Optimized new Shengmai powder(优化新生脉散方) inhibits myocardial fibrosis in heart failure by regulating the rat sarcoma/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase/extracellular regulated protein kinases signaling pathway

OBJECTIVE: Exploring the effect of Optimized New Shengmai powder(优化新生脉散方, ONSMP) on myocardial fibrosis in heart failure(HF) based on rat sarcoma(RAS)/rapidly accelerated fibrosarcoma(RAF)/mitogen-activated protein kinase kinase(MEK)/extracellular regulated protein kinases(ERK) signaling pathway. METHODS: Randomized 70 Sprague-Dawley rats into sham(n = 10) and operation(n = 60) groups, then established the HF rat by ligating the left anterior descending branch of the coronary artery. We randomly divided the operation group rats into the model, ONSMP [including low(L), medium(M), and high(H) dose], and enalapril groups. After the 4-week drug intervention, echocardiography examines the cardiac function and calculates the ratios of the whole/left heart to the rat's body weight. Finally, we observed the degree of myocardial fibrosis by pathological sections, determined myocardium collagen(COL) Ⅰ and COL Ⅲ content by enzyme-linked immunosorbent assay, detected the m RNA levels of COL Ⅰ, COL Ⅲ, α-smooth muscle actin(α-SMA), and c-Fos proto-oncogene(c-Fos) by universal real-time, and detected the protein expression of p-RAS, p-RAF, p-MEK1/2, p-ERK1/2, p-ETS-like-1 transcription factor(p-ELK1), p-c-Fos, α-SMA, COL Ⅰ, and COL Ⅲ by Western blot. RESULTS: ONSMP can effectively improve HF rat's cardiac function, decrease cardiac organ coefficient, COL volume fraction, and COL Ⅰ/Ⅲ content, down-regulate the m RNA of COL Ⅰ/Ⅲ, α-SMA and c-Fos, and the protein of p-RAS, p-RAF, p-MEK1/2, p-ERK1/2, p-ELK1, c-Fos, COL Ⅰ/Ⅲ, and α-SMA. CONCLUSIONS: ONSMP can effectively reduce myocardial fibrosis in HF rats, and the mechanism may be related to the inhibition of the RAS/RAF/MEK/ERK signaling pathway.

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.

N2L,a novel lipoic acid-niacin dimer,attenu⁃ates ferroptosis and decreases lipid peroxida⁃tion in HT22 cells

OBJECTIVE N2L is a novel lipoic acid-niacin dimer regulating lipid metabolism with multifunction,including antioxidant effect.We investigated the protective effect of N2L and the underlying mechanisms under the ferroptosis inducer RAS-selective lethality 3(RSL3)treat⁃ment in HT22 cells.METHODS HT22 cells were pretreated with N2L and then were treated with RSL3 to establish a ferroptosis cell model.MTT assay was used to detect the cell survival rate.Free radical probe(dihydroethidium,DHE)and ferrous probe FerroOrange were used to detect the contents of free radicals and ferrous ions in cells.The ultrastructure of mitochondria of treat⁃ed cells was observed by transmission electron microscope.The expression of ferroptosis-relat⁃ed proteins acyl-CoA synthetase long-chain family member 4(ACSL4),glutathione peroxidase 4(GPX4),cyclooxygenase-2(COX-2),ferritin Heavy Chain 1(FTH1),nuclear factor E2-related factor 2/heme oxygenase-1,and phosphoryla⁃tion levels of the c-Jun N-terminal kinase(JNK)/extracellular regulated protein kinases(ERK)pathway were detected by Western blotting.RE⁃SULTS RSL3 decreased the cell viability and induced excessive accumulation of(reactive oxy⁃gen species)ROS in HT22 cells.N2L pretreat⁃ment effectively protected HT22 cells against lipid peroxidation.What′s more,N2L recovered GPX4 protein expression and blocked the increase of COX-2 and ACSL4 expressions.Moreover,N2L also significantly prevented FTH1 from downregulation and maintained iron homeo⁃stasis.Finally,N2L pretreatment could decrease JNK/ERK activation induced by RSL3.CON⁃CLUSION N2L is an excellent ferroptosis inhibi⁃tor,and its anti-ferroptosis mechanism may be related to the reduction of lipid peroxidation and the regulation of iron homeostasis.

