Extracellular Regulated Protein Kinases Research Articles

Yeast β-glucan modulates macrophages and improves antitumor NK-cell responses in cancer.

As the largest proportion of myeloid immune cells in tumors, macrophages play an important role in tumor growth and regression according to their different phenotypes, thus reprogramming macrophages has become a new research direction for cancer immunotherapy. Yeast-derived whole β-glucan particles (WGPs) can induce M0 macrophages to differentiate into M1 macrophages and convert M2 macrophages and tumor-associated macrophages (TAMs) into M1 macrophages. In vitro, studies have confirmed that WGP-treated macrophages increase the activating receptors in natural killer cells (NK cells) and enhance the cytotoxicity of NK cells. The extracellular regulated protein kinases (ERK) signaling pathway is involved in WGP-mediated regulation of the macrophage phenotype. Further in vivo studies show that oral WGP can significantly delay tumor growth, which is related to the increased proportion of macrophages and NK cells, the macrophage phenotype reversal, and the enhancement of NK cell immune function. NK-cell depletion reduces the therapeutic efficacy of WGP in tumor-bearing mice. These findings revealed that in addition to T cells, NK cells also participate in the antitumor process of WGP. It was confirmed that WGP regulates the macrophage phenotype to regulate NK-cell function.

Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured. The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity. EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

The CaMKII inhibitory peptide AIP alleviates renal fibrosis through the TGF-β/Smad and RAF/ERK pathways.

Renal fibrosis is characterized by the excessive deposition of extracellular matrix that destroys and replaces the functional renal parenchyma, ultimately leading to organ failure. It is a common pathway by which chronic kidney disease (CKD) can develop into end-stage renal disease (ESRD), which has high global morbidity and mortality, and there are currently no good therapeutic agents available. CaMKII has been indicated to be closely related to the occurrence of renal fibrosis, and its specific inhibitory peptide AIP, was shown to directly bind the active site of CaMKII. In this study, we examined the effect of AIP on the progression of renal fibrosis and its possible mechanism. The results showed that AIP could inhibit the expression of the fibrosis markers Fibronectin, Collagen I, MMP2 and α-SMA in vivo and in vitro. Further analysis revealed that AIP could inhibit the expression of various epithelial-to-mesenchymal transformation (EMT)-related markers, such as Vimentin and Snail 1, in vivo and in vitro. Mechanistically, AIP could significantly inhibit the activation of CaMKII, Smad 2, Raf and ERK in vitro and in vivo and reduce the expression of TGF-β in vivo. These results suggested that AIP could alleviate renal fibrosis by inhibiting CaMKII and blocking activation of the TGF-β/Smad2 and RAF/ERK pathways. Our study provides a possible drug candidate and demonstrates that CaMKII is a potential pharmacological target for the treatment of renal fibrosis. Significance Statement We have demonstrated that AIP significantly attenuated TGF-β1-induced fibrogenesis and ameliorated UUO-induced renal fibrosis through the CaMKII/TGF-β/Smad and CaMKII/RAF/ERK signaling pathways in vitro and in vivo. Our study provides a possible drug candidate and demonstrates that CaMKII can be a potential pharmacological target for the treatment of renal fibrosis.

A herbal product inhibits carbon tetrachloride-induced liver fibrosis by suppressing the epidermal growth factor receptor signaling pathway.

