Caspase-3 Gene Expression Research Articles

Comparison of cytotoxic, apoptotic and oxidative properties of Akacid plus and chlorhexidine in corneal epithelial cell culture.

This study aims to compare the cytotoxic, apoptotic, and oxidative effects of a new cationic disinfectant, Akacid Plus, with chlorhexidine, on the human corneal epithelial cell line. Time-dependent cytotoxicity studies were performed with the Alamar Blue method. Apoptotic activity was investigated by flow cytometric methods. Reactive oxygen species levels were measured with the ROS cellular test kit. BAX, BCL2 and caspase 3, 9, 12 mRNA expressions were evaluated by PCR, as well as BAX and BCL2 protein expressions by Western-Blot. At the fifth minute of the treatment, the viability was 68.15% with Akacid Plus and 43.95% with chlorhexidine. At the 15th minute, no significant difference was observed with both solutions. In the apoptotic evaluation, Akacid Plus significantly increased the early and late apoptotic activity in the cell line (p < 0.0001), while a significant increase was observed in late apoptosis and necrosis levels with chlorhexidine (p < 0.001). Chlorhexidine also induced gene expression of BAX, BCL2, caspase 3, 9 and BAX proteins (p < 0.05), while reducing protein expression of BCL2 (p < 0.001). Akacid Plus induced the gene expressions of BCL2, CASP3 and caspase 9, reduced gene expressions of BAX and caspase 12 and protein expression of BCL2 (p < 0.05). No significant difference was observed in the ROS level with both solutions (p > 0.05). Due to the widespread use of cationic polymers in ophthalmology, this new molecule with high antimicrobial activity and relatively low cytotoxicity may be of interest for clinical use. Further investigations are necessary to fully understand the ophthalmologic potential of this solution.

Targeted delivery of oxaliplatin via folate-decorated niosomal nanoparticles potentiates resistance reversion of colon cancer cells

BackgroundColorectal cancer (CRC) is a prevalent type of cancer, ranking third in incidence and fourth in cancer-related deaths globally. The increase in mortality rates related to colorectal cancer among younger patients is a cause for concern. Chemotherapy is the primary approach for palliative care in colon cancer, but the development of drug resistance limits its effectiveness. Apoptosis is a process of programmed cell death that plays a crucial role in regulating normal cell death and abnormal tissue degeneration in cancer. Genes such as caspase-3, caspase-9, p53, and survivin are involved in apoptosis induction. The field of nanotechnology has presented exciting opportunities for controlled drug delivery and addressing drug resistance in cancer. Niosomes are among the nanocarriers known for their impressive features, making them excellent candidates for drug delivery. In the current study, we investigate whether niosomal nanoparticles coated with FA have the ability to deliver oxaliplatin to drug-resistant cells effectively and potentially resistance reversion in colon cancer cells. MethodsThe niosomal nanoparticles (NPs) were fabricated using the thin-film hydration method and characterized using DLS (Dynamic Light Scattering), FTIR (Fourier Transform Infrared Spectroscopy), SEM (Scanning Electron Microscopy), and AFM (Atomic Force Microscopy) systems. The drug release and drug encapsulation efficiency of the NPs were also determined. An MTT assay was performed on oxaliplatin-resistant cells to determine the IC50 values of the drug in its pure and nano-encapsulated forms. Gene expression of caspase-3, caspase-9, p53, and survivin was investigated using the qRT-PCR (quantitative Reverse Transcription Polymerase Chain Reaction) technique, and cell apoptosis or necrosis was quantified using flow cytometry. ResultsSize, PDI, zeta potential, morphology, drug release, and encapsulation efficiency of fabricated niosomal NPs were acceptable. Oxaliplatin anti-cancer drug showed a higher impact on cancerous cells in nano-encapsulated form. The expression level of caspase-3, caspase-9, and p53 was increased which was in confirmation by flow cytometry results. ConclusionTaken together, results of this study demonstrated potential effect of folate decorated oxaliplatin-loaded niosomal NPs to resistance-reversion of Oxaliplatin-resistance colon cancer cells.

