Sesamol as a potential candidate for the treatment of hepatic fibrosis, based on its regulation of FXR/LXR axis-mediated inhibition of autophagy through crosstalk between hepatic cells and macrophage.

BACKGROUND: Sesamol (SEM), a natural lignan compound isolated from sesame, has strong anti-oxidant property, regulating lipid metabolism, decreasing cholesterol and hepatoprotection. However, its anti-hepatic fibrosis effect and mechanisms have not been comprehensively elucidated. HYPOTHESIS/PURPOSE: This study aims to investigate the anti-hepatic fibrosis of SEM and its underlying mechanisms. METHOD: C57BL/6 mice with hepatic fibrosis were induced by TAA, then administrated with SEM or curcumin, respectively. HSCs were stimulated by TGF-ß or conditioned medium, and then cultured with SEM, GW4064, GW3965, Rapamycin (RA) or 3-methyladenine (3-MA), respectively. Mice with hepatic fibrosis also were administrated with SEM, RA or 3-MA to estimate the effect of SEM on autophagy. RESULTS: In vitro, SEM significantly inhibited extracellular matrix deposition, P2 × 7r-NLRP3, and inflammatory cytokines. SEM increased FXR and LXRα/ß expressions and decreased MAPLC3α/ß and P62 expressions, functioning as 3-MA (autophagy inhibitor). In vivo, SEM reduced serum transaminase, histopathology changes, fibrogenesis, autophagy markers and inflammatory cytokines caused by TAA. LX-2 were activated with conditioned medium from LPS-primed THP-1, which resulted in significant enhance of autophagy markers and inflammatory cytokines and decrease of FXR and LXRα/ß expressions. SEM could reverse above these changes and function as 3-MA, GW4064, or GW3965. Deficiency of FXR or LXR attenuated the regulation of SEM on α-SMA, MAPLC3α/ß, P62 and IL-1ß in activated LX-2. In activated THP-1, deficiency of FXR could decrease the expression of LXR, and vice versa. Deficiency of FXR or LXR in activated MΦ decreased the expressions of FXR and LXR in activated LX-2. Deficiency FXR or LXR in activated MΦ also attenuated the regulation of SEM on α-SMA, MAPLC3α/ß, P62, caspase-1 and IL-1ß. In vivo, SEM significantly reversed hepatic fibrosis via FXR/LXR and autophagy. CONCLUSION: SEM could regulate hepatic fibrosis by inhibiting fibrogenesis, autophagy and inflammation. FXR/LXR axis-mediated inhibition of autophagy contributed to the regulation of SEM against hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. SEM might be a prospective therapeutic candidate, and its mechanism would be a new direction or strategy for hepatic fibrosis treatment.

Pterostilbene exerts cytotoxicity on activated hepatic stellate cells by inhibiting excessive proliferation through the crosstalk of Sirt1 and STAT3 pathways.

Pterostilbene (PTE), a natural analogue of resveratrol, abundantly exists in blueberries and grapes and has several beneficial potentials against oxidative stress, inflammation, and cancer. In current study, we investigated the effects of PTE on hepatic fibrosis in vitro and in vivo. Activation of hepatic stellate cells (HSCs) is an initiating event in the initiation of hepatic fibrosis. MTT assay revealed that PTE (3.125-12.5 µM) displayed cytotoxicity on activated HSCs, no cytotoxicity on AML-12 and quiescent HSCs. PTE significantly inhibited the expressions of α-SMA, collagen Ⅰ and TIMP-1/MMP13 ratio; suppressed inflammatory cascade activation to reduce inflammatory cytokines release, such as Caspase-1, IL-1ß and IL-6. PTE activated Sirt1 and decreased STAT3 phosphorylation, functioning as SRT1720 and Niclosamide. Sirt1 deficiency significantly elevated p-STAT3 expression, while STAT3 deficiency resulted in Sirt1 increasing and inhibited fibrosis and inflammatory cytokines expressions. In mice with hepatic fibrosis induced by thioacetamide (TAA), PTE significantly decreased ALT and AST activities, reduced fibrosis markers, STAT3 phosphorylation and activated Sirt1 expression. PTE showed cytotoxicity on activated HSCs to ameliorate hepatic fibrosis via regulating fibrogenesis, energy metabolism and inflammation and targeting the crosstalk of Sirt1 and STAT3. In conclusion, PTE could be potentially beneficial as a natural plant metabolite in preventing and treating hepatic fibrosis.

Enhanced Cytotoxic Effects of Arenite in Combination with Active Bufadienolide Compounds against Human Glioblastoma Cell Line U-87.

The cytotoxicity of a trivalent arsenic derivative (arsenite, AsIII) combined with arenobufagin or gamabufotalin was evaluated in human U-87 glioblastoma cells. Synergistic cytotoxicity with upregulated intracellular arsenic levels was observed, when treated with AsIII combined with arenobufagin instead of gamabufotalin. Apoptosis and the activation of caspase-9/-8/-3 were induced by AsIII and further strengthened by arenobufagin. The magnitude of increase in the activities of caspase-9/-3 was much greater than that of caspase-8, suggesting that the intrinsic pathway played a much more important role in the apoptosis. An increase in the number of necrotic cells, enhanced LDH leakage, and intensified G2/M phase arrest were observed. A remarkable increase in the expression level of γH2AX, a DNA damage marker, was induced by AsIII+arenobufagin. Concomitantly, the activation of autophagy was observed, suggesting that autophagic cell death associated with DNA damage was partially attributed to the cytotoxicity of AsIII+arenobufagin. Suppression of Notch signaling was confirmed in the combined regimen-treated cells, suggesting that inactivation of Jagged1/Notch signaling would probably contribute to the synergistic cytotoxic effect of AsIII+arenobufagin. Given that both AsIII and arenobufagin are capable of penetrating into the blood-brain barrier, our findings may provide fundamental insight into the clinical application of the combined regimen for glioblastoma.

