Expression Of p-ERK Research Articles

Amphiregulin blockade decreases the levodopa-induced dyskinesia in a 6-hydroxydopamine Parkinson's disease mouse model.

Levodopa (L-DOPA) is considered the most reliable drug for treating Parkinson's disease (PD) clinical symptoms. Regrettably, long-term L-DOPA therapy results in the emergence of drug-induced abnormal involuntary movements (AIMs) in most PD patients. The mechanisms underlying motor fluctuations and dyskinesia induced by L-DOPA (LID) are still perplexing. Here, we first performed the analysis on the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs) using linear models for microarray analysis (Limma) R packages from the Bioconductor project. 12 genes (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, Lypd1) were found to be upregulated. Six genes were validated on quantitative polymerase chain reaction and subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Areg LV_shRNA was used to knock down Areg to explore its therapeutic role in the LID model. Western blotting and immunofluorescence results show that AREG is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. Moreover, Areg knockdown reduced the protein expression of P-ERK. In order to ascertain whether the inhibition of the ERK pathway (a common pathway known to mediate levodopa-induced dyskinesia) could also impede Areg, the animals were injected with an ERK inhibitor (PD98059). Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the control group. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group. Taken together, our results indicate unequivocal involvement of Areg in levodopa-induced dyskinesia, thus a target for therapy development.

Tumor Necrosis Factor-α Promotes the Tumorigenesis, Lymphangiogenesis, and Lymphatic Metastasis in Cervical Cancer via Activating VEGFC-Mediated AKT and ERK Pathways.

Lymphatic metastasis is a common phenomenon of cervical cancer. Tumor necrosis factor-α (TNF-α) was found to be closely associated with lymphatic cancer metastasis. However, the mechanism through which TNF-α regulates lymphatic metastasis in cervical cancer remains unclear. In this study, cervical cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with or without TNF-α for 48 h, and then the corresponding conditional medium (CM-TNF-α or CM) was collected. The level of vascular endothelial growth factor (VEGFC) in the corresponding CM was then detected using an enzyme-linked immunosorbent assay (ELISA). Next, human lymphatic endothelial cells (HLECs) were cultured in CM-TNF-α or CM for 48 h. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and angiogenesis was detected using a tube formation assay. Subsequently, the expressions of AKT, p-AKT, ERK, and p-ERK in HLECs were detected using western blotting. In addition, to further investigate the effect of TNF-α on the progression of cervical cancer, a C33A subcutaneous xenograft model was established in vivo. We found that TNF-α significantly stimulated cervical cancer cells to secrete VEGFC. Additionally, the CM collected from the TNF-α-treated cervical cancer cells notably promoted the proliferation, migration, and angiogenesis of HLECs; however, these changes were reversed by MAZ51, a VEGFR3 inhibitor. Moreover, TNF-α obviously elevated D2-40 and VEGFC protein expressions in tumor tissues, promoting lymphangiogenesis and lymphatic metastasis in vivo. Meanwhile, TNF-α markedly upregulated p-AKT and p-ERK expressions in tumor tissues, whereas these changes were reversed by MAZ51. Collectively, TNF-α could promote tumorigenesis, lymphangiogenesis, and lymphatic metastasis in vitro and in vivo in cervical cancer via activating VEGFC-mediated AKT and ERK pathways. These results may provide new directions for the treatment of cervical cancer.

American ginseng with different processing methods ameliorate immunosuppression induced by cyclophosphamide in mice via the MAPK signaling pathways.

This study aimed to clarify the effects of two processed forms of American ginseng (Panax quinquefolius L.) on immunosuppression caused by cyclophosphamide (CTX) in mice. In the CTX-induced immunosuppressive model, mice were given either steamed American ginseng (American ginseng red, AGR) or raw American ginseng (American ginseng soft branch, AGS) by intragastric administration. Serum and spleen tissues were collected, and the pathological changes in mice spleens were observed by conventional HE staining. The expression levels of cytokines were detected by ELISA, and the apoptosis of splenic cells was determined by western blotting. The results showed that AGR and AGS could relieve CTX-induced immunosuppression through the enhanced immune organ index, improved cell-mediated immune response, increased serum levels of cytokines (TNF-α, IFN-γ, and IL-2) and immunoglobulins (IgG, IgA, and IgM), as well as macrophage activities including carbon clearance and phagocytic index. AGR and AGS downregulated the expression of BAX and elevated the expression of Bcl-2, p-P38, p-JNK, and p-ERK in the spleens of CTX-injected animals. Compared to AGS, AGR significantly improved the number of CD4+CD8-T lymphocytes, the spleen index, and serum levels of IgA, IgG, TNF-α, and IFN-γ. The expression of the ERK/MAPK pathway was markedly increased. These findings support the hypothesis that AGR and AGS are effective immunomodulatory agents capable of preventing immune system hypofunction. Future research may investigate the exact mechanism to rule out any unforeseen effects of AGR and AGS.

