Study on Effects of Extracts from Salvia Miltiorrhiza and Curcuma Longa in Inhibiting Phosphorylated Extracellular Signal Regulated Kinase Expression in Rat's Hepatic Stellate Cells

Objective： To study the effect of salvianolic acid B （SAB） and curcumin, the extracts of Solvie Miltiorrhize and Curcume Longe, on the proliferation and activation of hepatic stellate cell （HSC）, and the extracellular signal regulated kinase （ERK） expression in it. Methods： Rat＇s HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-（4,5-dimthyl-2- 2thiazoly）-2,5-diphenyl-2H-tetrazolium bromide （MTT） colorimetry, and the expression levels of a smooth actin （a-SMA）, collagen type Ⅰ , and ERK were determined by Western blot. Results： SAB and curcumin inhibited the proliferation and activation of rat＇s HSC-T6 in dose-dependent fashion and significantly reduced the expression level of a-SMA （ P〈0.01 ）. Curcumin significantly reduced the expression of collagen type Ⅰ （P〈0.05）. Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group （ P〈0.01 and P〈0.05 respectively）. Conclusion： SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type Ⅰ collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.

Shenmai Injection(参麦注射液) Inhibiting the Extracellular Signal Regulated Kinase-lnduced Human Airway Smooth Muscle Proliferation in Asthma

Objective： To investigate the relationship between the proliferation of sensitized human airway smooth muscle cells （HASMCs） and the expression of extracellular signal regulated kinase （ERK） and the effect of Shenmai Injection （参麦注射液, SMI） on HASMCs. Methods： The HASMCs cultured in vitro were divided into three groups： （1） control group; （2） sensitized group： containing 10% asthmatic serum; （3） SMI group： further divided into three different concentration subgroups interferred with 10 μL/mL, 50 μL/mL, and 100 μL/mL SMI, respectively. The proliferation of HASMCs was detected using MTT method, the expression of proliferating cell nucleus antigen （PCNA） in HASMCs was detected using immunocytochemical staining, and the expression of phosphoration-ERK1/2 （p-ERK1/2） protein was detected using Western-blot. Results： After passive sensitization, the optical density value （A49o value） of HASMCs was significantly increased from 0.366± 0.086 to 0.839 ± 0.168 （P〈0.05）. In addition, the expression of PCNA was significantly increased from 28.7% ± 5.9% in the control group to 69.8% ±7.5% in the sensitized group （P〈0.05）. At the same time, the expression of p-ERK1/2 in passively sensitized HASMCs was significantly increased compared with the control group （all P〈0.05）. Affer application of 10 μL/mL, 50 μL/mL, and 100 μL/mL SMI to the cultured media of passively sensitized group, the A570 value was significantly decreased from 0.839 ±0.168 to 0.612 ±0.100, 0.412 ± 0.092, and 0.339 ± 0.077, respectively （P〈0.05）. Moreover, the expression of PCNA was significantly decreased from 69.8% ±7.5% to 57.8% ± 6.2%, 40.7%±5.4%, and 26.1% ± 5.2%, respectively. At the same time, the expression of p-ERK1/2 in each SMI group was significantly decreased compared with the sensitized group （all ,P〈0.05）. Conclusion： ERK signal transduction pathway may be involved in the airway remodeling in asthma. The expression of ERK can be inhibited by SMI in a dose-dependent manner, thus preventing the proliferation of HASMCs.

Expression of Extracellular Signal-regulated Kinase and Angiotensin-converting Enzyme in Human Atria during Atrial Fibrillation

In order to investigate the changes in the expression of extracellular signal regulated kinase (ERK1/ERK2) and angiotensin converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2 mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P <0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Effect of lead on ERK activity and the protective function of bFGF in rat primary culture astroglia

Objective： To observe the effects of lead on levels ofphosphorylated extracellular signal regulated kinase （p-ERK） in the cytoplasm of primary cultures of rat astroglial cells and the possible protective effect of basic fibroblast growth factor （bFGF） on lead-induced effects. Methods： The primary astroglia cells from 1-6 d old Wistar rats were cultured. The cells pretreated with the MEK1 （mitogen-activated protein kinase kinase 1） inhibitor PD98059 and bFGF, respectively, were exposed to Pb acetate of different concentrations for different times. Western blotting and reverse transcription polymerase chain reaction （RT-PCR） methods were used to detect the protein and mRNA expressions of ERK. Results： mRNA expression for ERK peaked 15 min after initiation of lead exposure （P〈0.05） and protein expression of p-ERK peaked at 30 min （P〈0.05）. ERK mRNA levels and p-ERK protein levels returned to baseline after 60 and 120 min of lead exposure, respectively （P〉0.05）. The increase in p-ERK levels in lead-treated cells could be inhibited by PD098059. Activation of ERK in the cells by lead was prevented by pretreatment with bFGF. Total ERK protein levels did not change under the same experimental conditions （P〉0.05）. Conclusion： Low-level lead exposure resulted in transient activation of ERK through the MEK pathway, which then returned to basal levels in the continued presence of lead. Exogenous bFGF protected ERK signaling components in astroglia from lead poisoning.