Fuzheng Huayu formula (FZHY), composed of Salvia miltiorrhiza Bunge, Cordyceps sinensis, the seed of Prunus persica (L.) Batsch, the pollen of Pinus massoniana Lamb, Gynostemma pentaphyllum (Thunb.) Makino and the fruit of Schisandra chinensis (Turcz.) Baill, is a Chinese herbal compound with demonstrated clinical benefits in liver fibrosis (LF). However, its potential mechanism and molecular targets remain to be elucidated. This study was designed to evaluate the anti-fibrotic role of FZHY in hepatic fibrosis and to elucidate the potential mechanisms. Network pharmacology was assayed to identify the interrelationships among compounds of FZHY, potential targets and putative pathways on anti-LF. Then the core pharmaceutical target for FZHY against LF was verified by serum proteomic analysis. Further in vivo and in vitro assays were performed to verify the prediction of the pharmaceutical network. The network pharmacology analysis revealed that a total of 175 FZHY-LF crossover proteins were filtered into a protein-protein interaction (PPI) network complex and designated as the potential targets of FZHY against LF, and the Epidermal Growth Factor Receptor (EGFR) signaling pathway was further explored according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then analytical studies were validated by carbon tetrachloride (CCl4)-induced model in vivo. We found FZHY could attenuate CCl4-induced LF, especially decrease p-EGFR expression in α-Smooth Muscle Actin (α-SMA)-positive hepatic stellate cell (HSC) and inhibit the downstream of the EGFR signaling pathway, especially Extracellular Regulated Protein Kinases (ERK) signaling pathway in liver tissue. We further demonstrate that FZHY could inhibit Epidermal Growth Factor (EGF)-induced HSC activation, as well as the expression of p-EGFR and the key protein of the ERK signaling pathway. FZHY has a good effect against CCl4-induced LF. The action mechanism was associated with the down-regulation of the EGFR signaling pathway in activated HSCs.

DUSP2 Deletion Inhibits Macrophage Migration by Inhibiting ERK Activation in Zebrafish

Dual-specificity phosphatase 2 (DUSP2) regulates the activation of members of the mitogen-activated protein kinase (MAPK) family, which is involved in a variety of cellular processes including cell proliferation, differentiation, apoptosis, and migration. DUSP2 also regulates the expression of inflammatory mediators in macrophages; however, it remains unknown whether DUSP2 participates in macrophage migration. Here, using the tail fin injury model in zebrafish larvae, we found that the deletion of DUSP2 inhibited the expression of pro-inflammatory cytokines and macrophage chemokines. Moreover, live imaging results showed that the migration of macrophages to the injury site was inhibited after DUSP2 deletion. This inhibitory effect was mediated through the reduced activation of extracellular regulated protein kinases (ERK) in DUSP2 knockout zebrafish.

Elevated retinal fibrosis in experimental myopia is involved in the activation of the PI3K/AKT/ERK signaling pathway

ObjectiveThis study aimed to investigate the regulatory role of the PI3K/AKT/ERK signaling pathway in retinal fibrosis in −6.0 diopter (D) lens-induced myopic (LIM) guinea pigs. MethodsBiological measurements of eye tissues were performed on guinea pigs to obtain their refraction, axial length, retinal thickness, physiological function, and fundus retinal status. In addition, Masson staining and immunohistochemical (IHC) assay were further done to explore the changes in retinal morphology after myopic induction. Meanwhile, hydroxyproline (HYP) content was measured to evaluate the degree of retinal fibrosis. Moreover, the levels of the PI3K/AKT/ERK signaling pathway and fibrosis-related molecules in retinal tissues including matrix metalloproteinase 2(MMP2), collagen type I (Collagen I), and α-smooth muscle actin (α-SMA) were detected by real-time quantitative PCR (qPCR) and Western blot. ResultsThe LIM guinea pigs showed a significant myopic shift in refractive error and an increase in axial length compared with those of the normal control (NC) group. Masson staining, hydroxyproline content determination, and IHC showed an increase in retinal fibrosis. After myopic induction, qPCR and western blot analyses showed that phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA), protein kinase B (AKT), extracellular regulated protein kinase 1/2 (ERK1/2), MMP2, Collagen I, and α-SMA were consistently elevated in the LIM group than those in the NC group. Conclusion: The PI3K/AKT/ERK signaling pathway was activated in the retinal tissues of myopic guinea pigs, which exaggerated fibrotic lesions and reduced retinal thickness, ultimately leading to retinal physiological dysfunctions in myopic guinea pigs.

