Extracellular Signal-regulated Protein Kinase Research Articles

Aggf1 Specifies Hemangioblasts at the Top of Regulatory Hierarchy via Npas4l and mTOR-S6K-Emp2-ERK Signaling.

Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.

Leptin-mediated ERK Signaling Pathway Promotes the Transformation of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust

Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust. Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R₂₀₀ cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method. Both the cells in the R group and R₂₀₀ group express leptin receptor OB-R. Compared to the R₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R₂₀₀ group (R₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R₂₀₀ (R₂₀₀LU) group. Additionally, the cells in the R₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R₂₀₀LU group and R₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R₂₀₀L group. However, when the cells in the R₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased. Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

The Effects of Injectable Platelet-Rich Fibrin and Advanced-Platelet Rich Fibrin on Gingival Fibroblast Cell Vitality, Proliferation, Differentiation.

Injectable Platelet Rich Fibrin (I-PRF) and Advanced-Platelet Rich Fibrin (A-PRF) are autologous materials derived from patients' blood and employed in periodontal regenerative surgery. Although I-PRF and A-PRF have different characteristics, their biological effects on gingival tissue fibroblasts remain unclear. This research aims to compare the in vitro capacity in inducing gene expression and proliferation of human gingival fibroblasts between A-PRF and I-PRF. Human donors undergoing dental implant surgery were sampled for normal human gingival fibroblasts (NHGFCs), followed by preparing A-PRF and I-PRF membranes. Enzyme-linked immunosorbent assay (ELISA) kit was used to assess the release of platelet-derived growth factor-AA (PDGF-AA), transforming growth factor-beta1 (TGF- β1), and insulin growth factor-1 (IGF-1) at different periods. Cell viability and proliferation of A-PRF and I-PRF were compared using CCK-8 assay. The impacts of platelet concentration on human gingival fibroblast cells (HGFCs) were evaluated by quantifying the level or amount of phosphorylated extracellular signal-regulated protein kinase (p-ERK), and Matrix metalloproteinases (MMPs), MMP-1 and MMP-3. The effects of PRF on aged human gingival fibroblast cells were examined retrospectively. Overall, A-PRF demonstrated a higher release of TGF-B1 and PDGF-AA, while I-PRF reflected higher levels of IGF-1. A significantly higher level of cell proliferation was induced by higher cell proliferation by A-PRF and I-PRF. Additionally, in comparison to I-PRF, the expression of ERK phosphorylation and MMP-1 &MMP-3 in HGFCs was demonstrated by I-PRF and A-PRF. The increase in A-PRF was time-dependent (p < 0.05). Both I-PRF and A-PRF induced a stimulatory biological impact on the proliferation of human gingiva fibroblasts, with the latter demonstrating better capacity in facilitating the release of different growth factors. A-PRF also induced higher gene expression of p-ERK, MMP-1 &MMP-3, and the proliferation of fibroblasts.

A ring N(CH3)2-based derivative of resveratrol inhibits pulmonary vascular remodeling in hypoxia pulmonary hypertension

Pulmonary artery smooth muscle cells (PASMCs) phenotypic switching and pulmonary artery endothelial cells (PAECs) endothelial-mesenchymal transition (EndMT) are important in promoting pulmonary hypertension (PH)-pulmonary vascular remodeling (PVR). Resveratrol can efficiently inhibit the proliferation of PASMCs, but its application is limited due to its low bioavailability and solubility. In this study, we modified resveratrol to assess the role of A ring N(CH3)2-based derivatives of resveratrol (Res4) in PVR-PASMCs phenotypic switching and PVR-PAECs EndMT. Chemical methods were used for the preparation of Res4; NMRS and HPLC were used to authenticate Res4. Mice developed PVR after 4 weeks of hypoxia (10% O2). Res4 (50 mg/kg/d) attenuated right ventricular systolic pressure, right ventricular hypertrophy, and PVR. PASMCs developed phenotypic switching and PAECs developed EndMT after 2 days of hypoxia (3% O2). Res4 (10 μM) could inhibit PASMCs and PAECs viability. Res4 could decrease proliferating cell nuclear antigen (PCNA) and osteopontin (OPN) expression, and increase α-smooth muscle actin (α-SMA) and vimentin expression in PASMCs. It could also decrease PCNA, α-SMA, vimentin expression and increase platelet endothelial cell adhesion molecule (CD31) expression in PAECs. Notably, Res4 inhibited the phosphorylation levels of mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated protein kinase (ERK), Jun-N-terminal kinase (JNK), and p38 kinase in hypoxia-treated PASMCs and PAECs, indicating MAPK pathway may be involved in Res4-induced inhibition of PASMCs phenotypic switching and PAECs EndMT. Our data demonstrated that Res4 exerts antiproliferative effects by regulating PASMCs phenotypic switching and PAECs EndMT. Res4 may be potentially used as a drug against PH-PVR.