Apigenin promotes cell death in NCI-H23 cells by upregulation of PTEN: potential involvement of the binding of apigenin with WWP2 protein

The tumour suppressor protein PTEN is often down-regulated in non-small cell lung cancer. A major protein promoting the lowering of the PTEN protein is WWP2. Polyphenols have been shown to promote the expression of tumour suppressor genes like PTEN. We carry out the study to check for the ability of apigenin to bind with the WWP2 protein using in-silico investigation comprising docking and simulation. We checked for the cytotoxic effect of apigenin upon the non-small cell lung cancer cell line NCI-H23. We checked the PTEN expression status at the gene and protein levels. The expression levels of the apoptotic regulators BCL2, BAX and CASPASE3 genes along with the activity levels of the caspase-3 protein were checked. The ultrastructure of the cells was analysed. Our Autodock analysis showed that apigenin bound favourably with the WWP2 protein. Molecular dynamics simulation revealed that apigenin increased the parameters of RMSD, Rg and SASA when bound with the WWP2 protein. The protein-ligand complex had hydrogen bonding and majorly van der Wal’s interactions. PCA analysis revealed greater fluctuations in the apigenin-bound state of the protein. The mutant form of the WWP2 revealed similar results in the presence of apigenin. Apigenin showed efficacy against the NCI-H23 cell line and promoted PTEN protein levels, lowered BCL2 gene expression and up-regulated BAX and CASPASE3 gene expression. Increased caspase-3 activity and ultra-structural analysis revealed the occurrence of apoptosis. Thus the binding of apigenin with WWP2 could promote PTEN protein levels and lead to apoptotic activity in NCI-H23 cells. Communicated by Ramaswamy H. Sarma

Andrographolide Reverses Doxorubicin Resistance in Human Breast Cancer Stem Cells by Regulating Apoptotic Gene Expressions

BACKGROUND: Breast cancer stem cells (BCSCs) have been identified as playing a crucial role in therapeutic resistance. This resistance can be attributed to the anti-apoptotic protein survivin and the antioxidant MnSOD high expression. To overcome the resistance to doxorubicin (DOX), this study proposed the utilization of andrographolide (ANDRO), the primary bioactive compound in Andrographis paniculata leaves. The objective was to examine the role of andrographolide in regulating survivin, caspase-9, and caspase-3 gene expressions to reverse doxorubicin resistance in human BCSCs.METHODS: BCSCs were exposed to 0.1 µM DOX every two days or 50 µM rotenone (ROT) for 6 hours, subsequently supplemented with 0.3 mM ANDRO. Superoxide and peroxide levels were measured using DHE and DCFH-DA assay. The MnSOD, survivin, caspase-9, and caspase-3 mRNA expression levels were analyzed using qRT-PCR. Protein expressions were evaluated using Western blotting assay. MnSOD activity was determined using xanthine oxidase inhibition assay. The apoptotic cells were determined using Annexin-V/PI staining.RESULTS: This study indicated that the cytotoxic mechanisms of DOX, similar to ROT, in BCSCs were attributed to oxidative stress, as evidenced by an elevation in superoxide rather than peroxide levels, accompanied by a decrease in MnSOD activity. This study also highlighted that ANDRO reversed DOX resistance in BCSCs subjected to repeated DOX treatment by downregulating survivin and upregulating caspase-9 and caspase-3 mRNA expressions, thereby activating the intrinsic apoptotic pathway.CONCLUSION: This study provides insights into the role of ANDRO in modulating the expression of apoptotic genes, such as survivin, caspase-9, and caspase-3, to overcome DOX resistance in BCSCs.KEYWORDS: breast cancer, breast cancer stem cell, andrographolide, doxorubicin, oxidative stress, apoptosis

Lentivirus-based shRNA of Caspase-3 gene silencing inhibits chondrocyte apoptosis and delays the progression of surgically induced osteoarthritis.

Chondrocyte apoptosis is an important pathological feature of osteoarthritis (OA). Excessive apoptosis of chondrocytes disrupts the dynamic balance of cell proliferation and apoptosis, with a marked reduction in chondrocytes and cartilage matrix disintegration, which represents the main pathology of OA. Caspases, especially Caspase-3, play a central role in cell apoptosis. In this study, a lentiviral vector was used to transduce caspase-3 short hairpin RNA (shRNA) into rat chondrocytes (RCs), and the apoptotic and phenotypic genes of RCs were analyzed using real-time PCR and western blotting in vitro. In addition, in vivo intra-articular injection of Caspase-3 shRNA lentivirus was performed in a surgically induced OA rat model. Our results showed that Caspase-3 gene silencing could down-regulate the TNF-α-mediated inflammatory gene expression of TNFR1, FADD, and IL-1β, apoptotic gene expression of APAF1, Caspase-3, and Caspase-9, thereby attenuating the apoptotic pathway in vitro. Caspase-3 gene silencing also attenuated TNF-α-mediated decreased gene expression of ACAN, Col1-a1, and Col2-a1. Furthermore, Caspase-3 gene silencing could effectively reduce the OARSI score, and gene expression of Caspase-3, Caspase-9, MMP13, and TNF-α in a surgically induced OA rat model. Caspase-3 gene silencing may serve as a novel therapeutic strategy for cartilage injury and OA.