Glyphosate induces autophagy in hepatic L8824 cell line through NO-mediated activation of RAS/RAF/MEK/ERK signaling pathway and energy metabolism disorders

Glyphosate is an herbicide commonly used worldwide, and its substantial use causes widespread pollution with runoff. However, research on glyphosate toxicity has mostly remained at the embryonic level and existing studies are limited. In the present study, we investigated whether glyphosate can induce autophagy in hepatic L8824cells by regulating energy metabolism and rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling by activating nitric oxide (NO). First, we selected 0, 50, 200, and 500μg/mL as the challenge doses, according to the inhibitory concentration of 50% (IC50) of glyphosate. The results showed that glyphosate exposure increased the enzyme activity of inducible nitric oxide synthase (iNOS), which in turn increased the NO content. The activity and expression of enzymes related to energy metabolism, such as hexokinase (HK)1, HK2, phosphofructokinase (PFK), phosphokinase (PK), succinate dehydrogenase (SDH), and nicotinamide adenine dinucleotide with hydrogen (NADH), were inhibited, and the RAS/RAF/MEK/ERK signaling pathway was activated. This led to the negative expression of mammalian target of rapamycin (mTOR) and P62 in hepatic L8824cells and the activation of the autophagy marker genes microtubule-associated proteins light chain 3 (LC3) and Beclin1 to induce autophagy. The above results were dependent on glyphosate concentration. To verify whether autophagy can be excited by the RAS/RAF/MEK/ERK signaling pathway, we treated L8824cells with the ERK inhibitor U0126 and found that the autophagy gene LC3 was reduced due to the inhibition of ERK, thus demonstrating the reliability of the results. In conclusion, our results demonstrate that glyphosate can induce autophagy in hepatic L8824cells by activating NO, thus regulating energy metabolism and the RAS/RAF/MEK/ERK signaling pathway.

Emodin attenuates high lipid-induced liver metastasis through the AKT and ERK pathways in vitro in breast cancer cells and in a mouse xenograft model

Emodin, a natural anthraquinone derivative, can inhibit lipid synthesis and breast cancer cell proliferation. We previously found that emodin decreased breast cancer liver metastasis via epithelial-to-mesenchymal transition (EMT) inhibition. However, the mechanism through which emodin affects breast cancer liver metastasis in high-fat diet-induced obese and hyperlipidemic mice has not been elucidated. Bioinformatics analysis was used to reveal the potential targets and pathways of emodin. The mouse model of liver metastasis was established by injecting breast cancer cells into the left ventricle in high-fat diet-induced obese mice. The effect of emodin on inhibiting liver metastasis of breast cancer was evaluated by animal experiments. The mechanisms through which emodin inhibits liver metastasis of breast cancer were studied by cell and molecular biological methods. Emodin reduced lipid synthesis by inhibiting the expression of triglyceride (TG) synthesis-related genes, such as fatty acid synthase (Fasn), glycerol-3-phosphate acyltransferase 1 (Gpat1), and stearoyl-CoA desaturase (Scd1), and ultimately reduced liver metastasis in breast cancer. In addition, emodin inhibited breast cancer cell proliferation and invasion through the serine/threonine kinase (AKT) signaling and extracellular-regulated protein kinase (ERK) pathways by interacting with CSNK2A1, ESR1, ESR2, PIM1 and PTP4A3. Our results indicate that emodin may have therapeutic potential in the prevention or treatment of breast cancer liver metastasis.

ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes.

