this bile acid to prevent, in the first case, and restore, in the last, the effects produced by the transfected hSOD1 on cell death by apoptosis and on the release of ATP, NO, as well as MMP-2 and MMP-9 into the extracellular medium, at 4 DIV. The dose of 50 μM GUDCA was chosen to mimic the concentration of GUDCA commonly found in the serum of patients after treatment with UDCA at a dose of 13 to 15 mg per kg of body weight per day. We previously evidenced that such concentration is not toxic to neurons and, most importantly, has beneficial properties in preventing neurodegeneration

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Cell culture and treatment [3]
To test whether UCB-induced injury to endothelial cells could be abrogated in the presence of UDCA and Glycoursodeoxycholic acid (GUDCA), we used a HBMEC line as a simplified model of the human BBB. This cell line was derived from primary cultures of HBMEC transfected with SV40 large T antigen (Stins et al., 2001) and was recently proved to be the most suitable human cell line for an in vitro BBB concerning barrier tightness (Eigenmann et al., 2013). Cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum, 10% NuSerum IV, 1% non-essential amino acids, 1% minimum essential medium vitamins, 1 mM sodium pyruvate, 2 mM l-glutamine, and 1% antibiotic-antimycotic solution, seeded at a density of 8 × 104 cell/mL in collagen I-coated coverslips or plates and treated after 2 days in culture, as previously described (Palmela et al., 2011). For integrity studies, based on the measurement of paracellular permeability to sodium fluorescein, cells were seeded on collagen I-coated polyester transwell inserts (0.4 μm) at a density of 8 × 104 cell/insert and treated after 8 days in culture (Palmela et al., 2012). Endothelial cultures were maintained at 37°C in a humid atmosphere enriched with 5% CO2, and all experiments were performed at confluence.
Co-incubation studies were also performed with the bile acids UDCA and GUDCA, molecules with octanol/water partition coefficients of 1000 for the unconjugated form and 105 for the glycine-amidated molecule, and logP values of 3.0 and 2.02 for the former and the later, respectively (Roda et al., 1990). In the co-incubation studies, UDCA or GUDCA were added at a final concentration of 50 μ M, which is found in the circulation of patients under therapy with UDCA. In particular, the concentration of 50 μM GUDCA is commonly found in the serum of patients after treatment with UDCA at a dose of 13–15 mg per kilogram of body weight per day (Podda et al., 1990; Poupon et al., 1994; Brites et al., 1998). We previously showed that such concentration is not toxic to neurons (Silva et al., 2001b) and, most importantly, has beneficial properties in preventing neurodegeneration (Brito et al., 2008; Vaz et al., 2010). The bile acids were added at three different time points: 1 h prior to UCB addition and at 4 or 8 h after UCB incubation. For short periods of UCB incubation only the effects of 1 h pre-incubation with the bile acids were evaluated. Appropriate controls including cells treated with UDCA and GUDCA (without UCB) were also included to ascertain the absence of toxicity of these molecules.

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Transmission electron microscopy [3]
Ultrastructural analysis was performed by transmission electron microscopy following 48 h exposure to UCB in HBMEC pre-treated with UDCA or Glycoursodeoxycholic acid (GUDCA). Cells were fixed with 1.2% glutaraldehyde in 0.1 M phosphate buffer and 1% osmium tetroxide in the same buffer, dehydrated with a graded series of ethanol, and then embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Hitachi H-7500 transmission electron microscope at an acceleration voltage of 80 kV.

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Evaluation of barrier integrity by permeability measurement [3]
The capacity of UDCA and Glycoursodeoxycholic acid (GUDCA) to modulate permeability was evaluated in cells treated with UCB for 48 h, the time-point resulting in the maximum disruption of the integrity state of HBMEC monolayer by UCB (Palmela et al., 2012).
In our previous studies, we found that UCB increases the permeability to sodium fluorescein (Palmela et al., 2012), a low molecular weight tracer (376 Da), but not to albumin-bound Evans blue, a high molecular weight tracer (68 kDa). So, in this study HBMEC paracellular permeability assay was conducted with sodium fluorescein as previously described (Veszelka et al., 2007; Cardoso et al., 2012; Palmela et al., 2012). Briefly, cell culture inserts were transferred to 12-well plates containing Ringer–Hepes solution (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 5.5 mM d-glucose, 20 mM Hepes, pH 7.4) in the basal compartments. The sodium fluorescein solution (10 mg/mL sodium fluorescein in Ringer–Hepes) was added to the upper chambers. The inserts were transferred to new wells at 20, 40, and 60 min. Lower chamber solutions were collected to determine sodium fluorescein levels (Hitachi F-2000 fluorescence spectrophotometer, excitation: 440 nm and emission: 525 nm). Flux across cell-free inserts was also measured. The endothelial permeability coefficient was calculated as previously described (Deli et al., 2005) and the average control permeability coefficient was 1.4 × 10−5 cm/s.

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Assessment of UDCA and Glycoursodeoxycholic acid (GUDCA) passage across the HBMEC monolayer [3]
To establish whether UDCA and Glycoursodeoxycholic acid (GUDCA) are able to cross the BBB endothelium, a two-chamber culture system was used. Bile acids were added to the upper chambers and media from the lower chambers were collected after 4 and 48 h of incubation. The bile acid passage across the HBMEC monolayer was evaluated by measuring the concentrations of UDCA and GUDCA by an enzymatic-fluorimetric assay (Brites et al., 1998). Results were shown as average concentration (μM) ± SEM.

[1]. Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic acid. J Neurochem. 2010 Jan;112(1):56-65.

