Expression Of Apoptosis-related Factors Research Articles

Scutellarin alleviates liver injury in type 2 diabetic mellitus by suppressing hepatocyte apoptosis in vitro and in vivo

ObjectiveScutellarin is a primary active composition come from Erigeronbreviscapus. It is well known that scutellarin has anti-inflammatory and antioxidant physiological functions. In this study, we detected the effects of scutellarin on hepatocyte cell apoptosis in type 2 diabetes mellitus (T2DM) rats. MethodsSprague Dawley (SD) (6–8 weeks, 160–180 g) rats were randomly divided into six groups: control, model, scutellarin low-dose, medium-dose, high-dose treatment, and rosiglitazone positive groups; with 10 SD rats in each group (n = 10). The changes of biochemical factors in serum were detected by automatic biochemical instrument, the pathological changes of liver tissue were detected by hematoxylin and eosin (HE) staining, the apoptosis of liver tissue and cells was detected by tissue staining and flow analyzer, and the expression of apoptosis-related factors were determined by qPCR, Western blot and immunohistochemistry in liver tissues or cells. ResultsThe results showed that scutellarin decreased the levels of fasting blood glucose, total cholesterol, triglyceride, and low-density lipoprotein and increased the levels of high-density lipoprotein. Meanwhile, scutellarin decreased the levels of alanine transaminase (ALT) and aspartate transaminase (AST) and improved liver function. In addition, scutellarin suppressed the secretion of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and reduced hepatocyte apoptosis. Furthermore, scutellarin inhibited the expression of cleaved Caspase-3, Bax, and cytochrome C (Cyt-C) and promoted the expression of Bcl-2. ConclusionScutellarin can inhibit the apoptotic pathway, thereby relieving T2DM.

Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis rats

To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion (combination) groups (12 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension （10 mL/kg）. EA (10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion (3 cones) was applied to "Zhongwan" (CV12), bilateral "Neiguan" (PC6) and bilateral "Sanyinjiao" (SP6) of the related groups, once a day for 3 weeks. The blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The apoptosis index of gastric antrum cells were observed by TUNEL staining. The protein and mRNA expressions of Caspase-3, B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in gastric antrum were detected by Wes-tern blot and real-time quantitative PCR, respectively. Compared with the normal group, the blood glucose, the apoptosis index, the protein and gene expressions of Caspase-3 and Bax were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsive rate, the protein and gene expressions of Bcl-2 were considerably decreased (P<0.01) in the model group. In contrast to the model group, the blood glucose in the EA, moxibustion and combination groups, the apoptosis index in the 4 treatment groups, as well as Caspase-3 protein, Bax protein and mRNA expressions in the medication, EA and combination groups, Caspase-3 protein and mRNA, Bax mRNA expressions in the moxibustion group were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate in the 4 treatment groups, and Bcl-2 protein and mRNA expressions in the medication and combination groups, Bcl-2 mRNA expressions in the EA and moxibustion groups were obviously increased (P<0.01). The effects of EA+moxibustion were significantly superior to those of simple EA, moxibustion or medication in increasing gastric emptying rate and intestinal propulsive rate, and in lowering blood glucose (P<0.05, P<0.01). And the effects of the combination treatment were better than those of EA in lowering Caspase-3 protein and Bax mRNA expressions (P<0.01), and in increasing Bcl-2 protein and mRNA expressions (P<0.05, P<0.01). Also the effects of the combination treatment were better than those of moxibustion in lowering the apoptosis index, Caspase-3 protein, and Bax protein and mRNA expressions (P<0.01, P<0.05), and in increasing Bcl-2 protein expression (P<0.05). EA combined with Zhuang-medicine-thread moxibustion can reduce blood glucose and improve gastrointestinal motility in DGP rats, which may be related to its effect in regulating of Caspase-3, Bax and Bcl-2 expression.

Synthesis and characterization of polypyridine ruthenium(II) complexes and anticancer efficacy studies in vivo and in vitro

In this article, ligand IPP (IPP = 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline) and its three Ru(II) complexes: [Ru(bpy)2(IPP)](ClO4)2 (1) (bpy = 2,2′-bipyridine), [Ru(dmbpy)2(IPP)](ClO4)2 (2) (dmbpy = 4,4′-dimethyl-2,2′-bipyridine), and [Ru(phen)2(IPP)](ClO4)2 (3) (phen = 1,10-phenanthroline) were synthesized and characterized. The anticancer activity in vitro of the complexes was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The scratching and colony-forming experiments confirmed the complexes 1, 2, 3 interfered with the proliferation and migration ability of cells. The accumulation of the complexes in cells was researched and we found that these complexes directly accumulated in mitochondria, then the complexes cause a decline of the mitochondrial membrane potential and induce an increase of intracellular reactive oxygen species (ROS) levels. The growth of B16 cells were inhibited by 1, 2 and 3 at G0/G1 phase. Apoptosis was induced through mitochondrial pathway and the expression of apoptosis-related factors was regulated. In addition, the complexes promoted the transition of poly(ADP-ribose)polymerase (PARP) into the cleaved form (Cleaved PARP), downregulated the anti-apoptotic proteins, and upregulated the pro-apoptotic proteins. Consequently, complexes 1, 2 and 3 exerted their anticancer activity by regulating B-cell lymphoma-2 (Bcl-2) family proteins. Complex 2 showed excellent antitumor effects with a high inhibitory rate of 65.95% in vivo. Taken together, the complexes cause apoptosis in B16 cells through a ROS-mediated mitochondrial dysfunction pathway.