This study aimed to investigate the potential role of pyruvate kinase M2 (PKM2) and extracellular regulated protein kinase (ERK) in arsenic-induced cell proliferation. L-02 cells were treated with 0.2 and 0.4 μmol/L As3+, glycolysis inhibitor (2-deoxy-D-glucose,2-DG), ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene, U0126] or transfected with PKM2 plasmid. Cell viability, proliferation, lactate acid production, and glucose intake capacity were determined by CCK-8 assay, EdU assay, lactic acid kit and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake kit, respectively. Also, levels of PKM2, phospho-PKM2S37, glucose transporter protein 1 (GLUT1), lactate dehydrogenase A (LDHA), ERK, and phospho-ERK were detected using Western blot and the subcellular localization of PKM2 in L-02 cells was detected by immunocytochemistry (ICC). Treatment with 0.2 and 0.4 μmol/L As3+ for 48 h increased the viability and proliferation of L-02 cells, the proportion of 2-NBDG+ cell and lactic acid in the culture medium, and GLUT1, LDHA, PKM2, phospho-PKM2S37, and phospho-ERK levels and PKM2 in nucleus. Compared with the 0.2 μmol/L As3+ treatment group, the lactic acid in the culture medium, cell proliferation and cell viability, and the expression of GLUT1 and LDHA were reduced in the group co-treated with siRNA-PKM2 and arsenic or in the group co-treated with U0126. Moreover, the arsenic-increased phospho-PKM2S37/PKM2 was decreased by U0126. Therefore, ERK/PKM2 plays a key role in the Warburg effect and proliferation of L-02 cells induced by arsenic, and also might be involved in arsenic-induced upregulation of GLUT1 and LDHA. This study provides a theoretical basis for further elucidating the carcinogenic mechanism of arsenic.

Astragaloside IV attenuates high glucose-induced NF-κB-mediated inflammation through activation of PI3K/AKT-ERK-dependent Nrf2/ARE signaling pathway in glomerular mesangial cells.

Inflammation is a key contributor to diabetic kidney disease pathogenesis, including reactive oxidation stress (ROS)-mediated nuclear factor-κB (NF-κB) signaling pathway. In this study, we examined the effect of Astragaloside IV (AS-IV) on anti-inflammatory and anti-oxidative properties under high glucose (HG) condition and the potential mechanism in glomerular mesangial cells (GMCs). We showed that AS-IV concentration-dependently reduced GMCs proliferation, restrained ROS release and hydrogen peroxide content, and suppressed pro-inflammatory cytokines as well as pro-fibrotic factors expression, which were associated with the inhibition of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling activation. Accordingly, both NF-κB overexpression by using RNA plasmid and Nrf2 gene silencing by using RNA interference weakened the ability of AS-IV to ameliorate HG-induced oxidative stress, inflammation, and cell proliferation. Furthermore, phosphatidylinositide 3-kinases (PI3K)/serine/threonine protein kinase (Akt) and extracellular regulated protein kinases (ERK) signaling pathway regulated the process of AS-IV-induced Nrf2 activation and antioxidant capacity, which evidenced by using PI3K inhibitor LY294002 or ERK inhibitor PD98059 that largely abolished the AS-IV efficacy. Taken together, these results indicated that AS-IV protected against HG-induced GMCs damage by inhibiting ROS/NF-kB-induced increases of inflammatory cytokines, fibrosis biomarkers, and cell proliferation via up-regulation of Nrf2-dependent antioxidant enzyme expression, which were mediated by PI3K/Akt and ERK signaling pathway activation.

Targeting KRAS Mutations

Targeting KRAS Mutations

Discovery of a novel Coumarin-Dihydroquinoxalone derivative MY-673 as a tubulin polymerization inhibitor capable of inhibiting the ERK pathway with potent anti-gastric cancer activities