Creation of myofascial pain syndrome-like muscle by artificial electrical stimulation and stretching treatment.

BackgroundMyofascial pain syndrome（MPS）is a common chronic pain disorder characterized by muscle hardness, low extensibility, restriction of range of motion (ROM) and pain with trigger point (TP). Eccentric contraction has been used in past animal studies of MPS. However, clinical experience suggests that concentric contraction is also involved in MPS formation. New methodsIn this study, we adopted artificial electrical stimulation to create artificial concentric contraction (ACC) in rat gastrocnemius muscle. After ACC, muscle hardness, torsion and range of motion (ROM) were compared between before ACC, after ACC, and stretching group. To clarify the association with pain, the expression of pERK in DRG were analyzed. Comparison to existing methodsPrevious animal studies have created MPS models by inducing eccentric contractions in muscles. In this study, full tetanus contraction of the gastrocnemius muscle was achieved via tibial nerve stimulation. This method substituted muscle contraction due to abnormal excitation. ResultsWe found that artificial abnormal contraction (ACC) induced muscle hardness and ROM restriction. The pERK expression in DRG was increased by ACC. Analysis of muscle tissue sections revealed a meandeling structure in muscle fibers. The stretching treatment improved these indicators. These results were similar to feature of the MPS muscles. ConclusionsThe ACC caused by artificial electrical stimulation leads to the characteristic of MPS in rat gastrocnemius muscle. This ACC model can be one of the useful options for MPS analysis.

ENPP2 alleviates hypoxia/reoxygenation injury and ferroptosis by regulating oxidative stress and mitochondrial function in human cardiac microvascular endothelial cells

This study aimed to elucidate the molecular mechanisms of hypoxia/reoxygenation (H/R) injury in human cardiac microvascular endothelial cells (HCMECs) by regulating ferroptosis. H/R model was established with HCMECs and before the reperfusion, ferroptosis inhibitor ferrostatin-1 or ferroptosis inducer erastin was all administered. Wound-healing assay was performed to detect the migration ability of cells in each group, and the angiogenesis ability was determined by tube formation assay. The level of reactive oxygen species (ROS) was detected by flow cytometry. Transmission electron microscopy (TEM) was used to observe the state of mitochondria. The expressions of related proteins in HCMECs were assessed by Western blot. From the results, H/R injury could inhibit the migration and angiogenesis, induce the ROS production, and cause the mitochondrial damage of HCMECs. Ferroptosis activator erastin could aggravate H/R injury in HCMECs, while the ferroptosis inhibitor ferrostatin-1 could reverse the effects of H/R on HCMECs. Western blot results showed that H/R or/and erastin treatment could significantly induce ACSL4, HGF, VEGF, p-ERK, and uPA protein expression and inhibit GPX4 expression. The addition of ferrostatin-1 resulted in the opposite trend of the proteins expression above to erastin treatment. What is more, overexpression of ENPP2 markedly suppressed the damaging effect of H/R on HCMECs and reversed the effects of H/R or erastin treatment on the expression of related proteins. These results demonstrated a great therapeutic efficacy of ENPP2 overexpression in preventing the development of H/R injury through inhibiting oxidative stress and ferroptosis.

Neuroprotective Effect of Mangiferin against Parkinson's Disease through G-Protein-Coupled Receptor-Interacting Protein 1 (GIT1)-Mediated Antioxidant Defense.