Oleanolic acid alleviates the extrapyramidal symptoms and cognitive impairment induced by haloperidol through the striatal PKA signaling pathway in mice

Haloperidol, one of the representative typical antipsychotics, is on the market for schizophrenia but shows severe adverse effects such as extrapyramidal symptoms (EPS) or cognitive impairments. Oleanolic acid (OA) is known to be effective for tardive dyskinesia which is induced by long-term treatment with L-DOPA. This study aimed to investigate whether OA could ameliorate EPS or cognitive impairment induced by haloperidol. The balance beam, catalepsy response, rotarod and vacuous chewing movement (VCM) tests were performed to measure EPS and the novel object recognition test was used to estimate haloperidol-induced cognitive impairment. Levels of dopamine and acetylcholine, the phosphorylation levels of c-AMP-dependent protein kinase A (PKA) and its downstream signaling molecules were measured in the striatum. OA significantly attenuated EPS and cognitive impairment induced by haloperidol without affecting its antipsychotic properties. Valbenazine only ameliorated VCM. Also, OA normalised the levels of dopamine and acetylcholine in the striatum which were increased by haloperidol. Furthermore, the increased phosphorylated PKA, extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) levels and c-FOS expression level induced by haloperidol were significantly decreased by OA in the striatum. In addition, cataleptic behaviour of haloperidol was reversed by sub-effective dose of H-89 with OA. These results suggest that OA can alleviate EPS and cognitive impairment induced by antipsychotics without interfering with antipsychotic properties via regulating neurotransmitter levels and the PKA signaling pathway in the striatum. Therefore, OA is a potential candidate for treating EPS and cognitive impairment induced by antipsychotics.

The mechanism of acrolein exposure inhibited the release of neutrophil extracellular traps: By reducing respiratory burst and Raf/MEK/ERK pathway and promote cell apoptosis

Acrolein (AC) is a highly toxic volatile substance in the environment, and studies have found that excessive AC had a toxic effect on the immune system. Neutrophils are the first line of defense against pathogen invasion. The release of neutrophil extracellular traps (NETs) is a protective mechanism for neutrophils, and its release is affected by environmental pollutants. However, the effect of AC on NETs release and its mechanism remains unclear. In this study, chicken peripheral blood neutrophils were pretreated with 20 μM AC and treated with 5 μM Phorbol 12-myristate 13-acetate (PMA) to stimulate the release of NETs. The results showed that AC exposure significantly inhibited the release of NETs induced by PMA, respiratory burst, and the expression levels of phospho-rapidly accelerated fibrosarcoma (p-Raf), phospho-mitogen-activated extracellular signal-regulated kinase (p-MEK) and phospho-extracellular regulated protein kinases (p-ERK). In addition, AC exposure significantly inhibited the expression of B-cell lymphoma-2 (Bcl-2) and promoted the expression of apoptotic factors Bcl2-Associated X (Bax), cytochrome c (Cyt C), cysteinyl aspartate specific proteinase 9 (Casp 9) and cysteinyl aspartate specific proteinase 3 (Casp 3). Further inhibition of neutrophil apoptosis significantly improved the release of NETs. The above results indicated that AC exposure led to a decrease in the formation of NETs, which is caused by excessive AC-induced neutrophil apoptosis. Our study clarified the immune toxicity mechanism of AC on chickens, which is of great significance and reference value for protecting the ecological environment and poultry health.

