[Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells].

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Therapeutic mechanism of Liangxue-Guyuan-Yishen decoction on intestinal stem cells and tight junction proteins in gastrointestinal acute radiation syndrome rats.

On the basis of the previous research, the Traditional Chinese Medicine theory was used to improve the drug composition for gastrointestinal acute radiation syndrome (GI-ARS). The purpose of this study was to study the therapeutic mechanism of Liangxue-Guyuan-Yishen decoction (LGYD) on GI-ARS and to provide a new scheme for the treatment of radiation injury. Here, we investigated the effects of LGYD on intestinal stem cells (ISCs) in a GI-ARS rat model. Rat health and survival and the protective efficacy of LGYD on the intestines were analyzed. The active principles in LGYD were detected using liquid chromatography-mass spectrometry (LC-MS). ISC proliferation, intestinal epithelial tight junction (TJ) protein expression and regulatory pathways were explored using immunohistochemistry, western blotting (WB) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Involvement of the WNT and MEK/ERK pathways in intestinal recovery was screened using network pharmacology analysis and validated by WB and RT-qPCR. LGYD administration significantly improved health and survival in GI-ARS rats. Pathological analysis showed that LGYD ameliorated radiation-induced intestinal injury and significantly promoted LGR5+ stem cell regeneration in the intestinal crypts, upregulated TJ protein, and accelerated crypt reconstruction in the irradiated rats. LC-MS revealed ≥13 constituents that might contribute to LGYD's protective effects. Collectively, LGYD can promote crypt cell proliferation and ISCs after radiation damage, the above effect may be related to WNT and MEK/ERK pathway.

Combination of the LARS1 Inhibitor, BC-LI-0186 with a MEK1/2 Inhibitor Enhances the Anti-Tumor Effect in Non-Small Cell Lung Cancer.

PURPOSE: The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and proliferation by growth factor coordination and amino acid availability. Leucyl-tRNA synthetase 1 (LARS1) senses the intracellular leucine concentration and mediates amino acid-induced activation of mTORC1. Thus, LARS1 inhibition could be useful in cancer treatment. However, the fact that mTORC1 can be stimulated by various growth factors and amino acids suggests that LARS1 inhibition alone has limitations in inhibiting cell growth and proliferation. We investigated the combined effects of BC-LI-0186, a LARS1 inhibitor, and trametinib, an MEK inhibitor, on non-small cell lung cancer (NSCLC). Materials and Methods: Protein expression and phosphorylation were observed by immunoblotting, and genes differentially expressed between BC-LI-0186-sensitive and -resistant cells were identified by RNA sequencing. The combined effect of the two drugs was inferred from the combination index values and a xenograft model. RESULTS: LARS1 expression was positively correlated with mTORC1 in NSCLC cell lines. BC-LI-0186 treatment of A549 and H460 cells maintained in media supplemented with fetal bovine serum revealed paradoxical phosphorylation of S6 and activation of mitogen- activated protein kinase (MAPK) signaling. Compared with BC-LI-0186-sensitive cells, -resistant cells showed enrichment of the MAPK gene set. The combination of trametinib and BC-LI-0186 inhibited the phosphorylation of S6, MEK, and extracellular signal-regulated kinase and their synergistic effects were confirmed in a mouse xenograft model. CONCLUSION: The combination of BC-LI-0186 and trametinib inhibited the non-canonical mTORC1-activating function of LARS1. Our study demonstrated a new therapeutic approach for NSCLC without targetable driver mutations.

Valtrate, an iridoid compound in Valeriana, elicits anti-glioblastoma activity through inhibition of the PDGFRA/MEK/ERK signaling pathway.

BACKGROUND: Valtrate, a natural compound isolated from the root of Valeriana, exhibits antitumor activity in many cancers through different mechanisms. However, its efficacy for the treatment of glioblastoma (GBM), a tumor type with a poor prognosis, has not yet been rigorously investigated. METHODS: GBM cell lines were treated with valtrate and CCK-8, colony formation and EdU assays, flow cytometry, and transwell, 3D tumor spheroid invasion and GBM-brain organoid co-culture invasion assays were performed to assess properties of proliferation, viability, apoptosis and invasion/migration. RNA sequencing analysis on valtrate-treated cells was performed to identify putative target genes underlying the antitumor activity of the drug in GBM cells. Western blot analysis, immunofluorescence and immunohistochemistry were performed to evaluate protein levels in valtrate-treated cell lines and in samples obtained from orthotopic xenografts. A specific activator of extracellular signal-regulated kinase (ERK) was used to identify the pathways mediating the effect. RESULTS: Valtrate significantly inhibited the proliferation of GBM cells in vitro by inducing mitochondrial apoptosis and suppressed invasion and migration of GBM cells by inhibiting levels of proteins associated with epithelial mesenchymal transition (EMT). RNA sequencing analysis of valtrate-treated GBM cells revealed platelet-derived growth factor receptor A (PDGFRA) as a potential target downregulated by the drug. Analysis of PDGFRA protein and downstream mediators demonstrated that valtrate inhibited PDGFRA/MEK/ERK signaling. Finally, treatment of tumor-bearing nude mice with valtrate led to decreased tumor volume (fivefold difference at day 28) and enhanced survival (day 27 vs day 36, control vs valtrate-treated) relative to controls. CONCLUSIONS: Taken together, our study demonstrated that the natural product valtrate elicits antitumor activity in GBM cells through targeting PDGFRA and thus provides a candidate therapeutic compound for the treatment of GBM.

Polyphyllin I improves myocardial damage in coronary artery disease via modulating lipid metabolism and myocardial apoptosis.