As a class of microtubule targeting agents, colchicine binding site inhibitors (CBSIs) are considered as promising drug candidates for cancer therapy. However, due to adverse reactions, there are currently no CBSIs approved by FDA for cancer treatment. Therefore, extensive efforts are still encouraged to find novel CBSIs with different chemical structures and better anticancer efficacies. In this work, we designed and synthesized a new coumarin-dihydroquinoxalone derivative, MY-673, and evaluated its anticancer potency in vitro and in vivo. We confirmed that MY-673 was a potent CBSI that it not only inhibited tubulin polymerization, but also exhibited significant inhibitory potency on the growth of 13 cancer cells with IC50 values from 11.7 nM to 395.9 nM. Based on the results of kinase panel screening, MY-673 could inhibit ERK (extracellular regulated protein kinases) pathways-related kinases. We further confirmed that MY-673 could inhibit ERK signaling pathway in MGC-803 and HGC-27 cells, and then affected the expression level of SMAD4 protein in TGF-β (transforming growth factor β) /SMAD (small mother against decapentaplegic) signaling pathway using the western blotting assay. In addition, compound MY-673 could effectively inhibit cell proliferation, migration and induce cell apoptosis. We also further confirmed the in vivo efficacy of MY-673 in inhibiting tumor growth using the MGC-803 xenograft tumor model. At 20 mg/kg, the TGI rate was 85.9%, and it did not cause obvious toxicity to the main organs of mice. Together, the results we report here indicated that MY-673 was a promising CBSI for cancer treatment, which was capable of inhibiting the ERK pathway with potent antiproliferative activities in vitro and in vivo.

Anti-inflammatory Activity of Gegen Decoction and Its Modulatory Mechanism on the NF-κB and MAPK Signaling Pathways.

Gegen Decoction as anti-inflammatory medicine is used in clinic widespread, however the specific anti-inflammatory molecular mechanism of Gegen Decoction is still unclear. The purpose was to study the anti-inflammatory activity of Gegen Decoction in vivo and to research its anti-inflammatory molecular mechanism. The content of main essential components in Gegen Decoction were determined by HPLC method. The anti-inflammatory activity of Gegen Decoction was confirmed through in vivo animal experiments. Furthermore, RAW 264.7 cells were stimulated by lipopolysaccharides to induce inflammatory reaction, the modulatory effect of Gegen Decoction on the activation process of mitogen-activated protein kinases and nuclear factor-κB signaling pathways was investigated. The content of puerarin was the highest among all the index components. Gegen Decoction inhibited carrageenan-induced paw edema in rats and xylene-induced ear swelling in mice. Gegen Decoction had no obvious toxicity against RAW 264.7 cells at the concentrations of 10-40 mg/mL; significantly inhibited the release of nitric oxide, prostaglandin E2, tumor necrosis factor-α and interleukin-6; down-regulated the high expression of inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2. It inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs)/extracellular regulated protein kinases (ERK)/c-Jun N-terminal kinase (JNK), the degradation of nuclear factor-κB (NF-κB)/inhibitor of NF-κB-α (IκB-α) and the nuclear translocation of NF-κB/p65 into nucleus. Gegen Decoction exerts significant anti-inflammatory activity, mainly by blocking the activation of both MAPKs and NF-κB pathway.

MASH1 induces neuron transdifferentiation of adrenal medulla chromaffin cells.

Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms. Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA. Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited. MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.

Effect of transcutaneous electrical acupoint stimulation on learning and memory ability of chronic fatigue syndrome rats and its mechanisms

To observe the effect of transcutaneous electrical acupoint stimulation (TEAS) on the histomorphological manifestations of hippocampal CA1 region and the expression of extracellular regulatory protein kinase (ERK), cyclic adenosine response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in chronic fatigue syndrome (CFS) rats, so as to explore the mechanisms of TEAS in improving the learning and memory abilities of CFS rats. Forty male Wistar rats were randomly divided into normal group (10 rats) and modeling group (30 rats); then after modeling, they were selected and randomly divided into model group (10 rats) and TEAS group (10 rats). CFS rats model was prepared by sleep deprivation combined with weight-bearing swimming. Rats in the TEAS group were stimulated with Han's acupoint nerve stimulator at bilateral "Zusanli" (ST36) and "Shenshu" (BL23) (2 Hz/15 Hz, 1-2 mA), 20 min each time, once a day for 4 weeks with 1 d rest every 6 d. The score of general conditions of rats was evaluated. The learning and memory ability was tested with Morris water maze. The morphology and ultrastructure of hippocampal CA1 region were observed by HE staining and transmission electron microscopy. The expression levels of ERK, CREB and BDNF mRNAs and proteins in hippocampus were detected by real time quantitative PCR and Western blot, respectively. Compared with the normal group, the score of general condition was increased (P<0.01); the escape latency was prolonged (P<0.05, P<0.01) and the times of crossing the original platform was decreased (P<0.05); the expression levels of ERK, CREB and BDNF mRNAs and proteins in hippocampus were decreased (P<0.05, P<0.01) in the model group. Compared with the model group, the scores of general condition on the 42nd and 49th day were decreased (P<0.05, P<0.01); the escape latency was shortened (P<0.01, P<0.05)and the times of crossing the original platform were increased (P<0.05); the expression levels of ERK, CREB and BDNF mRNAs and proteins in hippocampus were increased (P<0.01, P<0.05) in the TEAS group. The morphology of neurons in hippocampal CA1 region was normal in the normal group. In the model group, the number of neurons in hippocampal CA1 region decreased, the arrangement of nerve cells was scattered, the number of apoptotic cells increased, some nuclear structures disappeared, nuclear heterochromatin increased, the cell membrane wrinkled, the chromatin appeared empty bright area, and the crista was incomplete. Compared with the model group, the nerve cells morphology in hippocampal CA1 region was more regular, the number of apoptotic cells decreased, the chromatin and the cytoplasm were uniformly distributed, and the crista was relatively intact in the TEAS group. TEAS can improve the learning and memory ability of CFS rats, the mechanisms may be related to improving the neural structure of hippocampal CA1 region and up-regulating the expression levels of ERK/CREB/BDNF.

Y-box-binding protein 1 mediates sorafenib resistance via the extracellular signal regulated-protein kinase pathway in hepatoma cells

Objective: To investigate the effect and possible mechanism of Y-box-binding protein 1 (YB-1) on sorafenib resistance in hepatoma cells. Methods: Lentiviral vectors with YB-1 overexpression and knockdown were constructed, respectively, to stimulate human hepatoma cell lines (HepG2 and Huh7) alone or in combination with sorafenib.The overexpression part of the experiment was divided into four groups: overexpression control group (Lv-NC), YB-1 overexpression group (Lv-YB-1), overexpression control combined with sorafenib resistance group (Lv-NC+sorafenib), YB-1 overexpression combined with sorafenib resistance group (Lv-YB-1 + sorafenib). The knockdown part of the experiment was also divided into four groups: knockdown control group (Lv-shNC), YB-1 knockdown group (Lv-shYB-1), knockdown control combined with sorafenib resistance group (Lv-shNC + sorafenib), YB-1 knockdown combined with sorafenib resistance group (Lv-shYB-1 + sorafenib). The occurrence of cell apoptosis was detected by TUNEL. The protein expression levels of phosphorylated (p)-ERK and ERK, key proteins in the extracellular regulatory protein kinase (ERK) signaling pathway, were detected by Western blot and quantified by ImageJ software. Subcutaneous tumorigenesis experiments were performed in nude mice. The effect of YB-1 on the efficacy of sorafenib was verified in vivo. The comparison between the two sets of data was carried out by an independent sample t-test. One-way ANOVA was used for comparisons between the three groups of data above. Results: Sorafenib had accelerated the occurrence of apoptosis in hepatoma cells, while YB-1 overexpression had inhibited cell apoptosis, and at the same time also inhibited the apoptosis-accelerating impact of sorafenib. On the contrary, YB-1 knockdown accelerated cell apoptosis and amplified the induction effect of sorafenib on apoptosis. Furthermore, sorafenib resistance had down-regulated p-ERK levels (HepG2: Lv-NC 0.685 ± 0.143, Lv-NC + sorafenib 0.315 ± 0.168, P < 0.05; Huh7: Lv-NC 0.576 ± 0.078, Lv-NC + sorafenib 0.150 ± 0.131, P < 0.01), whereas YB-1 overexpression had inhibited sorafenib resistance p-ERK reduction (HepG2: Lv-NC + sorafenib 0.315 ± 0.168, Lv-YB-1 + sorafenib 0.688 ± 0.042, P < 0.05; Huh7: Lv-NC + sorafenib 0.150 ± 0.131, Lv-YB-1 + sorafenib 0.553 ± 0.041, P < 0.05). YB-1 knockdown further increased sorafenib-induced p-ERK downregulation (HepG2: Lv-shNC + sorafenib 0.911 ± 0.252, Lv-shYB-1 + sorafenib 0.500 ± 0.201, P < 0.05; Huh7: Lv-shNC + sorafenib 0.577 ± 0.082, Lv-shYB-1 + sorafenib 0.350 ± 0.143, P < 0.05), which was further verified in naked mice (Lv-shNC + sorafenib 0.812 ± 0.279, Lv-shYB-1 + sorafenib 0.352 ± 0.109, P < 0.05). Conclusion: YB-1 mediates the occurrence of sorafenib resistance via the ERK signaling pathway in hepatoma cells.