Schistosoma mansoni excretory-secretory products induce protein kinase signalling, hyperkinesia, and stem cell proliferation in the opposite sex

Adult male and female schistosomes in copula dwell within human blood vessels and lay eggs that cause the major Neglected Tropical Disease human schistosomiasis. How males and females communicate to each other is poorly understood; however, male-female physical interaction is known to be important. Here, we investigate whether excretory-secretory products (ESPs), released into the external milieu by mature Schistosoma mansoni, might induce responses in the opposite sex. We demonstrate that ESPs adhere to the surface of opposite sex worms inducing the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, particularly in the parasite tegument. Furthermore, we show that mature worms stimulated signalling in juvenile worms. Strikingly, we demonstrate that ESPs from the opposite sex promote stem cell proliferation, in an ERK- and p38 MAPK-dependent manner, in the tegument and within the testes of males, and the ovaries and vitellaria of females. Hyperkinesia also occurs following opposite sex ESP exposure. Our findings support the hypothesis that male and female schistosomes may communicate over distance to modulate key processes underlying worm development and disease progression, opening unique avenues for schistosomiasis control.

Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants.

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and –LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and –LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO–K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and –LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors.

Increased Expression of Long Noncoding RNA LOC100506314 in T cells from Patients with Nonsegmental Vitiligo and Its Contribution to Vitiligo Pathogenesis.

This study aimed to identify the abnormal expression of long noncoding RNAs (lncRNAs) in T cells from patients with vitiligo and to investigate their functional roles in the immune system. Using microarray analysis, the expression levels of RNA transcripts in T cells from patients with vitiligo and controls were compared. We identified several genes and validated their expression levels in T cells from 41 vitiligo patients and 41 controls. The biological functions of the lncRNAs were studied in a transfection study using an RNA pull-down assay, followed by proteomic analysis and western blotting. The expression levels of 134 genes were significantly increased, and those of 142 genes were significantly decreased in T cells from vitiligo patients. After validation, six genes had increased expression, and three genes had decreased expression in T cells from patients with vitiligo. T-cell expression of LOC100506314 was increased in vitiligo, especially CD4+, but not CD8+ T cells. The expression levels of LOC100506314 in CD4+ T cells was positively and significantly associated with the severity of vitiligo. LOC100506314 was bound to the signal transducer and activator of transcription 3 (STAT3) and macrophage migration inhibitory factor (MIF). Enhanced expression of LOC100506314 inhibited the phosphorylation of STAT3, protein kinase B (AKT), and extracellular signal-regulated protein kinases (ERK), as well as the levels of nuclear protein of p65 and the expression of IL-6 and IL-17 in Jurkat cells and T cells from patients with vitiligo. In conclusion, this study showed that the expression of LOC100506314 was elevated in CD4+ T cells from patients with vitiligo and associated the severity of vitiligo. LOC100506314 interacted with STAT3 and MIF and inhibited IL-6 and IL-17 expression by suppressing the STAT3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), AKT, and ERK pathways. Enhanced expression of LOC100506314 in T cells may be a potential treatment strategy for vitiligo.

Global miRNA profiling reveals key molecules that contribute to different chronic lymphocytic leukemia incidences in Asian and Western populations.