PCDH8 participates in the growth process of colorectal cancer cells by regulating the AKT/GSK3β/β-catenin signaling pathway

Protocadherin 8 (PCDH8) is lower-expressed in many cancers (gastric cancer, breast cancer, bladder cancer and nasopharyngeal cancer), while the molecular mechanism underlying its effects in human colorectal cancer has not been reported. Therefore, this study aims to investigate the mechanism of PCDH8 in colorectal cancer. After colorectal cancer cells transfected with overexpressing or silencing the gene PCDH8, then the effects of PCDH8 on cell viability, migration, invasion and apoptosis were determined by a series of molecular biological experiments, respectively. The expressions of apoptosis-related factors, AKT/GSK3β/β-catenin signaling pathway- and epithelial-mesenchymal transition (EMT)-related proteins were quantified by western blotting. PCDH8 expression was significantly reduced in the colorectal cancer tissue and cell lines. Overexpressed PCDH8 significantly reduced the proliferation, migration and invasion of colorectal cancer cells, while silent PCDH8 had the opposite effects. Moreover, overexpressed PCDH8 could induce apoptosis, significantly down-regulate expressions of B-cell lymphoma-2 (Bcl-2), N-Cadherin and Vimentin but up-regulate those of Bcl-2 associated X (Bax), cleaved caspase-3 and E-Cadherin, whereas the negative regulations on the apoptosis and the expressions of apoptosis- and EMT-related proteins were observed in colorectal cancer cells following the silence of PCDH8. Furthermore, overexpressed PCDH8 significantly inhibited the phosphorylation of AKT and GSK3β, and repressed the expression of β-catenin, while silent PCDH8 had the opposite effects. In short, PCDH8 overexpression inhibits proliferation, invasion, EMT, and induces apoptosis of colorectal cancer cells by regulating AKT/GSK3β/β-catenin signaling pathway.

Scopolin obtained from Smilax china L. against hepatocellular carcinoma by inhibiting glycolysis: A network pharmacology and experimental study

Ethnopharmacological relevanceSmilax china L. is a well-known traditional medicinal plant. In China, it is a common anti-cancer drug that has been inherited for thousands of years. Some in vitro and in vivo studies have confirmed its potential lipid-lowering, anti-inflammatory and anti-ovarian cancer effects. However, there is no research on the material basis and mechanism of the rhizome of Smilax china L. against hepatocellular carcinoma. Aim of the studyTo explore the material basis and mechanism of scopolin from Smilax china L. against hepatocellular carcinoma. MethodsThe potential targets and active components of Smilax china L. against hepatocellular carcinoma were screened by transcriptomics, network pharmacology and molecular docking. Microscale Thermophoresis (MST) detection was used to verify the affinity of small molecule compounds with potential proteins and protein-protein interaction. The Extract from HepG2 cells was used to measure the expression of glycolysis-related proteins, glucose consumption and lactate production. The expression of apoptosis-related factors and glycolysis-related proteins in vivo was detected by immunohistochemistry. ResultsThe glycolysis-related proteins glucose-6-phosphate isomerase (GPI), glycerol-3-phosphate dehydrogenase, mitochondrial (GPD2) and phosphoglycerate kinase 2 (PGK2) screened by transcriptomics, network pharmacology showed strongly binding with scopolin by molecular docking. MST detection has also verified the affinity of scopolin with GPI and GPD2. It was the first time found that Heat shock protein HSP 90-alpha (Hsp90α) bound strongly to GPI and GPD2 in the worldwide, while scopolin was able to affect the interaction between Hsp90α and GPD2. In vitro and in vivo experiments further demonstrated that scopolin may play an anti-cancer role by affecting the stability of tumor-associated proteins. The results showed that scopolin obtained from Smilax china L. could regulate the expression of GPI, GPD2 and PGK2 and inhibit the interaction of protein-protein, reduce the energy metabolism of tumor tissue, thereby inhibit tumor growth. ConclusionScopolin obtained from Smilax china L. plays the role of anti-hepatocellular carcinoma by regulating the expression of glycolysis proteins GPI, GPD2 and PGK2. Scopolin could affect the interaction between Hsp90α and GPD2 may provide a novel potential treatment direction for hepatocellular carcinoma.