Parkinson's disease (PD), known as a neurodegenerative disease, is characterized by movement disorders, with increasing age being the predominant risk factor for its development. Mangiferin, a bioactive compound isolated from mango, shows potent neuroprotection. In our work, we investigated the neuroprotection and mechanisms of mangiferin against PD. We established PD models by treating SH-SY5Y cells with rotenone and mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and investigated the therapeutic effects of mangiferin. Our results showed that mangiferin exhibited a cell-protective effect. Mangiferin also improved the motor behavior and attenuated the activation of microglia and astrocytes in MPTP mice. In addition, mangiferin decreased reactive oxygen species (ROS) levels and increased glutathione (GSH) and superoxide dismutase (SOD). Mangiferin also markedly activated GIT1, p-ERK, Nrf2, HO-1, and SOD expression and inhibited Keap1 expression in vitro and in vivo. To further investigate the role of GIT1, GIT1 siRNA was used. In the presence of GIT1 siRNA, the neuroprotection of mangiferin in PD was weakened. Our results indicate that mangiferin exhibited its therapeutic effect against PD by regulating GIT1 and its downstream Keap1/Nrf2 pathways. Our studies exhibited that mangiferin showed neuroprotection in PD, and its main target was GIT1. What is more, mangiferin could reduce the oxidative stress of PD by targeting GIT1 and its downstream Keap1/Nrf2 pathways. These indicated that mangiferin is a good candidate for PD therapy. However, the role of p-ERK in mangiferin-treated PD requires further investigation.

Protective effect of Cordyceps sinensis against diabetic kidney disease through promoting proliferation and inhibiting apoptosis of renal proximal tubular cells

BackgroundDiabetic kidney disease (DKD) has mainly been considered as a glomerular disease. Our previous study showed that the progression of DKD was highly correlated with the dysfunction of renal proximal tubular cells. Fermented Cordyceps sinensis (CS), a substitute for natural CS, is a prominent herb widely used in China, and has exhibited excellent efficacy on DKD. However, the underlying mechanisms remain poorly understood.MethodsThe database analysis was used to identify the main therapeutic targets and pathways of CS involved in DKD treatment. Next, the protective effects of fermented CS on high glucose (HG, 30 mM) induced HK-2 cell injury was validated through cell proliferation and apoptosis assay, including CCK-8, EdU and TUNEL. Finally, quantitative real‑time PCR (qRT-PCR) and western blotting were used to verify key target genes.ResultsOur results revealed that 9 main targets (RELA, JNK1, PTEN, VEGFA, EGF, ERK2, CASP3, AKT1, MMP9) were recognized as key therapeutic targets with excellent binding affinity screened by database analysis and molecular docking. The biological processes were identified by Gene Ontology (GO) enrichment, which appeared mainly involved in the positive regulation of cell proliferation as well as the negative regulation of apoptosis. The verification experiments in vitro revealed that fermented CS significantly attenuated the HG-induced cytotoxicity and apoptosis, and promoted the proliferation of HK-2 cells. Moreover, fermented CS significantly downregulated the expressions of Bax, Caspase-3, VEGFA, P-AKT and P-ERK, and upregulated the expression of PTEN compared with that of HG group.ConclusionOur results demonstrate that the fermented CS has nephroprotective effects significantly, which functions via promoting proliferation and inhibiting apoptosis of renal proximal tubular cells, likely by targeting Caspase-3, Bax, VEGFA and PTEN. Furthermore, AKT and ERK signaling pathway may be the critical mechanisms underlying the efficacy of fermented CS in DKD treatment.

Ginsenoside Rg1 treatment alleviates renal fibrosis by inhibiting the NOX4–MAPK pathway in T2DM mice

Diabetic kidney disease (DKD) is a severe complication of type 2 diabetes mellitus (T2DM). However, the pathogenesis of DKD remains unclear, and effective treatment strategies are still lacking. Ginsenoside Rg1 (Rg1) has been reported to improve DKD, but the mechanism is unclear. NADPH oxidase 4 (NOX4) is an essential reactive oxygen species (ROS) source in the kidney. The mitogen-activated protein kinase (MAPK) signaling may exacerbate renal fibrosis. Therefore, we hypothesized that Rg1 might alleviate renal injury and fibrosis by inhibiting NOX4 and MAPK signaling in T2DM-induced DKD. We found that Rg1 significantly improves lipid deposition, fibrosis, and ROS production and reduces NOX4, p22phox, p47phox, p-ERK, p-JNK, and p-P38 MAPK expressions in the T2DM mice kidneys. We also found that the high-fat diet treatment in mice and the palmitate (PA) and PA + HG (high glucose) exposure in human mesangial cells could significantly induce lipid deposition, ROS production, fibrosis, and the activation of NOX4–MAPK signaling. The results suggest that high lipid and glucose may play a significant role in DKD progression, while Rg1 may attenuate renal fibrosis by inhibiting NOX4–MAPK signaling.

