The Treatment of Tubal Inflammatory Infertility using Yinjia Tablets through EGFR/MEK/ERK Signaling Pathway based on Network Pharmacology.

Background: Salpingitis obstructive infertility (SOI) refers to infertility caused by abnormal conditions such as tubal adhesion and blockage caused by acute and chronic salpingitis. SOI has a serious impact on women's physical and mental health and family harmony, and it is a clinical problem that needs to be solved urgently.

Objective: The purpose of the present study was to explore the potential pharmacological mechanisms of the Yinjia tablets (Yin Jia Pian, YJP) on tubal inflammation.

Methods: Networks of YJP-associated targets and tubal inflammation-related genes were constructed through the STRING database. Potential targets and pathway enrichment analysis related to the therapeutic efficacy of YJP were identified using Cytoscape and Database for Annotation, Visualization, and Integrated Discovery (metascape). E. coli was used to establish a rat model of tubal inflammation and to validate the predictions of network pharmacology and the therapeutic efficacy of YJP. H&E staining was used to observe the pathological changes in fallopian tubes. TEM observation of the ultrastructure of the fallopian tubes. ELISA was used to detect the changes of IL-6 and TNF-α in fallopian tubes. Immunohistochemistry was used to detect the expression of ESR1. The changes of Bcl-2, ERK1/2, p-ERK1/2, MEK, p-MEK, EGFR, and p-EGFR were detected by western blot.

Results: Through database analysis, it was found that YJP shared 105 identical targets with the disease. Network pharmacology analysis showed that IL-6, TNF, and EGFR belong to the top 5 core proteins associated with salpingitis, and EGFR/MEK/ERK may be the main pathway involved. The E. coli-induced disease rat model of fallopian tube tissue showed damage, mitochondrial disruption, and increased levels of the inflammatory factors IL-6 and TNF-α. Tubal inflammatory infertility rats have increased expression of Bcl-2, p-ERK1/2, p-MEK, and p-EGFR, and decreased expression of ESR1. In vivo, experiments showed that YJP improved damage of tissue, inhibited shedding of tubal cilia, and suppressed the inflammatory response of the body. Furthermore, YJP inhibited EGFR/MEK/ERK signaling, inhibited the apoptotic protein Bcl-2, and upregulated ESR1.

Conclusion: This study revealed that YJP Reducing tubal inflammation and promoting tissue repair may be associated with inhibition of the EGFR/MEK/ERK signaling pathway.

Deregulated JNK signaling enhances apoptosis during hyperthermia.

PURPOSE: c-Jun N-terminal kinases (JNKs) comprise a subfamily of mitogen-activated protein kinases (MAPKs). The JNK group is known to be activated by a variety of stimuli. However, the molecular mechanism underlying heat-induced JNK activation is largely unknown. The aim of this study was to clarify how JNK activity is stimulated by heat. METHODS AND MATERIALS: The expression levels of various MAPK members in HeLa cells, with or without hyperthermia treatment, were evaluated via western blotting. The kinase activity of MAPK members was assessed through in vitro kinase assays. Cell death was assessed in the absence or presence of siRNAs targeting MAPK-related members. RESULTS: Hyperthermia decreased the levels of MAP3Ks, such as ASK1 and MLK3 which are JNK kinase kinase members, but not those of the downstream MAP2K/SEK1 and MAPK/JNK. Despite the reduced or transient phosphorylation of ASK1, MLK3, or SEK1, downstream JNK was phosphorylated in a temperature-dependent manner. In vitro kinase assays demonstrated that heat did not directly stimulate SEK1 or JNK. However, the expression levels of DUSP16, a JNK phosphatase, were decreased upon hyperthermia treatment. DUSP16 knockdown enhanced the heat-induced activation of ASK1-SEK1-JNK pathway and apoptosis. CONCLUSION: JNK was activated in a temperature-dependent manner despite reduced or transient phosphorylation of the upstream MAP3K and MAP2K. Hyperthermia-induced degradation of DUSP16 may induce activation of the ASK1-SEK1-JNK pathway and subsequent apoptosis.

Glycolysis-non-canonical glutamine dual-metabolism regulation nanodrug enhanced the phototherapy effect for pancreatic ductal adenocarcinoma treatment.