Protective effects of Panax ginseng berry extract on blue light-induced retinal damage in ARPE-19 cells and mouse retina

BackgroundAge-related macular degeneration (AMD) is a significant visual disease that induces impaired vision and irreversible blindness in the elderly. However, the effects of ginseng berry extract (GBE) on the retina have not been studied. Therefore, this study aimed to investigate the protective effects of GBE on blue light (BL)-induced retinal damage and elucidate its underlying mechanisms in human retinal pigment epithelial cells (ARPE-19 cells) and Balb/c retina. MethodsTo investigate the effects and underlying mechanisms of GBE on retinal damage in vitro, we performed cell viability assay, pre-and post-treatment of sample, reactive oxygen species (ROS) assay, quantitative real-time PCR (qRT-PCR), and western immunoblotting using A2E-laden ARPE-19 cells with BL exposure. In addition, Balb/c mice were irradiated with BL to induce retinal degeneration and orally administrated with GBE (50, 100, 200 mg/kg). Using the harvested retina, we performed histological analysis (thickness of retinal layers), qRT-PCR, and western immunoblotting to elucidate the effects and mechanisms of GBE against retinal damage in vivo. ResultsGBE significantly inhibited BL-induced cell damage in ARPE-19 cells by activating the SIRT1/PGC-1α pathway, regulating NF-kB translocation, caspase 3 activation, PARP cleavage, expressions of apoptosis-related factors (BAX/BCL-2, LC3-Ⅱ, and p62), and ROS production. Furthermore, GBE prevented BL-induced retinal degeneration by restoring the thickness of retinal layers and suppressed inflammation and apoptosis via regulation of NF-kB and SIRT1/PGC-1α pathway, cleavage of caspase 3 and PARP, and expressions of apoptosis-related factors in vivo. ConclusionsGBE could be a potential agent to prevent dry AMD and progression to wet AMD.

Cytosine-phosphate-guanine oligodeoxynucleotides regulate the cell cycle, apoptosis, and steroidogenesis of mouse ovarian granulosa cells by targeting inhibin alpha (1 ~ 32) fragments.

Cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODNs), which exist in vertebrate, bacterial, and viral genomes, are regarded as strong immune adjuvants. To date, the biological activities of CpG-ODNs in reproduction remain unknown. Here, we investigated the effects of CpG-ODNs on the cell cycle, apoptosis, and steroidogenesis in mouse granulosa cells (mGCs), in combination with inhibin alpha (1 ~ 32) fragments. mGCs were transfected with pEGFP (containing green fluorescent protein, as a control), pEGISI (containing inhibin alpha (1 ~ 32) fragments), or pEGISI-CpG-ODNs (containing inhibin alpha (1 ~ 32) fragments and CpG-ODNs motifs) plasmid for 48h in vitro. Our results showed that the mRNA and protein expression levels of inhibin alpha were downregulated in mGCs transfected with pEGISI-CpG-ODNs, compared to those transfected with pEGISI. Flow cytometry demonstrated that pEGISI-CpG-ODNs transfection promoted cell proliferation (for example, increasing the number of cells in S and G2 phases) and decreased apoptosis, compared to pEGISI transfection. The present study also indicated that the expression of cell cycle-related genes (cyclin D2, cyclin D3, cyclin E1, Cdk2, and Cdk6) was increased, while the expression of apoptosis-related factors (Fas, FasL, caspase-8, and caspase-3) decreased after pEGISI-CpG-ODNs treatment. Additionally, pEGISI-CpG-ODNs reversed the effect of pEGISI on the secretion of estradiol in mGCs, which was further validated by upregulating the levels of its synthesis-related factors (StAR, Cyp11a1, and 17β-HSD II). Nevertheless, pEGISI-CpG-ODNs or pEGISI did not affect the concentration of progesterone nor changed the expression levels of its synthesis-related factors (3β-HSD I and Cyp19a1). In conclusion, this study demonstrated that CpG-ODNs may affect the cell cycle, apoptosis, and steroidogenesis by targeting the effects of inhibin alpha (1 ~ 32) fragments, supporting the potential role of CpG-ODNs in the development of granulosa cells.

Long noncoding RNA CCAT2 reduces chemosensitivity to 5-fluorouracil in breast cancer cells by activating the mTOR axis.

