Pyrrole as an Important Scaffold of Anticancer Drugs: Recent Advances.

With the significant increase of patients suffering from different types of cancer, it is evident that prompt measures in the development of novel and effective agents need to be taken. Pyrrole moiety has been found in various active compounds with anti-inflammatory, antiseptic, antibacterial, lipid-lowering and anticancer properties. Recent advances in the exploration of highly active and selective cytotoxic structures containing pyrrole motifs have shown promising data for future investigations. Accordingly, this review presents an overview of recent developments in the pyrrole derivatives as anticancer agents, with a main focus towards the key moieties required for the anti-tumor activities. Pyrrole molecules comprising prominent targeting capacities against microtubule polymerization, tyrosine kinases, cytochrome p450 family 1, histone deacetylase and bcl-2 proteins were reported. In addition, several mechanisms of action, such as apoptosis, cell cycle arrest, inhibiting kinases, angiogenesis, disruption of cell migration, modulation of nuclear receptor responsiveness and others were analyzed. Furthermore, in most of the discussed cases we provided synthesis schemes of the mentioned molecules. Overall, the utilization of pyrrole scaffold for the design and synthesis of novel anticancer drugs could be a promising approach for future investigations.

in Vitro Effect of Methamphetamine on Proliferation, Differentiation and Apoptosis of Adipose Tissue Stem Cells.

PURPOSE: Among abused substances, methamphetamine is a psychostimulant drug widely used recreationally with public health importance. This study investigated the effect of methamphetamine on proliferation, differentiation, and apoptosis of human adipose tissue stem cells (AdSCs). METHODS: AdSCs were isolated from human abdominal adipose tissue and were characterized for mesenchymal properties and growth kinetics. MTT assay was undertaken to assess methamphetamine toxicity on proliferation and differentiation properties and apoptosis of hAdSCs. RESULTS: Isolated cells were shown to have mesenchymal properties and a population doubling time (PDT) of 40.1 h. Following methamphetamine treatment, expressions of KI-67 and TPX2 as proliferation genes and Col1A1 and PPARg as differentiation genes decreased. Methamphetamine administration increased the expression of Bax and decreased Bcl-2 genes responsible for apoptosis. CONCLUSIONS: Our data suggested when AdSCs were exposed to methamphetamine, it decreased proliferation and differentiation properties of stem cells together with an increase in apoptosis. These findings can be added to the literature, especially when methamphetamine is used recreationally for weight loss purposes.

Astragalus Polysaccharide Regulates miR-182/Bcl-2 Axis to Relieve Metabolic Memory through Suppressing Mitochondrial Damage-Mediated Apoptosis in Retinal Pigment Epithelial Cells.

INTRODUCTION: Metabolic memory is one of the causes of diabetic retinopathy, and astragalus polysaccharide (APS) has great advantages in the treatment of diabetes. However, the effect of APS on metabolic memory remains to be investigated. METHODS: Retinal pigment epithelial cell line ARPE-19 and primary retinal pigment epithelial cells were used to verify the effect of APS on mitochondria damage and apoptosis induced by high glucose-induced metabolic memory. The relationship between miR-182 and Bcl-2 was confirmed by a luciferase activity assay. Western blotting and quantitative reverse-transcriptase polymerase chain reaction were conducted to investigate the changes in mitochondrial damage- and apoptosis-associated markers. The cell mitochondrial membrane potential was assessed by JC-1 fluorescence. Terminal deoxynucleotidyl transferase dUTP nick end labelling staining and flow cytometry assays were performed to determine the occurrence of apoptosis. RESULTS: Treatment with high glucose followed by normal glucose significantly upregulated the expression of miR-182 and downregulated the expression of its target Bcl-2, and APS treatment reversed the above effects. Additionally, APS treatment restored mitochondrial function and inhibited apoptosis in cells in a state of metabolic memory. The effects of APS against mitochondrial damage and apoptosis were partially inhibited after miR-182 overexpression. CONCLUSION: APS alleviated mitochondrial damage and apoptosis induced by metabolic memory by regulating the miR-182/Bcl-2 axis, which might serve as a new strategy for the treatment of diabetic retinopathy.

