Phosphorylation Of Protein Kinase Research Articles

Protein Kinase C-β Inhibition and Survival Signaling after Simulated Cardioplegic Ischemia/Reperfusion in Non-Diabetic and Diabetic Human Coronary Arterial Endothelial Cells.

Cardioplegic ischemia/reperfusion (I/R) injury poses substantial challenges during postoperative recovery, with diabetic patients particularly susceptible to adverse events. Using a model entailing the subjection of human coronary artery endothelial cells (HCAECs) to simulated cardioplegic I/R, we investigated the potential of protein kinase c β (PKC-β) inhibition to augment cellular survival in this context. HCAECs were isolated from harvested coronary arteries of diabetic (D) and nondiabetic (C) patients (N = 4 per group). HCAECs were either cultured in normoxic conditions without drug (D and C), subjected to hypoxia/reoxygenation alone (DH and CH), or subjected to hypoxia/reoxygenation and the PKC-β inhibitor LY333531 (DHT and CHT). Molecular signaling was assessed using immunoblotting. Simulated I/R decreased anti-apoptotic phosphorylated protein kinase b (p-Akt, p = 0.04) and p-Akt:Akt ratio (p = 0.004) in CH versus C, with PKC-β inhibition restoring expression in CHT (p ≤ 0.04). Treatment also increased p-Akt:Akt ratio in DHT versus D (p = 0.03). Anti-apoptotic inducible nitric oxide synthase increased in CHT versus CH (p = 0.003), and pro-apoptotic phosphor-FOXO:FOXO ratio decreased in CHT versus CH (p = 0.001). I/R elevated Bcl-2-associated agonist of cell death (BAD) in CH versus C (p = 0.01), but PKC-β inhibition increased anti-apoptotic p-BAD (p = 0.001) and p-BAD:BAD ratio (p = 0.03) in CHT. I/R also increased cleaved PARP (p < 0.001) and cleaved caspase 3 (p < 0.001) in DH versus D, both of which were reversed by treatment (p < 0.001 for DHT versus DH). PKC-β inhibitor treatment increased pro-survival signaling and decreased pro-apoptotic signaling in nondiabetic and diabetic HCAECs subjected to simulated I/R, with mechanistic differences observed between these cohorts.

MicroRNAs Dependent G-ELNs Based Intervention Improves Glucose and Fatty Acid Metabolism While Protecting Pancreatic β-Cells in Type 2 Diabetic Mice.

Metabolic disorders such as Type 2 diabetes mellitus (T2DM) imposes a significant global health burden. Plant-derived exosome like nanoparticles (P-ELNs) have emerged as a promising therapeutic alternate for various diseases. Present data demonstrates that treatment with Ginger-derived exosome like nanoparticles (G-ELNs) enhance insulin dependent glucose uptake, downregulate gluconeogenesis and oxidative stress in insulin resistant HepG2 cells. Furthermore, oral administration of G-ELNs in T2DM mice decreases fasting blood glucose levels and improves glucose tolerance as effectively as metformin. These improvements are attributed to the enhanced phosphorylation of Protein kinase B (Akt-2), the phosphatidylinositol 3-kinase at serine 474 which consequently leads to increase in hepatic insulin sensitivity, improvement in glucose homeostasis and decrease in ectopic fat deposition. Oral administration of G-ELNs also exerts protective effect on Streptozotocin (STZ)-induced pancreatic β-cells damage, contributing to systemic amelioration of T2DM. Further, as per computational tools, miRNAs present in G-ELNs modulate the phosphatidylinositol 3-kinase (PI3K)/Akt-2pathway and exhibit strong interactions with various target mRNAs responsible for hepatic gluconeogenesis, ectopic fat deposition and oxidative stress. Furthermore, synthetic mimic of G-ELNs miRNA effectively downregulates its target mRNA in insulin resistant HepG2 cells. Overall, the results indicate that the miRNAs present in G-ELNs target hepatic metabolism thus, exerting therapeutic effects in T2DM.

Phytochemicals From Salvia substolonifera With Anti-Angiogenic Properties and Substolide H Decreased Oxygen-Induced Retinal Neovascularization.

Retinal neovascularization is a pathological feature of ischemic retinopathy. Current therapeutic approaches are limited, and additional treatment options are needed. This study aims to discover lead compounds from Salvia substolonifera that inhibit angiogenesis. As a result, an undescribed norditerpene lactone, substolide H (3), and eight known compounds (1, 2, and 4-9) have been isolated. The structure was elucidated using synthetic spectroscopy and electron circular dichroism. Compounds 2, 3, 5, 7, and 9 inhibited human umbilical vein endothelial cells (HUVEC) proliferation with IC50 values of 26.47, 6.10, 43.27, 36.81, and 35.11µM, respectively. Compounds 2, 3, and 7 inhibited HUVEC migration with IC50 values of 12.48, 8.37, and 7.63µM, respectively. Further studies have shown that the substolide H (3) suppresses tube formation in human umbilical vein endothelial cells and that intravitreous administration suppresses retinal neovascularization in oxygen-induced retinopathy mice. Turning to its mechanism of action, we have shown that the anti-angiogenic effect of the substolide H may be through the downregulation of vascular endothelial growth factor expression and the phosphorylation of VEGFR2, ERK1/2, and protein kinase B. Our study represents the first report of these anti-angiogenic compounds from this plant, and substolide H may be a potential candidate for the treatment of retinal neovascularization.

GLP-1 Receptor Agonists Alleviate Diabetic Kidney Injury via β-Klotho-Mediated Ferroptosis Inhibition.

Semaglutide (Smg), a GLP-1 receptor agonist (GLP-1RA), shows renal protective effects in patients with diabetic kidney disease (DKD). However, the exact underlying mechanism remains elusive. This study employs transcriptome sequencing and identifies β-Klotho (KLB) as the critical target responsible for the role of Smg in kidney protection. Smg treatment alleviates diabetic kidney injury by inhibiting ferroptosis in patients, animal models, and HK-2 cells. Notably, Smg treatment significantly increases the mRNA expression of KLB through the activation of the cyclic adenosine monophosphate (cAMP) signaling pathway, specifically through the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Subsequently, the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway is activated, reprograming the key metabolic processes of ferroptosis such as iron metabolism, fatty acid synthesis, and the antioxidant response against lipid peroxidation. Suppression of ferroptosis by Smg further attenuates renal inflammation and fibrosis. This work highlights the potential of GLP-1RAs and KLB targeting as promising therapeutic approaches for DKD management.

Protective effect of Apelin-13 on oxygen and glucose deprivation induced-damage in retinal ganglion cells cultured in vitro.

Ischemic-anoxic injury plays an important role in the pathophysiology of diabetes retinopathy, optic neuropathy, even glaucoma and other ocular diseases. It may ultimately cause damage to neuronal death like retinal ganglion cells (RGCs) and subsequent visual loss. RGCs are essential elements of the retina and optic nerve that are crucial to visual formation. Ischemic-anoxic injury, inflammation, and oxidative stress are vital causes of RGC death. Thus, neuroprotection is essential for the treatment of these ocular diseases. Recent studies have shown the neuroprotective property of apelin-13 in many disease models. In this study, we isolated RGCs and found that apelin-13 promoted the viability of RGCs and increased the phosphorylation of Protein kinase B (PKB, Akt) in an in vitro oxygen-glucose deprivation model. Moreover, apelin-13 increased the expressions of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADPH) and reduced the level of reactive oxygen species (ROS). And, we also found that apelin-13 could promote the expressions of glucose transporter-1 (GLUT1) and adenosine triphosphate (ATP). These results indicated that apelin-13 could delay or stop RGC death, which might be as potential therapeutic targets for treatment of diseases mediated by ischemic-anoxic damage like diabetes retinopathy, optic neuropathy, even glaucoma.

Menisoxoisoaporphine A, a novel oxoisoaporphine alkaloid from Menispermi Rhizoma, inhibits inflammation by targeting PDE4B.

Dysregulated and excessive inflammatory reactions can lead to tissue damage, which is the underlying cause of most human diseases. Menisoxoisoaporphine A (MA), a novel oxoisoaporphine alkaloid, was obtained from Menispermi Rhizoma, a traditional Chinese medicinal herb used in the treatment of inflammatory conditions in clinical practice. This suggests that MA has very promising potential for the development of anti-inflammatory therapeutics. Hence, this study aims to investigate the anti-inflammatory effects and underlying mechanisms of MA. The anti-inflammatory effects of MA were evaluated in lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 cells. Its underlying mechanisms were explored through RNA sequencing and Western blotting. The binding modes and interactions sites between MA and phosphodiesterase 4B (PDE4B) were predicted using molecular docking and validated by molecular dynamics simulation. MA treatment significantly reduced LPS-induced morphological changes, inflammatory cytokine relesae, and proinflammatory genes expression in RAW264.7 cells compared to the LPS-induced controls. Transcriptome sequencing analysis suggested that PDE4B might be a key target for MA to exert its therapeutic effect. Mechanismly, MA directly acted on Tyr405 site of PDE4B, thus leading to a sustained elevation of the cyclic adenosine monophosphate (cAMP) levels, which subsequently inactivated NF-κB signaling pathway by phosphorylating protein kinase A (PKA). MA inhibited the NF-κB-mediated inflammatory response depending on PDE4B. MA, a natural and novel compound, exerted anti-inflammatory effects in LPS-induced RAW264.7 macrophage cells. It demonstrated a strong binding ability to the Tyr405 sites of PDE4B, thereby inhibiting NF-κB signaling pathway by regulating the cAMP-PKA axis. Elucidating the interaction between MA and PDE4B holds significant potential for the advancement of innovative therapeutic strategies aimed at inflammatory diseases. By strategically modulating this interaction, it may be feasible to achieve more precise regulation of inflammatory responses, thereby offering promising therapeutic benefits for conditions such as rheumatoid arthritis, asthma, and inflammatory bowel disease.

C1q/tumor necrosis factor-related protein-6 suppresses the angiotensin II-induced differentiation of cardiac fibroblasts to myofibroblasts via activation of the AMPK pathway.

C1q/tumor necrosis factor-related protein-6 (CTRP6) has multiple protective effects against cardiovascular diseases. Myofibroblast differentiation plays a critical role in cardiac fibrosis under various cardiac pathological conditions. The aim of the present study was to determine the effects of CTRP6 on cardiac fibrosis, and to identify the possible mechanisms of action. Toward this end, we measured the expression of fibrotic markers, including collagen I, collagen III, CTGF, and TGFβ1, and assessed the effects of CTRP6 on cardiac fibroblast differentiation into myofibroblasts. CTRP6 inhibited the expression of the angiotensin II (Ang II)-induced myofibroblast markers α-SMA and SM22, and of profibrotic molecules, including collagen I, collagen III, CTGF, TGFβ1, MMP2, MMP9, and TIMP1. Furthermore, CTRP6 significantly attenuated the proliferation and migration of cardiac fibroblasts incubated with Ang II and activated the phosphorylation of AMP-activated protein kinase (AMPK). Incubation with an AMPK inhibitor reversed the subsequent inhibitory effects of CTRP6 on Ang II-induced myofibroblast differentiation. Therefore, CTRP6 suppresses cardiac fibrosis by inhibition of myofibroblast differentiation via AMPK pathway activation, suggesting CTRP6 as a target for the treatment of cardiac fibrosis.