Breast cancer (BC) is the most prevalent cancer in women and the second leading cause for cancer‐related death in women. LncRNA CCAT2 is involved in BC cell drug sensitivity. Drug resistance of BC cells after chemotherapy is the main obstacle to therapeutic effects. This study explored whether BC cell drug sensitivity to 5‐Fu was related to lncRNA CCAT2‐regulated mTOR pathway. Normal breast tissues and BC tissues before/after neoadjuvant chemotherapy were collected, and CCAT2 expression was detected by RT‐qPCR. Correlation between CCATA2 expression and neoadjuvant chemotherapy efficacy was analysed using the Kendall's tau‐b correlation analysis. Normal breast epithelial cells and BC cell lines were cultured. BC cell lines were treated with 5‐Fu, and CCAT2 mRNA level in cells was detected. The 5‐Fu‐resistant MCF‐7/5‐Fu and MDA‐MB‐231/5‐Fu cells were treated with CCAT2 overexpression/knockdown or CCI‐779 (the mTOR pathway inhibitor). The mTOR pathway levels were detected. Expression of apoptosis‐related factors was identified. A subcutaneous xenograft model was carried out. High CCAT2 expression was detected in BC tissues and BC drug‐resistant cells after neoadjuvant chemotherapy, and a negative link was revealed between CCAT2 expression and efficacy of neoadjuvant chemotherapy. p‐mTOR/mTOR in 5‐Fu‐resistant BC cells with inhibited CCAT2 was decreased, while CCAT2 overexpression activated the mTOR pathway. IC50 value, proliferation, cells in S phase increased and apoptosis reduced after CCAT2 overexpression. After si‐CCAT2 or CCI‐779 treatment, the growth rate of transplanted tumours was inhibited, while promoted after CCAT2 overexpression. CCAT2 may reduce BC cell chemosensitivity to 5‐Fu by activating the mTOR pathway.

Analyzing the Effect of Vitronectin on Cell Growth and Mesenchymal-Epithelial Transition of Pulmonary Fibroblast Cells.

Vitronectin (VTN), a multifunctional glycoprotein, is involved in various biological and pathological processes. The purpose of this study was to explore the effect of VTN on mesenchymal-epithelial transition (MET) of pulmonary fibroblast cells. Lentivirus encoding for VTN-specific shRNA was constructed and infected into the cultured fibroblast WI-38 cells. Real-time PCR and Western blot were applied to examine the expression of VTN in WI-38 cells. MTT assay was used to assess cell proliferation. Western blot was conducted to examine the expression of MET-related and apoptosis-related proteins. The knockdown of VTN significantly inhibited the growth of WI-38 cells compared to the control group. Meanwhile, knockdown of VTN remarkably increased the expression of Bax and Caspase 3 compared with the control group. Furthermore, knockdown of VTN significantly promoted the expression of E-cadherin in comparison to control group. Knockdown of VTN promoted the expression of apoptosis-related factors, meanwhile, facilitated the MET process of fibroblast cells by regulating the expression of relevant factors. In sum, VTN performed a potential regulator in cell growth and MET of pulmonary fibroblast cells, which can be considered as a potential target for diagnose and therapy of relevant diseases.

Pregnane Steroids from the Leaves of Melia Azedarach and Apoptotic Activity against T47D Cells.

Objective:Nature has provided us with many pharmaceutical resources so far. Breast cancer shows an increasing trend in the world for the last decade and becomes one of five leading causes of death. Among the plants, Melia azedarach L. has been used widely in traditional medicine for many ailments including breast cancer. Following our previous findings that the ethyl acetate fraction was the most active cytotoxic fraction against T47D cells, we aimed to isolate the cytotoxic compounds and further elucidate their apoptotic mechanisms. Methods:The compounds were isolated through a series of chromatography with cytotoxicity evaluations. Identification of the isolated compounds was achieved by intensive spectroscopic analysis such as NMR, MS, and IR spectra. Cytotoxicity was evaluated by MTT method using doxorubicin as a reference compound. The expression of apoptosis-related factors was quantified by flow cytometry and immunocytochemistry. Results:Two isomers of pregnane steroids with molecular weight 330.2087 (C21H30O3) were isolated from the EtOAc extract. Spectroscopic analysis revealed the structures as 17-ethylene-3,4-dihydroxy-14-methyl-18-norandrostene-16-one (1) and 17-ethylene-3,4-dihydroxy-5-pregnene-16-one (2), respectively. These compounds showed moderate cytotoxicity (IC50 172.9 and 62.2 µg/mL, respectively) comparable to doxorubicin (IC50 3.08 µg/mL). The execution of apoptosis may be related to the increase of the ratio of BAX/bcl-2 of the cells. Conclusion:The EtOAc fraction of Melia azedarach L. leaves and the isolated 5-pregnene-16-one steroids are promising reagents for breast cancer treatment by introducing apoptosis to tumor cells. However, further researches are required to highlight its safety and usage in vivo.

Silencing UHRF1 Enhances Radiosensitivity of Esophageal Squamous Cell Carcinoma by Inhibiting the PI3K/Akt/mTOR Signaling Pathway.

