Expression Of Apoptosis-related Proteins Research Articles

Hispidin Increases Cell Apoptosis and Ferroptosis in Prostate Cancer Cells Through Phosphatidylinositol-3-Kinase and Mitogen-activated Protein Kinase Signaling Pathway.

Chemotherapy is mainly used in the clinical treatment of prostate cancer. Different anticancer mechanisms can induce cell death in various cancers. Reactive oxygen species (ROS) play crucial roles in cell proliferation, differentiation, apoptosis, and signal transduction. It is widely accepted that ROS accumulation is closely related to chemical drug-induced cancer cell death. We utilized the MTT assay to detect changes in cell proliferation. Additionally, colony formation and wound healing assay were conducted to investigate the effect of hispidin on cell colony formation and migration ability. Fluorescence microscopy was used to detect intracellular and mitochondrial ROS levels, while western blot was used for detection of cell apoptosis. Hispidin treatment significantly decreased viability of PC3 and DU145 cancer cells but exhibited no cytotoxicity in WPMY-1 cells. Furthermore, hispidin treatment inhibited cell migration and colony formation and triggered cellular and mitochondrial ROS accumulation, leading to mitochondrial dysfunction and mitochondrion-dependent apoptosis. Moreover, hispidin treatment induced ferroptosis in PC3 cells. Scavenging of ROS with N-acetyl cysteine significantly inhibited hispidin-induced apoptosis by altering the expression of apoptosis-related proteins, such as cleaved caspase-3, 9, Bax, and Bcl2. Furthermore, hispidin treatment dramatically up-regulated MAPK (involving p38, ERK, and JNK proteins) and NF-kB signaling pathways while down-regulating AKT phosphorylation. Hispidin treatment also inhibited ferroptosis signaling pathways (involving P53, Nrf-2, and HO-1 proteins) in PC3 cells. In addition, inhibiting these signaling pathways via treatment with specific inhibitors significantly reversed hispidin-induced apoptosis, cellular ROS levels, mitochondrial dysfunction, and ferroptosis. Hispidin may represent a potential candidate for treating prostate cancer.

Role of C/EBP Homologous Protein (CHOP) and Nupr1 Interaction in Endoplasmic Reticulum Stress-Induced Apoptosis of Lens Epithelial Cells.

Our study mainly analyzed the mechanism of C/EBP homologous protein (CHOP) and its interacting protein Nupr1 on endoplasmic reticulum stress (ERS) induced lens epithelial cells (LEC) apoptosis. Cell proliferation was detected by CCK-8. Apoptosis was detected by flow cytometry and TUNEL. Nupr1 expressionwas detected byRT-qPCR. The expressions of CHOP, Nupr1, apoptosis-related protein, and ERS-related protein were detected by Western blot. DCFH-DA probe was used to detect cell ROS. The SOD, GSH-PX, and MDA contents were detected by the kit. Co-IP was used to detect the interaction between CHOP and Nupr1. The morphology of the lens was detected by HE staining. The result shows thatTunicamycin (TU) can induce endoplasmic reticulum stress and apoptosis in LEC in a concentration-dependent manner. TU induction leads to the occurrence of CHOP nuclear translocation. Overexpression of CHOP can further enhance the inhibitory effect of TU on LEC proliferation and the promotion of apoptosis, while knockdown of CHOP has the opposite effect. CHOP and Nupr1 are interacting proteins, and knockdown of Nupr1 or addition of Nupr1 inhibitor ZZW-115 can reverse the effects of TU and overexpression of CHOP, respectively. It has been observed in animal experiments that treatment with oe-CHOP can further aggravate the pathological lesions of the rat lens, while ZZW-115 can reverse the effect of oe-CHOP to a certain extent and improve the lesions of the rat lens. Overall,CHOP interacts with Nupr1 to regulate apoptosis caused by ERS and mediate cataract progression in rats, and this study provides a new potential therapeutic target for the treatment of cataract.

Radix Actinidiae chinensis induces the autophagy and apoptosis in renal cell carcinoma cells