Abstract 1725: Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma

Abstract Approximately 95% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, accounting for over 45,000 deaths per year in the US. In KRAS-mutant cells, KRAS protein exists predominantly in an active, GTP-bound state, leading to excessive oncogenic signaling via RAF/MEK/ERK and other effector pathways. After decades of anticipation, the first therapeutic inhibitors of this oncoprotein target a specific mutant allele (KRASG12C) and have shown promising results in this rare subset of human PDAC patients (1-2% of cases). While these encouraging clinical results bolster support for targeting RAS in human PDAC, most patients harbor KRASG12D and KRASG12V mutations and remain unserved by targeted therapies. Here we report preclinical studies of RM-042, a first-in-class, potent, oral, small molecule inhibitor of GTP-bound RAS proteins, designed to treat cancers driven by a variety of RAS mutations. RMC-6236, a clinical candidate and RM-042, a tool compound, both act through non-covalent binding to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages GTP-bound RAS to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors.We examined the anti-tumor activity of RM-042 in two complementary preclinical model systems of PDAC. Using primary human PDAC explant cultures, we found that RM-042 potently inhibits phospho-ERK expression (a marker of KRAS/RAF/MEK pathway activity), in a concentration-dependent manner and was associated with reduced viability and altered stromal content. Pilot short-term studies in the K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) mouse model of PDAC found that RM-042 induced initial tumor stabilizations and regressions associated with loss of pERK expression, in association with increased apoptosis and reduced proliferation, at tolerated doses. However, tumors collected at endpoints showed re-established pERK expression, suggesting activation of resistance/adaptation mechanisms. Ongoing studies will determine pharmacokinetic properties of RM-042 in the autochthonous PDAC mouse model, as well as the duration of tumor responses and evaluate a potential survival benefit provided by RM-042. Regulatory network analysis of single cell RNA sequencing performed on these tumors at multiple timepoints will provide a mechanistic understanding of the regulatory proteins that drive responses in each cell type in the tumors. Through this approach, we will assess the regulatory adaptations that occur as tumors initially respond and as they escape. Together, these studies provide complementary evidence from in vivo murine and ex vivo human models for promising anti-tumor activity in response to GTP-RAS inhibition, supporting the inclusion of PDAC patients in ongoing and future clinical trials of RMC-6236. Citation Format: Urszula N. Wasko, Stephen A. Sastra, Carmine F. Palermo, David Wildes, Mallika Singh, Kenneth P. Olive. Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1725.

Abstract 3648: MSU-42011, alone and in combination with selumetinib, reduces pERK levels in NF1 cancer cells and decreases CCL2 expression in THP-1 macrophages

Abstract Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes approximately 50% of affected individuals to develop plexiform neurofibromas (PNFs), which can progress to highly aggressive malignant peripheral nerve sheath tumors (MPNSTs) in approximately 10% of patients. NF1 is caused by mutations in the tumor suppressor gene NF1, which encodes for neurofibromin, a negative regulator of RAS activity. Selumetinib, a specific inhibitor of MEK1/2, is the only FDA-approved drug for NF1-associated PNFs. However, the anti-tumor effects of selumetinib are limited in MPNSTs and have dose-limiting side effects. Deficiency of the NF1 gene not only promotes tumorigenesis but also has broad effects on the immune cells and cytokine signaling driven by hyperactive RAS signaling. Because macrophages account for almost half of cells in NF1 lesions and their infiltration highly correlates with disease progression, we hypothesized that targeting tumor-promoting immune cells could be an alternative approach for NF1 treatment. The novel retinoid X receptor (RXR) agonist MSU-42011 reduces tumor growth in experimental Kras-driven cancers by decreasing pERK expression, reducing tumor-promoting immune cells like CD206+ macrophages and regulatory T cells, and increasing activated cytotoxic T cells. Here, we tested MSU-42011 and selumetinib, either alone or in combination, to evaluate their efficacy against NF1 cancer cells and macrophages using monoculture and conditioned media (CM) treatments. Treatment with 200 nM MSU-40211, 50 nM selumetinib, and the combination for 3 hours reduced pERK protein levels by approximately 40%, 70% and 90%, respectively vs. untreated controls. Treatment for 72 hours with selumetinib and the combination reduced the viability of PNF cells in a dose-dependent manner. Moreover, CM from human PNF cells increased monocyte chemoattractant CCL2 (C-C motif chemokine ligand 2) mRNA expression in human THP1 monocytes and THP1 macrophages differentiated by PMA. Notably, MSU-42011 and selumetinib alone similarly inhibited CCL2 mRNA expression by 25% in THP1 macrophages stimulated with CM from PNF cells, and the inhibition of CCL2 mRNA expression was enhanced to 50% with combination treatment. Taken together, our data suggest that MSU-42011 should be tested in relevant preclinical models of NF1. Citation Format: Pei-Yu Hung, Lizbeth Lockwood, Karen T. Liby. MSU-42011, alone and in combination with selumetinib, reduces pERK levels in NF1 cancer cells and decreases CCL2 expression in THP-1 macrophages. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3648.