4-Methoxycinnamic acid ameliorates post-traumatic stress disorder-like behavior in mice by antagonizing the CRF type 1 receptor

AimsPosttraumatic stress disorder (PTSD) is a debilitating neuropsychiatric illness caused by traumatic or life-threatening events and manifesting as various symptoms, including intrusive re-experiences of trauma, avoidance behaviors, hyperarousal, and negative changes in perception and mood. Main methodsCurrent monoamine-based medications commonly exhibit limited efficacy and significant side effects, which hamper their clinical utility. To address this unmet need, we explored 4-methoxycinnamic acid (4-MCA) as a potential novel treatment for PTSD in a single prolonged stress (SPS)-induced animal model. Key findingsAdministration of 4-MCA (3 and 10 mg/kg, p.o.) significantly mitigated anxiety-like behaviors, alleviated depression-like behaviors, and improved cognitive function in an SPS-treated PTSD mouse model. Further, 4-MCA treatment effectively rectified the fear extinction deficits in the fear conditioning test. Molecular analyses revealed that 4-MCA normalized the elevated corticotropin-releasing hormone (CRH) levels as well as the phosphorylation of protein kinase A (PKA) and cAMP response element-binding protein (CREB) in the amygdala, a pivotal region for fear memory formation. Co-administration of 4-MCA and the CRFR1 antagonist antalarmin at subeffective doses facilitated fear memory extinction. SignificanceThese findings suggest that 4-MCA alleviates SPS-induced PTSD-like behaviors by regulating the CRH-CRFR1-PKA-CREB signaling pathway in the amygdala, and that 4-MCA may be a potential candidate for future PTSD treatment.

Sparstolonin B Reduces Estrogen-Dependent Proliferation in Cancer Cells: Possible Role of Ceramide and PI3K/AKT/mTOR Inhibition

Background: The aim of this study was to determine the effect of Sparstolonin B (SsnB) on cell proliferation and apoptosis in human breast cancer (MCF-7) and human ovarian epithelial cancer (OVCAR-3) cell lines in the presence and absence of estradiol hemihydrate (ES). Phosphoinositol-3 kinase (PI3K), phosphorylated protein kinase B alpha (p-AKT), phosphorylated mTOR (mechanistic target of rapamycin) signaling proteins, and sphingomyelin/ceramide metabolites were also measured within the scope of the study. Methods: The anti-proliferative effects of SsnB therapy were evaluated over a range of times and concentrations. Cell proliferation was determined by measuring the Proliferating Cell Nuclear Antigen (PCNA). PCNA was quantified by ELISA and cell distribution was assessed by immunofluorescence microscopy. MTT analysis was used to test the vitality of the cells, while LC-MS/MS was used to analyze the amounts of ceramides (CERs), sphingosine-1-phosphate (S1P), and sphingomyelins (SMs). TUNEL labeling was used to assess apoptosis, while immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA) were used to measure the levels of PI3K, p-AKT, and p-mTOR proteins. Results: Sparstolonin B administration significantly decreased cell viability in MCF-7 and OVCAR-3 cells both in the presence and absence of ES, while it did not cause toxicity in healthy human fibroblasts. In comparison to controls, cancer cells treated with SsnB showed a significant drop in the levels of S1P, PI3K, p-AKT, and p-mTOR. In cancer cells cultured with SsnB, a significant increase in intracellular concentrations of C16-C24 CERs and apoptosis was observed. Conclusions: SsnB downregulated the levels of S1P, PI3K, p-AKT, and p-mTOR while reducing cell proliferation and promoting ceramide buildup and apoptosis.

Anti-Inflammatory Effects of Honeysuckle Leaf Against Lipopolysaccharide-Induced Neuroinflammation on BV2 Microglia.

Neurodegenerative disorders have emerged as a major global public health concern, and the burden is predicted to increase over time. Modulating neuroinflammation and microglial activity is considered a promising target for improving neurodegenerative disorders. The leaf of honeysuckle (LH), which has anti-inflammatory properties, has long been collected, regardless of the season, and used for medicinal purposes. However, research on its effects on neuroinflammation is scarce. In this study, we determined the neuroprotective effects of LH water extract by inhibiting microglial activation induced by lipopolysaccharide (LPS). The production or secretion of pro-inflammatory mediators was examined in LPS-exposed BV2 cells to ascertain the efficacy of LH water extract in improving neuroinflammation. In addition, the phosphorylation of mitogen-activated protein kinases (MAPKs) and the degradation of inhibitory κBα (IκBα) were analyzed to elucidate the regulatory mechanisms of LH water extract. Ultra-performance liquid chromatography (UPLC) analysis was conducted to identify the active component of the LH. LH water extract suppressed the formation of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, in LPS-activated BV2 cells. LH impeded the activation of c-Jun N-terminal kinase (JNK). Moreover, chlorogenic acid was found in LH. The above findings suggest that LH water extract could improve neuroinflammation.

Arachidonic acid impairs natural killer cell functions by disrupting signaling pathways driven by activating receptors and reactive oxygen species

BackgroundHigh levels of the polyunsaturated fatty acid arachidonic acid (AA) within the ovarian carcinoma (OC) microenvironment correlate with reduced relapse-free survival. Furthermore, OC progression is tied to compromised immunosurveillance, partially attributed to the impairment of natural killer (NK) cells. However, potential connections between AA and NK cell dysfunction in OC have not been studied.MethodsWe employed a combination of phosphoproteomics, transcriptional profiling and biological assays to investigate AA’s impact on NK cell functions.ResultsAA (i) disrupts interleukin-2/15-mediated expression of pro-inflammatory genes by inhibiting STAT1-dependent signaling, (ii) hampers signaling by cytotoxicity receptors through disruption of their surface expression, (iii) diminishes phosphorylation of NKG2D-induced protein kinases, including ERK1/2, LYN, MSK1/2 and STAT1, and (iv) alters reactive oxygen species production by transcriptionally upregulating detoxification. These modifications lead to a cessation of NK cell proliferation and a reduction in cytotoxicity.ConclusionOur findings highlight significant AA-induced alterations in the signaling network that regulates NK cell activity. As low expression of several NK cell receptors correlates with shorter OC patient survival, these findings suggest a functional linkage between AA, NK cell dysfunction and OC progression.

Kallikrein-Related Peptidase 6 Contributes to Murine Intestinal Tumorigenesis Driven by a Mutant Adenomatous polyposis coli Gene.

The objective of this study was to assess the role of a secreted serine protease, kallikrein-related peptidase 6 (KLK6), during colorectal tumorigenesis driven by a mutant Adenomatous polyposis coli (APC) tumor suppressor gene. A first analysis of KLK6 expression in the intestinal tract of Apc-mutant multiple intestinal neoplasia (ApcMin/+) mice revealed up to four-fold induction of Klk6 mRNA levels in adenomas relative to its level in the adjacent mucosa. The presence of KLK6 protein in the adenomatous areas was confirmed by immunohistochemistry and optical coherence tomography/laser-induced fluorescence (OCT/LIF) imaging. To assess the contribution of the KLK6 expression on the Apc-mutant intestinal and colon tumorigenesis, we engineered a mouse with floxed alleles of the Klk6 gene (Klk6lox/lox) and crossed it with a mouse expressing the truncated APC protein under control of the intestinal tract-specific human CDX2P9.5-NLS Cre transgene (CPC;Apcfl/fl;Klk6+/+). We found that CPC;Apcfl/fl mice with disrupted Klk6 gene expression (CPC;Apcfl/fl;Klk6fl/fl) had a significantly smaller average size of the small intestinal and colon crypts (p < 0.001 and p = 0.04, respectively) and developed a significantly fewer adenomas (p = 0.01). Moreover, a decrease in high-grade adenomas (p = 0.03) and adenomas with a diameter above 2 mm (p < 0.0001) was noted in CPC;Apcfl/fl;Klk6fl/fl mice. Further molecular analysis showed that Klk6 gene inactivation in the small intestine and colon tissues of CPC;Apcfl/fl;Klk6fl/fl mice resulted in a significant suppression of transforming growth factor β2 (TGF-β2) protein (p ≤ 0.02) and mitogen-activated protein kinase (MAPK) phosphorylation (p ≤ 0.01). These findings demonstrate the oncogenic role of KLK6 in the mutant Apc-mediated intestinal tumorigenesis and suggest the utility of KLK6 for early diagnosis of colorectal tumors.

