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Endotoxin-induced acute liver injury (ALI) is a severe disease associated with a poor prognosis. Therefore, it is urgent to discover new effective therapies to prevent ALI. Daidzein, extracted from leguminous plants, possess anti-inflammatory and antioxidative bioactivities. However, little is known about whether daidzein could attenuate lipopolysaccharide (LPS)-induced ALI. We investigated the effects of daidzein on hepatocyte injury and its underlying mechanisms. In LPS-induced hepatocyte supernatant, 100 μM daidzein decreased ALT and AST expression levels by 49.3% ± 5.6% and 39.3% ± 3.5%, respectively, with no cytotoxicity. In addition, the expression of inflammatory factors, including interleukin-1β (IL-lβ), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were decreased by 100 μM daidzein (73.8% ± 5.3%, 58.8 ± 9.0% and 55.5% ± 7.2%, respectively) in LPS-treated hepatocytes. Western blot analysis showed that daidzein inhibited LPS-induced p-ERK1/2, p-IκBα and p-p65 expression levels. Moreover, 100 μM daidzein reduced the LPS-induced production of Reactive oxygen species by 23.9 ± 7.8% and increased SOD activity by 88.4% ± 18.9% by downregulating Keap-1 and upregulating Nrf2 expression. In conclusion, these data indicate that daidzein ameliorates LPS-induced hepatocyte injury by inhibiting inflammation and oxidative stress.

Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor progression, but the mechanisms underlying are poorly understood. Here, we report that platelet releasates exerted a proliferative effect on hepatocellular carcinoma (HCC) cells both in vitro and in vivo. This effect depended on a reduction of KLF6 expression in HCC cells. After incubation with either platelets or platelet granule contents, SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis. However, no effect was observed when incubating cancer cells with resuspended activated platelet pellet which exhausted of releasates. Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase. Moreover, knocking down KLF6 expression significantly diminished the platelet-mediated enhancement of HCC growth. In addition, blocking TGF-β signaling with the TGF-β receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells. Based on these findings, we conclude that platelet releasates, especially TGF-β, promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6. This discovery identifies a potential new therapeutic target for the prevention and treatment of hepatocellular carcinoma.

Aims It has been proven that carbon nanoparticles or diesel exhaust particles stimulate platelet activation. However, the effect of fine particle matter (PM2.5) on platelet activation remains unknown, which motivates this study. Methods PM2.5 samples were collected in an urban area of Zhengzhou, China. To study the morphological characteristics and the mass concentrations of trace elements of PM2.5 samples, a filed-emission scanning electron microscope, the Image-J software, and an inductively coupled plasma mass spectrometry were used. Washed human platelets or platelet-rich-plasma were used to study the effect of PM2.5 on platelet aggregation, P-selectin expression, or platelet signaling pathways. The cytotoxicity in platelets exposed to PM2.5 was evaluated by a lactate dehydrogenase assay kit. In addition, platelet adhesion and spreading were studied on collagen-coated surfaces in stable conditions. Results The filed-emission scanning electron microscope scanning showed that PM2.5 samples varied in shape and size distributions. The mean equivalent spherical diameter of these particles was 1.97 ± 0.04 μm, of which 82.40% were particles with equivalent spherical diameters of less than 2.5 μm. The mass concentration of Ca was higher than that of other elements. The other elements followed the trend of Al>Fe>Zn>Mg>Pb>K>Mn>Cu>Ti>Ba>As>Sr>Sn>Sb>Cd>B>Se>Mo>Ag>Ni>TI>V>Co. Furthermore, pretreatment of PM2.5 significantly inhibited rather than potentiated collagen-induced platelet aggregation and P-selectin expression, whereas it had no significant effect on ADP-induced platelet aggregation and P-selectin expression. The lactate dehydrogenase analysis showed trivial cytotoxic effect of PM2.5 exposure on platelets. Pretreatment of PM2.5 inhibited platelet adhesion on immobilized collagen-coated surfaces; however, it almost did not impact the platelet spreading. Immunoblotting analysis indicated that PM2.5 reduced collagen-induced phosphorylation of phospholipase C gamma-2 (PLCγ2) at Tyr759, Akt at Ser473, and glycogen synthase kinase 3β (GSK3β) at Ser9. Conclusions PM2.5 attenuated collagen-induced platelet aggregation, α-granule secretion and adhesion, with the potential mechanism of impairing PLCγ2, Akt, and GSK3β signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol, 2016.

Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100μM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50μM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling.

cope Propolis is thought to help prevent thrombotic and related cardiovascular diseases in humans. Chrysin, a bioflavonoids compound found in high levels in propolis and in honey, has been reported to possess antiplatelet activity. However, the mechanism by which it inhibits platelet function is unclear. Methods and results The effects of chrysin on agonist-activated platelet-aggregation, granule-secretion, and integrin αIIbβ3 activation were examined. Its effects on the phosphorylation of Akt, GSK3β, MAPKs, and several proteins of the glycoprotein VI (GPVI) signaling pathway were also studied on collaged-activated platelets. In addition, human platelet spreading on immobilized fibrinogen was also tested. We found that chrysin dose dependently inhibited platelet aggregation and granule secretion induced by collagen, as well as platelet aggregation induced by ADP, thrombin, and U46619. Chrysin also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen. Biochemical analysis revealed that chrysin inhibited collagen-induced activation of Syk, PLCγ2, PKC, as well as the phosphorylation of Akt and ERK1/2. Additionally, chrysin attenuated phosphorylation of molecules such as FcγRIIa, FAK, Akt, and GSK3β in platelet spreading on immobilized fibrinogen. Conclusions Our findings indicate that chrysin suppresses not only integrin αIIbβ3-mediated “inside-out” signaling, but also the “outside-in” signal transmission. This implies that chrysin may represent a potential candidate for an antiplatelet agent.

It has been proven that carbon nanoparticles or diesel exhaust particles stimulate platelet activation. However, the effect of fine particle matter (PMPMThe filed-emission scanning electron microscope scanning showed that PMPM

Flavonoids exert both anti-oxidant and anti-platelet activities in vitro and in vivo. Pentamethylquercetin (PMQ), a polymethoxylated flavone derivative, has been screened for anti-carcinogenic and cardioprotective effects. However, it is unclear whether PMQ has anti-thrombotic effects. In the present study, PMQ (20 mg/kg) significantly inhibited thrombus formation in the collagen- epinephrine- induced acute pulmonary thrombosis mouse model and the ferric chloride-induced carotid injury model. To explore the mechanism, we evaluated the effects of PMQ on platelet function. We found that PMQ inhibited platelet aggregation and granule secretion induced by low dose agonists, including ADP, collagen, thrombin and U46619. Biochemical analysis revealed that PMQ inhibited collagen-, thrombin- and U46619-induced activation of Syk, PLCγ2, Akt, GSK3β and Erk1/2. Therefore, we provide the first report to show that PMQ possesses anti-thrombotic activity in vivo and inhibited platelet function in vitro, suggesting that PMQ may represent a potential therapeutic candidate for the prevention or treatment of thrombotic disorders.

Background: Platelet in the primary tumor microenvironment plays a crucial role in tumor cells angiogenesis, growth, and metastasis. Clinical and experimental evidences support that platelets and their extracts influence hepatocellular carcinoma (HCC) growth and biology. But the mechanism is still not fully clarified. The aim of present study was to elucidate an unperceived mechanism of the proliferative effect of platelet on HCC cells. Methods: Human blood was collected from health volunteers, washed platelets were prepared and resuspended by fresh medium. The ability of HepG2 cells to induce platelet aggregation was analyzed using a Chrono-Log Lumi-aggregometer. HepG2 cells were incubated with platelets activated by thrombin (0.08 U/ml) and collagen-related peptide (CRP, 0.8μg/ml), or releasates isolated from CRP-stimulated platelets. The effect of platelet releasate on HepG2 cell proliferation was determined with the colorimetric 3-(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT) assay. Western blot was used to measure expression of Krüppel-like factor 6 (KLF6) in HepG2 cells. Anti-FcγRIIa monoclonal antibody IV.3 (10μg/ml) and transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542 (10μM) were used. Furthermore, KLF6 gene silence was also conducted in HepG2 cells by transfected with KLF6 siRNA. Results: Our data showed HepG2 cells (1.0×105/ml) could induce human washed platelet (3.0×108/ml) aggregation in vitro, indicating that HepG2 cells could activate platelets. We further verified that releasate from CRP-activated platelets could promote the proliferation of HepG2 cells. Importantly, this effect exhibits on the down expression of KLF6 in HepG2 cells. In presence and absence of platelet stimulator thrombin (0.08 U/ml) or collagen-related peptide (CRP, 0.8μg/ml), washed platelets could reduce KLF6 expression in HepG2 cells after incubated for 12 and 24 hours. Meanwhile, supernatant from CRP-activated platelets exhibited the same effect. On the other hand, the resuspended CRP-activated platelet pellet showed no significant influence on KLF6 expression. And platelets incubated with anti- FcγRIIa monoclonal antibody IV.3 (10μg/ml) and transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542 (10μM) abolished the effects. Furthermore, the platelet’s promoting proliferation effect was attenuated in HepG2 cells with silencing KLF6 expression. Conclusion: Tumor cells could activate platelet, and activated platelet could regulate cancer cell progression in turn. We further verified that platelet, a main source of bioavailable TGF-β1, has a promoting effect on the proliferation of HepG2 cells. Importantly, this effect exhibits on the down expression of KLF6 in HepG2 cells, in which FcγRIIa and TGF-β1 involved. These results extend our understanding of mechanisms by which platelets contribute to tumor progression, which may provide a new therapeutic target for the prevention and treatment of HCC. Disclosures No relevant conflicts of interest to declare.