[2]. Glycoursodeoxycholic acid reduces matrix metalloproteinase-9 and caspase-9 activation in a cellular model of superoxide dismutase-1 neurodegeneration.

[3]. Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study. Front Neurosci. 2015 Mar 13;9:80.

Glycoursodeoxycholic acid is a bile acid glycine conjugate derived from ursoodeoxycholic acid. It has a role as a neuroprotective agent and a human blood serum metabolite. It is a bile acid glycine conjugate and a N-acylglycine. It is functionally related to an ursodeoxycholic acid. It is a conjugate acid of a glycoursodeoxycholate.

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High levels of unconjugated bilirubin (UCB) may initiate encephalopathy in neonatal life, mainly in pre-mature infants. The molecular mechanisms of this bilirubin-induced neurologic dysfunction (BIND) are not yet clarified and no neuroprotective strategy is currently worldwide accepted. Here, we show that UCB, at conditions mimicking those of hyperbilirubinemic newborns (50 microM UCB in the presence of 100 muM human serum albumin), rapidly (within 1 h) inhibited cytochrome c oxidase activity and ascorbate-driven oxygen consumption in 3 days in vitro rat cortical neurons. This was accompanied by a bioenergetic and oxidative crisis, and apoptotic cell death, as judged by the collapse of the inner-mitochondrial membrane potential, increased glycolytic activity, superoxide anion radical production, and ATP release, as well as disruption of glutathione redox status. Furthermore, the antioxidant compound Glycoursodeoxycholic acid (GUDCA) fully abrogated UCB-induced cytochrome c oxidase inhibition and significantly prevented oxidative stress, metabolic alterations, and cell demise. These results suggest that the neurotoxicity associated with neonatal bilirubin-induced encephalopathy occur through a dysregulation of energy metabolism, and supports the notion that GUDCA may be useful in the treatment of BIND. [1]

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects mainly motor neurons (MNs). NSC-34 MN-like cells carrying the G93A mutation in human superoxide dismutase-1 (hSOD1(G93A)) are a common model to study the molecular mechanisms of neurodegeneration in ALS. Although the underlying pathways of MN failure still remain elusive, increased apoptosis and oxidative stress seem to be implicated. Riluzole, the only approved drug, only slightly delays ALS progression. Ursodeoxycholic acid (UDCA), as well as its glycine (Glycoursodeoxycholic acid (GUDCA)) and taurine (TUDCA) conjugated species, have shown therapeutic efficacy in neurodegenerative models and diseases. Pilot studies in ALS patients indicate safety and tolerability for UDCA oral administration. We explored the mechanisms associated with superoxide dismutase-1 (SOD1) accumulation and MN degeneration in NSC-34/hSOD1(G93A) cells differentiated for 4 days in vitro (DIV). We examined GUDCA efficacy in preventing such pathological events and in restoring MN functionality by incubating cells with 50 μM GUDCA at 0 DIV and at 2 DIV, respectively. Increased cytosolic SOD1 inclusions were observed in 4 DIV NSC-34/hSOD1(G93A) cells together with decreased mitochondria viability (1.2-fold, p < 0.01), caspase-9 activation (1.8-fold, p < 0.05), and apoptosis (2.1-fold, p < 0.01). GUDCA exerted preventive effects (p < 0.05) while also reduced caspase-9 levels when added at 2 DIV (p < 0.05). ATP depletion (2-fold, p < 0.05), increased nitrites (1.6-fold, p < 0.05) and metalloproteinase-9 (MMP-9) activation (1.8-fold, p < 0.05), but no changes in MMP-2, were observed in the extracellular media of 4 DIV NSC-34/hSOD1(G93A) cells. GUDCA inhibited nitrite production (p < 0.05) while simultaneously prevented and reverted MMP-9 activation (p < 0.05), but not ATP depletion. Data highlight caspase-9 and MMP-9 activation as key pathomechanisms in ALS and GUDCA as a promising therapeutic strategy for slowing disease onset and progression. [2]

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Ursodeoxycholic acid and its main conjugate Glycoursodeoxycholic acid (GUDCA) are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells. [3]

C26H43NO5

449.6233

449.314

C, 58.96; H, 8.18; N, 2.64; O, 24.16; S, 6.05

64480-66-6

12310288

White to off-white solid powder

232-235ºC

3.985

4

5

6

32

727

10

O([H])[C@@]1([H])C([H])([H])[C@]2([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[C@]2(C([H])([H])[H])[C@]2([H])C([H])([H])C([H])([H])[C@]3(C([H])([H])[H])[C@@]([H])([C@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])C(N([H])C([H])([H])C(=O)O[H])=O)C([H])([H])C([H])([H])[C@@]3([H])[C@@]21[H])O[H]

GHCZAUBVMUEKKP-XROMFQGDSA-N

InChI=1S/C26H43NO5/c1-15(4-7-22(30)27-14-23(31)32)18-5-6-19-24-20(9-11-26(18,19)3)25(2)10-8-17(28)12-16(25)13-21(24)29/h15-21,24,28-29H,4-14H2,1-3H3,(H,27,30)(H,31,32)/t15-,16+,17-,18-,19+,20+,21+,24+,25+,26-/m1/s1

2-[[(4R)-4-[(3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid

Glycoursodeoxycholic acid; 64480-66-6; Ursodeoxycholylglycine; Glycoursodeoxycholate; GUDCA; Glycine ursodeoxycholic acid; Glycylursodeoxycholic acid; UNII-PF1G5J2X2A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~222.41 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)