Role of Raf-1/ERK Signaling Pathway in Estradiol and Propranolol in the Intervention of Xenograft Hemangioma In Vivo

The pathogenesis and the mechanism of orally administered propranolol in the treatment of hemangioma are unclear. In this study, we evaluated the changes of xenograft hemangioma in nude mice after intervention with estradiol and propranolol. Raf-1 and p-ERK expression in xenograft hemangiomas was assessed to evaluate their role in hemangioma proliferation and regression after treatment. A hemangioma xenograft model in nude mice was established. The successful xenograft specimens were selected and then randomized into control group, estradiol group and propranolol group. At the date of injection, and on day 7 and 21 after injection, the morphological changes of xenograft hemangiomas were visually characterized and imaged by light microscopy. The distribution and expression Raf-1 and p-ERK protein was determined by immunohistochemical detection. In control group, the xenografts increased gradually in volume, had a soft texture and their colors gradually turned red with observation of proliferation of endothelial cells and a capillary lumen that contained monolayer endothelial cells. In Estradiol group, the xenografts grew fast and increased significantly in volume, had a soft texture and their colors were dark red with a hyperplasia of endothelial cells, irregular volume, and deranged and compact endothelial cells. More capillary lumens and sinuses were also seen. Raf-1 and p-ERK expression in estradiol group was significantly increased (P < 0.05). In Propranolol group, the xenografts volume decreased, had a soft texture, and their colors turned gradually white with decreased number of proliferative endothelial cells. The vascular lumens, composed of endothelial cells, were larger, and some of them disappeared and were replaced by fibrous connective tissue and vascular adipose tissue. Raf-1 and p-ERK expression in propranolol group was lower than estradiol and control group (P < 0.05). In conclusion, Raf-1/ERK signaling pathway may be involved in hemangioma. Estrogen and propranolol may regulate the proliferation or regression of hemangioma through Raf-1/ERK signaling pathway.

Mechanism of Triptolide Liposome Nanoparticles on Apoptosis of Multiple Myeloma Cells Induced by Bortezomib

The aim of this research was to investigated the effects of triptolide (TP) and its liposome nanoparticles (TP-LNP) on the apoptosis of multiple myeloma (MM) cells induced by Bortezomi. The encapsulation rate (ER), drug loading (DL), particle size (PS), and Zeta potential characteristics of the TP-LNP were evaluated. MMT assay was employed to detect the inhibitory effect of TP single agent and TP-LNP on proliferation of MM cells. MM cells were enrolled into a control group (Ctrl group, no intervention), a Bortezomi group (75 nM Bortezomi intervention 12 h), a Bortezomi +TP group (Bortezomi combined with TP monotherapy), and a Bortezomi+TP-LNP group (Bortezomi combined with TP-LNP). CCK-8, flow cytometry, and Western blot were utilized to detect the cell survival rate (SR), apoptosis, and MAPK/ERK pathway related protein, respectively. The results revealed that the shape of TP-LNP was similar to circular and uniformly distributed. Its average PS was (181.2±6.3) nm, the average Zeta potential was −29.15 mV, the average ER was 82.1%, and the average DL was 1.09%. When the drug concentration was 100 nmol/L, the inhibition rates of cell proliferation of TP and TP-LNP were (66.8±0.5)% and (81.4±0.6)%, respectively; and their median inhibitory concentrations (MICs) after 72 h were (89.5±1.8) nmol/L and (55.3±2.2) nmol/L, respectively. Based on the Ctrl group, the SRs in the Bortezomi, Bortezomi+TP, and Bortezomi+TP-LNP groups were decreased obviously, while the apoptosis rates were increased, and the comparisons herein exhibited great differences with P <0.05. The SR in the Bortezomi+TP-LNP group was the lowest and the apoptosis rate was the highest, showing great differences among the three groups (P < 0.05). Based on the Ctrl group, the expressions of P-P38 and P-ErK in the Bortezomi, Bortezomi+TP, and Bortezomi+TP-LNP groups were decreased (P <0.05). The levels of P-P38 and P-Erk in the Bortezomi+TP-LNP group were the lowest, and the differences in those in this group and the Bortezomi and the Bortezomi+TP groups statistically great (P <0.05). TP-LNP could inhibit the activation of MAPK/ERK pathway, enhance the apoptosis of MM cells induced by Bortezomi, and inhibit the cell proliferation.