Phosphorylated protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) expression in breast cancer is correlated with malignant proliferation and histological grading.

This study aims to detect the expression of phosphorylated PERK in breast cancer using immunohistochemistry and explore its significance. We examined 134 cases of formalin-fixed and paraffin-embedded breast cancer tissues. It was found that the expression of phosphorylated PERK in ductal carcinoma was higher than that in lobular carcinoma, and the difference between them was statistically significant, suggesting that phosphorylated PERK played different roles in the occurrence and development of different types of breast cancer. Compared with Ki-67-negative breast cancer tissues, phosphorylated PERK has higher expression in Ki-67-positive tissues and is positively correlated with Ki67 expression, indicating that phosphorylated PERK plays an important role in breast cancer's malignant proliferation and progression. We also found a positive correlation between phosphorylated PERK expression and the histological grading of invasive ductal carcinoma, indicating that phosphorylated PERK plays an important role in the differentiation of invasive ductal carcinoma. Our study revealed the differential expression of phosphorylated PERK in subtypes of breast cancer. It contributed to the malignant proliferation of breast cancer and tissue differentiation of invasive ductal carcinoma of the breast.

Avenanthramides Ameliorate Insulin Resistance by Modulating Gluconeogenesis and Glycogen Synthesis in HepG2 Cells.

Diabetes mellitus (DM) is a multifaceted metabolic condition, mainly defined by elevated blood glucose levels. A feature of type 2 DM includes insulin resistance (IR), which involves impairments within the insulin signaling pathways. Avenanthramides (AVNs) are phenolic alkaloids found in Avena sativa L. The major AVNs are AVN A, AVN B, and AVN C. They have been reported to offer benefits in preventing inflammation, cancer, and cardiovascular diseases. However, the effects of AVNs on the liver glucose metabolism pathways remain unknown. This study examined the effects and underlying mechanisms through which AVNs alleviate IR induced by free fatty acid (FFA) in HepG2 cells. The results indicated that FFA treatment significantly decreased glucose consumption by 34.54% compared to the control. However, treatments with AVN A, B, and C at 100 μM increased glucose uptake by 57.93%, 58.28%, and 53.10%, respectively, compared to FFA treatment alone. This effect occurs through the increased expression of glucose transporter 4. Furthermore, AVNs significantly enhanced the glycogen content. AVNs induced increased phosphorylation of insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt). AVNs treatment decreased the levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in HepG2 cells. This effect was attributed to AMP-activated protein kinase activation and inhibition of forkhead box protein O1. Collectively, these results suggest that AVNs regulate glucose metabolism by activating the IRS-1/PI3K/Akt pathway, which is related to glycogen synthesis, and by inhibiting key molecules that promote gluconeogenesis.

Metformin inhibits migration and epithelial-to-mesenchymal transition in non-small cell lung cancer cells through AMPK-mediated GDF15 induction