Sorafenib inhibits growth and metastasis of hepatocellular carcinoma by blocking STAT3

AIM: To investigate the inhibitory role and the underlying mechanisms of sorafenib on signal transducer and activator of transcription 3 (STAT3) activity in hepatocellular carcinoma (HCC).METHODS: Human and rat HCC cell lines were treated with sorafenib. Proliferation and STAT3 dephosphorylation were assessed. Potential molecular mechanisms of STAT3 pathway inhibition by sorafenib were evaluated. In vivo antitumor action and STAT3 inhibition were investigated in an immunocompetent orthotopic rat HCC model.RESULTS: Sorafenib decreased STAT3 phosphorylationat the tyrosine and serine residues (Y705 and S727), but did not affect Janus kinase 2 (JAK2) and phosphatase shatterproof 2 (SHP2), which is associated with growth inhibition in HCC cells. Dephosphorylation of S727 was associated with attenuated extracellular signal-regulated kinase (ERK) phosphorylation, similar to the effects of a mitogen-activated protein kinase (MEK) inhibitor U0126, suggesting that sorafenib induced S727 dephosphorylation by inhibiting MEK/ERK signaling. Meanwhile, sorafenib could also inhibit Akt phosphorylation, and both the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and Akt knockdown resulted in Y705 dephosphorylation, indicating that Y705 dephosphorylation by sorafenib was mediated by inhibiting the PI3K/Akt pathway. Finally, in the rat HCC model, sorafenib signifi cantly inhibited STAT3 activity, reducing tumor growth and metastasis.CONCLUSION: Sorafenib inhibits growth and metastasis of HCC in part by blocking the MEK/ERK/STAT3 and PI3K/Akt/STAT3 signaling pathways, but independent of JAK2 and SHP2 activation.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

The regulatory effect of ERK1/2 signal pathway on production of TNFα induced by LPS in mice Kupffer cells

Objective: To study the rule of ERK1/2 activity and regulative effect of ERK1/2 pathway on the production of pro inflammatory cytokine TNFα in mice Kupffer cells (mKC) induced by LPS, and to exploring novel methods to prevent and treat clinical patients of endotoxemia. Methods: Immunoprecipitate kinase assay and Western blotting analysis were used to detect the phosphorylated ERK1/2 kinase activity in mKC stimulated by LPS, and ELISA was used to study the effect of ERK1/2 signaling cascade on LPS induced TNFα production in mKC. Results: In mKC, LPS treatment resulted in transient and rapid increase of kinase activity of ERK1/2 that phosphorylated their specific substrate ELK 1, with maximal value at 30 minutes and a return near to baseline within 2 hours, and LPS induced ERK1/2 activity from LPS concentration of 10 pg/ml to the top activity at 100 ng/ml . No activity was observed in unstimulated mKC. Inhibition of the ERK1/2 pathway using the specific ERK 1/2 signal pathway inhibitor PD98059 caused a marked and concentration dependent reduction of TNFα production. Conclusions: The results show that LPS can markedly activate ERK1/2 pathway in mKC. PD98059 causes a significant and concentration dependent reduction of TNFα production. ERK1/2 may be a novel target to treat clinical patient of endotoxemia.

Activation of glycine site and GluN2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats:Implications for affective pain

Objective The rostral anterior cingulate cortex （rACC） is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors （NMDARs） are highly expressed in the rACC and mediate painrelated affect by activating a signaling pathway that involves cyclic adenosine monophosphate （cAMP）/protein ki- nase A （PKA） and/or extracellular regulated kinase （ERK）/cAMP-response element-binding protein （CREB）. The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC. Methods Immunohistochemistry and Western blot analy- sis were used to separately assess the expression of phospho-ERK （pERK） and phospho-CREB （pCREB） in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB. Results Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antago- nist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine- binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up- regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB. Conclusion Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.