Clinical pancreatic ductal adenocarcinoma (PDAC) treatment is severely limited by lack of effective KRAS suppression strategies. To address this dilemma, a reactive oxygen species (ROS)-responsive and PDAC-targeted nanodrug named Z/B-PLS was constructed to confront KRAS through dual-blockade of its downstream PI3K/AKT/mTOR and RAF/MEK/ERK for enhanced PDAC treatment. Specifically, photosensitizer zinc phthalocyanine (ZnPc) and PI3K/mTOR inhibitor BEZ235 (BEZ) were co-loaded into PLS which was constructed by click chemistry conjugating MEK inhibitor selumetinib (SEL) to low molecular weight heparin with ROS-responsive oxalate bond. The BEZ and SEL blocked PI3K/AKT/mTOR and RAF/MEK/ERK respectively to remodel glycolysis and non-canonical glutamine metabolism. ZnPc mediated photodynamic therapy (PDT) could enhance drug release through ROS generation, further facilitating KRAS downstream dual-blockade to create treatment-promoting drug delivery-therapeutic positive feedback. Benefiting from this broad metabolic modulation cascade, the metabolic symbiosis between normoxic and hypoxic tumor cells was also cut off simultaneously and effective tumor vascular normalization effects could be achieved. As a result, PDT was dramatically promoted through glycolysis-non-canonical glutamine dual-metabolism regulation, achieving complete elimination of tumors in vivo. Above all, this study achieved effective multidimensional metabolic modulation based on integrated smart nanodrug delivery, helping overcome the therapeutic challenges posed by KRAS mutations of PDAC.

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide.

BACKGROUND: Precision treatment of glioblastoma is increasingly focused on molecular subtyping, with the mesenchymal subtype particularly resistant to temozolomide. Here, we aim to develop a targeted therapy for temozolomide resensitization in the mesenchymal subtype. METHODS: We integrated kinomic profiles and kinase inhibitor screens from patient-derived proneural and mesenchymal glioma-propagating cells and public clinical datasets to identify key protein kinases implicated in temozolomide resistance. RNAseq, apoptosis assays, and comet assays were used to examine the role of p38MAPK signaling and adaptive chemoresistance in mesenchymal cells. The efficacy of dual p38MAPK and MEK/ERK inhibition using ralimetinib (selective orally active p38MAPK inhibitor; phase I/II for glioblastoma) and binimetinib (approved MEK1/2 inhibitor for melanoma; phase II for high-grade glioma) in primary and recurrent mesenchymal tumors was evaluated using an intracranial patient-derived tumor xenograft model, focusing on survival analysis. RESULTS: Our transcriptomic-kinomic integrative analysis revealed p38MAPK as the prime target whose gene signature enables patient stratification based on their molecular subtypes and provides prognostic value. Repurposed p38MAPK inhibitors synergize favorably with temozolomide to promote intracellular retention of temozolomide and exacerbate DNA damage. Mesenchymal cells exhibit adaptive chemoresistance to p38MAPK inhibition through a pH-/calcium-mediated MEK/ERK pathway. Dual p38MAPK and MEK inhibition effectively maintain temozolomide sensitivity in primary and recurrent intracranial mesenchymal glioblastoma xenografts. CONCLUSIONS: Temozolomide resistance in mesenchymal glioblastoma is associated with p38MAPK activation. Adaptive chemoresistance in p38MAPK-resistant cells is mediated by MEK/ERK signaling. Adjuvant therapy with dual p38MAPK and MEK inhibition prolongs temozolomide sensitivity, which can be developed into a precision therapy for the mesenchymal subtype.

[Buyang Huanwu Decoction promotes arteriogenesis after hindlimb ischemia in mice by activating PDGF signaling pathway].

This study aims to investigate the effect of Buyang Huanwu Decoction on blood flow recovery and arteriogenesis after hindlimb ischemia in mice via the platelet-derived growth factor(PDGF) signaling pathway. Forty C57BL/6 mice were randomized into model(clean water, 10 mL·kg~(-1)·d~(-1)), beraprost sodium(positive control, 18 µg·kg~(-1)·d~(-1)), and low-, medium-, and high-dose(10, 20, and 40 g·kg~(-1)·d~(-1), respectively) Buyang Huanwu Decoction groups(n=8). The hindlimb ischemia model was established by femoral artery ligation. The mice were administrated with corresponding agents by gavage daily for 14 days after ligation. For laser Doppler perfusion imaging, the mice were anesthetized and measured under a Periscan PSI imager. The density of capillary and arterio-le in the ischemic gastrocnemius was measured using immunofluorescence staining of the frozen tissue sections. Western blot was employed to determine the expression of PDGF subunit B(PDGFB), phosphorylated mitogen extracellular kinase(p-MEK), MEK, phosphorylated extracellular signal-regulated kinase(p-ERK), and ERK. Real-time PCR was employed to determine the mRNA level of PDGFB. The Buyang Huanwu Decoction-containing serum was used to treat the vascular smooth muscle cells(VSMCs) in hypoxia at doses of 10% and 20%. The proliferation and migration of VSMCs was assessed in vitro. The results showed that compared with the model group, beraprost sodium and Buyang Huanwu Decoction enhanced the blood flow recovery, increased the capillary and arteriole density, and up-regulated the protein levels of PDGFB, p-MEK, p-ERK, and mRNA levels of PDGFB, with the medium-dose Buyang Huanwu Decoction demonstrating the most significant effect. The 10% Buyang Huanwu Decoction-containing serum enhanced the proliferation and migration of VSMCs. Our findings demonstrate that Buyang Huanwu Decoction up-regulates PDGFB transcription and activates PDGF signaling pathway to promote arteriogenesis and blood flow recovery in ischemic gastrocnemius.

Classical prescription Floris Sophorae Powder treat colorectal cancer by regulating KRAS/MEK-ERK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Colorectal cancer (CRC) belongs to the category of intestinal wind, anal ulcer, abdominal mass and other diseases in traditional Chinese medicine (TCM). Floris Sophorae Powder (F.S), is a classical prescription is recorded in Puji Benshi Fang for the treatment of intestinal carbuncle. It has been incorporated into the prescriptions for the treatment of intestinal diseases and achieved remarkable results in modern medicine. However, the mechanism of F.S in the treatment of colorectal cancer remains unclear and requires further study. AIM OF THE STUDY: To investigate F.S in treating CRC and clarify the underlying mechanism. MATERIALS AND METHODS: This study was based on Dextran Sulfate Sodium Salt (DSS) combined with Azoxymethane (AOM) induced CRC mouse model to clarify the pharmacological effects of F.S. The serum metabolomics was used to study the mechanism of action, and the chemical composition of F.S was found by UPLC-Q-TOF-MS. The rationality of serm metabolomics results was verified through the clinical target database of network pharmacology, and the upstream and downstream targets of related pathways were found. The mechanism pathway was verified by Western blot to clarify its mechanism of action. RESULTS: In vivo pharmacological experiments showed that F.S inhibited tumor growth and improved hematochezia. The vital signs of mice in the high-dose F.S group approached to those in the control group. A total of 43 differential metabolites were found to be significantly changed by serum metabolomics. F.S could modulate and recover most of the differential metabolites, which proved to be closely related to the KRAS/MEK-ERK signaling pathway. A total of 46 compounds in F.S were identified, and the rationality of serm metabolic pathway was verified by network pharmacology. Western blot results also verified that the expression of KRAS, E2F1, p-MEK and p-ERK were significantly decreased after F.S treatment. CONCLUSION: Classical prescription Floris Sophorae Powder treat colorectal cancer by regulating KRAS/MEK-ERK signaling pathway.

Oral administration of punicalagin attenuates imiquimod-induced psoriasis by reducing ROS generation and inflammation via MAPK/ERK and NF-κB signaling pathways.

Psoriasis, an immune-mediated chronic inflammatory skin disease, imposes a huge mental and physical burden on patients and severely affects their quality of life. Punicalagin (PU), the most abundant ellagitannin in pomegranates, has become a research hotspot owing to its diverse biological activities. However, its effects on psoriasis remain unclear. We explored the impact and molecular mechanism of PU on M5-stimulated keratinocyte cell lines and imiquimod (IMQ)-induced psoriasis-like skin inflammation in BABL/c mice using western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), hematoxylin and eosin (H&E) stain, immunohistochemistry, and immunofluorescent. Administration of PU-enriched pomegranate extract at dosages of 150 and 250 mg/kg/day markedly attenuated psoriatic severity, abrogated splenomegaly, and reduced IMQ-induced abnormal epidermal proliferation, CD4+ T-cell infiltration, and inflammatory factor expression. Moreover, PU could decrease expression levels of pro-inflammatory cytokines, such as IL-1ß, IL-1α, IL-6, IL-8, TNF-α, IL-17A, IL-22, IL-23A, and reactive oxygen species (ROS), followed by keratinocyte proliferation inhibition in the M5-stimulated cell line model of inflammation through inhibition of mitogen-activated protein kinases/extracellular regulated protein kinases (MAPK/ERK) and nuclear factor kappaB (NF-κB) signaling pathways. Our results indicate that PU may serve as a promising nutritional intervention for psoriasis by ameliorating cellular oxidative stress and inflammation.

Polyphenol-enriched Penthorum chinense Pursh ameliorates alcohol-related liver injury through Ras/Raf/MEK/ERK pathway: Integrating network pharmacology and experiment validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense Pursh (PCP) has acknowledged as an edible herbal medicinal plant for the prevention and treatment of alcoholic liver injury (ALI). However, only few of researches focus on the chemical material basis and potential mechanisms of PCP against ALI. AIM OF THE STUDY: Herein, we explored the therapeutic effects of PCP extract against ALI based on the integration of network pharmacology, molecular docking, and experiment validation. METHODS: Based on the standard quality control of PCP herbs by UPLC fingerprint and quantitative determination, 80% ethanol extract fraction of PCP containing more polyphenols, compared to aqueous extract fraction of PCP, were chosen for further experiments. After oral administration of PCP ethanol extract, serum pharmacochemistry based on UPLC-Q-Exactive-MS analysis was implemented to evaluate the potential effective compounds. These absorbed prototypes in PCP were used to construct network pharmacology and predict the potential mechanisms of PCP extract against ALI. Then, the predicted targets and biological mechanisms of PCP extract were validated using animal experiments and molecular docking analysis. RESULTS: Although totally 19 polyphenol compounds were identified in PCP ethanol extract by UPLC-MS analysis, only 18 absorbed prototypes were found in the serum collected from mice at 1 h post-administration with PCP extract. These candidate active compounds were further screened into 13 compounds to construct network pharmacology and 433 targets were identified as PCP targets. GO and KEGG pathway enrichment analyses indicated that the effects of PCP extract would involve in Ras signaling pathway. The animal experiments on chronic ALI model mice shown that the oral administration of PCP can alleviate ALI by attenuating hepatic oxidative stress, inflammation and down-regulating the target proteins in Ras/Raf/MEK/ERK pathway. Molecular docking analysis revealed the good binding ability between the three polyphenols (i.e. quercetin, apigenin, thonningianin B) in PCP with the top contribution in network pharmacology, and these target proteins (Ras, Raf, MEK1/2, and ERK1/2). CONCLUSION: Our results clarified that PCP ethanol extract could effectively alleviate ALI by down-regulating Ras/Raf/MEK/ERK signaling pathway promisingly. Quercetin, apigenin, and thonningianin B may be the active compounds of PCP, attributing to the intervention benefits of PCP against ALI.

Effect of electroacupuncture on urodynamics and Raf/MEK/ERK signaling pathway in spinal cord tissue of rats with detrusor hyperreflexia after suprasacral spinal cord injury. / ç”µé’ˆå¯¹éª¶ä¸Šè„Šé«“æŸä¼¤åŽé€¼å°¿è‚Œåå°„äº¢è¿›åž‹å¤§é¼ å°¿æµåŠ¨åŠ›å­¦åŠè„Šé«“Raf/MEK/ERK信号通路的影响.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on urodynamics and Raf/MEK/ERK signaling pathway in spine cord tissue of rats after suprasacral spinal cord injury (SSCI), so as to explore its possible mechanism in improving bladder function in rats with detrusor hyperreflexia after SSCI. METHODS: Female SD rats were randomly divided into blank, sham operation, model, EA and EA+PD98059 groups, with 12 rats in each group. Thorax (T) 10 spinal cord transection was performed by surgery. Rats in the EA group were given EA (10 Hz/50 Hz, 20 min) at "Ciliao" (BL32), "Zhongji" (CV3), "Sanyinjiao" (SP6) and "Dazhui" (GV14) once daily for 7 d. Rats of the EA+PD98059 group received intraperitoneal injection of PD98059 (5 mg/kg) 2 h before EA intervention. The urodyna-mics was used to measure the base pressure, leak point pressure, maximum pressure, maximum capacity and comp-liance of bladder, and the morphology of bladder detrusor tissue was observed with HE staining. The TUNEL staining was used to detect the cell apoptosis of the spinal cord tissue. The expression levels of exchange protein directly activated by cAMP 2 (Epac2), Rap, phosphorylated rapidly accelerated fibrosarcoma (p-Raf), phosphorylated mitogen-activated extracellular signal-regulated kinase (p-MEK), phosphorylated extracellular signal regulated kinase 1 and 2 (p-ERK1/2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax) were determined by Western blot. RESULTS: Compared with the sham operation group, the base pressure, leak point pressure and maximum pressure of bladder were significantly increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), the cell apoptosis rate of spinal cord tissue was increased (P<0.01), and the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were decreased (P<0.01), while the expression level of Bax protein was increased (P<0.01) in the model group. After the treatment and compared with the model group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate of spinal cord tissue, the expression level of Bax protein were decreased (P<0.05) in the EA group, while the maximum bladder capacity and bladder compliance, the expression levels of Epac2, Rap, p-Raf, p-MEK, p-ERK1/2, and Bcl-2 protein in spinal cord tissue were all increased (P<0.05, P<0.01). In comparison with the EA group, the base pressure, leak point pressure and maximum pressure of bladder, the cell apoptosis rate, the expression level of Bax protein were significantly increased (P<0.05), whereas the maximum bladder capacity, bladder compliance, and the expression levels of p-MEK, p-ERK1/2, and Bcl-2 protein were decreased (P<0.05) in the EA+PD98059 group. Results of HE staining showed disordered transitional epithelial cells and destroyed lamina propria in bladder detrusor tissue, with the infiltration of monocytes in the model group, which was obviously milder in both EA and EA+PD98059 groups, especially in the EA group. CONCLUSIONS: EA can improve the bladder function in detrusor hyperreflexia rats after SSCI, which may be related to its effect in up-regulating Epac2 and Rap, activating the Raf-MEK-ERK pathway, and reducing the cell apoptosis of spinal cord tissue.

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.

Polyphyllin VII induces autophagy-dependent ferroptosis in human gastric cancer through targeting T-lymphokine-activated killer cell-originated protein kinase.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is a serine-threonine kinase that is overexpressed in gastric cancer (GC) and promotes tumor progression. Polyphyllin VII (PPVII), a pennogenin isolated from the rhizomes of Paris polyphylla, shows anticancer effects. Here, we explored the antitumor activity and mechanism of PPVII in GC. Ferroptosis was detected by transmission electron microscope, malondialdehyde, and iron determination assays. Autophagy and its upstream signaling pathway were detected by Western blot, and gene alterations. The binding of PPVII and TOPK was examined through microscale thermophoresis and drug affinity responsive target stability assays. An in vivo mouse model was performed to evaluate the therapeutic of PPVII. PPVII inhibits GC by inducing autophagy-mediated ferroptosis. PPVII promotes the degradation of ferritin heavy chain 1, which is responsible for autophagy-mediated ferroptosis. PPVII activates the Unc-51-like autophagy-activating kinase 1 (ULK1) upstream of autophagy. PPVII inhibits the activity of TOPK, thereby weakening the inhibition of downstream ULK1. PPVII stabilizes the dimer of the inactive form of TOPK by direct binding. PPVII inhibits tumor growth without causing obvious toxicity in vivo. Collectively, this study suggests that PPVII is a potential agent for the treatment of GC by targeting TOPK to activate autophagy-mediated ferroptosis.

The Role of Activation of PI3K/AKT/mTOR and RAF/MEK/ERK Pathways in Aggressive Pituitary Adenomas-New Potential Therapeutic Approach-A Systematic Review.

Pituitary tumors (PT) are mostly benign, although occasionally they demonstrate aggressive behavior, invasion of surrounding tissues, rapid growth, resistance to conventional treatments, and multiple recurrences. The pathogenesis of PT is still not fully understood, and the factors responsible for its invasiveness, aggressiveness, and potential for metastasis are unknown. RAF/MEK/ERK and mTOR signaling are significant pathways in the regulation of cell growth, proliferation, and survival, its importance in tumorigenesis has been highlighted. The aim of our review is to determine the role of the activation of PI3K/AKT/mTOR and RAF/MEK/ERK pathways in the pathogenesis of pituitary tumors. Additionally, we evaluate their potential in a new therapeutic approach to provide alternative therapies and improved outcomes for patients with aggressive pituitary tumors that do not respond to standard treatment. We perform a systematic literature search using the PubMed, Embase, and Scopus databases (search date was 2012-2023). Out of the 529 screened studies, 13 met the inclusion criteria, 7 related to the PI3K/AKT/mTOR pathway, and 7 to the RAF/MEK/ERK pathway (one study was used in both analyses). Understanding the specific factors involved in PT tumorigenesis provides opportunities for targeted therapies. We also review the possible new targeted therapies and the use of mTOR inhibitors and TKI in PT management. Although the RAF/MEK/ERK and PI3K/AKT/mTOR pathways play a pivotal role in the complex signaling network along with many interactions, further research is urgently needed to clarify the exact functions and the underlying mechanisms of these signaling pathways in the pathogenesis of pituitary adenomas and their role in its invasiveness and aggressive clinical outcome.

Pharmacodynamic structure of deer antler base protein and its mammary gland hyperplasia inhibition mechanism by mediating Raf-1/MEK/ERK signaling pathway activation.

Mammary gland hyperplasia (MGH) is a common mammary disease whose main pathogenesis is the disruption of estradiol (E2) and progesterone (P) secretion, thereby causing overproliferation of mammary epithelial cells and mammary gland tissue hyperplasia. Deer antler base is a traditional Chinese medicine that has been used for many years to treat MGH. However, its pharmacological mechanism and pharmacodynamic material basis are unclear. In this study, we for the first time used the graded salting method to classify deer antler base protein (CNCP) as CNCP-A, CNCP-B, and CNCP-C and explored the pharmacological mechanism of the anti-MGH properties of CNCP. We found that CNCP could regulate the hormonal levels of E2, P, and follicle stimulating hormone (FSH) and improve the histopathological condition. The potential mechanism might be related to the recombinant C-Raf proto oncogene serine/threonine protein kinase/mitogen-activated protein/extracellular regulated protein kinase (Raf-1/MEK/ERK) signaling pathway. By upregulating the protein expression of the follicle stimulating hormone receptor (FSHR), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) inhibited the activation of the downstream Raf-1/MEK/ERK signaling pathway, which in turn inhibited the proliferation of mammary epithelial cells. We analyzed the physicochemical properties of CNCP-A, CNCP-B, and CNCP-C and obtained CNCP-C-I by column chromatographic purification of the best pharmacophore protein CNCP. Using high-performance liquid gel filtration chromatography (HPGFC), we determined the molecular weight of CNCP-C-I and identified it by high-performance liquid tandem mass spectrometry (LC-MS/MS) to obtain the first match for a high confidence protein KRT1. This study provides a theoretical basis for the development of effective traditional Chinese medicines with low toxicity levels for the prevention and treatment of mammary gland diseases.

Xanthohumol inhibits non-small cell lung cancer via directly targeting T-lymphokine-activated killer cell-originated protein kinase.

Xanthohumol is a principal prenylated chalcone isolated from hops. Previous studies have shown that xanthohumol was effective against various types of cancer, but the mechanisms, especially the direct targets for xanthohumol to exert an anticancer effect, remain elusive. Overexpression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) promotes tumorigenesis, invasion and metastasis, implying the likely potential for targeting TOPK in cancer prevention and treatment. In the present study, we found that xanthohumol significantly inhibited the cell proliferation, migration and invasion of non-small cell lung cancer (NSCLC) in vitro and suppressed tumor growth in vivo, which is well correlated with inactivating TOPK, evidenced by reduced phosphorylation of TOPK and its downstream signaling histone H3 and Akt, and decreased its kinase activity. Moreover, molecular docking and biomolecular interaction analysis showed that xanthohumol was able to directly bind to the TOPK protein, suggesting that TOPK inactivation by xanthohumol is attributed to its ability to directly interact with TOPK. The findings of the present study identified TOPK as a direct target for xanthohumol to exert its anticancer activity, revealing novel insight into the mechanisms underlying the anticancer activity of xanthohumol.

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.

Ebracteolatain A exerts anti-proliferation of breast cancer by inhibiting Protein kinase D 1 in MEK/ERK and PI3K/AKT signaling pathways.

BACKGROUND: Ebracteolatain A (EA) is an acetyl-phloroglucinol compound extracted from Euphorbiae Ebracteolatae Radix, which has been shown to have antitumor activity. PURPOSE: Current research addressed the antitumor activity of EA in breast cancer and further clarified its mechanism. STUDY DESIGN: Based on the pharmacodynamic evaluation in breast cancer cells and animal models, the antitumor effects of EA will be validated in vitro and in vivo. METHODS: Breast cancer cells were processed with increasing concentrations of EA. CCK-8 and colony formation assays were employed to examine the effects of EA on proliferation and survival. Flow cytometry detected the blocking function of EA on the cell cycle. The specific mechanism of EA in breast cancer was studied by transfection experiments and Western Blot analysis. Finally, a nude mice xenograft tumor model was constructed to assess the therapeutic and potential mechanism of EA. RESULTS: We proved that EA caused a dose-dependent inhibition on MCF-7 and MDA-MB-415 cells with IC50 of 6.164 and 6.623 µmol/l, respectively. While EA reduced cell proliferation and clone formation, and markedly arrested cells in the G0/G1 phase. In vivo, EA remarkably suppressed the tumor weight and volume in xenograft nude mice. Besides, PKD1 reversed the inhibition of EA on breast cancer cell proliferation, clone formation, and cycle arrest, and restored tumor growth in xenograft nude mice. Western Blot confirmed that EA regulates breast cancer by suppressing PKD1 in MEK/ERK and PI3K/AKT signaling pathways. CONCLUSION: Herein, we first confirmed EA exerts anti-proliferation by inhibiting PKD1 in MEK/ERK and PI3K/AKT signaling pathways, indicating that EA is a prodigious breast cancer drug candidate.

Tongluo Shenggu capsule promotes angiogenesis to ameliorate glucocorticoid-induced femoral head necrosis via upregulating VEGF signaling pathway.

BACKGROUND: Tongluo Shenggu Capsule (TLSGC) is a product of Traditional Chinese patent medicine that has been effective in glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) clinically for many years. It is made from water extracts of a well-used herbal and dietary supplement-pigeon pea leaves. Nevertheless, the material basis and pharmacological mechanisms of TLSGC ameliorating GIONFH needed to be better defined. PURPOSE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH. METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a "drug targets-disease genes" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs). RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro. CONCLUSION: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.

MicroRNA-143-3p Inhibits Wilms' Tumor Cell Growth By Targeting the Ras/Raf/MEK/ERK Pathway.

Background: A previous study found that microRNA-143-3p (miR-143-3p) is a tumor suppressor in various types of human cancer. However, the roles and molecular mechanisms of miR-143-3p in the progression of Wilms' tumor (WT) remain to be clarified. The aim of the present study was to determine the expression and biological functions of miR-143-3p in WT. Material and Methods: The expression levels of miR-143-3p in primary WT tissues and adjacent tissues were determined using quantitative-reverse transcription polymerase chain reaction (qRT-PCR), and the association of the miR-143-3p expression level with various clinicopathological features of WT was investigated. Western blotting was used to evaluate the protein expression of the related signaling pathway. Results: The expression of miR-143-3p was significantly downregulated in WT tissues and its expression levels were closely associated with tumor stage and lymph node metastasis. Overexpression of miR-143-3p in SK-NEP-1 and G401 cell lines inhibited cell proliferation by G0/G1 cell cycle phase arrest and induction of apoptosis. Moreover, k-Ras, a unique oncogene, was confirmed as a direct target of miR-143-3p, and k-Ras messenger RNA (mRNA) expression was increased in WT tissues and inversely correlated with miR-143-3p. Knockdown of k-Ras by si-k-Ras could inhibit, whereas overexpression of k-Ras could promote. cell proliferation in WT cells. Meanwhile, overexpression of k-Ras reversed the inhibitory effects on WT cells induced by miR-143-3p mimics. Conclusion: Our findings indicate that miR-143-3p may be a potential novel prognostic biomarker and therapeutic target for WT.