The growth differentiation factor 15 (GDF15) may serve as a biomarker of metformin, which mediates the bodyweight lowering effect of metformin. However, whether GDF15 also serves as a molecular target of metformin to inhibit carcinogenesis remains largely unknown. This study examined the role and molecular mechanisms of GDF15 in the anticancer effects of metformin in non-small cell lung cancer (NSCLC) cells, which has never been reported before. We found that metformin significantly inhibited the migration of NSCLC A549 and NCI-H460 cells and reduced the expression of epithelial-to-mesenchymal transition (EMT)-related molecules, including neuro-cadherin (N-cadherin), matrix metalloproteinase 2 (MMP2), and the zinc finger transcription factor Snail, but increased epithelial cadherin (E-cadherin) expression. Furthermore, metformin increased GDF15 and its upstream transcription factors activated transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) expressions and increased AMP-activated protein kinase (AMPK) phosphorylation in NSCLC cells. GDF15 siRNA partially reverses the inhibitory effect of metformin on NSCLC cell migration. Moreover, metformin-induced increases in GDF15, CHOP, and ATF4 expression and the inhibition of migration were partially reversed by treatment with Compound C, a specific AMPK inhibitor. Meanwhile, metformin significantly inhibited NCI-H460 xenograft tumor growth in nude mice, increased GDF15 expression, and regulated EMT- and migration-related protein expression in xenograft tumors. In conclusion, our results provide novel insights into revealing that GDF15 can serve as a potential molecular target of metformin owing to its anti-cancer effect in NSCLC, which is mediated by AMPK activation.

GPER-1 Rapid Regulation Influences p-Akt Expression to Resist Stress-Induced Injuries in a Sex-Specific Manner.

G protein-coupled estrogen receptor 1 (GPER-1) has gained recognition for its role in conferring cardioprotection. However, the extent to which GPER-1 exerts equally important effects in both sexes remains unclear. The study found similar expressions of GPER-1 in rat heart apex in both sexes. In male rats, administering epinephrine (Epi) at a dose of 31.36 microg/100 g resulted in a rapid decline in cardiac function, accompanied by a sharp increase in bax/bcl-2 levels. In contrast, female rats did not display significant changes in cardiac function under the same conditions. Additionally, compared to the injection of Epi alone (at a dose of 15.68 microg/100 g), the administration of G15 (GPER-1 antagonist) further decreased cardiac function in both male and female rats. However, it only increased mortality and lung coefficient in male rats. Conversely, G1 (GPER-1 agonist) administration improved cardiac function in both sexes. Notably, the apex of the male heart exhibited lower levels of inhibitory G protein (Galphai). Furthermore, female and male rats treated with Epi displayed elevated phosphorylated protein kinase B (p-Akt). Compared to their respective Epi groups, the administration of G15 increased p-Akt levels in female rat hearts but decreased them in male rat hearts. Conversely, the administration of G1 decreased p-Akt levels in females but rapidly increased them in male rats. Our study uncovers the vital role of GPER-1 in protecting against stress-induced heart injuries in a sex-specific manner. These findings hold immense potential for advancing targeted cardiac therapies and enhancing outcomes for both females and males.

Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer.

Bladder cancer (BC) is a common malignancy and its most prevalent type is urothelial carcinoma, which accounts for ~90% of all cases of BC. The current treatment options for BC are limited, which necessitates the development of alternative treatment strategies. Hyperthermia (HT), as an adjuvant cancer therapy, is known to improve the efficacy of chemotherapy or radiotherapy. The present study aimed to investigate the anti‑tumor effects of HT on cell survival, invasiveness, chemoresistance and immune evasion in human BC cell lines (5637, T24 and UMUC3). Calcein AM staining was performed to analyze the cytotoxicity of natural killer (NK) cells against human BC cells following HT treatment. Cell migration and invasion affected by HT were analyzed using Transwell migration and invasion assays. It was found that HT inhibited the proliferation of BC cells by downregulating the phosphorylation of protein kinase B. Moreover, HT effectively enhanced the sensitivity of BC cells to the chemotherapy drug cisplatin (DDP) and reduced the chemoresistance of DDP‑resistant cells by downregulating the expression of cadherin‑11. It was further demonstrated that HT inhibited the migration and invasion of BC cells and enhanced the cytotoxic effects of NK cells. In summary, the antineoplastic effects of HT were mediated through three main mechanisms: Enhancement of the chemosensitivity of BC cells and mitigation of DDP‑induced chemoresistance, suppression of the invasive potential of BC cells and reinforcement of the anticancer response of NK cells. Thus, HT appears to be a promising adjunctive therapy for human BC.

