8-bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK

AIM:To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin(BrMC) involve reactive oxygen species(ROS) generation and c-Jun N-terminal kinase(JNK) activation in human hepatocellular carcinoma cells(HCC).METHODS:HepG2,Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry(FCM) after propidium iodide(PI) staining,caspase-3 activity using enzymelinked immunosorbent assay(ELISA),and DNA agarose gel electrophoresis.ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate(DCHFDA) probe labeling.The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.RESULTS:FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell pop-ulation(P < 0.05),reaching 39.0% ± 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 μmol/L.The potency of BrMC to HepG2 and Bel-7402(32.1% ± 2.6%) cells was found to be more effective than the lead compound,chrysin(16.2% ± 1.6% for HepG2 cells and 11.0% ± 1.3% for Bel-7402 cell) at 40 μmol/L and similar to 5-flurouracil(33.0% ± 2.1% for HepG2 cells and 29.3% ± 2.3% for Bel-7402 cells) at 10 μmol/L.BrMC had little effect on human embryo liver L-02 cells,with the percentage of sub-G1 cell population 5.4% ± 1.8%.Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3,in a concentration-dependent manner.z-DEVD-fmk,a caspase-3specific inhibitor,prevented the activation of caspase-3.Treatment with BrMC at 10 μmol/L for 48 h resulted in the formation of a DNA ladder.Treatment of cells with BrMC(10 μmol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 ± 1.12 at 0 h to 79.8 ± 3.9 at 3 h and 89.7 ± 4.7 at 6 h.BrMC did not affect ROS generation in L-02 cells.BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine(10 mmol/L).In addition,in HepG2 cells treated with BrMC(2.5,5.0,10.0 μmol/L) for 12 h,JNK activation was observed.Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h.The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment,but GW9662 had no effect.SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells.N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.CONCLUSION:BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.

Role of Jun amino-terminal kinase （JNK） in apoptosis of cavernosal tissue during acute phase after cavernosal nerve injury

The present study aimed to identify which mitogen-activated protein kinase （p38 or Jun amino-terminal kinase [JNK]） was involved in cavernosal apoptosis during the acute phase after cavernosal nerve crush injury （CNCI） in rats to ameliorate apoptosis of cavernosal tissue, such as smooth muscle （SM）. A total of twenty 10-week-old male Sprague-Dawley rats were divided equally into two groups： sham surgery （S） and CNCI （I）. The I group approximated the clinical situation of men undergoing radical prostatectomy using two 60-second compressions of both CNs with a microsurgical vascular clamp. At 2-week postinjury, erectile response was assessed using electrostimulation. Penile tissues were harvested for immunohistochemistry analysis of alpha-SM actin （（x-SMA）, western blot analysis, and double immunofluorescence analysis of （x-SMA and phosphorylated p38 or JNK, as well as double immunofluorescent of TUNEL and phosphorylated p38 or JNK. At 2-week postinjury, the I group had a significantly lower intracavernous pressure （ICP）/mean arterial pressure （MAP） and a lower area under the curve （AUC）/MAP than the S group. The I group also exhibited decreased immunohistochemical staining of α-SMA, an increase in the number of SM cells positive for phosphorylated JNK, an increased number of apoptotic cells positive for phosphorylated JNK, and increased JNK phosphorylation compared with the S group. However, there was no significant difference in p38 phosphorylation expression or the number of SM cells positive for phosphorylated p38 between the two groups. In conclusion, our data suggest that JNK, not p38, is involved in cavernosal apoptosis during the acute phase after partial CN damage.

Hypoxia Inhibits Proliferation of Human Dermal Lymphatic Endothelial Cells via Downregulation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 Expression

Objective:Lymphatic endothelial cell(LEC)proliferation is essential for lymphangiogenesis.Hypoxia induces lymphangiogenesis,but it directly inhibits LEC proliferation and the underlying mechanisms have not been fully understood.The aim of this study was to investigate the role of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1)in hypoxia-repressed LEC proliferation.Methods:Human dermal lymphatic endothelial cells(HDLECs)were cultured under normoxic or hypoxic conditions,and cell proliferation was determined using MTT or CCK-8 assays.CEACAM1 expression was silenced by siRNA transfection.Activation of mitogen-activated protein kinases(MAPKs)was examined by Western blotting and blocked by specific inhibitors.Results:Under hypoxia,HDLECs proliferation was suppressed and CEACAM1 expression was downregulated.Silence of CEACAM1 in normoxia inhibited HDLECs proliferation and did not further decrease proliferation in HDLECs in response to hypoxia,suggesting that CEACAM1 may mediate hypoxia-induced inhibition of HDLECs proliferation.In addition,silence of CEACAM1 increased phosphorylation of MAPK molecules:extracellular signal-regulated kinase(ERK),p38 MAPK and Jun N-terminal kinase(JNK)in HDLECs.However,only inhibition of the JNK pathway rescued the reduction of HDLEC proliferation induced by CEACAM1 silence.Conclusion:Our results suggested that hypoxia downregulates CEACAM1 expression by activation of the JNK pathway,leading to inhibition of HDLEC proliferation.These findings may help to understand the mechanisms of LEC-specific response to hypoxia and develop novel therapies for pathological lymphangiogenesis.