PurposeResistance to radiotherapy results in a high treatment failure rate for locally advanced esophageal squamous cell carcinoma (ESCC). Ubiquitin-like with plant homeodomain and ring-finger domains 1 (UHRF1), is associated with poor prognosis in ESCC. The present study aims to characterize the effect of UHRF1 silencing on the radiosensitivity of ESCC and its potential mechanism.MethodsBoth in vitro and in vivo experiments were conducted to observe the effects of UHRF1 silencing on the radiosensitivity of ESCC. The effects of UHRF1 silencing on the apoptosis of ESCC cells were assessed by flow cytometry. The expression of apoptosis-related factors (caspase-3 and Bcl-2), PI3K/Akt/mTOR signaling pathway-related factors (PTEN, p-Akt and Akt, p-mTOR and mTOR), and DNMT1 were measured via Western blot, and the status of PTEN methylation was detected by methylation-specific PCR. Immunohistochemistry was used to detect the expressions of PTEN, p-AKT, and p-mTOR in xenograft tumor tissues.ResultsIn vitro and in vivo experiments showed that UHRF1 knock-down inhibited ESCC cell growth and enhanced their radiosensitivity. shUHRF1 combined with radiation significantly increased ESCC cell apoptosis. Meanwhile, it activated the expression of caspase-3 and inhibited the expression of Bcl-2. shUHRF1 inhibited the expression of DNMT1 and reduced the methylation of PTEN, and then upregulated the expression of PTEN to inhibit the PI3K/Akt/mTOR signaling pathway. On the contrary, the PI3K/Akt/mTOR signaling pathway can be activated by upregulation of UHRF1.ConclusionOur findings provide a theoretical basis for UHRF1 as a target to improve the radiosensitivity of ESCC.

The role and mechanism of PKM2 in the development of LPS-induced acute kidney injury.

A previous study suggested that pyruvate kinase M2 (PKM2) plays a vital role of metabolic reprogramming in the regulation of the innate inflammatory response, while PKM2 is a sensitive biomarker for nephrotoxicity. In this study, we investigated the role and mechanism of PKM2 in development of LPS-induced acute kidney injury. The AKI model of mice was established using LPS. The serum levels of blood urea nitrogen (Bun), creatinine (Cr), and Cystatin C (CysC) were identified using the enzyme-linked immunosorbent assay (ELISA). Hematoxylin and Eosin staining (H&E) was employed to assess pathological changes in kidney tissues of LPS-induced AKI model. Immunohistochemical staining and Western blot analysis were carried out to determine the expression of apoptosis-related factors at protein levels. We found that Bun, CysC, and Cr were significantly increased in the LPS group compared with the control group. The histopathological assay showed model swollen tubular epithelial cells and the presence of vacuolar degeneration in the LPS-induced AKI. In addition, expression levels of PKM2 significantly increased in the LPS group compared with the control group at both protein and mRNA levels (P<0.01). The inhibition of PKM2 by shikonin notably suppressed the expression of HIF-1α and apoptosis-related factors such as BNIP3, Bax, and Caspase-3, while the inhibition of PKM2 by shikonin significantly improved the histopathological symptoms of LPS-induced AKI. This study demonstrated the potential role of PKM2 in LPS-induced AKI and identified PKM2 as a promising therapeutic target in the treatment of AKI.

Phosphatidylethanolamine binding protein 1 enhances sensitivity of gastric cancer cell to 5-fluorouracil via inhibition of cell proliferation, migration and invasion

Purpose: To determine the association between phosphatidylethanolamine binding protein 1, which is an Raf kinase inhibitor protein (RKIP), and 5-fluorouracil (5-FU) via analysis of the association between RKIP and clinical responses in individuals treated using fluorouracil-based chemotherapy.Methods: Human gastric cancer cell lines MGC-803 and SGC-7901 were used in this study. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and migration were determined with flow cytometry and Transwell chamber assays, respectively. The mRNA and protein expressions of apoptosis-related factors were assayed using realtime polymerase chain reaction (RT-PCR) and Western blotting, respectively, while the expression of RKIP was determined by immunohistochemical staining.Results: Chemotherapeutic drug (5-FU) treatment induced low RKIP expression levels in tumorigenic GC cells, thereby sensitizing the cells to apoptosis (8.57 vs 1.25 %, p &lt; 0.01). The highest RKIP level correlated well with initiation of apoptosis (4.20 vs 1.25 %, p &lt; 0.01). Following in vitro downregulation of RKIP, there was increase in the viability and proliferation of RKIP-inhibited cells over time, and these changes were linked to alterations in cell cycle phases and increased optical density in MTT proliferation assay (1.55 vs 1.18, p &lt; 0.01). In vitro Transwell assay measurement revealed an association between RKIP downregulation and enhancement of cell migration potential (652 vs 436, p &lt; 0.01). Ectopic RKIP expression restored the apoptotic sensitivity of resistant cells (14.30 vs 1.36 %, p &lt;0.01). This sensitization was annulled by upregulation of survival routes. Reduction of RKIP by expression of antisense and siRNA conferred resistance on cancer cells sensitive to 5-FU-mediated apoptosis (6.88 vs 2.13 %, p &lt; 0.01).Conclusion: Thus, RKIP is a promising therapeutic strategy for improving the efficacy of clinically relevant chemotherapeutic drugs for GC.&#x0D; Keywords: Gastric cancer, Raf kinase inhibitor protein, Cell proliferation, Invasion, Apoptosis, Chemotherapy, Phosphatidylethanolamine binding protein 1

HDAC9 Silencing Exerts Neuroprotection Against Ischemic Brain Injury via miR-20a-Dependent Downregulation of NeuroD1.