Characterization of 6-bromoferulic acid as a novel common-use matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: Ferulic acid (FA) is a standard matrix used for analyzing proteins. In this study, the ability of a halogenated FA to serve as an effective MALDI matrix was investigated. Various halogenated FAs were synthesized, and the characteristics and performance of each were compared with those of the standard matrices α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydrobenzoic acid (DHBA). METHODS: The abilities of 6-bromoferulic acid (6-BFA), ferulic acid (FA), and eight other halogenated FA derivatives to ionize eight synthetic peptides were examined. Absorption measurements, MM2 structure optimizations, and proton affinity (PA) calculations were also performed for 6-BFA and FA. The suitabilities of these compounds as matrices for matrix-assisted laser desorption/ionization (MALDI) for lipids, sugar chains, polymers, cyanocobalamin, synthetic peptides, and tryptic peptides originating from two types of serum proteins were also tested. RESULTS: The 6-position of FA was found to be the best site for introducing a bromine because the generated compound allowed facile detection of cyanocobalamin and several peptides. 6-BFA exhibited good sensitivity for large peptides (3-5 kDa) and peptides containing acidic amino acids or proline. 6-BFA was also shown to be a suitable matrix for tandem mass spectrometry (MS/MS) analysis when using MALDI time-of-flight (TOF) mass spectrometry (MS) with a quadrupole ion trap (QIT) system. CONCLUSIONS: The properties of 6-BFA as a MALDI matrix differed from those of DHBA and CHCA. 6-BFA appears to be a useful matrix for de novo sequencing using MALDI-QIT-TOF-MS.

Effects of Etanercept against Transient Cerebral Ischemia in Diabetic Rats.

Diabetes mellitus is known to exacerbate acute cerebral ischemic injury. Previous studies have demonstrated that infarction volumes caused by transient cerebral ischemia were greater in diabetic rats than in nondiabetic rats. Tumor necrosis factor-α (TNF-α) is a proinflammatory protein produced in the brain in response to cerebral ischemia that promotes apoptosis. Etanercept (ETN), a recombinant TNF receptor (p75)-Fc fusion protein, competitively inhibits TNF-α. Therefore, we evaluated the neuroprotective effects of chronic or acute treatment with ETN on cerebral injury caused by middle cerebral artery occlusion/reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. Furthermore, we evaluated the effects of ETN against the apoptosis and myeloperoxidase activity. Single administration of ETN before MCAO significantly suppressed exacerbation of cerebral damage in nondiabetic rats, as assessed by infarct volume. In contrast, the diabetic state markedly aggravated MCAO/Re-induced cerebral damage despite ETN treatment within 24 h before MCAO. However, the damage was improved by repeated administration of ETN at 900 µg/kg/daily in rats in an induced diabetic state. These results suggested that repeated administration of ETN can prevent exacerbation of cerebral ischemic injury in the diabetic state and is mainly attributed to anti-inflammatory effects.

Orally administrated ascorbic acid suppresses neuronal damage and modifies expression of SVCT2 and GLUT1 in the brain of diabetic rats with cerebral ischemia-reperfusion.

Diabetes mellitus is known to exacerbate cerebral ischemic injury. In the present study, we investigated antiapoptotic and anti-inflammatory effects of oral supplementation of ascorbic acid (AA) on cerebral injury caused by middle cerebral artery occlusion and reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. We also evaluated the effects of AA on expression of sodium-dependent vitamin C transporter 2 (SVCT2) and glucose transporter 1 (GLUT1) after MCAO/Re in the brain. The diabetic state markedly aggravated MCAO/Re-induced cerebral damage, as assessed by infarct volume and edema. Pretreatment with AA (100 mg/kg, p.o.) for two weeks significantly suppressed the exacerbation of damage in the brain of diabetic rats. AA also suppressed the production of superoxide radical, activation of caspase-3, and expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) in the ischemic penumbra. Immunohistochemical staining revealed that expression of SVCT2 was upregulated primarily in neurons and capillary endothelial cells after MCAO/Re in the nondiabetic cortex, accompanied by an increase in total AA (AA + dehydroascorbic acid) in the tissue, and that these responses were suppressed in the diabetic rats. AA supplementation to the diabetic rats restored these responses to the levels of the nondiabetic rats. Furthermore, AA markedly upregulated the basal expression of GLUT1 in endothelial cells of nondiabetic and diabetic cortex, which did not affect total AA levels in the cortex. These results suggest that daily intake of AA attenuates the exacerbation of cerebral ischemic injury in a diabetic state, which may be attributed to anti-apoptotic and anti-inflammatory effects via the improvement of augmented oxidative stress in the brain. AA supplementation may protect endothelial function against the exacerbated ischemic oxidative injury in the diabetic state and improve AA transport through SVCT2 in the cortex.