BackgroundRenal cell carcinoma (RCC) is a malignant tumor. Radix Actinidiaechinensis (RAC) is the root of Actinidia arguta (Sieb. et Zucc) Planch. ex Miq. In clinical research, RAC was confirmed to have a certain anti-tumor effect, including liver cancer and cholangiocarcinoma. This study investigated the anticancer effect and mechanism of RAC in RCC cells.MethodsThe 786-O and A498 cells were intervened with varying concentrations of RAC (0–100 mg/mL) to detect the half maximal inhibitory concentration (IC50) of RAC. The cells were then co-cultured with 0–50 mg/mL RAC for 0–72 h to assess the effect of RAC on cell viability using the cell counting kit-8. The effects on cell proliferation, cell cycle or apoptosis, migration or invasion, and autophagy were detected using cloning, flow cytometry, Transwell, AOPI assay and Western blot. The number of autophagolysosomes was quantified using a transmission electron microscope. PI3K/AKT/mTOR pathway-related proteins were detected by Western blot. Additionally, an autophagy inhibitor 3-MA was used to explore the underlying mechanism of RAC.ResultsIC50 values of RAC in 786-O and A498 were 14.76 mg/mL and 13.09 mg/mL, respectively. RAC demonstrated the ability to reduce the cell malignant phenotype of RCC cells, blocked the S phase of cells, promoted apoptosis and autophagy in cells. Furthermore, RAC was observed to increase autophagy-related proteins LC3II/I and Beclin-1, while decreasing the level of P62. The expression of apoptosis-related proteins was increased, while the ratios of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, p-P38/P38 and p-ERK/ERK were reduced by RAC. However, the addition of 3-MA reduced the apoptosis and autophagy- promotion effects of RAC on RCC cells.ConclusionRAC induced the apoptosis and autophagy, to inhibit the progression of RCC cells. This study may provide a theoretical and experimental basis for clinical anti-cancer application of RAC for RCC.

PRG4 represses the genesis and metastasis of osteosarcoma by inhibiting PDL1 expression

BackgroundOsteosarcoma is originated from skeletal system. Recombinant human proteoglycan 4 (rhPRG4) can inhibit cell proliferation and migration in multiple cancers. This research is designed to dig out the role and mechanism of PRG4 in osteosarcoma. MethodsHuman osteosarcoma cell lines, MG63 and 143B, were transfected with programmed death 1 (PD-L1) overexpression vectors and/or treated with 20, 50, and 100 μg/mL rhPRG4, followed by the determination of cell viability, colony formation, sphere formation, invasion, migration, apoptosis, and the expressions of matrix metalloproteinases (MMPs), PD-L1 and apoptosis-related proteins. Tumor-bearing mouse models were constructed by injection of 143B cells and treatment of anti-PD-L1 antibody and/or adenovirus PRG4 (AdPRG4). Tumor volume was monitored, and immunohistochemical location of Ki67 was performed. Expressions of MMPs, transforming growth factor-β (TGF-β), PD-L1, and epithelial mesenchymal transition (EMT)-related proteins were measured in tumors. ResultsRhPRG4 (20, 50, and 100 μg/mL) inhibited the viability, colony formation, sphere formation, invasion, migration, and the expressions of MMP2, MMP9 and Bcl2 in osteosarcoma cells, while promoting cell apoptosis as well as Bax and c-caspase3 expressions, at a dose-dependent manner; by contrast, PD-L1 overexpression reversed the above effects of 100 μg/mL rhPRG4. AdPRG4 or anti-PD-L1 antibody decreased tumor volume, number of pulmonary metastasis nodule, Ki67 location, and expressions of TGF-β, PD-L1, MMP2, MMP9, Vimentin, and Snail, but increased E-cadherin expression in tumor cells. Moreover, anti-PD-L1 antibody and AdPRG4 together functioned more effectively than them alone in reducing tumor burden. ConclusionPRG4 represses the genesis and metastasis of osteosarcoma via inhibiting PD-L1 expression, and AdPRG4 enhances the effectiveness of anti-PD-L1 therapy.

Synthesis of triazole AD-1 derivatives and its mechanism of mediating DNA damage of ROS in lung cancer cells

Based on the significant biological activities and the remarkable physical and chemical properties of 1H-1,2,3-triazole pharmacophore, we herein adopted the strategy of click chemistry to combine the triazole fragment and the unique scaffold of 25-OCH3-PPD (AD-1) to design a series of potent compounds inducing apoptosis and DNA damage. The anti-proliferative effect was verified by MTT assay and colony formation assay. DNA double-stand breaks (DSBs) were obtained by observing the nuclear focus formation and the protein expression of γ-H2AX. Cell cycle arrest was evaluated by the cycle-related proteins such as CDK2, CDK4, CDK6, Cyclin D1 and P21. Apoptosis was assessed by flow cytometry, mitochondrial membrane potential (MMP) detection and the expression of apoptosis-related proteins. Reactive oxygen species (ROS) generation was measured with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) staining. According to SAR analysis, the most potent compound 6a exhibited great inhibitory effect against A549 cells, which IC50 value of 2.84 ± 0.68 μM. Furthermore, 6a remarkably induced DNA damage, cell cycle arrest and apoptosis in A549 cells. 6a treatment increased the levels of ROS. Network pharmacology and molecular docking predicted the potential signaling pathways and ligand-receptor interactions, and the results of western blotting showed that 6a inhibited the PI3K/Akt/Bcl-2 signaling pathway by decreasing PI3K and Bcl-2 and total level of Akt expression, while Bax and Cyt c were increasing in 6a-treated A549 cells. As mentioned above, 6a has a potent inhibitory effect in A549 cells through induction of DNA damage, apoptosis via ROS generation and modulation of PI3K/Akt/Bcl-2 signaling pathway.

Mechanistic exploration and experimental validation of the Xiaochaihu decoction for the treatment of breast cancer by network pharmacology.

Xiaochaihu (XCH) decoction is a traditional Chinese prescription that has been recorded in the pharmacopeia of the People's Republic of China. In China, the XCH decoction is used clinically to treat a variety of tumors, including breast cancer. However, its potential mechanism of action is still undefined. The chemical compounds in the XCH decoction were identified via Q Exactive Orbitrap LC-MS/MS. Then, we screened the active ingredients and targets in the XCH decoction from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Next, Cytoscape and Metascape were used to construct an active ingredient-target-disease network, which included a protein-protein interaction (PPI) network, GO enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, we used molecular docking and in vitro experiments to verify the results of network pharmacology analysis. More than 70 major compounds were identified by Q Exactive Orbitrap LC-MS/MS analysis from the XCH decoction. A total of 162 active ingredients and 153 targets related to the XCH decoction and breast cancer were identified, and a compound-target-disease network was constructed. GO and KEGG analyses revealed that the XCH decoction regulated the drug response, apoptosis process, cancer pathway, and PI3K/Akt signaling pathway. Molecular docking and experimental validation indicated that the XCH decoction suppressed proliferation and induced apoptosis in breast cancer cells by regulating the expression of apoptosis-related proteins and inhibiting the PI3K/Akt pathway. This study suggested that the XCH decoction can be used to treat breast cancer by inhibiting cell proliferation, inducing apoptosis and downregulating the PI3K/Akt signaling pathway.

MiR-30a-5p attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by regulating PTEN protein expression and activating PI3K/Akt signaling pathway

BackgroundThis study was designed to investigate the mechanism by which miR-30a-5p mediates cardiomyocyte apoptosis after acute myocardial infarction (AMI) induced by hypoxia/reoxygenation (H/R).MethodsDifferentially expressed miRNAs were analyzed by RNA high-throughput sequencing in acute myocardial infarction (ST-elevation myocardial infarction) patients versus healthy individuals (controls). The H/R model was used to assess the regulatory mechanism of miRNAs in AMI. Lentivirus-associated vectors were used to overexpress or knock down miR-30a-5p in cellular models. The pathological mechanisms of miR-30a-5p regulating the development of acute myocardial infarction were serially explored by qPCR, bioinformatics, target gene prediction, dual luciferase, enzyme-linked immunosorbent assays (ELISAs) and Western blotting.ResultsThe results showed that the expression of miR-30a-5p was significantly increased in AMI patients and H9C2 cells. Hypoxia decreased cardiomyocyte survival over time, and reoxygenation further reduced cell survival. Bax and Phosphatase and tensin homolog (PTEN)were suppressed, while Bcl-2 was upregulated. Additionally, miR-30a-5p specifically targeted the PTEN gene. According to the GO and KEGG analyses, miR-30a-5p may participate in apoptosis by interacting with PTEN. The miR-30a-5p mimic decreased the expression of apoptosis-related proteins and the levels of the proinflammatory markers IL-1β, IL-6, and TNF-α by activating the PTEN/PI3K/Akt signaling pathway. Conversely, anti-miR-30a-5p treatment attenuated these effects. Additionally, silencing PTEN and anti-miR-30a-5p had opposite effects on H/R-induced cell apoptosis.ConclusionsmiR-30a-5p plays a crucial role in cardiomyocyte apoptosis after hypoxia-induced acute myocardial infarction. Our findings provide translational evidence that miR-30a-5p is a novel potential therapeutic target for AMI.

CCR5 antagonist maraviroc alleviates doxorubicin-induced neuroinflammation and neurobehavioral deficiency by regulating NF-κB/NLRP3 signaling in a breast cancer mouse model

The chemotherapeutic agent Doxorubicin (DOX) is known to cause chemotherapy-induced cognitive impairment (CICI). Maraviroc, a potent C–C chemokine receptor 5 (CCR5) antagonist, shows neuroprotective properties, while its role in CICI remains unclear. This study determined the therapeutic potential of maraviroc on CICI. Adult C57BL/6J mice with implanted breast cancer cells received four weekly intraperitoneal injections of saline (Control group), 5 mg/kg DOX (DOX group), 10 mg/kg maraviroc (MVC group), or 5 mg/kg DOX with 10 mg/kg maraviroc (DOX + MVC group). The Morris Water Maze (MWM) was used for neurobehavioural test. Western blot analysis and immunofluorescence were used to evaluate the expressions of inflammatory markers, apoptosis-related proteins, and synaptic-related proteins. The volume and weight of tumor were also evaluated after treatments. DOX treatment significantly increased chemokines (CCL3, CCL4) and inflammatory cytokines (IL-1β, TNF-α) in tumor-bearing mice hippocampus. While maraviroc administration reduced hippocampal proinflammatory factors compared to the DOX group. Furthermore, it also lowered apoptosis markers, restored synaptic proteins levels, and inhibited the NF-κB/NLRP3 pathway. Accordingly, maraviroc treatment significantly improved DOX-induced neurobehavioural impairments as evidenced by an increased number of platform crossings and percentage of target quadrant time in the MWM test. Additionally, when combined with DOX, maraviroc had additional inhibitory effects on tumor growth. These findings suggest that maraviroc can mitigate DOX-induced CICI by suppressing elevated proinflammatory chemokines and cytokines through the NF-κB/NLRP3 pathway, potentially offering an anti-tumor benefit. This research presents a promising therapeutic approach for DOX-induced CICI, enhancing the safety and efficacy of cancer treatments.

Downregulation of miR-4284 can Inhibit the Apoptosis of Human Arterial Smooth Muscle Cells (HASMCs) in Arteriosclerosis Obliterans (ASO).

The disease arteriosclerosis obliterans (ASO) affects the lower extremities. ASO's mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). The miR-4284 is involved in several biological processes of the cardiovascular system, including VSMC proliferation, migration, and death. However, it is unknown if the miR-4284 gene is involved in the control of ASO. Furthermore, the molecular processes behind the contribution of human arterial smooth muscle cells (HASMCs), one of the most significant components of the arterial wall, to arteriosclerosis obliterans (ASO) pathogenesis remain unknown. Previously, we explored the alterations of miRNAs in the blood of ASO patients, and now we wanted to test further whether these changes also take place in the HASMCs that are responsible for the pathogenesis of ASO. The expression levels of miR-29a in arterial walls were analyzed via a real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR- 4284 on HASMCs. The Transwell system and CCK-8 detection were used to assess the migration and proliferation of HASMCs. The proportion of apoptotic cells as well as the concentrations of apoptotic signal protein production were assessed using flow cytometry. A Western blot technique was used to identify B cell lymphoma-2 (Bcl2), Bcl2-associated X protein (BAX), as well as Xlinked inhibitors of apoptosis protein (XIAP). The results showed that PCR confirmed that the qualified production or expression of miR-4284 was significantly reduced in HASMCs after they were cultured without FBS and in an atmosphere of 1% O2 + 5% CO2 + 94% N2 and that glucose had no effect on its expression. MiR- 4284 has no effect on migration and proliferation, but downregulation of miR-4284 can decrease the apoptotic rate of HASMCs, as revealed by flow cytometry. Furthermore, western blot experiments showed that the expression of BAX was low, while the expression of the other two proteins, viz., Bcl2 and XIAP, was over-expressed. We found that miR-4284 downregulation enhanced Bcl2, as well as XIAP, and decreased Bax. This shows that downregulated miR-4284 regulates apoptosis-related protein expression in HASMCs. The mechanism is not clear, and we need further study to confirm it.

YBX1 inhibits mitochondrial-mediated apoptosis in ischemic heart through the PI3K/AKT signaling pathway

Abstract Background Myocardial infarction (MI) is associated with higher morbidity and mortality in the world, especially in cold weather. YBX1 is an RNA-binding protein that is required for pathological growth of cardiomyocyte by regulating cell growth and protein synthesis. But YBX1, as an individual RNA-binding protein, regulates cardiomyocytes through signaling cascades during myocardial infarction remain largely unexplored. Methods In vivo, the mouse MI model was induced by ligating the left anterior descending coronary artery (LAD), and randomly divided into sham operation group, MI group, MI+ YBX1 knockdown/overexpression group and MI+ negative control (NC) group. The protective effect of YBX1 was verified by echocardiography and triphenyltetrazolium chloride staining. In vitro, mitochondrial-dependent apoptosis was investigated by using CCK8, TUNEL staining, reactive oxygen species (ROS) staining and JC-1 staining in hypoxic neonatal mouse cardiomyocytes (NMCMs). Results YBX1 expression of cardiomyocytes was downregulated in a mouse model and a cellular model on the ischemic condition. Compared to mice induced by MI, YBX1 overexpression mediated by adeno-associated virus serotype 9 (AAV9) vector reduced the infarcted size and improved cardiac function. Knockdown of endogenous YBX1 by shRNA partially aggravated ischemia-induced cardiac dysfunction. In hypoxic cardiomyocytes, YBX1 overexpression decreased lactic dehydrogenase (LDH) release, increased cell viability, and inhibited apoptosis by affecting the expression of apoptosis related proteins, while knockdown of endogenous YBX1 by siRNA had the opposite effect. Overexpression of YBX1 restored mitochondrial dysfunction in hypoxic NMCMs by increasing mitochondrial membrane potential and ATP content and decreasing ROS. In hypoxic NMCMs, YBX1 overexpression increased the expression of phosphorylated phosphatidylinositol 3 kinase (PI3K)/AKT, and the antiapoptosis effect of YBX1 was eliminated t by LY294002, PI3K/AKT inhibitor. Conclusion YBX1 protected the heart from ischemic damage by inhibiting the mitochondrial-dependent apoptosis through PI3K/AKT pathway. It is anticipated that YBX1 may serve as a novel therapeutic target for MI.

SBFI-26 enhances apoptosis in docetaxel-treated triple-negative breast cancer cells by increasing ROS levels

Introduction: Fatty acid binding protein 5 (FABP5) exhibits heightened expression levels in triple-negative breast cancer. The inhibitor of FABP5, Stony Brook fatty acid‐binding protein inhibitor 26 (SBFI-26), has demonstrated the capacity to suppress cell proliferation, migration, and invasion. This study delves into the functional mechanism and impact of combining SBFI-26 with docetaxel in treating MDA-MB-231 cells of triple-negative breast cancer. Methods: Various concentrations of docetaxel and SBFI-26 were chosen for individual or combined treatments. The effects of SBFI-26, docetaxel, or their combination on cell cycle arrest and apoptosis were assessed using flow cytometry. Western blotting was utilised to detect the expression of apoptosis-related proteins, namely cysteinyl aspartate-specific proteases 3 (Caspase3), B cell leukemia/lymphoma 2 (Bcl-2), and Bcl-2 associated X (Bax), while intracellular reactive oxygen species (ROS) levels were determined using a fluorescence spectrophotometer. Results: The IC50 values for SBFI-26 and docetaxel in inhibiting MDA-MB-231 cells were determined to be 106.1 μM and 86.14 nM, respectively. Significantly, the combination treatment augmented the proportion of G1 phase (apoptotic) cells by 3.67-fold compared to the control group (P < 0.0001). Furthermore, the apoptosis rate in the combination group was 2.59-fold higher than that in the docetaxel group (P < 0.0001) and demonstrated a significant increase of 1.82-fold compared with the SBFI-26 group (P < 0.001). Analyses revealed a decrease in the protein expression of Bcl-2, while Bax and Caspase3 exhibited an increase in the combination group for MDA-MB-231 cells. Moreover, the combined treatment group demonstrated a 2.97-fold increase (P < 0.0001) in ROS fluorescence intensity compared to the control group, a noteworthy 1.39-fold increase (P < 0.01) compared to the SBFI-26 treatment group, and a substantial 1.70-fold increase (P < 0.0001) compared to the docetaxel treatment group. Conclusion: These findings suggest that the co-administration of SBFI-26 with docetaxel effectively enhances apoptosis in triple-negative breast cancer MDA-MB-231 cells by elevating intracellular ROS levels.

Anlotinib Inhibiting Mantle Cell Lymphoma Proliferation and Inducing Apoptosis through PI3K/AKT/mTOR Pathway.

This study investigates the inhibitory mechanism of anlotinib on human Mantle Cell Lymphoma (MCL) cells through in vitro and in vivo experiments. In vitro cellular experiments validate the effects of anlotinib on MCL cell proliferation and apoptosis. Moreover, a subcutaneous xenograft nude mice model of Mino MCL cells was established to assess the anti-tumour effect and tumour microenvironment regulation of anlotinib in vivo. The results indicate that MCL cell proliferation was significantly inhibited upon anlotinib exposure. The alterations in the expression of apoptosis-related proteins further confirm that anlotinib can induce apoptosis in MCL cells. Additionally, anlotinib significantly reduced the PI3K/Akt/mTOR phosphorylation level in MCL cells. The administration of a PI3K phosphorylation agonist, 740YP, could reverse the inhibitory effect of anlotinib on MCL. In the xenograft mouse model using Mino MCL cells, anlotinib treatment led to a gradual reduction in body weight and a significant increase in survival time compared to the control group. Additionally, anlotinib attenuated PD-1 expression and elevated inflammatory factors, CD4, and CD8 levels in tumour tissues. Anlotinib effectively inhibits proliferation and induces apoptosis in MCL both in vitro and in vivo. This inhibition is likely linked to suppressing phosphorylation in the PI3K/Akt/mTOR pathway.

Ethanolic extract of Euphorbia Gypsicola induces differentiation and apoptosis in human myeloid leukemia K562 cells

IntroductionThe Euphorbiaceae family, widely distributed and long utilized in traditional medicine, has been recognized for its potential in developing anti-cancer drugs. The current study investigates the capacity of Euphorbia gypsicola extract to induce apoptosis and differentiation in the human myeloid leukemia K562 cell line. MethodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic activity was visually evaluated through acridine orange/ethidium bromide (AO/EB) dye staining and annexin V/PI double labeling procedures. Induction of differentiation was examined using nitro blue tetrazolium (NBT) reduction and Wright-Giemsa staining assays. The expression of apoptosis-related proteins including Bax, Bcl2 and Survivin was evaluated by western blotting method. ResultsThe E. gypsicola extract exhibited potent medication properties with IC50 values of 207.59, 48.58, and 7.08 ng/mL at 24, 48, and 72 h, respectively. Fluorescence microscopy, DNA fragmentation, annexin V/PI double staining, and sub-G1 cell cycle arrest confirmed apoptosis occurrence in K562 cells treated with the E. gypsicola extract. Our findings demonstrated that differentiated cells reduced NBT yellow solution after endocytosis, forming dark crystals. Wright Giemsa staining observations indicated a decrease in nuclear volume-to-cytoplasm ratio in K562 cells treated with the crude extract, signifying Induction of differentiation. Furthermore, western blot analysis revealed down-regulation of Survivin protein expression and up-regulation of Bax protein level following treatment with the E. gypsicola extract. However, no significant decrease in Bcl2 protein expression level was observed in K562 cells. ConclusionThese findings indicate the growth inhibitory effects of the E. gypsicola extract on leukemia K562 cells, which supports the further study of this plant to discover novel therapeutic compounds for the treatment of leukemia.

An herbal formulation “Shugan Xiaozhi decoction” ameliorates methionine/choline deficiency-induced nonalcoholic steatohepatitis through regulating inflammation and apoptosis-related pathways

Ethnopharmacological relevanceShugan Xiaozhi (SGXZ) decoction is a traditional Chinese medicine used for treating nonalcoholic steatohepatitis (NASH). It has been used clinically for over 20 years and proved to be effective; however, the molecular mechanism underlying the effects of SGXZ decoction remains unclear. Aim of the studyWe analyzed the chemical components, core targets, and molecular mechanisms of SGXZ decoction to improve NASH through network pharmacology and in vivo experiments. Materials and methodsThe chemical components, core targets, and related signaling pathways of SGXZ decoction intervention in NASH were predicted using network pharmacology. Molecular docking was performed to verify chemical components and their core targets. The results were validated in the NASH model treated with SGXZ decoction. Mouse liver function was assessed by measuring ALT and AST levels. TC and TG levels were determined to evaluate lipid metabolism, and lipid deposition was assessed via oil red O staining. Mouse liver damage was determined via microscopy following hematoxylin and eosin staining. Liver fibrosis was assessed via Masson staining. Western blot (WB) and immunohistochemical (IHC) analyses were performed to detect inflammation and the expression of apoptosis-related proteins, including IL-1β, IL-6, IL-18, TNF-α, MCP1, p53, FAS, Caspase-8, Caspase-3, Caspase-9, Bax, Bid, Cytochrome c, Bcl-2, and Bcl-XL. In addition, WB and IHC were used to assess protein expression associated with the TLR4/MyD88/NF-κB pathway. ResultsQuercetin, luteolin, kaempferol, naringenin, and nobiletin in SGXZ decoction were effective chemical components in improving NASH, and TNF-α, IL-6, and IL-1β were the major core targets. Molecular docking indicated that these chemical components and major core targets might interact. KEGG pathway analysis showed that the pathways affected by SGXZ decoction, primarily including apoptosis and TLR4/NF-κB signaling pathways, interfere with NASH. In vivo experiments indicated that SGXZ decoction considerably ameliorated liver damage, fibrosis, and lipid metabolism disorder in MCD-induced NASH mouse models. In addition, WB and IHC verified the underlying molecular mechanisms of SGXZ decoction as predicted via network pharmacology. SGXZ decoction inhibited the activation of apoptosis-related pathways in MCD-induced NASH mice. Moreover, SGXZ decoction suppressed the activation of TLR4/MyD88/NF-κB pathway in MCD-induced NASH mice. ConclusionSGXZ decoction can treat NASH through multiple targets and pathways. These findings provide new insights into the effective treatment of NASH using SGXZ decoction.

LINC00942 Alleviates NaAsO2-induced Apoptosis by Promoting GSH Synthesis Through Targeting miR-214-5p.

The mechanism of arsenic-induced liver toxicity is not fully understood. This study aimed to investigate the role of LINC00942 in arsenic-induced hepatotoxicity by regulating miR-214-5p. As the exposure dose of NaAsO2 gradually increases, cell viability, intracellular GSH content, ΔΨm, and the protein levels of GCLC and GCLM were reduced significantly. Apoptosis rate, ROS, and expression of apoptosis-related and NF-κB pathway proteins increased. The expression of LINC00942 was increased, while the expression of miR-214-5p was decreased. After suppressing LINC00942 levels, NaAsO2 exposure further decreased cell viability, intracellular GSH content, ΔΨm, GCLC protein, and miR-214-5p expression. The apoptosis rate, ROS, and apoptosis-related and NF-κB pathway proteins further increased. miR-214-5p is targeted and negatively regulated by LINC00942. After miR-214-5p was overexpressed, NaAsO2 further decreased cell viability, intracellular GSH content, ΔΨm, and GCLC protein expression compared to NaAsO2 exposure. The apoptosis rate, ROS, apoptosis-related and NF-κB pathway proteins p65, and IKKβ were higher than those exposed to NaAsO2. LINC00942 inhibitor along with miR-214-5p inhibitor combined with NaAsO2 treatment resulted in increased cell viability, GSH, Bcl-2, and GCLC protein expression and decreased apoptosis rate, apoptosis related, p65, IKKβ protein, and ΔΨm, as compared to the combined NaAsO2 and si LINC00942 group. NaAsO2 exposure induces oxidative damage and apoptosis in LX-2 cells by activating NF-κB and inhibiting GSH synthesis. During this process, the expression level of LINC00942 increases, targeting to reduce the level of miR-214-5p, then weakening the effect of NaAsO2 on NF-κB, thereby alleviating cellular oxidative damage and playing a protective role.

Honokiol regulates ovarian cancer cell malignant behavior through YAP/TAZ pathway modulation.

Ovarian cancer (OVCA) stands as one of the most fatal gynecological malignancies. Honokiol (HNK) has been substantiated by numerous studies for its anti-tumor activity against malignancies including OVCA. Consequently, this work was designed to elucidate the impact of HNK-mediated modulation of the YAP/TAZ pathway on the biological functions of OVCA cells. OVCA cells were subjected to treatment with varying concentrations (0, 25, 50, 75, and 100 μM) of HNK, concomitant with the administration of YAP agonist (XMU). Assessment of cellular viability was executed employing the CCK-8 assay, while quantification of cellular proliferation transpired via colony formation assays. Apoptosis was ascertained using flow cytometry, and expression of apoptosis-related proteins (caspase-3, Bcl-2, Bax), EMT-related proteins (E-cadherin, N-cadherin), migration-associated proteins (MMP-2, MMP-9), and YAP/TAZ pathway-related proteins was evaluated by western blot. Transwell experiments were conducted to assess cellular migratory and invasive propensities. Xenograft tumor models were built to observe tumor growth (volume and weight), apoptosis was assessed by TUNEL staining, and Ki67 expression was evaluated through IHC. HNK exerted inhibitory effects on the viability and proliferative capacity of OVCA cells, elicited apoptotic responses, curtailed the migratory and invasive tendencies of cells, and downregulated the YAP/TAZ pathway. Stimulation with YAP agonist (XMU-MP-1) partially attenuated the impacts of HNK on OVCA cell biology. Experiments in vivo confirmed that HNK inhibited OVCA tumor growth. The outcomes of this investigation conclusively established that HNK orchestrated the modulation of the YAP/TAZ pathway, thereby exerting control over the malignant phenotypic manifestations of OVCA cells. The ascertained function of HNK in restraining cellular proliferation and tumor progression provided novel evidence of its anti-proliferative activity within OVCA cells.

Effect of Celastrus Orbiculatus Extract on proliferation and apoptosis of human Burkitt lymphoma cells.

The lymphoma incidence rate is on the rise, with invasive forms particularly prone to relapse following conventional treatment, posing a significant threat to human life and wellbeing. Numerous studies have shown that traditional Chinese botanical drug medicine offers promising therapeutic benefits for various malignancies, with previous experimental findings indicating that Celastrus orbiculatus extract effectively combats digestive tract tumors. However, its impact on lymphoma remains unexplored. This study aims to investigate the impact and underlying mechanisms of COE on the proliferation and apoptosis of Burkitt lymphoma cells. We diluted COE in RPMI-1640 medium to create various working concentrations and introduced it to human Burkitt lymphoma Raji and Ramos cells. To evaluate cell viability, we used the CCK-8 assay, and we observed morphological changes using HE staining. We also conducted Annexin V-PI and JC-1 staining experiments to assess apoptosis. By combining the cell cycle experiment with the EDU assay, we gained insights into the effects of COE on DNA replication in lymphoma cells. Using Western blotting, we detected alterations in apoptosis-related proteins. In vivo experiments revealed that following COE intervention, tumor volume decreased, survival time was prolonged, spleen size reduced, and the expression of tumor apoptosis-related proteins changed. Our findings indicate that COE effectively inhibits lymphoma cell proliferation and promotes apoptosis by regulating these apoptosis-related proteins.

Studies Related to the Involvement of EsA in Improving Intestinal Inflammation in Acute Pancreatitis via the NF-κB Pathway.

Acute pancreatitis (AP) is a clinically frequent acute abdominal condition, which refers to an inflammatory response syndrome of edema, bleeding, and even necrosis caused by abnormal activation of the pancreas's own digestive enzymes. Intestinal damage can occur early in the course of AP and is manifested by impaired intestinal mucosal barrier function, and inflammatory reactions of the intestinal mucosa, among other factors. It can cause translocation of intestinal bacteria and endotoxins, further aggravating the condition of AP. Therefore, actively protecting the intestinal mucosal barrier, controlling the progression of intestinal inflammation, and improving intestinal dynamics in the early stages of AP play an important role in enhancing the prognosis of AP. The viability and apoptosis of RAW264.7 cells treated with Esculentoside A (EsA) and/or lipopolysaccharide were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The expression of apoptosis-related proteins and NF-κB signaling pathway-related proteins were detected by western blot (WB). An enzyme-linked immunosorbent assay was used to measure TNF-α and IL-6 secretion. In vitro experiments demonstrated that EsA not only promoted the apoptosis of inflammatory cells but also reduced the secretion of TNF-α and IL-6 in a dose-dependent manner. Additionally, it inhibited the activation of the NF-κB signaling pathway by decreasing the expression of phosphorylated-p65(p-p65) and elevating the expression of IκBα. Similarly, in vivo experiments using a rat AP model showed that EsA inhibited the expression of p-p65 elevating the expression of IκBα in the intestinal tissues of the rat AP model and promoting the apoptosis of inflammatory cells in the intestinal mucosa in vivo experiments, while improving the pathological outcome of the pancreatic and intestinal tissues. Our results suggest that EsA can reduce intestinal inflammation in the rat AP model and that EsA may be a candidate for treating intestinal inflammation in AP and further arresting AP progression.

Effect of Shoutai Pills on expression of key glycolytic proteins and apoptosis related factors at maternal fetal interface in mouse model of threatened abortion with syndrome of kidney deficiency

This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P<0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P<0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P<0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.

Stellera chamaejasme against multidrug resistance of triple-negative breast cancer MDA-MB-231 cell through Nrf2

This study aims to investigate the effect and mechanism of Stellera chamaejasme extract(SCL) on multidrug resistance(MDR) in breast cancer. Human triple-negative breast cancer cell line MDA-MB-231 and its adriamycin-resistant cell line MDA-MB-231/ADR were used in the experiment. Cell viability was detected by methyl thiazolyl tetrazolium(MTT) assay, and cell apoptosis was detected by DAPI staining and Annexin-V/Pi double staining. Western blot(WB) was used to detect the expression levels of Keap1, Nrf2, HO-1, Bcl-2, Bax, caspase-9, and caspase-3. Immunofluorescence staining was used to observe the distribution of Nrf2 in the cell, and flow cytometry was used to detect the level of reactive oxygen species(ROS) in the cell. The results showed that the resis-tance factor of SCL was 0.69, and that of adriamycin and paclitaxel was 8.40 and 16.36, respectively. DAPI staining showed that SCL could cause nuclear shrinkage and fragmentation of breast cancer cells. Annexin-V/Pi double staining showed that the average apoptosis rate of the drug-resistant cells was 32.64% and 50.29%, respectively under medium and high doses of SCL. WB results showed that SCL could significantly reduce the expression levels of anti-apoptotic proteins Bcl-2, caspase-9, and caspase-3 and significantly increase the expression level of pro-apoptotic protein Bax. Further studies showed that SCL could significantly promote the expression of Keap1, significantly inhibit the expression of Nrf2 and HO-1, and significantly reduce the expression level of Nrf2 in the nucleus. Correspondingly, flow cytometry showed that the intracellular ROS level was significantly increased. In conclusion, SCL can significantly inhibit the proliferation of MDA-MB-231 multidrug-resistant cells of triple-negative breast cancer and cause cell apoptosis, and the mechanism is related to inhibiting Keap1/Nrf2 signaling pathway, leading to ROS accumulation in drug-resistant cells and increasing the expression of apoptosis-related proteins.