Vernodalin Suppresses Tumor Proliferation and Increases Apoptosis of Gastric Cancer Cells Through Attenuation of FAK/PI3K/AKT/mTOR and MAPKs Signaling Pathways.

Gastric cancer (GC) is the most aggressive malignant tumor with limited treatment alternatives post metastasis. Vernodalin (VN) induced apoptosis has been reported in various types of human cancer cells. However, the precise molecular mechanisms underlying the anti-metastasis action of VN on GC cells are yet to be elucidated. In this study, we investigated the anti-metastatic and apoptotic effects of VN on SGC- 7901 and AGS cells, with a purpose of gaining a deeper understanding of the anti-metastatic mechanisms of VN on gastric carcinoma. To attenuate the activation of PI3K/AKT/mTOR and mitogen-activated protein kinase (MAPK) signaling pathways by VN in GC cells. We employed VN and gastric cancer cells in experiments such as MTT assay, apoptosis, MMP, DAPI, Rh-123, cell adhesion assay, and western blot analysis on GC SGC-7901 and AGS cells. Our results revealed that VN inhibits cell proliferation, adhesion, and metastasis and induces apoptosis of both GC cells. VN potentially reduced the protein expressions of MMP-2, MMP-9, and uPA, whereas intensified expressions of TIMP-1 and TIMP-2. Also, VN attenuates the expression of FAK, p-PI3K, p-AKT, p-mTOR, p-JNK, p-p38MAPK, and p-ERK. Thus, it is inferred that VN treatment reduced the activities of MMP-2 and MMP-9 via the FAK/PI3K/AKT/ mTOR, and MAPKs signaling pathways. Our results confirm that VN prevented GC growth, invasion and metastasis and induce apoptosis in GC cells. Our findings suggest that VN is a potential natural therapeutic compound as a new remedy for GC chemotherapy treatment.

Eriocitrin suppresses proliferation and migration of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPK pathway

To investigate the role of the ROS/MAPK signaling axis in mediating the inhibitory effect of eriocitrin on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells. SMMC-7721 cells were treated with different concentrations of eriocitrin for 24 h, and the changes in cell viability were detected with CCK-8 assay. The migration and invasion abilities of the treated cells were evaluated using Transwell and scratch healing assays, the cell proliferation was assessed with colony-forming assay, and changes in nuclear morphology were observed with DAPI staining. Western blotting was performed to examine the changes in the expressions of E-cadherin, N-cadherin, MMP-2, MMP-9, PARP, Pro-caspase 3, pJNK, p-P38, and p-ERK. The effect of eriocitrin on PARP cleavage in SMMC-7721 cells pretreated with ERK, JNK and P38 inhibitors (U0126, SB203580 and SP600125, respectively) was detected using Western blotting. The effect of treatment with Nacetyl-cysteine (NAC, 30 μmol/L) and eriocitrin (100, 200, and 300 μg/mL), alone or in combination, on reactive oxygen species (ROS) levels in the cells was examined using a DCFH-DA fluorescent probe. Eriocitrin below 50 μg/mL did not produce significant effect on the viability of SMMC-7721 cells (P>0.05). Treatment with eriocitrin significantly inhibited scratch healing, migration, and colony formation of the cells (P < 0.01), reduced the protein expressions of N-cadherin, MMP-2, and MMP-9 (P < 0.01), and up-regulated E-cadherin protein expression (P < 0.05). Eriocitrin-treated SMMC-7721 cells showed obvious apoptotic morphologies with decreased Procaspase 3 expression and increased PARP cleavage (P < 0.01) and phosphorylation levels of JNK, P38, and ERK (P < 0.01); Eriocitrin-induced PAPR cleavage was obviously enhanced by U0126 and SB203580 but attenuated by SP600125. Treatment with 300 μg/mL eriocitrin for 30 min significantly increased ROS level in the cells, and this effect was obviously suppressed by NAC. Eriocitrin can suppress the proliferation and migration and promote apoptosis of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPKs signaling pathway.