It has been known for decades that the incidence of chronic lymphocytic leukemia (CLL) is significantly lower in Asia than in Western countries, but the reason responsible for this difference still remains a major knowledge gap. Using GeneChip® miRNA array to analyze the global microRNA expression in B lymphocytes from Asian and Western CLL patients and healthy individuals, we have identified microRNA with CLL-promoting or suppressive functions that are differentially expressed in Asian and Western individuals. In particular, miR-4485 is upregulated in CLL patients of both ethnic groups, and its expression is significantly lower in Asian healthy individuals. Genetic silencing of miR-4485 in CLL cells suppresses leukemia cell growth, whereas ectopic expression of miR-4485 promotes cell proliferation. Mechanistically, miR-4485 exerts its CLL-promoting activity by inhibiting the expression of TGR5 and activating the ERK1/2 pathway. In contrast, miR-138, miR-181a, miR- 181c, miR-181d, and miR-363 with tumor-suppressive function are highly expressed in Asian healthy individuals. Our study suggests that differential expression of several important microRNA with pro- or anti-CLL functions in Asian and Western B lymphocytes likely contributes to the difference in CLL incidence between the two ethnic groups, and that miR-4485 and its downstream molecule TGR5 could be potential therapeutic targets.

Differential Effects of the Processed and Unprocessed Garlic (Allium sativum L.) Ethanol Extracts on Neuritogenesis and Synaptogenesis in Rat Primary Hippocampal Neurons.

Garlic (Allium sativum L.) is an aromatic herb known for its culinary and medicinal uses for centuries. Both unprocessed (white) and processed (black) garlic are known to protect against the pathobiology of neurological disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD), which has been attributed to their anti-inflammatory and antioxidant properties. The information on the effects of processed and unprocessed garlic on neuronal process outgrowth, maturation, and synaptic development is limited. This study aimed at investigating and comparing the effects of the ethanol extracts of unprocessed (white garlic extract, WGE) and processed (black garlic extract, BGE) garlic on the maturation of primary hippocampal neurons. Neurite outgrowth was stimulated in a dose-dependent manner by both WGE and BGE and the most effective doses were 15 μg/mL and 60 μg/mL, respectively, without showing cytotoxicity. At this optimal concentration, both extracts promoted axonal and dendritic growth and maturation. Furthermore, both extracts substantially increased the formation of functional synapses. However, the effect of WGE was more robust at every developmental stage of neurons. In addition, the gas chromatography and mass spectrometry (GC-MS) analysis revealed a chemical profile of various bioactives in both BGE and WGE. Linalool, a compound that was found in both extracts, has shown neurite outgrowth-promoting activity in neuronal cultures, suggesting that the neurotrophic activity of garlic extracts is attributed, at least in part, to this compound. By using network pharmacology, linalool's role in neuronal development can also be observed through its modulatory effect on the signaling molecules of neurotrophic signaling pathways such as glycogen synthase kinase 3 (GSK3β), extracellular signal-regulated protein kinase (Erk1/2), which was further verified by immunocytochemistry. Overall, these findings provide information on the molecular mechanism of processed and unprocessed garlic for neuronal growth, survival, and memory function which may have the potential for the prevention of several neurological disorders.

TLR2 mediates autophagy through ERK signaling pathway in Chlamydia psittaci CPSIT_p7 protein-stimulated RAW264.7 cells.

Chlamydia psittaci is a zoonotic pathogen found in birds and humans. Macrophages, major components of the innate immune system, can resist chlamydial infections and trigger adaptive immune responses. However, the molecular mechanisms underlying the action of macrophages against C. psittaci infection are not well understood. This study investigated the roles and mechanisms of plasmid-encoded protein CPSIT_p7 of C. psittaci in regulating autophagy in RAW264.7 cells. The results demonstrated that stimulation of RAW264.7 with C. psittaci plasmid protein CPSIT_p7 induced the expressions of the autophagy signaling primary regulators LC3 and Beclin1, which could also significantly induce the phosphorylation levels of ERK, JNK, p38, and Akt. Next, siRNA knockdown of TLR2 resulted in significant downregulation of CPSIT_p7-triggered autophagy in RAW264.7 cells. Moreover, the extracellular regulated protein kinase (ERK) inhibitor PD98059 markedly reduced autophagy in CPSIT_p7-stimulated macrophages. In summary, these results indicated that TLR2 plays an essential role in the induction of autophagy through the ERK signaling pathway in CPSIT_p7-stimulated RAW264.7 cells.