Cerebral stroke is an acute cerebrovascular disease that is a leading cause of death and disability worldwide. Stroke includes ischemic stroke and hemorrhagic strokes, of which the incidence of ischemic stroke accounts for 60–70% of the total number of strokes. Existing preclinical evidence suggests that inhibitors of histone deacetylases (HDACs) are a promising therapeutic intervention for stroke. In this study, the purpose was to investigate the possible effect of HDAC9 on ischemic brain injury, with the underlying mechanism related to microRNA-20a (miR-20a)/neurogenic differentiation 1 (NeuroD1) explored. The expression of HDAC9 was first detected in the constructed middle cerebral artery occlusion (MCAO)-provoked mouse model and oxygen-glucose deprivation (OGD)-induced cell model. Next, primary neuronal apoptosis, expression of apoptosis-related factors (Bax, cleaved caspase3 and bcl-2), LDH leakage rate, as well as the release of inflammatory factors (TNF-α, IL-1β, and IL-6) were evaluated by assays of TUNEL, Western blot, and ELISA. The relationships among HDAC9, miR-20a, and NeuroD1 were validated by in silico analysis and ChIP assay. HDAC9 was highly-expressed in MCAO mice and OGD-stimulated cells. Silencing of HDAC9 inhibited neuronal apoptosis and inflammatory factor release in vitro. HDAC9 downregulated miR-20a by enriching in its promoter region, while silencing of HDCA9 promoted miR-20a expression. miR-20a targeted Neurod1 and down-regulated its expression. Silencing of HDAC9 diminished OGD-induced neuronal apoptosis and inflammatory factor release in vitro as well as ischemic brain injury in vivo by regulating the miR-20a/NeuroD1 signaling. Overall, our study revealed that HDAC9 silencing could retard ischemic brain injury through the miR-20a/Neurod1 signaling.

Tectorigenin protect HUVECs from H2O2-induced oxidative stress injury by regulating PI3K/Akt pathway

Oxidative stress injury (OSI) occurs in many cardiovascular diseases, and the OSI of endothelial cells is the main pathological basis of these diseases. Tectorigenin has an effect on oxidative stress in fibroblasts, keratinocytes, and neuroblastoma. This study attempted to reveal the effect of Tectorigenin on OSI in endothelial cells.An OSI cell model was firstly established by treating human umbilical vein endothelial cells (HUVECs) with H2O2. The H2O2-induced HUVECs were further pre-treated with Tectorigenin or PI3K inhibitor. Then the viability and apoptosis of HUVECs were evaluated using MTT, Hochest 33258 staining and TUNEL staining. Lactate dehydrogenase (LDH) leakage, enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) level were measured through colorimetric assays. The expressions of apoptosis-related factors and the activation of the PI3K/Akt pathway in HUVECs were detected by RT-qPCR or Western blot.Tectorigenin had no inhibiting effect on the viability of HUVECs at the concentrations of 0.1, 0.5, 0.5, 1, and 10 μmol/L. Tectorigenin reversed the H2O2 induced-destruction of HUVECs morphology. Tectorigenin increased the viability and decreased the apoptosis of H2O2-induced HUVECs. Tectorigenin increased Bcl-2 expression and the enzyme activities of SOD and GSH-Px, but decreased LDH leakage, MDA level, and the expressions of Bax and Cleaved Caspase-3 in H2O2-induced HUVECs. Furthermore, Tectorigenin increased the ratios of p-PI3K to PI3K and p-Akt to Akt in H2O2-induced HUVECs. PI3K inhibitor had an opposite effect of Tectorigenin on the OSI in H2O2-induced HUVECs and its effect was further reversed by Tectorigenin.Tectorigenin protected HUVECs against H2O2-induced OSI via PI3K/Akt pathway.

Synergistic Tumoricidal Effects of Alpha-Lipoic Acid and Radiotherapy on Human Breast Cancer Cells via HMGB1.