Lactiplantibacillus plantarum Postbiotics Suppress Salmonella Infection via Modulating Bacterial Pathogenicity, Autophagy and Inflammasome in Mice.

Our study aimed to explore the effects of postbiotics on protecting against Salmonella infection in mice and clarify the underlying mechanisms. Eighty 5-week-old C57BL/6 mice were gavaged daily with Lactiplantibacillus plantarum (LP)-derived postbiotics (heat-killed bacteria, LPBinactive; culture supernatant, LPC) or the active bacteria (LPBactive), and gavaged with Salmonella enterica Typhimurium (ST). The Turbidimetry test and agar diffusion assay indicated that LPC directly inhibited Salmonella growth. Real-time PCR and biofilm inhibition assay showed that LPC had a strong ability in suppressing Salmonella pathogenicity by reducing virulence genes (SopE, SopB, InvA, InvF, SipB, HilA, SipA and SopD2), pili genes (FilF, SefA, LpfA, FimF), flagellum genes (FlhD, FliC, FliD) and biofilm formation. LP postbiotics were more effective than LP on attenuating ST-induced intestinal damage in mice, as indicated by increasing villus/crypt ratio and increasing the expression levels of tight junction proteins (Occludin and Claudin-1). Elisa assay showed that LP postbiotics significantly reduced ST-induced inflammation by regulating the levels of inflammatory cytokines (the increased IL-4 and IL-10 and the decreased TNF-α) in serum and ileum (p < 0.05). Furthermore, LP postbiotics inhibited the activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome by decreasing the protein expression of NLRP3 and Caspase-1, and the gene expression of Caspase-1, IL-1β and IL-18. Meanwhile, both LPC and LPB observably activated autophagy under ST infection, as indicated by the up-regulated expression of LC3 and Beclin1 and the downregulated p62 level (p < 0.05). Finally, we found that LP postbiotics could trigger an AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy. In summary, Lactiplantibacillus plantarum-derived postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and NLRP3 inflammasome in mice. Our results confirmed the effectiveness of postbiotics agents in the control of Salmonella infection.

Curcumin promotes apoptosis of human melanoma cells by caspase 3.

Cutaneous melanoma (CM) is a malignant neoplasm with a high metastatic rate that shows poor response to systemic treatments in patients with advanced stages. Recently, studies have highlighted the antineoplastic potential of natural compounds, such as polyphenols, in the adjuvant therapy context to treat CM. The objective of the present study was to evaluate the effect of different concentrations of curcumin (0.1-100 µM) on the metastatic CM cell line SK-MEL-28. The cells were treated for 6 and 24 h with different concentrations of curcumin. Cell viability was assessed by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and fluorescence microscopy. The apoptotic-inducing potential was detected by annexin V flow cytometry. The wound healing assay was used to verify cell migration after the curcumin exposition. The redox profile was evaluated by levels of the pro-oxidant markers reactive oxygen species (ROS) and Nitric oxide (NOx) and antioxidants of total thiols (PSH) and nonprotein thiols. The gene expression and enzymatic activity of caspase 3 were evaluated by reverse transcription-quantitative polymerase chain reaction and a sensitive fluorescence assay, respectively. Curcumin significantly decreased the cell viability of SK-MEL-28 cells at both exposure times. It also induced apoptosis at the highest concentration tested (p < .0001). SK-MEL-28 cell migration was inhibited by curcumin after treatment with 10 µM (p < .0001) and 100 µM (p < .0001) for 6 and 24 h (p = .0006 and p < .0001, respectively). Furthermore, curcumin significantly increased levels of ROS and NOx. Finally, curcumin was capable of increasing the gene expression at 10 µM (p = .0344) and 100 µM (p = .0067) and enzymatic activity at 10 µM (p = .0086) and 100 µM (p < .0001) of caspase 3 after 24 h. For the first time, we elucidated in our study that curcumin increases ROS levels, promoting oxidative stress that activates the caspase pathway and culminates in SK-MEL-28 metastatic CM cell death.