Polyphyllin I (PPI) is a famous traditional medicine ingredient, which has been explored in wide range of areas. Nevertheless, whether PPI exerts any functions in coronary artery disease (CAD) is still uncertified. Herein, we probed the effect and mechanism of PPI on lipid metabolism and myocardial dysfunction in myocardial cells and CAD rat model. Hypoxia/reoxygenation (H/R)-treated H9c2 cells model was constructed for the in vitro experiments, and CAD model in vivo was established by high-fat feeding. After management with PPI, the correlated factors of lipid metabolism and myocardial function were investigated. The apoptosis of myocardial cells was assessed by Annexin V-FITC/PI kit and TUNEL staining. The apoptosis-associated factors (caspase 3, cleaved caspase 3, Bax, and Bcl-2) were tested by Western blot analysis. The MEK/ERK inhibitor was applied and the functions of MEK/ERK pathway in myocardial damage were investigated. H/R-treated H9c2 cells model was constructed for the in vitro experiments, and CAD model in vivo was established by high-fat feeding. After management with PPI, the correlated factors of lipid metabolism and myocardial function were investigated. The apoptosis of myocardial cells was assessed by Annexin V-FITC/PI kit and TUNEL staining. The apoptosis-associated factors (caspase 3, cleaved caspase 3, Bax, and Bcl-2) were tested by Western blot analysis. The MEK/ERK inhibitor was applied and the functions of MEK/ERK pathway in myocardial damage were investigated. PPI improved lipid metabolism disorder in H/R-induced H9c2 cells or in CAD rat model. Additionally, PPI attenuated myocardial dysfunction in CAD rats via enhancing left ventricular systolic pressure, maximum rate of change of left ventricular pressure (±dp/dtmax ), and arterial blood flow (CF). The apoptosis of myocardial cells was lessened by PPI management, which was further verified by reducing Bax and cleaved caspase 3 expression. Furthermore, PD0325901 (MEK/ERK inhibitor) weakened the effect of PPI on myocardial dysfunction, lipid metabolism, and myocardial cell apoptosis in CAD rats. The research confirmed the protective effect of PPI on myocardial damage in CAD, which was regulated by MEK/ERK pathway.

Electroacupuncture-Regulated miR-34a-3p/PDCD6 Axis Promotes Post-Spinal Cord Injury Recovery in Both In Vitro and In Vivo Settings.

Electroacupuncture (EA) could enhance neuroregeneration and posttraumatic conditions; however, the underlying regulatory mechanisms remain ambiguous. PDCD6 (programmed cell death 6) is an established proapoptotic regulator which is responsible for motoneuronal death. However, its potential regulatory role in post-spinal cord injury (SCI) regeneration has remained largely unknown. Further investigations are warranted to clarify the involvement of PDCD6 post-SCI recovery and the underlying mechanisms. In our study, based on bioinformatics prediction, we found that miR-34a-3p might be an upstream regulator miRNA for PDCD6, which was subsequently validated through combined utilization of the qRT-PCR, western blot, and dual-luciferase reporter system. Our in vitro results showed that miR-34a-3p might promote the in vitro differentiation of neural stem cell (NSC) through suppressing PDCD6 and regulating other important neural markers such as fibroblast growth factor receptor 1 (FGFR1), MAP1/2 (MAP kinase kinases 1/2), myelin basic protein (MBP), ßIII-tubulin Class III ß-tubulin (ßIII tubulin), and glial fibrillary acidic protein (GFAP). Notably, in the post-SCI rat model, exogenous miR-34a-3p agomir obviously inhibited the expression of PDCD6 at the protein level and promoted neuronal proliferation, motoneurons regeneration, and axonal myelination. The restorations at cellular level might contribute to the improved hindlimbs functions of post-SCI rats, which was manifested by the Basso-Beattie-Bresnahan (BBB) locomotor test. The impact of miR-34a-3p was further promoted by EA treatment in vivo. Conclusively, this paper argues that a miR-34a-3p/PDCD6 axis might be a candidate therapeutic target for treating SCI and that the therapeutic effect of EA is driven through this pathway.

NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP.

BACKGROUND: NeiyiKangfu tablets (NYKF) are widely used clinically for the treatment of endometriosis (EMS), whose mechanism of action has been extensively studied. Researchers have found that NYKF may control the development of ectopic lesions by inhibiting angiogenesis and inflammatory cytokine secretion. Nevertheless, NYKF's mechanism of action remains unclear. METHODS: In the present study, the function of NYKF in the progression of EMS and the associated underlying mechanism was investigated by in vivo and in vitro experiments. EMS model mice were treated with NYKF and the pro-inflammatory factors and apoptosis of ectopic endometrium as well as RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling activation were assessed. In addition, human endometriosis-derived immortalized entopic stromal (hEM15A) cells transfected with or without RAF kinase inhibitor protein (RKIP)-small-interfering RNA (siRNA) were also treated with NYKF and the proliferation, migration, apoptosis, and RAF/MEK/ERK signaling activation were measured by Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, and western blot, respectively. RESULTS: Results showed that NYKF increased the expression of RKIP, inhibited RAF/MEK/ERK signaling activation, and induced apoptosis while inhibiting proliferation and migration both in EMS mice and hEM15A cells. RKIP knockdown could inhibit the effect of NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and the proliferation and migration of hEM15A cells. CONCLUSIONS: In conclusion, these results suggest that NYKF treatment promotes apoptosis and inhibits proliferation and migration in EMS by inhibiting the RAF/MEK/ERK signaling pathway by targeting RKIP.