Secreted Frizzled-Related Protein 5 Mediates Wnt5a Expression in Microcystin-Leucine-Arginine-Induced Liver Lipid Metabolism Disorder in Mice

Objective Microcystin-leucine-arginine(MC-LR)exposure induces lipid metabolism disorders in the liver.Secreted frizzled-related protein 5(SFRP5)is a natural antagonist of winglesstype MMTV integration site family,member 5A(Wnt5a)and an anti-inflammatory adipocytokine.In this study,we aimed to investigate whether MC-LR can induce lipid metabolism disorders in hepatocytes and whether SFRP5,which has anti-inflammatory effects,can alleviate the effects of hepatic lipid metabolism by inhibiting the Wnt5a/Jun N-terminal kinase(JNK)pathway.Methods We exposed mice to MC-LR in vivo to induce liver lipid metabolism disorders.Subsequently,mouse hepatocytes that overexpressed SFRP5 or did not express SFRP5 were exposed to MC-LR,and the effects of SFRP5 overexpression on inflammation and Wnt5a/JNK activation by MC-LR were observed.Results MC-LR exposure induced liver lipid metabolism disorders in mice and significantly decreased SFRP5 mRNA and protein levels in a concentration-dependent manner.SFRP5 overexpression in AML12cells suppressed MC-LR-induced inflammation.Overexpression of SFRP5 also inhibited Wnt5a and phosphorylation of JNK.Conclusion MC-LR can induce lipid metabolism disorders in mice,and SFRP5 can attenuate lipid metabolism disorders in the mouse liver by inhibiting Wnt5a/JNK signaling.

Buyang Huanwu Decoction Ameliorates Damage of Erectile Tissue and Function Following Bilateral Cavernous Nerve Injury

Objective To verify the effect of Buyang Huanwu Decoction(BHD)in ameliorating erectile dysfunction(ED)after radical prostatectomy(RP).Methods The composition of BHD was verified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS/MS)analysis.Bilateral cavernous nerve crush injury(BCNI)in rats was used to mimic the neurovascular injury occurring after RP.By the envelope method,forty rats were randomly divided into 4 groups as follows:sham(cavernous nerves exposed only),model(BCNI),low-dosage BHD[LBHD,12.8 g/(kg·d)],and high-dosage BHD[HBHD,51.2 g/(kg·d)]groups,10 rats in each group,feeding for 3 weeks respectively.Erectile function was evaluated by measuring intracavernosal pressure(ICP).Changes in the histopathology of corpus cavernosum(CC)were examined by hematoxylin-eosin staining.Meanwhile,the fibrosis of CC was measured by Masson’s trichrome staining and Western blot was used to detect the expressions of collagen I,transforming growth factor beta 1(TGF-β1)andα-smooth muscle actin(α-SMA).Apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)and Western blot for determining the expressions of B-cell lymphoma 2(Bcl-2)and Bcl-2-associated X(Bax).The oxidative stress in the CC were assessed by the superoxide dismutase(SOD),malondialdehyde(MDA)and reactive oxygen species(ROS)levels.The proteins expression of c-Jun N-terminal kinase(JNK)and c-Jun were detected by Western blot.In addition,the expression ofα-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining.Results The UPLC-QTOF-MS/MS analysis showed that BHD contained calycosin-7-O-β-D-glucoside,ononin,calycosin and formononetin.Compared with the model group,LBHD and HBHD treatment improved the ICP and the circumference,area,and weight of CC(P<0.05 or P<0.01).Furthermore,LBHD and HBHD treatments increased CC smooth muscle content and decreased apoptosis index(P<0.05 or P<0.01).LBHD and HBHD also elevated SOD and expression level ofα-SMA and Bcl-2,and reduced MDA and ROS levels,as well as expression of TGF-β1,collagen I,Bax,p-c-JNK,p-JNK in the CC compared with the model group(P<0.05 or P<0.01).The double immunofluorescence staining showed that the fluorescence degree of p-c-Jun in both LBHD and HBHD treatment groups was significantly reduced,whereas theα-SMA expression increased(P<0.05 or P<0.01).Conclusions BHD can improve ED of rats with BCNI,which is related to inhibiting fibrosis,apoptosis,and oxidative stress of CC.The ROS/JNK/c-Jun signaling pathway may play an important role in the process.

Oxidation resistance 1 （OXR1） participates in silkworm defense against bacterial infection through the JNK pathway

Bacterial infection causes enhanced reactive oxygen species （ROS） levels in insects. Oxidation resistance 1 （OXR1） plays an antioxidant role in eukaryotic organisms, including insects. In this report, we demonstrated that Pseudomonas aeruginosa and Staphylococcus aureus infection and hydrogen peroxide （H2O2） injection induced the expression of specific transcriptional isoforms of OXR1 in larval silkworms. We further showed that a Jun kinase （JNK） pathway inhibitor, SP600125, down-regulated expression of OXRI during infection, leading to elevated H2O2 levels in the hemolymph, resulting in lower viability of the injected bacteria inside the silkworm larvae. Our study suggests that OXR1 participates in protecting larval silkworms from oxidative stress and bacterial infection through the JNK pathway.