Statins enhances antitumor effect of oxaliplatin in KRAS-mutated colorectal cancer cells and inhibits oxaliplatin-induced neuropathy

BackgroundKRAS mutations are fraught with the progression of colorectal cancer and resistance to chemotherapy. There are pathways such as extracellular regulated protein kinase 1/2 (ERK1/2) and Akt downstream and farnesylation and geranylgeranylation upstream that are activated upon mutated KRAS. Previous studies have shown that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are effective to treat KRAS mutated colorectal cancer cells. Increased doses of oxaliplatin (L-OHP), a well-known alkylating chemotherapeutic drug, causes side effects such as peripheral neuropathy due to ERK1/2 activation in spinal cords. Hence, we examined the combinatorial therapeutic efficacy of statins and L-OHP to reduce colorectal cancer cell growth and abrogate neuropathy in mice.MethodsCell survival and confirmed apoptosis was assessed using WST-8 assay and Annexin V detection kit. Detection of phosphorylated and total proteins was analyzed the western blotting. Combined effect of simvastatin and L-OHP was examined the allograft mouse model and L-OHP-induced neuropathy was assessed using cold plate and von Frey filament test.ResultsIn this study, we examined the effect of combining statins with L-OHP on induction of cell death in colorectal cancer cell lines and improvement of L-OHP-induced neuropathy in vivo. We demonstrated that combined administration with statins and L-OHP significantly induced apoptosis and elevated the sensitivity of KRAS-mutated colorectal cancer cells to L-OHP. In addition, simvastatin suppressed KRAS prenylation, thereby enhancing antitumor effect of L-OHP through downregulation of survivin, XIAP, Bcl-xL, and Bcl-2, and upregulation of p53 and PUMA via inhibition of nuclear factor of κB (NF-κB) and Akt activation, and induction of c-Jun N-terminal kinase (JNK) activation in KRAS-mutated colorectal cancer cells. Moreover, simvastatin enhanced the antitumor effects of L-OHP and suppressed L-OHP-induced neuropathy via ERK1/2 activation in vivo.ConclusionTherefore, statins may be therapeutically useful as adjuvants to L-OHP in KRAS-mutated colorectal cancer and may also be useful in the treatment of L-OHP-induced neuropathy.

Protective effect of Lactiplantibacillus pentosus CQZC01 in Kunming mice of subacute alcoholic liver injury.