Xiebai Zengye decoction improves respiratory function and attenuates inflammation in juvenile rats with postinfection cough via regulating ERK signaling pathway.

This study aimed to determine the effects of Xiebai Zengye decoction (XBZY)on airway inflammation and respiratory function in rats with postinfectious cough (PIC),and its regulatory effects on the extracellular signal-regulated kinase (ERK)signaling pathway. Compared with the normal group, the rats from thePIC group had significantly shortened expiratory time (TE)and enhanced pause (EEP),increased resistance (RT),and enhanced pause (Penh), along with increased levels of serum interleukin-4 (IL-4)and IL-6,and decreased levels of IL-10. The lung and colon tissues of rats from the PIC group showed histopathological changes, including inflammatory cell infiltration, damaged mucosal epithelium, and crypt structure, with significantly increased ERK mRNA and protein expression levels. Treatment with XBZY and montelukast sodium (MAS)improved the respiratory function and serum cytokine levels, reduced tissue inflammation, and decreased ERK mRNA and protein expression levels in the lung and colon tissues. In the lung tissues, XBZY treatment significantly decreased the expression of phosphorylated-ERK (p-ERK)protein, as well as p-MEK1/2, p-ERK1/2, and p-c-Fos proteins, while in the colon tissues, XBZY significantly decreased the expression of p-ERK1/2and p-c-Fos proteins. However, MAS treatment only showed significant improvement in the lung tissue inflammation score, and the expression level of p-ERK protein in the lung tissue was decreased. In conclusion, the present study suggests that XBZY has a potential therapeutic effect on PIC by improving respiratory function and attenuating inflammation, and this effect may be associated with the inhibition of the ERK signaling pathway. These findings could provide a new direction for the development of treatments for PIC. However, further research is needed to elucidate the underlying molecular mechanisms of XBZY and to confirm its safety and efficacy in clinical trials.

Emerging roles of PHLPP phosphatases in lung cancer.

Pleckstrin homologous domain leucine-rich repeating protein phosphatases (PHLPPs) were originally identified as protein kinase B (Akt) kinase hydrophobic motif specific phosphatases to maintain the cellular homeostasis. With the continuous expansion of PHLPPs research, imbalanced-PHLPPs were mainly found as a tumor suppressor gene of a variety of solid tumors. In this review, we simply described the history and structures of PHLPPs and summarized the recent achievements in emerging roles of PHLPPs in lung cancer by 1) the signaling pathways affected by PHLPPs including Phosphoinositide 3-kinase (PI3K)/AKT, RAS/RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and Protein kinase C (PKC) signaling cascades. 2) function of PHLPPs regulatory factor USP46 and miR-190/miR-215, 3) the potential roles of PHLPPs in disease prognosis, Epidermal growth factor receptors (EGFR)- tyrosine kinase inhibitor (TKI) resistance and DNA damage, 4) and the possible function of PHLPPs in radiotherapy, ferroptosis and inflammation response. Therefore, PHLPPs can be considered as either biomarker or prognostic marker for lung cancer treatment.