Effects of tetrahydroxy stilbene glycoside derivatives on free radical damage and apoptosis in APP695V717I transgenic mice

ABSTRACTMitochondrial dysfunction leading to overproduction of oxygen free radicals is an important event in the development of Alzheimer's disease. Tetrahydroxy stilbene glycoside (TSG) is one of the main effective components of Polygonum multiflorum and has a certain free radical scavenging effect. We synthesized tetrahydroxy stilbene glycoside derivatives (Mito-TSGs) that can cross the mitochondrial membrane and may provide effective protection against Alzheimer's disease. This experiment investigates the protective mechanism of tetrahydroxy stilbene glycoside derivatives against mitochondrial free radical damage and apoptosis in APP695V717I transgenic model mice. The experimental subjects were healthy 3-month-old APP695V717I transgenic model mice, while C57BL/6J mice of the same age and genetic background served as controls. The results demonstrated that the tetrahydroxy stilbene glycoside derivatives significantly improved mouse behavioral performance. It also led to a decrease in the levels of H2O2, NO, MDA, and LD, along with an increase in LDH activity and in the antioxidant enzyme activity of SOD, CAT, and GSH-Px. Moreover, it elevated the mitochondrial membrane potential, decreased the gene and protein expression of Caspase-3 and Bax, and increased the gene and protein expression of Bcl-2. Notably, the effectiveness of tetrahydroxy stilbene glycoside derivatives was superior to that of traditional tetrahydroxy stilbene glycoside.

Dual anticancer activity of Aspergillus nidulans pigment and Ionizing γ-Radiation on human larynx carcinoma cell line

BackgroundFungi are a readily available source of naturally generated colored compounds. These compounds might be used as radiosensitizers for treating cancer cells.MethodsAspergillus nidulans was examined for its color-producing ability in Potato dextrose agar (PDA) broth medium. The pigment was characterized by Ultraviolet (UV) spectrophotometer and Gas Chromatography Mass Spectrometry (GC/MS). Pigment extracts from A. nidulans were studied for their cytotoxic effects on the growth of human larynx carcinoma cell line (HEp-2) with or without exposure to γ-radiation at three different doses (5, 10, and 15 Gy). A. nidulans pigment cytotoxic activity was tested against normal Vero cells. Cell apoptosis was studied using flow cytometry. Gene expression of P53, Caspase 3 and Bcl-2 were quantified.ResultsUltraviolet spectrum and GC/MS revealed the ability of Aspergillus nidulans to produce Rhodopin pigment. HEp-2 cells treated with A. nidulans pigment only give IC50 about 208 µg/ml. In contrast, when treated with the pigment +10 Gy γ-radiation, it give about 115 µg/ml. However, for normal cells, lower cytotoxic activity was detected. Treatment with pigment (208 g/mL) caused about 50% ± 1.0 total apoptosis level and gene expression of P53: 2.3 fold and Caspase 3: 1.84 fold in respect to untreated HEp-2), while Bcl-2 was decreased (Bcl-2: 0.63 fold in respect to untreated HEp-2). Furthermore, treated with pigment (115 µg/mL) + 10Gy caused about 47.41% ± 1.7 total apoptosis level and P53: 2.53 fold and Caspase 3: 2.0 fold in respect to untreated HEp-2, while Bcl-2 was downregulated (Bcl-2: 0.61 fold in respect to untreated HEp-2).ConclusionThis study concluded that the anti-cancer activity of Aspergillus nidulans pigment was enhanced by ionizing radiation at 10 Gy, as well as its low cytotoxic activity against normal Vero cells.

Cepabiflas B and C as Novel Anti-Inflammatory and Anti-Apoptotic Agents against Endotoxin-Induced Acute Kidney and Hepatic Injury in Mice: Impact on Bax/Bcl2 and Nrf2/NF-κB Signalling Pathways.

Cepabiflas B and C (CBs) are flavonoid dimers separated from Allium cepa. They demonstrated antioxidant and α-glucosidase and protein tyrosine phosphatase 1B inhibition capacities. However, their anti-inflammatory activities and their effects on endotoxemia are unknown. The current study aimed at exploring the protective activities of CBs on lipopolysaccharide (LPS)-induced kidney and liver damage in mice and investigating the possible molecular mechanisms. Mice were orally treated with a low (40 mg/kg) or high (60 mg/kg) dose of CBs for five days prior to a single intraperitoneal injection of LPS (10 mg/kg). Samples of serum and hepatic and kidney tissues were collected 24 h after the LPS challenge. Changes in serum indices of hepatic and renal injury, pathological changes, molecular biological parameters, and proteins/genes related to inflammation and apoptosis of these organs were estimated. LPS injection resulted in deleterious injury to both organs as indicated by elevation of serum ALT, AST, creatinine, and BUN. The deteriorated histopathology of hepatic and renal tissues confirmed the biochemical indices. CBs treated groups showed a reduction in these parameters and improved histopathological injurious effects of LPS. LPS-induced hepatorenal injury was linked to elevated oxidative stress as indicated by high levels of MDA, 4-HNE, as well as repressed antioxidants (TAC, SOD, and GSH) in hepatic and kidney tissues. This was accompanied with suppressed Nrf2/HO-1 activity. Additionally, there was a remarkable inflammatory response in both organs as NF-κB signalling was activated and high levels of downstream cytokines were produced following the LPS challenge. Apoptotic changes were observed as the level and gene expression of Bax and caspase-3 were elevated along with declined level and gene expression of Bcl2. Interestingly, CBs reversed all these molecular and genetic changes and restricted oxidative inflammatory and apoptotic parameters after LPS-injection. Collectedly, our findings suggested the marked anti-inflammatory and anti-apoptotic activity of CBs which encouraged its use as a new candidate for septic patients.

Co-delivery of curcumin and PTTG1 siRNA by galactose receptor-targeted liposomes for enhanced anti-tumor effects in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies posing a serious threat to human health worldwide. To date, the efficacy of chemotherapy in HCC has been limited because of drug resistance. To overcome this, combination therapy with chemotherapeutic drugs and siRNA represents a new strategy to improve anticancer effects through synergistic effects. Therefore, this study examined the efficacy of combination therapy for HCC, wherein two types of hepatoma-targeting ligand-based liposomes were used to co-deliver curcumin and pituitary tumor modifying gene-1 (PTTG1) siRNA. First, curcumin (Cur) was encapsulated in POPC-based neutral liposomes with surface-exposed galactose residues (Gal-POPC/Cur). Second, PTTG1 siRNA (siPTTG1) was complexed with liposomes containing a galactose ligand based on cationic lipids (Gal-DOTAP/siPTTG1). Under optimized experimental conditions, curcumin and siPTTG1 were effectively loaded into the respective liposomes (Gal-POPC/Cur or Gal-DOTAP/siPTTG1). These liposomes with selected sizes and zeta potentials effectively accumulated at the tumor sites and entered HCC cells. Moreover, they showed higher cytotoxic effects on cancer cells in the Gal-POPC/Cur and Gal-DOTAP/siPTTG1 groups than in the group treated with non-target liposomes. The two combination treatments exhibited excellent anticancer effects by inhibiting the expression of Bcl-2 gene and increasing the expression of Caspase-3 gene in hepatocellular carcinoma. Tumor growth was effectively inhibited by intravenous injection of Gal-POPC/Cur and Gal-DOTAP/siPTTG1 into nude mice carrying subcutaneous human Huh-7 xenografts. Overall, these results demonstrate the high potential of liver-targeted liposome nano-pharmaceuticals carrying curcumin and PTTG1 siRNA to improve HCC treatment.

Limosilactobacillus johnsoni and Limosilactobacillus mucosae and Their Extracellular Vesicles Alleviate Gut Inflammatory Injury by Mediating Macrophage Polarization in a Lipopolysaccharide-Challenged Piglet Model

BackgroundLimosilactobacillus johnsoni (L. j) and Limosilactobacillus mucosae (L. m) can alleviate the inflammatory response. ObjectivesThis study aimed to elucidate the underlying mechanisms by which L. j- and L. m-derived extracellular vesicles (EVs) mitigate lipopolysaccharide (LPS)-induced intestinal injury. MethodsPiglets were assigned to 4 groups: oral phosphate-buffered saline inoculation for 2 wk prior to intraperitoneal injection of physiological saline or LPS, and oral L. j/L. m inoculation for 2 wk prior to intraperitoneal injection of LPS. The intestinal integrity, macrophage markers, cytokine levels, and microbiota were determined. The cytokine levels and macrophage phenotype were detected after L. j/L. m and their EVs were coincubated with macrophages. The levels of cytokines, tight junction proteins, and apoptosis were measured after intestinal epithelial cells were cocultured with macrophages. ResultsLPS challenge decreased jejunal villus length; expression levels of zonula occludens-1 (ZO-1), occludin, arginase-1 (Arg1), and interleukin (IL)-10; and number of CD163+ cells and increased the expression levels of inducible nitric oxide synthase (iNOS), IL-1β, IL-6, and tumor necrosis factor (TNF)-α compared with that in the control. L. j and L. m pretreatment rescued the aforementioned indicators compared with LPS challenge. Pretreatment of L. j and L. m and their EVs reversed the levels of IL-1β, IL-6, TNF-α, and IL-10 and the gene expression of iNOS and Arg1 in the LPS group in macrophages. Pretreatment with L. j and L. m-derived EVs increased ZO-1 and occludin mRNA expression and reduced IL-1β, caspase-3, and bax gene expression in intestinal epithelial cells of the coculture system. Enzyme-treated EVs were less effective than native EVs. ConclusionsThis study suggests that EVs secreted by L. j and L. m control inflammation by modulating macrophage polarization, thereby improving intestinal barrier function.

Elastin-targeted nanoparticles delivering doxycycline mitigate cytokine storm and reduce immune cell infiltration in LPS-mediated lung inflammation.

Cytokine storm invoked during acute and chronic lung injury promotes alveolar damage and remodeling. The current study shows that degraded elastin-targeted nanoparticles releasing doxycycline (Doxy NPs) are potent in mitigating cytokines storm, migration of immune cells in the lungs, and inhibiting inflammasome pathways in the LPS mouse model. Cytokine storm and lung injury were induced using LPS and elastase in C57BL/6 mice (rodent model for emphysema). The mice were then treated with I.V. Doxy NPs, blank NPs, or Doxy a day before LPS administration. Cytokine levels, immune cell population, and MMP activity were analyzed in broncheo-alveolar lavage fluid (BALF) 4 hours after LPS administration. Additionally, gene expression of IL-6, IL-1beta, MCP-1, NLRP3, Caspase 1 and MMPs were investigated in alveolar cells on day 3 after LPS administration. Doxycycline NPs but not Doxycycline significantly decreased IL-6, TNF-α, IL-23 and were significantly more effective in decreasing the percentage of immune cells in the BALF. This is the first in-vivo study to demonstrate that Doxycycline can effectively inhibit inflammasome pathways in the lungs. IV administration of elastin antibody conjugated Doxycycline-loaded albumin NPs can effectively modulate the local immune environment in the lungs, which is not achieved by IV Doxycycline even at 100-fold higher dose. This novel method of drug delivery can effectively lead to the repurposing of traditional Doxycycline as a potential adjunct treatment for managing the cytokine storm in the lungs in COPD and viral infections.

Cyberlindnera jadinii yeast as a functional protein source for Atlantic salmon (Salmo salar L.): Early response of intestinal mucosal compartments in the distal intestine.

The interplay between nutrition and the immune system is well recognized, and several studies show that experimental diets elicit local morphological changes and alteration of gene and protein expression in the intestinal mucosa of Atlantic salmon. In this study the pathophysiological effects of experimental diets on mucosal responses in the distal intestine of Atlantic salmon were investigated. Atlantic salmon were fed diets with inclusion of soybean meal (SBM) and Cyberlindnera jadinii (CJ) yeast for 7 days. A standard fish meal (FM) diet was used as a control. Morphological, immunohistochemical and gene expression analyses were used to evaluate the presence of immune cells, proliferating cells, and stem cell populations in mucosal compartments of the simple folds in the distal intestine. Fish fed SBM developed morphological changes consistent with SBM induced enteritis. Immunohistochemistry showed an increased presence of apoptotic cells, CD3ϵ and CD8α labelled cells in the simple fold epithelium of SBM group compared with the CJ group. For the investigated genes, expression levels in all three groups were mostly higher in the epithelial compartment of the simple fold than in the compartment beneath the folds. Most changes within the epithelial compartment were observed in fish fed SBM, where expression of CD3ζ, CD8α, MHC I and MHC II were lower than the FM control group. The CJ group had an increased expression of the stem cell marker Lgr5 in the epithelial compartment compared with SBM group. The division of the simple fold into an apical and basal compartment showed that the increase in Lgr5 was evident along the whole length of the simple folds and not confined to the base of the folds. Similarly, proliferation (PCNA, MCM2) and apoptosis (Caspase-3) gene expression was present in the entire length of the simple folds, suggesting that intestinal epithelial cell turnover is not confined to the basal or apical part of the fold. This study shows that the epithelial compartment is active in the early immunoregulatory response towards dietary stimuli and that the level of an intestinal stem cell marker in salmon was influenced by a diet containing CJ yeast.

Astragalus adscendens extract shows antidiabetic effects through controlling oxidative stress, inflammation and apoptosis in streptozotocin-induced diabetic rats

Objective: To assess the effect of oral treatment of methanolic extract of the aerial parts of Astragalus adscendens in streptozotocin-induced diabetic rats. Methods: In order to induce diabetes, rats intraperitoneally received streptozotocin at 65 mg/kg. Sixty adult male Wistar rats were allocated into six groups (10 rats per each) including the healthy control group, the diabetic group as well as the diabetic group treated with Astragalus adscendens methanolic extract at 50, 100, and 200 mg/kg per day or glibenclamide (0.6 mg/kg/day) for 28 d. The effects of Astragalus adscendens methanolic extract on the levels of glucose, insulin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, creatinine, urea, uric acid, total protein, albumin, triglyceride, cholesterol, α-amylase, oxidant/antioxidant enzymes, and inflammatory cytokines were evaluated. Real time-PCR was also used for measuring the gene expression of caspase-3, Bcl2, and Bax. Results: The levels of glucose, cholesterol, triglyceride, creatinine, urea, uric acid, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, and malondialdehyde considerably declined (P&lt;0.001) in diabetic rats after treatment with Astragalus adscendens methanolic extract especially at a dose of 200 mg/kg. In addition, treatment with Astragalus adscendens methanolic extract noticeably increased the level of insulin, total protein, and albumin as well as improved the activities of catalase, glutathione peroxidase, and superoxide dismutase, as well as the expression levels of TNF-α, IL-1β, caspase-3, Bcl2 and Bax (P&lt;0.001) compared to the diabetic control group. The extract also inhibited α-amylase in a dose-dependent manner with an IC50 value of 19.6 µg/mL. Conclusions: Astragalus adscendens methanolic extract shows potent antidiabetic, anti-inflammatory, anti-apoptotic, and antioxidant effects in diabetic rats. However, more studies are needed to verify the underlying mechanism of the effect of this plant extract and test its efficacy in clinical trials.

Cytotoxic effects of walnut hull extracts against human osteosarcoma cell line (U2OS)

This study aimed to evaluate the total phenolic, total flavonoid content and the antioxidant activity of walnut (Juglans regia L.) hull (JRH) extracts and investigate the cytotoxic effect of different walnut hull extracts on osteosarcoma U2OS cell line and study their impact on apoptosis, autophagy and heat shock protein 90. For this purpose, the dried walnut hulls were extracted by soxhlet apparatus using methanol, ethanol, ethyl acetate and hexane as extraction solvents. The total phenolic content, total flavonoid content and antioxidant activity of each extract were determined by three colorimetric methods: Folin-Ciocalteu method, AlCl3 method and DPPH radical scavenging assay, respectively. In order to investigate the osteosarcoma U2OS cells viability in response to treatment with each JRH extracts, MTT assays were performed. qRT-PCR was used to investigate the cytotoxic effect of the extracts. Caspase-3 and Bcl-2 were checked to determine their role in apoptosis, Beclin 1 for autophagy and HSP90 for viability/proliferation. Overall, the highest extraction yield (41.9 %.), total phenolic content (85.16 ± 5.43 mg GAE/g), total flavonoid content (105.49 ± 6.44 mg RE/g), the antioxidant activity (IC50 = 15.484 μg/mL) and cytotoxic effect on osteosarcoma cell line (IC50 = 180.20 ± 27 μg/mL) were determined in JRH methanol, ethanol, ethyl acetate, ethanol, methanol extracts respectively. The assessments of the present investigation demonstrated that all JRH extracts reduced the viability of osteosarcoma U2OS cells in a dose dependent manner and methanol extract showed the highest anticancer activity by upregulating the gene expression of Caspase-3 and downregulating the gene expressions of Beclin-1, Bcl-2 and HSP-90.

Inhibition of discoidin domain receptor (DDR)-1 with nilotinib alters CSF miRNAs and is associated with reduced inflammation and vascular fibrosis in Alzheimer’s disease

Discoidin Domain Receptor (DDR)-1 is activated by collagen. Nilotinib is a tyrosine kinase inhibitor that is FDA-approved for leukemia and potently inhibits DDR-1. Individuals diagnosed with mild–moderate Alzheimer’s disease (AD) treated with nilotinib (versus placebo) for 12 months showed reduction of amyloid plaque and cerebrospinal fluid (CSF) amyloid, and attenuation of hippocampal volume loss. However, the mechanisms are unclear. Here, we explored unbiased next generation whole genome miRNA sequencing from AD patients CSF and miRNAs were matched with their corresponding mRNAs using gene ontology. Changes in CSF miRNAs were confirmed via measurement of CSF DDR1 activity and plasma levels of AD biomarkers. Approximately 1050 miRNAs are detected in the CSF but only 17 miRNAs are specifically altered between baseline and 12-month treatment with nilotinib versus placebo. Treatment with nilotinib significantly reduces collagen and DDR1 gene expression (upregulated in AD brain), in association with inhibition of CSF DDR1. Pro-inflammatory cytokines, including interleukins and chemokines are reduced along with caspase-3 gene expression. Specific genes that indicate vascular fibrosis, e.g., collagen, Transforming Growth Factors (TGFs) and Tissue Inhibitors of Metalloproteases (TIMPs) are altered by DDR1 inhibition with nilotinib. Specific changes in vesicular transport, including the neurotransmitters dopamine and acetylcholine, and autophagy genes, including ATGs, indicate facilitation of autophagic flux and cellular trafficking. Inhibition of DDR1 with nilotinib may be a safe and effective adjunct treatment strategy involving an oral drug that enters the CNS and adequately engages its target. DDR1 inhibition with nilotinib exhibits multi-modal effects not only on amyloid and tau clearance but also on anti-inflammatory markers that may reduce cerebrovascular fibrosis.

Autophagy and unfolded protein response induction: a crosstalk between street rabies virus and the host.

The endoplasmic reticulum (ER) response mechanism to cellular stress is mediated by the unfolded protein response/ER-associated degradation (UPR/ERAD) pathway. A viral infection can trigger ER stress and engage some transcription factors, depending on the host cell and virus type, activating or inhibiting autophagy. The relationship between ER response and autophagy in rabies has not been investigated yet. In the present study, the mouse brain was infected with street rabies virus (SRABV). Total RNA was extracted from the brains of animals, and cDNA was synthesized. Next, real-time PCR assay was performed using specific primers. The expression of hypoxanthine-guanine phosphoribosyltransferase (Hprt), CCAAT/enhancer binding protein homologous protein (CHOP), apoptosis signal-regulating kinase 1 (ASK1), activating transcription factor 6 (ATF6), and caspase 3 (CASP3) genes was also investigated. Based on the results, SRABV caused significant changes in the mRNA expression of ATF6, CHOP, and ASK1 genes in the brains of infected mice in the control group (group V). Treatment of infected cells with the pIRES-EGFP-Beclin-1 vector and rapamycin caused changes in nearly most of the parameters. However, alterations in CASP3 gene expression were only observed when the vector and the virus were simultaneously injected into the cells. Overall, protection and autophagy against cell death induced by SRABV infection can be achieved by activating the ER stress pathway, followed by a marked increase in the expression of ATF6, CHOP, ASK1, and CASP3 genes.

Antiproliferative Effect of Polysaccharide Fraction of Astragalus Ovinus Against MCF-7 Cancer Cell Line

Background: The effect of Astragalus has been recognized in traditional Chinese medicine, and the roots are believed to have anti-cancer properties. This study investigated the effects of the ethanolic extract and polysaccharide fraction of Astragalus ovinus roots on MCF7 cell line. Methods: We used MTT assay to evaluate the cytotoxicity of A. ovinus roots. The status of cell cycle and apoptosis were examined, using flow cytometry. The gene expressions related to the extrinsic and intrinsic apoptotic pathways (caspases 8 &amp; 9) were also examined by real-time polymerase chain reaction (PCR). Results: The cytotoxicity data showed that the A. ovinus extract inhibited the proliferation of MCF7 cancer cells in a dose-dependent manner. The IC50 of Cisplatin , ethanolic extract and polysaccharides fraction were 22.42, 560.9, and 961.2 in µg/ml, respectively. Also, the cell cycle analysis showed that the ethanolic extract and polysaccharide fractions arrested the cell cycle in the G1 phase. Examination of the apoptotic effects showed that treatment with either the A. ovinus extract or the polysaccharide fraction induced apoptosis in MCF-7 cancer cells. The expression of caspases 8 and 9 genes was inhibited after exposure to either cisplatin or the polysaccharide fraction (PFA). However, treatment with the extract inhibited only the caspase-8 gene expression. Conclusion: The results confirmed that treatment with the extract and polysaccharide fraction caused anti-proliferative effect and apoptosis of MCF-7 cell line. Therefore, it can be concluded that the A. ovinus extract and the polysaccharides have potential therapeutic effects against human breast cancer cells.

Synthesis, DFT calculations, and anti-proliferative evaluation of pyrimidine and selenadiazolopyrimidine derivatives as dual Topoisomerase II and HSP90 inhibitors

Novel series of aminopyrimidines bearing a biologically active cyclohexenone 3a–f and oxo-selaneylidene moiety 4, besides selenadiazolopyrimidines (5a–e and 7), were synthesised using 5,6-diaminouracils as starting materials. Compound 3a exhibited strong anti-proliferative activity against three cell lines: HepG-2 (IC50 14.31 ± 0.83 µM), A-549 (IC50 30.74 ± 0.76 µM), and MCF-7 (IC50 27.14 ± 1.91 µM). Also, it was four times more selectively cytotoxic against WI-38 cell lines than doxorubicin. Furthermore, Topoisomerase II (IC50 4.48 ± 0.65 µM) and HSP90 (IC50 1.78 ± 0.11 µM) were both strongly inhibited in vitro by 3a. The cell cycle was halted at the G1-S phase, and total apoptotic cells were 65 times more than control Hep-G2 cells. Besides, it increased caspase-3 gene expression, triggering mitochondrial cell death. Molecular docking study indicated that it could bind to Topoisomerase II and HSP90 binding sites in an inhibitory mode. Its geometric properties were investigated using the density functional theory (DFT). Furthermore, compound 3a demonstrated in silico good oral bioavailability.