TLR3 activation enhances antitumor effects of sorafenib in hepatocellular carcinoma by activating NK cell functions through ERK and NF-κB pathways

Background Sorafenib is a standard therapeutic agent for advanced hepatocellular carcinoma (HCC). However, its efficacy is moderate, as the survival of patients is prolonged for only a few months, and the response rate is low. The mechanism of low efficacy remains unclear. In this study, we investigated the effect of Toll-like receptor 3 (TLR3) on the effects of sorafenib on HCC. Methods Polyinosinic-polycytidylic acid [poly(I: C)] was used as a double-stranded RNA analog and TLR3 agonist in subsequent experiments. After orthotopic implantation of HCC tumors in BALBc nu/nu or C57BL/6 mice, survival time, tumor growth, and metastasis in the abdomen and lungs were analyzed. Flow cytometry and cytotoxicity assays were used to analyze NK cells isolated from the spleen or peripheral blood. ELISA was used to detect the expression of plasma interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1. In addition, the expression of phosphorylated-extracellular regulated kinase 1/2 (pERK1/2), phosphorylated-protein kinase B (pAKT), ERK1/2 and AKT was analyzed by Western blotting. Results Sorafenib reduced the number and activity of NK cells in tumor-bearing mice and simultaneously decreased the levels of MCP-1 and IFN-γ in the plasma. The combination of sorafenib and poly(I: C) synergistically inhibited tumor growth and metastasis in tumor xenograft mice and prolonged survival. Poly(I: C) not only exerts a direct inhibitory effect on tumor growth and metastasis by targeting the TLR3 receptor on tumor cells but also facilitates the proliferation and activation of NK cells, indirectly impeding tumor progression. Mechanistically, poly(I: C) decreased the sorafenib-induced inhibition of ERK phosphorylation and increased the phosphorylation of IκB in NK cells, thereby enhancing NK cell function. Conclusion Activation of TLR3 can enhance the antitumor effect of sorafenib on HCC. The combination of a TLR3 activator and sorafenib may be a new strategy for the treatment of HCC.

Chitosan oligosaccharides: A potential therapeutic agent for inhibiting foam cell formation in atherosclerosis

Foam cell formation is a key hallmark in atherosclerosis and associated cardiovascular diseases (CVDs). The potential anti-atherosclerotic potential of chitosan oligosaccharides (COS) was investigated using oxLDL-treated RAW264.7 murine cells. COS treatment led to a significant inhibition of lipid accumulation, as demonstrated by Oil Red O staining, and reduced levels of total cholesterol, free cholesterol, cholesterol esters, and triglycerides in.oxLDL-treated RAW264.7 cells. COS blocked cholesterol influx through down-regulating class A1 scavenger receptors (SR-A1) and cluster of differentiation 36 (CD36) expression and stimulated cholesterol efflux through up-regulating ABC transporters ABCA-1 and ABCG-1 expressions. Additionally, COS treatment stimulated nuclear signaling pathways involving peroxisome proliferator-activated receptor-γ (PPAR-γ) and liver X receptor α (LXR-α), and also led to the phosphorylation of AMP-activated protein kinase (AMPK). COS further demonstrated anti-inflammatory effects by inhibiting the production of pro-inflammatory cytokines and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in oxLDL-treated RAW264.7 cells, through suppression of NF-κB signaling. Furthermore, COS alleviated oxidative stress induced by oxLDL by activating Nrf2 signaling and enhancing the expression of antioxidant genes, including heme oxygenase-1 (HO-1), superoxide dismutase (SOD), glutathione peroxidase (Gpx), and catalase (CAT). In conclusion, COS can be beneficial in preventing atherosclerosis and related diseases.

A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

Exvivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering exvivo culture necessary yet detrimental. Mechanistically, exvivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.