Tongmai Yangxin pill alleviates myocardial no-reflow by activating GPER to regulate HIF-1α signaling and downstream potassium channels

Context The Tongmai Yangxin pill (TMYX) has potential clinical effects on no-reflow (NR); however, the effective substances and mechanisms remain unclear. Objective This study evaluates the cardioprotective effects and molecular mechanisms of TMYX against NR. Materials and methods We used a myocardial NR rat model to confirm the effect and mechanism of action of TMYX in alleviating NR. Sprague-Dawley (SD) rats were divided into Control (Con), sham, NR, TMYX (4.0 g/kg), and sodium nitroprusside (SNP, 5.0 mg/kg), and received their treatments once a day for one week. In vitro studies in isolated coronary microvasculature of NR rats and in silico network pharmacology analyses were performed to reveal the underlying mechanisms of TMYX and determine the main components, targets, and pathways of TMYX, respectively. Results TMYX (4.0 g/kg) showed therapeutic effects on NR by improving the cardiac structure and function, reducing NR, ischemic areas, and cardiomyocyte injury, and decreasing the expression of cardiac troponin I (cTnI). Moreover, the mechanism of TMYX predicted by network pharmacology is related to the HIF-1, NF-κB, and TNF signaling pathways. In vivo, TMYX decreased the expression of MPO, NF-κB, and TNF-α and increased the expression of GPER, p-ERK, and HIF-1α. In vitro, TMYX enhanced the diastolic function of coronary microvascular cells; however, this effect was inhibited by G-15, H-89, L-NAME, ODQ and four K+ channel inhibitors. Conclusions TMYX exerts its pharmacological effects in the treatment of NR via multiple targets. However, the contribution of each pathway was not detected, and the mechanisms should be further investigated.

Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Acid-sensing ion channel 3 is required for agmatine-induced histamine-independent itch in mice.

Itch is a common symptom of many skin and systemic diseases. Identifying novel endogenous itch mediators and the downstream signaling pathways involved will contribute to the development of new strategies for the treatment of chronic itch. In the present study, we adopted behavioral testing, patch clamp recording and metabonomics analysis to investigate the role of agmatine in itch and the underlying mechanism. Behavioral analysis was used to evaluate the establishing of acute and chronic itch mice model, and to test the effects of different drugs or agents on mice itch behavior. Western blotting analysis was used to test the effect of agmatine on phosphorylation of ERK (p-ERK) expression in the spinal cord. Patch clamp recording was used to determine the effect agmatine on the excitability of DRG neurons and the role of ASIC3. Finally, the metabonomics analysis was performed to detect the concentration of agmatine in the affected skin under atopic dermatitis or psoriasis conditions. We fused a mouse model and found that an intradermal injection of agmatine (an endogenous polyamine) into the nape of the neck or cheek induced histamine-independent scratching behavior in a dose-dependent manner. In addition, the ablation of nociceptive C-fibers by resiniferatoxin (RTX) abolished agmatine-induced scratching behavior. However, agmatine-induced itch was not affected by the pharmacological inhibition of either transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1); similar results were obtained from TRPV1-/- or TRPA1-/- mice. Furthermore, agmatine-induced itch was significantly suppressed by the administration of acid-sensing ion channel 3 (ASIC3) inhibitors, APETx2 or amiloride. Agmatine also induced the upregulation of p-ERK in the spinal cord; this effect was inhibited by amiloride. Current clamp recording showed that the acute perfusion of agmatine reduced the rheobase and increased the number of evoked action potentials in acute dissociated dorsal root ganglion (DRG) neurons while amiloride reversed agmatine-induced neuronal hyperexcitability. Finally, we identified significantly higher levels of agmatine in the affected skin of a mouse model of atopic dermatitis (AD) when compared to controls, and the scratching behavior of AD mice was significantly attenuated by blocking ASIC3. Collectively, these results provide evidence that agmatine is a novel mediator of itch and induces itch via the activation of ASIC3. Targeting neuronal ASIC3 signaling may represent a novel strategy for the treatment of itch.