Integrated miRNA and mRNA omics reveal dioscin suppresses migration and invasion via MEK/ERK and JNK signaling pathways in human endometrial carcinoma in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonatum sibiricum Redouté (PS, also called Huangjing in traditional Chinese medicine), is a perennial herb as homology of medicine and food. According to the traditional Chinese medicine theory "Special Records of Famous Doctors", its functions include invigorating qi and nourishing yin, tonifying spleen and kidney. Traditionally, qi and blood therapy has been believed as most applicable to the treatment of uterine disease. The current research has focused on the effect and mechanism of dioscin, the main active component of PS, on Endometrial carcinoma (EC). AIM OF THE STUDY: To study the efficacy of dioscin on proliferation and migration of Endometrial carcinoma cell line, we conducted experiments by using xenograft model and Ishikawa cells, and explored the potential molecular mechanism. MATERIALS AND METHODS: mRNA and miRNA omics techniques were employed to investigate the regulatory mechanism of dioscin on EC Ishikawa cells. Based on in vivo and in vitro experiments, cell clone formation, cell scratching, Transwell, H&E staining, immunohistochemistry, q-PCR, and Western blot techniques were used to determine the molecular effects and mechanisms of dioscin on cell migration. RESULTS: Integrated miRNA and mRNA omics data showed that 513 significantly different genes marked enrichment in MAPK signaling pathway. The in vivo data showed that dioscin (24 mg/kg) significantly inhibited tumor growth. The in vitro proliferation and invasiveness of dioscin on Ishikawa cells showed that dioscin could significantly decrease the colony numbers, and suppress the Ishikawa cell wound healing, migration and invasion. Molecular data revealed that dioscin decreased the MMP2 and MMP9 expression in vitro and in vivo. The p-MEK, p-ERK, and p-JNK expression levels were also confirmed to be significantly reduced. Key regulators in the MAPK signaling pathway were further validated in xenograft tumors. CONCLUSION: Our data indicated that dioscin inhibited Ishikawa cell migration and invasion mediated through MEK/ERK and JNK signaling. More importantly, screened hub miRNAs and genes can be regarded as potential molecular targets for future EC treatment.

Carthami flos extract against carbon tetrachloride-induced liver fibrosis via alleviating angiogenesis in mice.

BACKGROUND: Angiogenesis is a pathological phenomenon contribute to the development of chronic liver diseases, and anti-angiogenic therapy is an effective strategy to alleviate liver fibrosis. Carthami flos, a medicinal and edible herb, has the effects of improving blood circulation and regulating angiogenesis. However, the anti-angiogenic effect of Carthami flos in liver fibrosis remains unknown. METHODS: We investigated the protective effect and therapeutic mechanism of Carthami flos extract (CFE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. The liver injury and collagen deposition were observed and evaluated by conducting HE, Masson, and Sirius red staining, testing the serum biochemical indexes (ALT, AST, ALP, γ-GT), and measuring the contents of HYP and four indexes of liver fiber (Col-IV, LN, HA, PC-III). Simultaneously, the expressions of α-SMA and Collagen-I were detected to determine the activation of hepatic stellate cells (HSCs). Subsequently, we measured the expressions of angiogenesis-related proteins such as PDGFRB, ERK1/2, p-ERK1/2, MEK, p-MEK, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, and the mRNA levels of PDGFRB and VEGFA. Additionally, immunofluorescence staining and RT-qPCR assays were carried out to ascertain the expressions of continuous endothelial markers CD31, CD34 and vWF, and scanning electron microscope analysis was performed to observe the number of sinusoidal endothelial fenestrations. RESULTS: Herein, we found that CFE could significantly reduce liver injury and collagen deposition, like the same effect of colchicine. CFE significantly alleviated CCl4-induced liver injury and fibrosis, mainly manifested by reducing the levels of ALT, AST, ALP and γ-GT and decreasing the contents of HYP, Col-IV, LN, HA and PC-III. Additionally, CCl4 promoted the activation of HSCs by increasing the expressions of α-SMA and Collagen-I, while CFE could rectify the condition. Moreover, CFE treatment prevented the CCl4-induced the up-regulation of PDGFRB, p-MEK, p-ERK1/2, HIF-1α, VEGFA, VEGFR2, AKT and eNOS, suggesting that CFE might provide the protection against abnormal angiogenesis. In the meantime, the gradual disappearance of sinusoidal capillarization after CFE treatment was supported by the decreased the contents of CD31, CD34 and vWF, as well as the increased number of sinusoidal endothelial fenestrae. CONCLUSION: In this study, the reduction of collagen deposition, the obstruction of HSCs activation, the inactivation of angiogenic signaling pathways and the weakening of hepatic sinusoidal capillarization jointly confirmed that CFE might be promising to resist angiogenesis in liver fibrosis via the PDGFRB/ERK/HIF-1α and VEGFA/AKT/eNOS signaling pathways. Nevertheless, as a potential therapeutic drug, the deeper mechanism of Carthami flos still needs to be further elucidated.