PurposeRadiotherapy (RT) is one of main strategies of cancer treatment. However, some cancer cells are resistant to radiation-induced cell death, including apoptosis. Therefore, alternative approaches targeting different anti-tumor mechanisms such as cell senescence are required. This study aimed to investigate the synergistic effect of alpha-lipoic acid (ALA) on radiation-induced cell death and senescence in MDA-MB-231 human breast cancer cells.Materials and MethodsThe cells were divided into four groups depending on the cell treatment (control, ALA, RT, and ALA+RT). Cells were analyzed for morphology, apoptotic cell death, mitochondrial reactive oxygen species, membrane potential, cellular senescence, and cell cycle.ResultsOur data showed that ALA significantly promoted apoptotic cell death when combined with RT, as reflected by Annexin V staining, expression of apoptosis-related factors, mitochondrial damages as well as cell morphological changes and reduction of cell numbers. In addition, ALA significantly enhanced radiation-induced cellular senescence, which was shown by increased HMGB1 expression in the cytosol fraction compared to the control, increased p53 expression compared to the control, activation of p38 as well as nuclear factor κB, and G2/M cell cycle arrest.ConclusionThe current study is the first report showing a new mode of action (senescence induction) of ALA beyond apoptotic cell death in MDA-MB-231 cancer cells known to be resistant to RT.

Effects and Mechanism of Berberine on the Dexamethasone-Induced Injury of Human Tendon Cells.

Objective To investigate the effects of berberine (Berb) on dexamethasone- (Dex-) induced injury of human tendon cells and its potential mechanism. Methods CCK-8 assay was used to explore the appropriate concentration of Dex-induced injury of tendon cells and the doses of Berb attenuates Dex cytotoxicity; cell wound healing assay was used to detect the effects (P < 0.05) of Berb and Dex on the migration ability of tendon cells; flow cytometry was used to measure cell apoptosis; DCF DA fluorescent probe was used to measure the ROS activity of cells. Western blotting was used to detect the expression of phenotype related factors including smooth muscle actin α (SMA-α), type I collagen (Col I), col III, apoptosis-related factors, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and PI3K/AKT. Results CCK-8 assay showed that 1–100 μM Dex significantly inhibited the proliferation of tendon cells in a concentration-dependent manner (P < 0.05), where the inhibitory effect of 100 μM Dex was most significant (P < 0.005), and the pretreatment of 150, 200 μM Berb could reverse those inhibitions (all P < 0.05). Compared with the control group, Dex significantly inhibited cell migration (P < 0.05), while Berb pretreatment could enhance cell migration (P < 0.05). Flow cytometry and ROS assay showed that Dex could induce apoptosis and oxidative stress response of tendon cells (all P < 0.05), while Berb could reverse those responses (P < 0.05). Western blot showed that Dex could inhibit the expression of the col I and III as well as α-SMA (all P < 0.05) and enhance the expression of apoptosis-related factors including cleaved caspase-3 and cleaved caspase-9 (all P < 0.05). Besides, Dex could also inhibit the activation of the PI3K/AKT signaling pathway (all P < 0.05), thus affecting cell function, while Berb treatment significantly reversed the expression of those above proteins (all P < 0.05). Conclusion Berb attenuated DEX induced reduction of proliferation and migration, oxidative stress, and apoptosis of tendon cells by activating the PI3K/AKT signaling pathway and regulated the expression of phenotype related biomarkers in tendon cells. However, further studies are still needed to clarify the protective effects of Berb in vivo.

Effect of manual acupuncture of "Shuigou" (GV26)"Neiguan" (PC6) on neurological function and expression of apoptosis-related factors in brain tissues surrounding hematoma in intracerebral hemorrhage rats

To observe the effect of manual acupuncture stimulation of "Shuigou" (GV26) and "Neiguan" (PC6) on neurological function and expression of Caspase-3 and Caspase-9 in brain tissues around the intracerebral hematoma in rats with acute intracerebral hemorrhage (ICH), so as to explore its possible mechanisms underlying improvement of ICH. Ninety-six male SD rats were randomly divided into 4 groups: control, model, acupoint and non-acupoint (24 rats in each group). The ICH model was established by injection of the rat's autologous blood into the caudate nucleus. According to the time-points of 6, 24, 48 and 72 h after ICH, each of the 4 groups was further divided into 4 subgroups. For rats of the acupoint group, the PC6 on both sides was manually stimulated by manipulating the needle with lifting-thrusting-twisting reducing techniques, while the GV26 was stimulated with strong "sparrow-pecking" method for 10 times, then, left the needles in the acupoints for 30 min. For rats of the non-acupoint group, two non-acupoints: mid-spot below the bilateral axilla and the spot 3 mm above the left side of the coccyx tip were stimulated with the same methods to PC6 and GV26, respectively. For rats of the 6 h and 24 h subgroups, the intervention was given once after waking up from modeling, and for those of the 48 and 72 h subgroups, the intervention was conducted once a day for 2 or 3 times, respectively. The neurological severity score (NSS) was used to evaluate the degree of neurological function. The immunoactivity (expression) of Caspase-3 and Caspase-9 proteins of the hematoma focus of the brain was detected by immunohistochemistry. Following modeling, the NSS and the expression levels of Caspase-3 and Caspase-9 proteins in the brain tissues surrounding the hematoma at each time-points (6, 24, 48 and 72h) after modeling were significantly increased in the model group relevant to the control group (P<0.01, P<0.05). Compared with the model group, the NSS at 72h and the expression levels of Caspase-3 and Caspase-9 proteins at 6, 24, 48 and 72h were significantly down-regulated in the acupoint group (P<0.05) rather than in the non-acupoint group (P>0.05). Acupuncture of GV26 and PC6 can improve the neurological function in rats with ICH, which may be related to its function in reducing the expression of Caspase-3 and Caspase-9 proteins (apoptosis-related proteins) in the brain.