P38 mediates T-2 toxin-induced Leydig cell testosterone synthesis disorder

T-2 toxin is an unavoidable food and feed contaminant that seriously threatens human and animal health. Exposure to T-2 toxin can cause testosterone synthesis disorder in male animals, but the molecular mechanism is still not completely clear. The MAPK pathway participates in the regulation of testosterone synthesis by Leydig cells, but it is unclear whether the MAPK pathway participates in T-2 toxin-induced testosterone synthesis disorders. In this research, testosterone synthesis capacity, testosterone synthase expression and MAPK pathway activation were examined in male mice and TM3 cells exposed to T-2 toxin. The results showed that T-2 toxin exposure decreased testicular volume and caused pathological changes in the microstructure and ultrastructure of testicular Leydig cells. T-2 toxin exposure also decreased testicular testosterone content and the protein expression of testosterone synthase. In vitro, T-2 toxin inhibited cell viability and decreased the expression of testosterone synthase in TM3 cells, and it decreased the testosterone contents in cell culture supernatants. Moreover, T-2 toxin activated the MAPK pathway by increasing the expression of p38, JNK and ERK as well as the expression of p-p38, p-JNK and p-ERK in testis and TM3 cells. The p38 molecular inhibitor (SB203580) significantly alleviated the T-2 toxin-induced decrease in testosterone synthase expression in TM3 cells and the T-2 toxin-induced reduction in testosterone content in TM3 cell culture supernatants. In summary, p38 mediates T-2 toxin-induced Leydig cell testosterone synthesis disorder.

GABAB receptor activation alters astrocyte phenotype changes induced by trimethyltin via ERK signaling in the dentate gyrus of mice

AimsWe examined the effect of γ-aminobutyric acid (GABA)B receptor activation on astrocyte phenotype changes induced by trimethyltin (TMT) in the dentate gyrus of mice. Main methodsMale C57BL/6N mice received TMT (2.6 mg/kg, i.p.), and the expression of GABAB receptors was evaluated in the hippocampus. The GABAB receptor agonist baclofen (2.5, 5, or 10 mg/kg, i.p. × 5 at 12-h intervals) was administered 3–5 days after TMT treatment, and the expression of Iba-1, GFAP, and astrocyte phenotype markers was evaluated 6 days after TMT. SL327 (30 mg/kg, i.p.), an extracellular signal-related kinase (ERK) inhibitor, was administered 1 h after each baclofen treatment. Key findingsTMT insult significantly induced the astroglial expression of GABAB receptors in the dentate molecular layer. Baclofen significantly promoted the expression of S100A10, EMP1, and CD109, but not that of C3, GGTA1, and MX1 induced by TMT. In addition, baclofen significantly increased the TMT-induced expression of p-ERK in the dentate molecular layer. Interestingly, p-ERK was more colocalized with S100A10 than with C3 after TMT insult, and a significant positive correlation was found between the expression of p-ERK and S100A10. Consistently, SL327 reversed the effect of baclofen on astrocyte phenotype changes. Baclofen also enhanced the TMT-induced astroglial expression of glial cell-derived neurotrophic factor (GDNF), an anti-inflammatory astrocytes-to-microglia mediator, and consequently attenuated Iba-1 expression and delayed apoptotic neuronal death. SignificanceOur results suggest that GABAB receptor activation increases S100A10-positive anti-inflammatory astrocytes and astroglial GDNF expression via ERK signaling after TMT excitotoxicity in the dentate molecular layer of mice.

Moringa oleifera leaf attenuate osteoporosis in ovariectomized rats by modulating gut microbiota composition and MAPK signaling pathway

Moringa oleifera leaf (MLP) contains abundant complex nutrients with anti-osteoporosis potential. However, its efficacy and mechanisms against osteoporosis remain unknown. The purpose of this research is to investigate MLP's anti-osteoporotic effects and mechanisms. Animal experiments were used in this work to validate MLP's anti-osteoporotic efficacy. We investigated the mode of action of MLP, analyzed its impact on the gut microbiota, and predicted and validated its anti-osteoporosis-related molecular targets and pathways through network pharmacology, molecular docking, and western blotting. In an ovariectomized osteoporosis rat model, MLP significantly increased bone mineral density and improved bone metabolism-related indicators, bone microstructure, and lipid profile. Moreover, it improved gut microbiota composition and increased the expression of Occludin and Claudin-1 protein in the duodenum. Network pharmacology identified a total of 97 active ingredients and 478 core anti-osteoporosis targets. Of these, MAPK1 (also known as ERK2), MAPK3 (also known as ERK1), and MAPK8 (also known as JNK) were successfully docked with the active constituents of MLP. Interestingly, MLP increased ERK and VAV3 protein expression and decreased p-ERK and JNK protein expression in the femur. These findings confirm MLP's anti-osteoporotic efficacy, which could be mediated via regulation of gut microbiota and MAPK signaling.