To decrease the climbing rate of alcoholic liver disease, the protective effect in subacute alcoholic liver injury of newly isolated Lactiplantibacillus pentosus CQZC01 has been investigated. Lactiplantibacillus pentosus CQZC01 (1 × 109 CFU/kgbw) administered orally could keep weight of mice at 30.54±1.15g; alleviate alcoholic damage on hepatic morphology; decrease the activities of hyaluronidase (147±19U/L), procollagen III (4.82±0.54ng/mL), alanine transaminase (10.66±2.32U/L), and aspartate aminotransferase (15.18±1.98U/L); enhance the activities of alcohol dehydrogenase (65.15±3.2 U/mgprot), aldehyde dehydrogenase (16.50±0.96 U/mgprot), superoxide dismutase (623±39 U/mgprot), and glutathione (19.54±2.46µmol/gprot); and decrease liver total cholesterol (3.59±0.50mmol/gprot) and triglyceride (0.88±0.24mmol/gprot) (p<0.05). Moreover, L. pentosus CQZC01 elevated the level of interleukin-10 (IL-10; 807±44 pg/mL) but significantly decreased the levels of IL-1β (29.75±5.27pg/mL), IL-6 (58±8 pg/mL), and tumor necrosis factor-α (TNF-α, 564±13 pg/mL). Liver malondialdehyde was also significantly decreased by treatment with L. pentosus CQZC01 from 3.61±0.14 to 2.03±0.49nmol/mgprot. The relative expression of C-Jun N-terminal kinase, extracellular regulated protein kinases, and cyclooxygenase-1 was downregulated, and the SOD1, SOD2, peroxisome proliferator-activated receptor-α, glutathione peroxidase, catalase, nuclear factor erythroid-2-related factor 2, heme oxygenase-1 and nicotinamide adenine dinucleotide phosphate were upregulated by L. pentosus CQZC01. The overall protective effect of L. pentosus CQZC01 was comparable to commercial Lactobacillus delbrueckii subsp. Bulgaricus. Lactobacillus pentosus CQZC01 might be a suitable hepatoprotective measure for people who frequently ingest alcoholic drinks. PRACTICAL APPLICATION: L. pentosus CQZC01 can alleviate subacute alcoholic liver injury by raising the antioxidant status and upregulating the antioxidant-related genes.

Low-Intensity Pulsed Ultrasound Attenuates Postoperative Neurocognitive Impairment and Salvages Hippocampal Synaptogenesis in Aged Mice

Postoperative neurocognitive impairment is an urgent problem with global aging accelerating. The prevention and treatment of postoperative neurocognitive impairment have been widely investigated but lack effective strategies. Low-intensity pulsed ultrasound (LIPUS), a non-invasive tool, has shown an effect on neuroprotection, but whether it could attenuate the postoperative neurocognitive impairment and the underlying mechanisms remains unknown. An experimental setup for LIPUS stimulation of the hippocampus was well established. A laparotomy model in aged mice was applied, and a Morris water maze was used to assess cognitive function. RT-qPCR and western blotting were used to detect levels of Piezo1, synapse-associated proteins in the hippocampus, respectively. Immunofluorescent staining was also used to determine the neural activation and Piezo1 expression. The results showed that LIPUS increased synapse-related proteins of the hippocampus and attenuated cognitive impairment in aged mice. Meanwhile, LIPUS suppressed the overexpression of Piezo1 in the hippocampus. We further found that LIPUS promoted Calpain1 activity and increased extracellular regulated protein kinases (Erk) phosphorylation. Our results suggested that LIPUS could improve cognitive impairment and increase hippocampal synaptogenesis through the Piezo1-mediated Calpain1/ Erk pathway. LIPUS could be used as an effective physical intervention to alleviate postoperative cognitive dysfunction in the aged population.

CD74: a prospective marker for reactive microglia?

CD74: a prospective marker for reactive microglia?