Pro-inflammatory action of formoterol in human bronchial epithelia

Despite the wide usage of β2-adrenoceptor agonists in asthma treatment, they do have side effects such as aggravating inflammation. We previously reported that isoprenaline induced Cl- secretion and IL-6 release via cAMP-dependent pathways in human bronchial epithelia, but the mechanisms underlying the inflammation-aggravation effects of β2-adrenoceptor agonists remain pooly understood. In this study, we investigated formoterol, a more specific β2-adrenoceptor agonist, -mediated signaling pathways involved in the production of IL-6 and IL-8 in 16HBE14o- human bronchial epithelia. The effects of formoterol were detected in the presence of PKA, exchange protein directly activated by cAMP (EPAC), cystic fibrosis transmembrane conductance regulator (CFTR), extracellular signal-regulated protein kinase (ERK)1/2 and Src inhibitors. The involvement of β-arrestin2 was determined using siRNA knockdown. Our results indicate that formoterol can induce IL-6 and IL-8 secretion in concentration-dependent manner. The PKA-specific inhibitor, H89, partially inhibited IL-6 release, but not IL-8. Another intracellular cAMP receptor, EPAC, was not involved in either IL-6 or IL-8 release. PD98059 and U0126, two ERK1/2 inhibitors, blocked IL-8 while attenuated IL-6 secretion induced by formoterol. Furthermore, formoterol-induced IL-6 and IL-8 release was attenuated by Src inhibitors, namely dasatinib and PP1, and CFTRinh172, a CFTR inhibitor. In addition, knockdown of β-arrestin2 by siRNA only suppressed IL-8 release when a high concentration of formoterol (1 μM) was used. Taken together, our results suggest that formoterol stimulates IL-6 and IL-8 release which involves PKA/Src/ERK1/2 and/or β-arrestin2 signaling pathways.

Pers reverse angiotensin II -induced vascular smooth muscle cell proliferation by targeting cyclin E expression via inhibition of the MAPK signaling pathway

ABSTRACT The circadian rhythm of blood pressure (BP) is believed to be regulated by the clock system, which is closely linked to levels of angiotensin II (Ang II). This study aimed to investigate whether Ang II mediates the proliferation of vascular smooth muscle cells (VSMCs) through the interaction between the clock system and the mitogen-activated protein kinase (MAPK) signaling pathway. Primary rat aortic VSMCs were treated with Ang II, with or without MAPK inhibitors. VSMC proliferation, expression of clock genes, CYCLIN E, and MAPK pathways were assessed. Ang II treatment resulted in increased VSMC proliferation and rapid upregulation of clock gene Periods (Pers) expression. Compared to the non-diseased control (NC) group, VSMCs incubated with Ang II displayed a noticeable delay in the G1/S phase transition and downregulation of CYCLIN E upon silencing of Per1 and Per2 genes. Importantly, silencing Per1 or Per2 in VSMCs led to decreased expression of key MAPK pathway proteins, including RAS, phosphorylated mitogen-activated protein kinase (P-MEK), and phosphorylated extracellular signal-regulated protein kinase (P-ERK). Moreover, the MEK and ERK inhibitors, U0126 and SCH772986, significantly attenuated the Ang II-induced proliferation of VSMCs, as evidenced by an increased G1/S phase transition and decreased CYCLIN E expression. The MAPK pathway plays a critical role in regulating VSMC proliferation in response to Ang II stimulation. This regulation is controlled by the expression of circadian clock genes involved in the cell cycle. These findings provide novel insights for further research on diseases associated with abnormal VSMC proliferation.

DMSO Alleviates LPS-Induced Inflammatory Responses in RAW264.7 Macrophages by Inhibiting NF-κB and MAPK Activation

Dimethyl sulfoxide (DMSO), an amphipathic molecule composed of one highly polar sulfinyl group and two nonpolar methyl groups, is considered an excellent solvent due to its capability to dissolve many polar and nonpolar compounds. Therefore, DMSO is widely used to solubilize drugs for therapeutic applications. DMSO is reported to possess anti-inflammatory, anticancer, and antioxidative capacities, and the anti-inflammatory efficacy of DMSO has been intensively studied in various cell lines and animal models. An in vitro model of mouse macrophage RAW 264.7 cells has been widely used, among several experimental designs, for evaluation during the development of new anti-inflammatory drugs. DMSO, which is used to dissolve samples, is also prone to experimental errors because of its anti-inflammatory properties. Therefore, we systematically confirmed the cytotoxic and anti-inflammatory effects of DMSO and the related signaling pathways in RAW 264.7 cells. The results show that DMSO at 0.25% to 1.5% did not result in cellular toxicity, with results comparable to the control group where DMSO is absent; at concentrations 2.0%, however, it inhibited the viability of RAW264.7 cells (13.25%). The results demonstrate that pretreatment with DMSO profoundly attenuates the lipopolysaccharide (LPS)-stimulated levels of nitric oxide (NO) and prostaglandin (PG)E2, as well as the levels of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2) protein, and inducible nitric oxide synthase (iNOS). Collectively, the DMSO pretreatments appear to notably alleviate LPS-induced damage by reducing phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase proteins (ERKs), nuclear factor-kappa-B (NF-κB) in addition to NF-κB/p65 nuclear translocation. Taken together, the results clearly show that DMSO attenuates the inflammatory response in LPS-induced RAW264.7 cells by regulating the activation of the MAPK and NF-κB signaling pathways. These results contribute to potentially reducing experimental errors or misjudgments when using the LPS-induced RAW 264.7 macrophage cell model for evaluation during the development of new anti-inflammatory drugs.