SIGNAL MECHANISM OF INHIBITION OF BIFIDOBACTERIA ON GROWTH OF COLON CANCER

Objective: To explore the antitumor mechanisms of bifidobacteria adolescence in vivo. Methods: The content of extracellular signal regulated proteins (ERK)1/2, C-Jun N-terminal kinase (JNK), p38, c-fos and c-jun in nude mouse transplanted large bowel carcinoma was detected by using laser confocal microscopy. The expression of NF-κB was determined by immunohistochemistry. Results: After the nude mouse transplanted tumor was treated with bifidobacteria, the average fluorescent strength of ERK1/2, JNK, c-fos and c-jun was significantly lower than that in tumor control group (P<0.01). The average fluorescent strength of p38 was not obvious difference in the two groups (P>0.05). The positive cell density of NF-κB in large bowel carcinoma transplantation tumors in Bifidobacterium injection group was markedly lower than that in tumor group(P<0.01). Conclusion: bifidobacteria adolescence could markedly decrease the activity of ERK1/2 and JNK, the expression c-fos and c-jun, and the activity of NF-κB.

Sphingosine-1-Phosphate Protects Against the Development of Cardiac Remodeling via Sphingosine Kinase 2 and the S1PR2/ERK Pathway

Objective:Cardiac remodeling is a common pathological change in various cardiovascular diseases and can ultimately result in heart failure.Thus,there is an urgent need for more effective strategies to aid in cardiac protection.Our previous work found that sphingosine-1-phosphate(S1P)could ameliorate cardiac hypertrophy.In this study,we aimed to investigate whether S1P could prevent cardiac fibrosis and the associated mechanisms in cardiac remodeling.Methods:Eight-week-old male C57BL/6 mice were randomly divided into a sham,transverse aortic constriction(TAC)or a TAC+S1P treatment group.Results:We found that S1P treatment improved cardiac function in TAC mice and that the cardiac fibrosis ratio in the TAC+S1P group was significantly lower and was accompanied by a decrease inα-smooth muscle actin(α-SMA)and collagen type I(COL I)expression compared with the TAC group.We also found that one of the key S1P enzymes,sphingosine kinase 2(SphK2),which was mainly distributed in cytoblasts,was downregulated in the cardiac remodeling case and recovered after S1P treatment in vivo and in vitro.In addition,our in vitro results showed that S1P treatment activated extracellular regulated protein kinases(ERK)phosphorylation mainly through the S1P receptor 2(S1PR2)and spurred p-ERK transposition from the cytoplasm to cytoblast in H9c2 cells exposed to phenylephrine.Conclusion:These findings suggest that SphK2 and the S1PR2/ERK pathway may participate in the anti-remodeling effect of S1P on the heart.This work therefore uncovers a novel potential therapy for the prevention of cardiac remodeling.

Effect of Artemisia decoction on liver function and pERK/eIF2a signaling pathway in rats with alcoholic liver fibrosis

Objective: To investigate the effects of Artemisia decoction on liver function and phosphorylation of extracellular regulated protein kinase/eukaryotic translation initiation factor 2α (pERK/eIF2a) signaling pathway in rats with alcoholic liver fibrosis. Methods: A total of 40 healthy Sprague-Dole (SD) rats were randomly divided into 4 groups: the normal group, the sham operation group, the model group and the Artemisia decoction group, with 10 rats in each group. Alcoholic liver fibrosis model was established by "alcohol-corn oil-pyrazole" combined with a 12-week high-fat diet. After successful modeling, the normal group was not treated, and the sham operation group was given saline. The model group and the Artemisia decoction group were given the same amount of wormwood soup at the same time. The serum hydroxyproline (HYP), hyaluronidase (HA), laminin (LN), type IV collagen (CIV), type III procollagen (PIIINP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltranspeptidase (γ-TG), total bile acid (TBA), total bilirubin (TB), albumin (ALB), total cholesterol (CHOL), triglyceride (TG), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured after 12 weeks of continuous treatment. The degree of liver fibrosis was observed by hematoxylin-eosin staining (HE). The expression of pERK/eIF2a signaling pathway in liver tissue was detected by enzyme-linked immunosorbent assay (ELISA). Results: Compared with the normal group, the levels of serum HYP, HA, LN, CIV, PIIINP, AST, ALT, γ-GT, ALP, TBL and TB in the sham operation group were not significantly changed (P>0.05), while these indexes in the model group were significantly elevated (P<0.01). After treatment with Artemisia decoction, the levels of serum HYP, HA, LN, CIV, PIIINP, AST, ALT, γ-GT, ALP, TBL and TB were significantly lower than those in the model group (P<0.01). The serum albumin, lipid metabolism and oxidative damage indicators showed that there was no significant change in serum ALB, CHOL, TG, GSH, SOD and MDA levels in the sham operation group compared with the normal group (P>0.01). The levels of GSH and SOD in the model group were significantly decreased (P<0.01), and the levels of CHOL, TG and MDA in the model group were significantly increased (P<0.01). Compared with the model group, the serum ALB, GSH and SOD levels were significantly increased (P<0.01), and CHOL, TG and MDA levels were significantly decreased after giving intervention with Artemisia decoction (P<0.01). The results of HE staining showed that compared with the control group, the morphology of the liver sections of the sham operation group was normal, while the liver sections of the model group showed obvious vacuolization changes. The liver sections of the rats treated with Artemisia decoction were significantly improved. The results of ELISA showed that there was no significant change in the levels of pERK and eIF2a in the liver tissue of the sham operation group compared with the normal group (P>0.05). The levels of pERK and eIF2a in the liver tissue of the model group were significantly increased (P<0.01). After treatment with Artemisia decoction, the levels of pERK and eIF2a in rat liver tissues were significantly lower than those in the model group (P<0.01). Conclusion: Artemisia decoction can effectively block the degree of liver fibrosis in rats with alcoholic liver fibrosis, reduce liver fibrosis index and improve hepatobiliary function. This effect may be related to inhibition of the pERK/eIF2a signaling pathway.

Hepatitis B virus X protein upregulates tumor necrosis factor-α expression of rat mesangial cell line via ERKs pathway

Hepatitis B virus X protein(HBx),a 17-kd protein encoded by X gene of hepatitis B virus(HBV),has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements.The aim of the study is to investigate the extracellular regulated protein kinases(ERKs)pathway of HBx on glomerular mesangial cell(GMC)proliferation and tumor necrosis factor-α(TNF-α)expression.The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector pCI-neo and confirmed by restriction endonuclease digestion and sequence analysis.PCI-neo containing HBV X gene(pCI-neo-X)was then transfected into cultured GMC line via liposome.GMC proliferation,TNF-αand its mRNA expression were compared in the condition of with or without U0126 in culture media.HBx,ERK1/2 and p-ERK1/2 expression in GMCs was assessed by Western blotting.TNF-αmRNA expression was assessed by semi-quantitative reverse transcription-PCR(RT-PCR).TNF-αlevel in supernatants was measured by ELISA.GMC proliferation was detected by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT)kit.The results showed that HBx expression was found in transfected GMCs and became prominent at 36th and 48th h after transfection whether with or without U0126 in culture media.TNF-αmRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-αlevels in supernatants in PCI-neo-X transfection without U0126 group were(189.0�18.1)and(172.3�24.3)pg/mL at 36th and 48th h after transfec-tion,respectively.In contrast,TNF-αlevels in supernatants with U0126 were(65.6�11.6)and(84.0�24.6)pg/mL at 36th and 48th h,respectively.The TNF-αlevels in the latter groups were significantly lower than those in the former groups(P<0.05).GMCs proliferation was also lower in added U0126 group at 36th and 48th h after transfection.From above,we can conclude that HBx could induce GMC proliferation and increase TNF-αmRNA expression and its protein production.HBx upregulates TNF-αexpression and induces cell proliferation of GMC line partly through ERK1/2 signal transduction pathway.

1,5-dicaffeoylquinic acid protects primary neurons from amyloid β1-42-induced apoptosis via PI3K/Akt signaling pathway

Background Recently, 1,5-dicaffeoylquinic acid （1,5-DQA）, a caffeoylquinic acid derivative isolated from Aster scaber, was found to have neuroprotective effects. However, the protective mechanisms of 1,5-DQA have not yet been clearly identified. The purpose of this study was to explore the protective mechanisms of 1,5-DQA on neuronal culture. Methods We investigated the neuroprotective effects of 1,5-DQA against amyloid IB1-42 （Aβ42）-induced neurotoxicity in primary neuronal culture. To evaluate the neuroprotective effects of 1,5-DQA, primary cultured cortical neurons from neonate rats were pretreated with 1,5-DQA for 2 hours and then treated with 40 pmol/L Aβ42 for 6 hours. Cell counting kit-8, Hoechst staining and Western blotting were used for detecting the protective mechanism. Comparisons between two groups were evaluated by independent t test, and multiple comparisons were analyzed by one-way analysis of variance （ANOVA）. Results 1,5-DQA treated neurons showed increased neuronal cell viability against Aβ42 toxicity in a concentration- dependent manner, both phosphoinositide 3-kinase （PI3K）/Akt and extracellular regulated protein kinase 1/2 （Erkl/2） were activated by 1,5-DQA with stimulating their upstream tyrosine kinase A （Trk A）. However, the neuroprotective effects of 1,5-DQA were blocked by LY294002, a PI3K inhibitor, but not by PD98059, an inhibitor of mitogen-activated protein kinase kinase. Furthermore, 1,5-DQA＇s anti-apoptotic potential was related to the enhanced inactivating phosphorylation of glycogen synthase kinase 313 （GSK3β） and the modulation of expression of apoptosis-related protein Bcl-2/Bax. Conclusion These resutts suggest that 1,5-DQA prevents AI342-induced neurotoxicity through the activation of PI3K/Akt followed by the stimulation of Trk A, then the inhibition of GSK313 as well as the modulation of Bcl-2/Bax.

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.