Inhibitory effect on the apoptosis of disc nucleus pulposus cells in rats by silencing COL9A3 gene to mediate MAPK signaling pathway: a study on the function and mechanism.

To explore the effect of collagen IX alpha 3 chain (COL9A3) gene silencing on apoptosis of nucleus pulposus cells in rats with intervertebral disc degeneration (IVDD) and its regulatory mechanism, so as to provide potential reference for the treatment of IVDD. The model of IVDD in rats were constructed to isolate, culture and identify nucleus pulposus cells for subsequent experiment. With the construction of lentivirus vectors, cells were divided into Blank group, negative control (NC) group, COL9A3 shRNA group, COL9A3 overexpression group, mitogen-activated protein kinase (MAPK) pathway inhibitor (Theaflavin 3,3'-digallate, TF3) group and COL9A3 shRNA+TF3 group according to different transfection treatments. After cell transfection, the expression of COL9A3, extracellular regulated protein kinase 1/2 (ERK1/2) and phosphorylated-ERK1/2 (p-ERK1/2), MEK1/2 and p-MEK1/2, as well apoptosis related indexes were detected by using quantitative real-time PCR (qRT-PCR) and Western Blot. Furthermore, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of transfected cells, and flow cytometry to detect changes of cell cycle and apoptosis. The model of IVDD in rats was established successfully, and rat nucleus pulposus cells were cultured subsequently. After transfection, compared with NC group, there was significant downregulation in COL9A3 expression, significant upregulation in ERK1/ERK2 and MEK1/MEK2 expressions, evident increase in Bax and Caspase3 expressions, and a decrease Bcl-2 expression in transfected cells of COL9A3 shRNA group (all p<0.05). The opposite trends were detected in the above indexes in cells of COL9A3 overexpression group (all p<0.05). Furthermore, in TF3 group, there was significant decrease in ERK1/ERK2 and MEK1/MEK2 expressions, reduction in Bax and Caspase3 expressions, increase in Bcl-2 expression (all p<0.05), yet without evident change in the expression of COL9A3. Meanwhile, COL9A3 expression was decreased in COL9A3 shRNA+TF3 group (p<0.05); however, there was no significant statistical difference in the remaining indexes when compared with those in the NC group (all p>0.05). Subsequently, compared with NC group, COL9A3 overexpression group and TF3 group revealed evident increased cell proliferation and significant decreased cell apoptosis (all p<0.05); whereas it was remarkably the opposite in COL9A3 shRNA group (all p<0.05). However, no evident difference was detected in the comparison of cell proliferation and apoptosis among Blank group, NC group and COL9A3 shRNA+TF3 group (all p>0.05). Overexpression of COL9A3 gene and inhibition of MAPK signaling pathway can induce proliferation and inhibit apoptosis of nucleus pulposus cells. Significantly, silence of COL9A3 gene expression can activate MAPK signaling pathway and affect the expression of apoptosis related factors, so as to inhibit the proliferation of nucleus pulposus cells and promote cell apoptosis in rats with IVDD.

Celastrol ameliorates endoplasmic stress-mediated apoptosis of osteoarthritis via regulating ATF-6/CHOP signalling pathway.

Osteoarthritis (OA) is a common degenerative joint disease with the pathological features of the reduced cartilage cellularity. Celastrol, a compound from Tripterygium wilfordii, exerted therapeutic effects on arthritis, but the potential mechanism remains unclear. Tunicamycin was used to establish a model of OA in vitro, and ACLT surgery model in rats was applied to verify the mechanism. Chondrocytes were isolated from the knee articular cartilage of rabbit. MTT and flow cytometry assay were used to detect cell viability and apoptosis rate. Haematoxylin-eosin staining was used to assess for the histopathological changes. The activity and expression of apoptosis-related factors and ERs (endoplasmic reticulum stress)-related factors were detected by ELISA, WB, PCR and IHC, respectively. Celastrol exhibited significant enhancement on cell viability and reduced the rate of apoptosis in Tm-exposed chondrocytes. Celastrol reduced enzyme activity and protein expression of caspase-3, caspase-6 and caspase-9, decreased Bip, Atf6, Chop and Xbp-1 expression both at protein and mRNA levels. Celastrol showed a more significant effect on cell apoptosis rate and mRNA expression in the combination with 4-PBA. This study reveals that celastrol may prevent OA by inhibiting the ERs-mediated apoptosis. All these might supply beneficial hints for celastrol on OA treatment.