Shaoyao Gancao decoction alleviates the central hyperalgesia of recurrent NTG-induced migraine in rats by regulating the NGF/TRPV1/COX-2 signal pathway

Ethnopharmacological relevanceShaoyao Gancao Decoction (SGD) is well known as an effective prescription for analgesia composed of two herbs, and is noted as traditional Chinese medicine morphine. It is widely used in various conditions causing pain, including migraine. However, there is currently no research exploring the mechanism of action in the treatment of migraines. Aim of the studyThe current research was devised to determine the underlying regulatory mechanism of SGD, by verifying its role in the NGF/TRPV1/COX-2 signal pathway. Materials and methodsThe active components in SGD were identified by UHPLC-MS. A migraine model was prepared by subcutaneous (s.c.) injection of nitroglycerin (NTG) into the neck to detect migraine-like behavior, orbital hyperalgesia threshold changes, and the therapeutic effect of SGD. The mechanism of SGD in remedying migraine was studied through transcriptome sequencing (RNA-seq), which was further validated utilizing Elisa, Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) experiments. ResultsIn the SGD chemical composition analysis, 45 components were identified including gallic acid, paeoniflorin and albiforin. In the behavioral experiments, SGD treatment significantly decreased the score of migraine-like head scratching in the NTG-induced migraine model (Mod) rats, while the hyperalgesia threshold increased outstandingly on days 10, 12, and 14 (P < 0.01, P < 0.001 or P < 0.0001). In migraine biomarkers experiment, compared with the Mod group, the 5-hydroxytryptamine (5-HT) contents were outstandingly enhanced by SGD treatment, while nitric oxide (NO) contents were markedly declined (P < 0.01). In the RNA-seq test, the down-regulated genes of SGD inhibiting hyperalgesia migraine included the neurotrophic factor (NGF) and transient receptor potential vanillic acid subfamily protein 1 receptor (TRPV1). The down-regulation pathway is the inflammatory mediator regulation of TRP channels. In gene set enrichment analysis (GSEA), SGD decreased the over-expression of protooncogene tyrosine-protein kinase Src (SRC) and TRPV1 in this pathway, and the two genes clustered at its lower end, with similar functions. PPI network results show that NGF interacts with TRPV1. Further verification shows that when compared with Mod group, the plasma cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) protein expression levels and the dura mater calcitonin gene-related peptide (CGRP), extracellular signal-regulated kinase (ERK), p-ERK, SRC and NGF protein expression levels in the SGD group were remarkably decreased (P < 0.01, P < 0.001 or P < 0.0001), and the expression level of TRPV1 protein showed a downward trend (P = 0.06). The expression levels of COX-2, NO, CGRP, TRPV1, SRC and NGF mRNA in the dura mater was overtly down-regulated (P < 0.05, P < 0.01 or P < 0.001). ConclusionsSGD has a significant inhibitory effect on the NGF/TRPV1/COX-2 signaling pathway that mediates central hyperalgesia migraine, thus suggesting the molecular mechanism of SGD in improving the symptoms of migraine may be related to the central hyperalgesia neurotransmitter that regulates the pathogenesis of migraine.

Single-Cell Heterogeneity in Snake Venom Expression Is Hardwired by Co-Option of Regulators from Progressively Activated Pathways.

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression.