Exploring the Efficacy Enhancement Mechanism of Qixue Shuangbu prescription after TCM processing for treating chronic heart failure by regulating ERK/Bcl-2/Bax/Caspases-3 signaling pathway.

Qixue Shuangbu prescription (QSP) has been used for the treatment of chronic heart failure (CHF) with remarkable curative effect. Processed QSP (PQSP) could significantly improve the treatment of CHF after traditional Chinese medicine (TCM) processing. This study elucidated the underlying efficacy enhancement mechanism of QSP after TCM processing for treating CHF in vitro and in vivo. The injury of rat cardiomyoblast H9c2 cells was induced by anoxia/reoxygenation to mimic CHF state in vitro. Sixty Sprague-Dawley rats were used to established CHF model by intraperitoneally injecting doxorubicin (the accumulative dose 15 mg/kg). Biochemical examinations were performed in serum and cellular supernatant, respectively. Cardiac functions and histopathological changes were evaluated in CHF model rats. The protein and mRNA levels of ERK1/2, Bcl-2, Bax and Caspase-3 were evaluated by Western blot and RT-PCR, respectively. All above results of low dose crude QSP-treated group (L-CQSP), high dose CQSP-treated group (H-CQSP), low dose PQSP-treated group (L-PQSP), high dose PQSP-treated group (H-PQSP) were compared to systematically explore correlations between TCM processing and the efficacy enhancement for treating CHF of PQSP. Compared with the model group, the L-CQSP group showed significant improvement in cardiac function at 8th weeks, while no significant improvement in cardiomyocyte apoptosis and fibrosis. Both H-CQSP, L-PQSP and H-PQSP exerted beneficial therapeutic effects in injured H9c2 cardiomyocytes and CHF model rats. L-PQSP and H-PQSP significantly increased cell viability and the activity of SOD, decreased the activities of LDH, MDA and NO, up-regulated the expression of ERK1/2 and Bcl-2, down-regulated the expression of Bax and Caspase-3 compared to the same dosage of CQSP. The efficacy enhancement mechanism of PQSP after TCM processing for treating CHF was directly related to the regulation of ERK/Bcl-2/Bax/Caspases-3 signaling pathway.

Structural characterization and improves cognitive disorder in ageing mice of a glucomannan from Dendrobium huoshanense.

In the present work, a neutral polysaccharide (DHP-2W) with attenuating cognitive disorder was identified from Dendrobium huoshanense and its structure was clarified. The polysaccharide was successfully purified from D. huoshanense by column chromatography and its activity was evaluated. With a molecular weight of 508.934kDa, this polysaccharide is composed of mannose and glucose at a molar ratio of 75.81: 24.19. Structural characterization revealed that DHP-2W has a backbone consisting of 4)-ß-D-Manp-(1 and 4)-ß-D-Glcp-(1. In vivo experiments revealed that DHP-2W improved cognitive disorder in D-galactose treated mice and relieved oxidative stress and inflammation. DHP-2W attenuates D-galactose-induced cognitive disorder by inhibiting the BCL2/BAX/CASP3 pathway and activating the AMPK/SIRT pathway, thereby inhibiting apoptosis. Furthermore, DHP-2W had a significant effect on regulating the serum levels of Flavin adenine dinucleotide, Shikimic acid, and Kynurenic acid in aged mice. These, in turn, had a positive impact on AMPK/SIRT1 and BCL2/BAX/CASP3, resulting in protective effects against cognitive disorder.

Exosomes of endothelial progenitor cells repair injured vascular endothelial cells through the Bcl2/Bax/Caspase-3 pathway.

The main objective of this study is to evaluate the influence of exosomes derived from endothelial progenitor cells (EPC-Exo) on neointimal formation induced by balloon injury in rats. Furthermore, the study aims to investigate the potential of EPC-Exo to promote proliferation, migration, and anti-apoptotic effects of vascular endothelial cells (VECs) in vitro. The underlying mechanisms responsible for these observed effects will also be thoroughly explored and analyzed. Endothelial progenitor cells (EPCs) was isolated aseptically from Sprague-Dawley (SD) rats and cultured in complete medium. The cells were then identified using immunofluorescence and flow cytometry. The EPC-Exo were isolated and confirmed the identities by western-blot, transmission electron microscope, and nanoparticle analysis. The effects of EPC-Exo on the rat carotid artery balloon injury (BI) were detected by hematoxylin and eosin (H&E) staining, ELISA, immunohistochemistry, immunofluorescence, western-blot and qPCR. LPS was used to establish an oxidative damage model of VECs. The mechanism of EPC-Exo repairing injured vascular endothelial cells was detected by measuring the proliferation, migration, and tube function of VECs, actin cytoskeleton staining, TUNEL staining, immunofluorescence, western-blot and qPCR. In vivo, EPC-Exo exhibit inhibitory effects on neointima formation following carotid artery injury and reduce the levels of inflammatory factors, including TNF-α and IL-6. Additionally, EPC-Exo downregulate the expression of adhesion molecules on the injured vascular wall. Notably, EPC-Exo can adhere to the injured vascular area, promoting enhanced endothelial function and inhibiting vascular endothelial hyperplasia Moreover, they regulate the expression of proteins and genes associated with apoptosis, including B-cell lymphoma-2 (Bcl2), Bcl2-associated x (Bax), and Caspase-3. In vitro, experiments further confirmed that EPC-Exo treatment significantly enhances the proliferation, migration, and tube formation of VECs. Furthermore, EPC-Exo effectively attenuate lipopolysaccharides (LPS)-induced apoptosis of VECs and regulate the Bcl2/Bax/Caspase-3 signaling pathway. This study demonstrates that exosomes derived from EPCs have the ability to inhibit excessive carotid intimal hyperplasia after BI, promote the repair of endothelial cells in the area of intimal injury, and enhance endothelial function. The underlying mechanism involves the suppression of inflammation and anti-apoptotic effects. The fundamental mechanism for this anti-apoptotic effect involves the regulation of the Bcl2/Bax/Caspase-3 signaling pathway.

Portulaca oleracea L. extract relieve mice liver fibrosis by inhibiting TLR-4/NF-κB, Bcl-2/Bax and TGF-ß1/Smad2 signalling transduction.

Portulaca oleracea L. are annual herb, which has various pharmacological effects including hepatoprotective property. However, the effect of Portulaca oleracea L. (POL-1) in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and its mechanism of action have not been clarified. POL-1 ameliorated the CCl4-induced liver fibrosis in mice, as shown by decreased collagen deposition and the decreased expression of liver fibrosis marker collagen I and α-smooth muscle actin (α-SMA) mRNA. In addition, treatment with POL-1 suppressed the proliferation of activated human hepatic stellate cell line (LX-2). POL-1 inhibited the oxidative stress and inflammation in fibrotic livers of mice. Mechanistically, POL-1 inhibited the CCl4-induced expression of toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B (NF-κBp65) p65, Bcl2-associated X (Bax), transforming growth factor-ß1 (TGF-ß1) and drosophila mothers against decapentaplegic 2 (Smad2) proteins, upregulated B-cell lymphoma -2 (Bcl-2) proteins in livers of mice. These findings suggested that POL-1 attenuated liver fibrosis.

Circadian gene Per3 promotes astroblastoma progression through the P53/BCL2/BAX signalling pathway.

The key circadian genes, Period1(Per1), Period2(Per2), and Period3(Per3), constitute the mammalian Period gene family. The abnormal expression of Per1 and Per2 is closely related to tumor development, but there are few reports on Per3 and tumorigenesis. This study was conducted to determine whether the abnormal expression of Per3 could influence the progression of astroblastoma. The results indicated that the expression level of Per3 was increased in astroblastoma cells, and the high expression of Per3 was correlated with the poor overall survival time of glioma patients. The role of Per3 in astroblastoma cells was then investigated using two approaches: interference and overexpression. The interference of Per3 inhibited astroblastoma cell proliferation by inducing the cell cycle at the S phase. The interference of Per3 inhibited the migration and invasion of astroblastoma cells, while promoted the astroblastoma cell apoptosis and the expression of the apoptosis genes Cleaved-CASP3, P53, and BAX. The overexpression of Per3 promoted proliferation by affecting the S phase distribution of the astroblastoma cell cycle. The overexpression of Per3 promoted the migration and invasion of astroblastoma cells, while inhibited the astroblastoma cell apoptosis and the expression of apoptosis genes Cleaved-CASP3, P53, and BAX. RNA-seq analysis showed that the interference of Per3 in astrocytoma cells resulted in significant changes in the expression levels of 764 genes. Among the differentially expressed genes enriched in apoptosis-related pathways, the interference of Per3 resulted in significant upregulation of MARCKSL1 expression, in contrast to significant downregulation of SFRP4, EPB41L3, and GPC5 expression. Taken together, our results suggest that Per3 appears to be a pro-cancer gene by altering the proliferation, migration, invasion, and apoptosis of astroblastoma cells. As a result, the Per3 gene may be a promising therapeutic target in the treatment of astroblastoma.

Mechanism of Fufang Congrong Yizhi Capsules in treatment of mild cognitive impairment based on network pharmacology / 中国药理学通报

Aim To predict the mechanism of Fufang Congrong Yizhi Capsules (FCYC) in the treatment of mild cognitive impairment (MCI) by network pharmacology method, and further validate it in combination with cellular experiments. Methods TCMSP, Gene-Cards, OMIM and TTD databases, Chinese Pharmacopoeia and related literature were used to screen the active ingredients of FCYC and the targets of MCI treatment. The TCM-compound-target-disease network and PPI of intersection targets were constructed, and the GO and KEGG analysis were performed by the Ehamb bioinformation platform. GO and KEGG analysis were performed through Yihanbo biological information platform. Cell model of MCI was established by PC-12 injury induced by Aβ

Effects of the Supercritical Fluid Extract of Magnolia figo on Inducing the Apoptosis of Human Non-Small-Cell Lung Cancer Cells.

Lung cancer has a high incidence rate worldwide, necessitating the development of new drugs. Although Magnolia figo (Lour.) DC. is known for its medicinal properties, studies on its efficacy against lung cancer are lacking. This study investigated whether the supercritical fluid extract of M. figo (FMO) can induce apoptosis in A549, a human non-small-cell lung cancer cell line. The cell viability was assessed using an MTT assay. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis and flow cytometry analysis were conducted. The expression of factors was assessed through Western blotting analyses. Gas chromatography-mass spectrometry (GC-MS) was performed. The results revealed that FMO treatment exhibited cytotoxicity, demonstrating dose-dependent effects. The TUNEL analysis and flow cytometry analysis revealed that FMO induced apoptosis in A549 cells. The Western blotting analysis revealed that FMO upregulated the expression of p53 and Bax protein, and downregulated the expression of Bcl-2 protein. The GC-MS analysis revealed eight components identified in FMO. These findings indicate that FMO can induce A549 apoptosis through the p53/Bcl-2/Bax pathways, confirming the apoptotic effects of M. figo on lung cancer cells. These results highlight the potential, for the first time, of M. figo as a source for developing novel drugs for lung cancer treatment.

The influence of BCL2, BAX, and ABCB1 gene expression on prognosis of adult de novo acute myeloid leukemia with normal karyotype patients.

BACKGROUND: Deregulation of the apoptotic process underlies the pathogenesis of many cancers, including leukemia, but is also very important for the success of chemotherapy treatment. Therefore, the gene expression profile of main apoptotic factors, such as anti-apoptotic BCL2 (B-cell lymphoma protein 2) and pro-apoptotic BAX (BCL2-associated X), as well as genes involved in the multi-drug resistance (ABCB1), could have significant impact on the prognosis and could be used as targets for specific therapy. PATIENTS AND METHODS: We analyzed the expression of BCL2, BAX, and ABCB1 in bone-marrow samples collected at diagnosis from 51 adult patients with acute myeloid leukemia with normal karyotype (AML-NK) using real-time polymerase chain reaction method, and examined their prognostic potential. RESULTS: Increased expression of BCL2 (BCL2 +) was associated with the presence of chemoresistance (p = 0.024), while patients with low BAX expression were more prone to relapse (p = 0.047). Analysis of the combined effect of BCL2 and BAX expression showed that 87% of patients with BAX/BCL2 low status were resistant to therapy (p = 0.044). High expression of ABCB1 was associated with BCL2 + status (p < 0.001), and with absence FLT3-ITD mutations (p = 0.019). CONCLUSIONS: The present analysis of BCL2, BAX, and ABCB1 gene expression profiles is the first study focusing solely on AML-NK patients. Preliminary results showed that patients with high BCL2 expression are likely to experience resistance to chemotherapy, and may benefit from specific anti-BCL2 treatment. Further investigations conducted on a larger number of patients could elucidate actual prognostic significance of these genes in AML-NK patients.

Antioxidant capacity of N-acetylcysteine against the molecular and cytotoxic implications of cadmium chloride leading to hepatotoxicity and vital progression.

Many studies have reported that cadmium (Cd) can induce liver cell injury; however, the toxicity mechanisms of Cd on the liver have not been fully explained. Thirty-two male albino rats were divided into four groups: the control group, the N-acetylcysteine (NAC) group orally as effervescent instant sachets with a concentration of 200 mg dissolved in distilled water and dosage was 200 mg/kg body weight freshly prepared, the cadmium chloride (CdCl2) group (treated with 3 mg/kg orally), and the N-acetylcysteine (NAC) + cadmium chloride group (treated with 200 mg/kg orally post to CdCl2) for 60 days. The NAC alone did not make notable changes in most of the parameters. The CdCl2 alone, compared to control, induced significant alterations in oxidative stress markers (increment in lipid peroxidation (LPO) and nitric oxide (NO)) and antioxidant defense system (decrement in superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx)), which resulted in a downregulation of pro-apoptotic Bcl2-associated X protein (Bax) and caspase-3 and upregulation of anti-apoptotic B-cell leukemia/lymphoma 2 (Bcl2) protein as well as the survival fate of hepatic cells. Post-administration of NAC to CdCl2 resulted in a reduction in oxidative stress markers, shifting of cells from the G2/M phase to the G0/G1 inhibiting signal-regulated kinase activation, and impairment of the anti-apoptotic signaling pathway when compared to the CdCl2 group alone. Accordingly, the Bcl2/Bax ratio was reduced to 1.17-fold change, as an adaptive process to hepatic tissue injury. These findings demonstrated that NAC would attenuate the possibility of oxidative stress and cytotoxicity of hepatic tissue induced by CdCl2.

[Effects of Qilan Prescription on the proliferation and apoptosis of human prostate cancer DU145 cells and its action mechanism].

OBJECTIVE: To investigate the effects of different concentrations of Qilan Prescription (QLP) on the proliferation and apoptosis of human PCa DU145 cells and its underlying mechanism. METHODS: We treated human PCa DU145 cells with QLP at 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 or 1.56 µg/ml for 24, 48 and 72 hours respectively. Then we observed the morphological changes of the cells, examined their viability by CCK-8 assay, detected their cell cycle and apoptosis by flow cytometry, and determined the protein expressions of cyclin D1, Bax, Bcl-2 and cleaved-caspase 3 in the DU145 cells by Western blot, followed by comparison of the parameters with those obtained from the blank control group. RESULTS: QLP significantly inhibited the growth, reduced the contour clarity and adhesion ability of the DU145 cells at the concentrations of 100, 200 and 400 µg/ml, and markedly decreased the activity of the cells at 200 and 400 µg/ml, most significantly at 400 µg/ml. The number of the G2-phase DU145 cells was dramatically increased in all the concentration groups (P < 0.01), so was the total number of apoptotic DU145 cells (P < 0.01), while that of the S-phase cells remarkably decreased in the 400 µg/ml QLP (P < 0.01) and 200 µg/ml QLP (P < 0.05) groups. The expression of the cyclin D1 protein was significantly down-regulated in the 400 µg/ml QLP group (P < 0.01). That of Bcl-2 was also down-regulated (P < 0.01) while those of Bax and cleaved-caspase 3 up-regulated in the 400 and 200 µg/ml QLP groups (P < 0.01). CONCLUSION: QLP can inhibit the proliferation and promote the apoptosis of human PCa DU145 cells, which may be associated with its effects of down-regulating the expression of the cell cycle-related protein cyclin D1, disrupting the Bax-Bcl-2 balance, and up-regulating the expression of cleaved-caspase 3.

The protective effect of trigonelline on H / 中国药理学通报

Aim To study the protective effect of trigonelline on H

Effect of SMAC Gene on Sensitivity of Lung Adenocarcinoma Cells to Paclitaxel and Cell Viability Based on caspase-3/Bcl-2/Bax Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect of the SMAC gene on paclitaxel sensitivity and cellular activity in lung adenocarcinoma cells based on the caspase-3/Bcl-2/Bax signaling pathway. Methods A paclitaxel-resistant cell line A549/Taxol was established for lung adenocarcinoma, and the cells were divided into four following groups: pcDNA-NC (transfected with pcDNA-NC blank vector), pcDNA-SMAC (transfected with pcDNA-SMAC vector), siRNA-NC (transfected with siRNA-NC empty virus vector), and siRNA-SMAC groups (transfected with siRNA-SMAC lentiviral vector). The SMAC mRNA expression in cells was detected by qRT-PCR; cell sensitivity was detected by MTT; cell proliferation ability was detected by cloning assay; cell invasion ability was detected by Transwell; apoptosis ability was detected by flow cytometry assay; and caspase-3, Bcl-2 and Bax protein expression in cells were detected by Western blot analysis. Results The SMAC mRNA expression was significantly lower in A549 cells compared with BEAS-2B cells (P < 0.05). The SMAC mRNA expression was significantly higher in the pcDNA-SMAC group than that in the pcDNA-NC group cells (P < 0.05). The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than that in the siRNA-NC group. The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than in the siRNA-NC group. Compared with the pcDNA-NC group, the cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly lower in the pcDNA-SMAC group, the cell resistance index reversal was 2.51-fold, and the apoptosis ability and caspase-3, as well as Bax protein expression, were significantly higher (P < 0.05). Compared with the siRNA-NC group, cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly higher in the siRNA-SMAC group, and apoptosis ability and caspase-3 and Bax protein expression were significantly lower (P < 0.05). Conclusion High expression of SMAC increases paclitaxel sensitivity, inhibits cell growth and invasion, promotes apoptosis in lung adenocarcinoma cells, and has a regulatory effect on the caspase-3/Bcl-2/Bax signaling pathway.

[Xanthoceras sorbifolium Bunge flower extract inhibits benign prostatic hyperplasia in rats].

OBJECTIVE: To assess the inhibitory effect of the extract of Xanthoceras sorbifolium Bunge flower against benign prostatic hyperplasia (BPH) and explore its possible mechanism. METHODS: MTT assay was used to examine the effect of the extract of Xanthoceras sorbifolium Bunge flower on proliferation of benign prostatic hyperplasia cells (BPH-1), and cell apoptosis and cell cycle changes following the treatment were analyzed using annexin V/PI double staining and flow cytometry. The protein expression levels of Bcl-2, Bax, caspase-3, PI3K and AKT in the treated cells were detected using Western blotting. A rat model of BPH established by subcutaneous injection of testosterone propionate was treated with the flower extract for 28 days, and pathological changes in the prostate tissue were observed with HE staining. The protein expression levels of Bcl-2, Bax, caspase3 and PI3K/AKT in the prostate tissue were detected with Western blotting. RESULTS: Within the concentration range of 125-1000 µg/mL, the flower extract of Xanthoceras sorbifolium Bunge significantly inhibited the proliferation of BPH-1 cells and caused obvious cell cycle arrest at G0/G1 phase; the apoptotic rate of the cells was positively correlated with the concentration of the flower extract (P < 0.05). Bcl-2, p-PI3K and p-AKT expression levels were significantly down-regulated and Bax and caspase-3 expression levels were significantly increased in the cells after treatment with the flowers extract (P < 0.05). In the rat models of BPH, the rats treated with the flowers extract at moderate and high doses showed obviously decreased expressions of p-AKT and Bcl-2 and an increased expression of Bax in the prostate tissue; a significantly lowered p-AKT expression was observed in the prostate tissue of rats receiving the low-dose treatment (P < 0.05). CONCLUSION: The flower extract of Xanthoceras sorbifolium Bunge has a inhibitory effect on BPH both in vitro and in rats, suggesting its potential value in the development of medicinal plant preparations for treatment of BPH.

Para­hydroxybenzaldehyde against transient focal cerebral ischemia in rats via mitochondrial preservation.

Gastrodia elata (GE) Blume has been widely used for thousands of years to treat various central and peripheral nervous disorders. P-hydroxybenzaldehyde (PHBA) is a chemical component of GE. However, its role and mechanism in transient focal cerebral ischemia remain unclear. The present study aimed to investigate the protective effect of PHBA on middle cerebral artery occlusion (MCAO) rats. A total of 56 Sprague-Dawley male rats were randomly divided into control, model, PHBA-high dose (PHBA-H) and PHBA-low dose (PHBA-L) groups. The MCAO injury model was replicated in all rats except for the control group. In the control group, only the right common carotid artery was isolated without embolization. After treatment with PHBA, the protective effects (neurological deficit score, cerebral index, weight and cerebral infarct) were analyzed. Western blotting was performed to estimate the protein levels of Bcl-2, Bax and Caspase-3. Apoptotic cells were detected using hematoxylin-eosin staining and TUNEL immunofluorescence assay. Mitochondrial oxidative stress indicators, including reactive oxygen species (ROS), malondialdehyde (MDA) and total superoxide dismutase (T-SOD), while dysfunction indicators, including mitochondrial permeability transition pore (MPTP), ATP and cytochrome C oxidase, were measured using commercial kits. The ultrastructure of mitochondria was observed under an electron microscope. Once the model was successful established, the rats in the MCAO group suffered neurological damage (P<0.001), increased cerebral index (P<0.001), decreased body weight (P<0.001) and had severe cerebral infarction (P<0.001). Moreover, the number of apoptotic cells and the levels of ROS (P<0.001) and MDA (P<0.05) in mitochondria and the protein levels of Bax (P<0.001) and cleaved caspase-3 (P<0.001) were increased. The activities of T-SOD (P<0.001) and cytochrome C oxidase (P<0.001) in the mitochondria, ATP content (P<0.05) and Bcl-2 protein level (P<0.001) decreased, MPTP was stimulated to open and mitochondrial structures were damaged (P<0.001). PHBA treatment resulted in a decrease of the neurological deficit score (PHBA-H 24 h, P<0.001; PHBA-H 6 h and PHBA-L 24 h, P<0.01; PHBA-L 6 h, P<0.05), apoptotic cell number (P<0.001), mitochondrial ROS (P<0.001) and MPTP opening (P<0.001), Bax (P<0.01, P<0.001) and cleaved caspase-3 protein expression (P<0.001) in rats. And the expression of Bcl-2 protein (P<0.001) was increased. In addition, the cerebral index (P<0.05), weight loss (P<0.05), infarction rate (P<0.01) and MDA content (P<0.001) were decrease in the PHBA-H group. The level of ATP (P<0.05) and cytochrome C oxidase (P<0.05) and T-SOD activity (P<0.05) of PHBA-H group rats increased, but no significant difference was observed in the PHBA-L group. Overall, PHBA had a protective effect on transient focal cerebral ischemia in normal rats, regulated the expression of Bcl-2, Bax and cleaved caspase-3 proteins and improved the oxidative stress and dysfunction of mitochondria.

Pseudogene CSPG4P12 affects the biological behavior of non­small cell lung cancer by Bcl­2/Bax mitochondrial apoptosis pathway.

Increasing evidence has shown that chondroitin sulfate proteoglycan 4 (CSPG4) serve a critical role in tumor progression. However, the roles of chondroitin sulfate proteoglycan 4 pseudogene 12 (CSPG4P12) remain to be elucidated. The present study aimed to investigate the potential effects of CSPG4P12 on the physiological behaviors of non-small cell lung cancer (NSCLC) and its underlying biological mechanism. The expression levels of CSPG4P12 in NSCLC tissues and adjacent normal tissues were analyzed using the gene expression profiling interactive analysis 2 database and reverse transcription-quantitative PCR. Cell Counting Kit-8 and colony formation assays were performed to measure cell proliferation. In addition, Transwell and wound healing assays were performed to assess cell invasion and migration. Cell adhesion was measured by cell-extracellular matrix adhesion assay. Hoechst 33342 staining assay was performed to detect nucleoli of apoptotic cells, and transmission electron microscopy (TEM) was utilized for apoptosis detection. Immunofluorescence and western blot assays were performed to measure the expression levels of apoptosis-related proteins. The present results revealed that the expression levels of CSPG4P12 in NSCLC tissues were significantly lower compared with those in adjacent normal tissues. Overexpression of CSPG4P12 inhibited cell proliferation, invasion, migration and adhesion whilst promoting apoptosis. There were missing mitochondrial cristae and mitochondrial vacuoles in the CSPG4P12-overexpressed cells when observed under TEM. Overexpression of CSPG4P12 also increased the expression of Bax and p53, whereas it inhibited the expression of Bcl2. In conclusion, CSPG4P12 could inhibit NSCLC development and tumorigenesis by activating the p53/Bcl2/Bax mitochondrial apoptotic pathway.

Immunosuppression and apoptosis activation mediated by p53-Bcl2/Bax signaling pathway -The potential mechanism of goldfish (Carassius auratus Linnaeus) gill disease caused by Myxobolus ampullicapsulatus.

Myxobolus, a major harmful type of myxospora, is one of the main parasitic pathogens of freshwater fish. Once myxoboliosis occurs, treatment can be extremely difficult. Therefore, clear understandings of the etiology of myxoboliosis and its pathological mechanism are keys for prevention and control. Here, histology, transmission electron microscopy, transcriptome study, tunel assay, and immunohistochemistry were carried out, revealing the morphology, pathological effects as well as host response mechanism of goldfish gill to Myxobolus ampullicapsulatus. Histological studies showed that the mature spores of Myxobolus ampullicapsulatus were composed of three parts, the spore shell, sporoplasm and bottle shaped polar capsule containing double S-shaped polar filaments. Transcriptome analysis revealed that Myxobolus ampullicapsulatus -infected (Myx) goldfish gills were characterized by apoptosis activation mediated by "p53 signaling pathway" with significantly up-regulated apoptosis-related differential genes dominated by p53-Bcl2/Bax signaling pathway. In addition, tunel assay revealed severe gill apoptosis in the Myx group. Transcriptome analysis also revealed that Myx group showed changes in immune response and significantly down-regulated immune-related differential genes. Beyond that, immunohistochemistry showed that there was no significant increase in the number of gill lymphocyte after parasite infection. These results suggest that the pathological mechanism of Myxobolus ampullicapsulatus infection on gills of goldfish may be related to apoptosis and immunosuppression. Subsequent qRT-PCR showed that apoptosis-related genes (Caspase3,Bad, Bax) and anti-inflammatory gene IL-10 were significantly increased, while immune-related pro-inflammatory genes (IL-1ß, IL-8) were markedly down-regulated, further verifying the transcriptome results. Based on the above results, we concluded that p53-Bcl2/Bax related networks that dominant the expression of apoptosis genes were activated while immunity was suppressed in the gills of Myxobolus ampullicapsulatus infected goldfish. Our study is not only of benefit to enrich the taxonomy of Myxobolus but also clarifies its pathogenic mechanism, thus providing targets for prevention and control of myxoboliosis.

Role of Lactiplantibacillus plantarum strain RD1 (Lpb RD1) in mitochondria-mediated apoptosis: an in vitro analysis.

The purpose of this study was to determine the cytotoxicity of Lactiplantibacillus plantarum strain RD1 (Lpb RD1), which was isolated and identified from the curd by 16 S rRNA sequencing. The probiotic properties of the isolated strain were studied by bile and NaCl tolerance and the ethyl acetate extract of Ea-LpRD1, was used to determine the toxicity against human breast cancer (MCF-7) cell lines and human embryonic kidney (HEK-293) cell lines by MTT assay. DNA fragmentation assay was carried out to study apoptosis induction. Flow cytometry analysis was done to determine the % of a cell population using the FTIC-Annexin V staining method. RT-PCR was used to assess gene expression levels in both cell lines. The IC50 concentration of the Ea-LpRD1 in MCF-7 cells was 0.30 mg/ml and in HEK-293 was 0.47 mg/ml. The expression levels of the BCL-2 gene anti-apoptotic genes in humans were reduced and BAX, caspase-8, caspase-3, and caspase-9 were an increased expression in MCF-7 cell lines.

Protective effects of Ligularia fischeri root extracts against ulcerative colitis in mice through activation of Bcl-2/Bax signalings.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by high levels of proinflammatory cytokines and epithelial barrier dysfunction. The root of Ligularia fischeri (Ledeb.) Turcz. is a traditional Chinese medicinal herb with diverse therapeutic properties, which has been successfully used to treat inflammation-related diseases. However, little is known about its effect and mechanism against UC. PURPOSE: To investigate the efficacy and mechanism of L. fischeri root extracts against UC. METHODS: L. fischeri root samples were prepared using the alcohol extraction method and liquid-liquid extraction method. A dextran sodium sulfate-induced UC mouse model and a lipopolysaccharide (LPS)-induced inflammatory cell model were employed in the present study. Cell apoptosis was detected by TUNEL staining, and an enzyme-linked immunosorbent assay was used to quantify the abundance of inflammatory factors in tissues. Hematoxylin and eosin staining and Masson staining were employed to analyze drug toxicity to the liver and kidney. A myeloperoxidase (MPO) assay kit was used to detect neutrophil infiltration in colon tissues. RT-qPCR was then employed to quantify the transcriptional levels of proinflammatory and apoptotic-related genes, while tight junction and apoptosis-related proteins were quantified via western blotting. Gas Chromatography/Mass Spectrometry analysis was then performed to identify the natural compounds in L. fischeri root extracts. RESULTS: The water decoction extract, methanol extract, and especially the chloroform extract (CE) exerted potent therapeutic effects in UC mice. Similar to the positive control group (5-aminosalicylic acid), oral administration of CE (30, 60, and 90 mg/kg/d) elicited distinct therapeutic effects on UC mice in the medium- and high-dose groups. CE decreased disease activity index, histopathological score, and MPO level significantly, and effectively retained the colon length. Furthermore, CE significantly reduced the levels of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and enhanced the expression of tight junction proteins, such as zonula occludens (ZO)-1, ZO-2, claudin-1, and occludin, as well as the transcriptional levels of mucins, such as MUC-1 and MUC-2, in UC mice. Notably, CE prevented apoptosis of colonic epithelial cells by up-regulating Bcl-2 and down-regulating Bax. Also, CE inhibited the secretion of pro-inflammatory cytokines and apoptosis in LPS-induced RAW264.7 macrophages via the activation of Bcl-2/Bax signals. CONCLUSIONS: Collectively, L. fischeri root extracts, especially CE, have obvious therapeutic effects against UC. CE reduces inflammation and protects the intestinal epithelial cells and intestinal epithelial barrier via activation of the Bcl-2/Bax signaling pathway, and may be a promising therapeutic agent for UC treatment.

In vivo Study of a Newly Synthesized Chromen-4-one Derivative as an Antitumor Agent against HCC.

BACKGROUND: Chromenes are a wide group of natural compounds that can be synthesized chemically. The chromen-4-one nucleus acts as a skeleton for varieties of additional active groups that makes the chromene activity vary between antioxidant and anti-inflammatory agents. In the present study, a newly synthesized chromene compound exhibits different behaviors other than anti-inflammatory and antioxidant activities that it is the first time that a member of chromen-4-one compound can control the cancer progress. Inflammation is the first step in tumor development where the severity grade can potentiate tumor growth and progression. In many tumors, pro-inflammatory genes record high expression level such as tumor necrosis factor (TNF-α) and vascular endothelial growth factors (VEGF). These pro-inflammatory factors act as rate limiting steps in tumor initiation, and controlling its expression acts as an early therapeutic way to control the tumor proliferation. The chromone derivatives have biological activities such as anti-inflammatory and anti-tumor activity. METHODS: In the present study, hepatocellular cancer (HCC) induced by diethylnitrosamine (DEN) in rats and then treated with the new chromene derivative and the parameters TNF-α, VEGF, p53, Cyt C, MMP-9, Bcl2, and Bax were measured. RESULTS: The treatment strategy Ch compound is to downregulate pro-inflammatory gene expression of early genes as TNF-α as well as VEGF and subsequently control other factors such as p53, Cyt C, and MMP-9. Also, retrieve the balance between Bcl2 and Bax proteins in DEN-induced HCC in rats. CONCLUSION: The ability of the new Ch derivative to control the primary initiators of HCC such as TNF-α offers this derivative an anti-tumor activity and encourages further researches to follow and monitor its effect on the molecular level.

Curcumin represses lipid accumulation through inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis in porcine subcutaneous preadipocytes.

OBJECTIVE: Excessive lipid accumulation in adipocytes results in prevalence of obesity and metabolic syndrome. Curcumin (CUR), a naturally phenolic active ingredient, has been shown to have lipid-lowering effects. However, its underlying mechanisms have remained largely unknown. Therefore, the study aims to determine the effect of CUR on cellular lipid accumulation in porcine subcutaneous preadipocytes (PSPA) and to clarify novel mechanisms. METHODS: The PSPA were cultured and treated with or without CUR. Both cell counting Kit-8 and lactate dehydrogenase release assays were used to examine cytotoxicity. Intracellular lipid contents were measured by oil-red-o staining extraction and triglyceride quantification. Apoptosis was determined by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling assay. Adipogenic and apoptosis genes were analyzed by quantitative polymerase chain reaction and Western blot. RESULTS: The CUR dose-dependently reduced the proliferation and lipid accumulation of PSPA. Noncytotoxic doses of CUR (10 to 20 µM) significantly inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and expression of adipogenic genes peroxisome proliferation-activity receptor-γ (PPAR-γ), CCAAT/enhancer binding protein-α, sterol regulatory element-binding protein-1c, adipocyte protein-2, glucose transporter-4 as well as key lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase, while ERK1/2 activation significantly reversed CUR-reduced lipid accumulation by increasing PPAR-γ. Furthermore, compared with differentiation induced media treated cells, higher dose of CUR (30 µM) significantly decreased the expression of AKT and B-cell lymphoma-2 (BCL2), while increased the expression of BCL-2-associated X (BAX) and the BAX/BCL-2 expression ratio, suggesting triggered apoptosis by inactivating AKT and increasing BAX/BCL-2 ratio and Caspase-3 expression. Moreover, AKT activation significantly rescued CUR inhibiting lipid accumulation via repressing apoptosis. CONCLUSION: These results demonstrate that CUR is capable of suppressing differentiation by inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis via decreasing AKT and subsequently increasing BAX/BCL-2 ratio and Caspase-3, suggesting that CUR provides an important method for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.