Actein Antagonizes Oral Squamous Cell Carcinoma Proliferation through Activating FoxO1.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is among the most prevalent head and neck malignancies globally, and it is associated with high mortality rates. Actein is one of the primary active components extractable from the rhizomes of Cimicifuga foetida. This study aimed to evaluate the anti-OSCC effects of actein and evaluate the potential underlying mechanisms. METHODS AND RESULTS: CCK-8 cell proliferation experiments demonstrated significant dose- and time-dependent anti-OSCC effects of actein, while actein had weak cytotoxic effects on normal oral cell lines. Flow cytometry for cell cycle evaluation revealed that actein could induce cell cycle arrest at the G1 phase among OSCC cell lines. In our Annexin V/PI double staining apoptosis analysis, actein induced significant apoptosis among OSCC cells, with upregulation of Bax and downregulation of Bcl-2. Our mechanistic study implicated the involvement of the Akt/FoxO1 pathway in the anti-OSCC effects of actein. Akt1 and Akt2 expression significantly decreased in association with the FoxO1 upregulation. Furthermore, Bim and p21 were significantly upregulated, while survivin expression was downregulated. Finally, actein treatment was associated with significant p-Akt downregulation and p-FoxO1 upregulation in OSCC cells, demonstrating the validated roles of Akt/FoxO1 in actein-mediated OSCC cell apoptosis and cell cycle arrest. FoxO1 knockdown significantly reversed the anti-OSCC effects of actein. Additionally, a xenograft model indicated that actein could inhibit OSCC cell growth in vivo. CONCLUSIONS: Our findings demonstrated that actein could be a strong anti-OSCC candidate. Further evaluations of its safety and effectiveness are necessary before it can be considered for clinical use.

Zhoushi Qiling decoction induces apoptosis of human prostate cancer cells via miR-143/Bcl-2 axis.

A number of traditional Chinese medicines (TCMs) are widely used in prostate cancer treatment in China. The aim of this study was to test the efficacy of a TCM, Zhoushi Qiling Decoction (ZQD), in combination with androgen deprivation therapy (ADT) and explore its underlying mechanism. A total of 151 patients were recruited to receive ADT treatment or ADT+ZQD treatment. The survival of patients who received ADT+ZQD treatment was significantly higher than those who received ADT therapy only. DU145 prostate cancer cells were treated with ZQD (50 mg/mL) for 24 h in vitro and expression levels of an array of miRNAs were examined. Our results suggested that miR-143 demonstrated prominent upregulation in DU145 cells after treatment with ZQD. In patient serum samples, miR-143 expression was also significantly upregulated after ADT+ZQE treatment, which was however absent in patients treated with ADT only. In DU145 cells, ZQD treatment led to a dose-dependent increase in apoptosis, which could be reduced by anti-miR-143 treatment. There was a binding site between miR-143 and B cell CLL/lymphoma-2 (Bcl-2) and ZQD treatment reduced Bcl-2 expression. ZQD treatment led to increased caspase-3 and Bax expression. ZQD treatment could promote apoptosis of prostate cancer cells by promoting miR-143 upregulation, which could be a possible mechanism underlying the inhibitory effect of ZQD in prostate cancer in patient.

Reproductive toxicity of cadmium in pubertal male rats induced by cell apoptosis.

Cadmium (Cd) is a heavy metal that is widely present in modern industrial production. It is a known, highly toxic environmental endocrine disruptor. Long-term exposure to Cd can cause varying degrees of damage to the liver, kidney, and reproductive system of organisms, especially the male reproductive system. This study aimed to explore the mechanism of Cd toxicity in the male reproductive system during puberty. Eighteen healthy 6-week-old male Sprague-Dawley rats were randomly divided into three groups (control group, low-dose group, and high-dose group) according to their body weight, with six in each group. Cd (0, 1, and 3 mg/kg/day) was given by gavage for 28 consecutive days. The results showed that Cd exposure to each dose group caused a decrease in the testicular organ coefficient and sperm count, compared with the control group. Cd exposure resulted in significant changes in testicular morphology in the 3 mg/kg/day Cd group. In the 1 and 3 mg/kg/day Cd groups, serum testosterone decreased and apoptosis of testicular cells increased significantly (p < 0.05). In addition, compared with the control group, the activity of glutathione peroxidase and superoxide dismutase in each Cd exposure dose group decreased, but the content of malondialdehyde in the high-dose, 3 mg/kg/day Cd treatment group significantly increased (p < 0.05). Although Cd exposure caused an increase in the messenger RNA (mRNA) levels of Bcl-2, Caspase-3 and Caspase-9 in the testicular tissues (p < 0.05), Bcl-2 expression was unchanged (p > 0.05). The expression level of Akt mRNA in testicular tissue of rats in the high-dose 3 mg/kg/day Cd group was increased (p < 0.05). Our data suggest that Cd affected testosterone levels, and apoptosis was observed in spermatids.

CD34 and Bcl-2 as predictors for the efficacy of neoadjuvant chemotherapy in cervical cancer.

BACKGROUND: Successful neoadjuvant chemotherapy (NACT) could improve the surgical resection rate and radical curability of patients with cervical cancer, but only a subset of patients benefits. Therefore, identifying predictive biomarkers are urgently needed. The aim of this study was to evaluate the predictive value of CD34 and Bcl-2 in the NACT effectiveness of cervical cancer. METHODS: Sixty-seven patients with locally advanced cervical cancer (FIGO stages IB3, IIA2 or IIB) were classified into two groups based on effective (n = 48) and ineffective (n = 19) response to NACT. Immunohistochemistry was employed to identify CD34 and Bcl-2 expression before and after NACT. We analyzed the associations between the pre-NACT expression of these two biomarkers and the response of NACT. The expression of these two biomarkers before and after NACT was also assessed and compared. RESULTS: More patients were CD34 positive expression before NACT in effective group compared to ineffective group (p = 0.005). However, no statistically significant difference in Bcl-2 expression before NACT were found between two groups (p = 0.084). In NACT effective group, the expression of both CD34 and Bcl-2 after NACT are down-regulated (p < 0.001 and p < 0.001, respectively), while there are no statistical differences between the pre- and post-NACT expression of CD34 and Bcl-2 in NACT ineffective group (p = 0.453 and p = 0.317, respectively). CONCLUSION: The positive CD34 expression before NACT may serve as a predictive biomarker for NACT of cervical cancer, but the pre-NACT expression of Bcl-2 is not an independent predictor. The down-regulated expression of these two indicators after NACT may indicate effective NACT.

Thymol induces mitochondrial pathway-mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in A549 cells.

BACKGROUND: Thymol is a monoterpene phenol found in thyme species plants. The present study was carried out to investigate the effect of thymol and its molecular mechanism on non-small lung cancer (A549) cells. METHODS: The cytotoxic effect of thymol on A549 cells was assessed via MTT assay. ROS production, macromolecular damage, apoptosis were determined using DCF-DA, PI, AO/EtBr stains, respectively. ROS-dependent effect of thymol was confirmed using NAC. The expression of caspase-9, Bcl-2, Bax and cell cycle profile was analyzed via western blot and FACS, respectively. RESULTS: The antiproliferative effect of thymol on A549 cells was found to be both dose and time dependent with IC50 values of 112 µg/ml (745 µM) at 24 h. Thymol treatment favored apoptotic cell death and caused G0/G1 cell cycle arrest. It mediated cellular and nuclear morphological changes, phosphatidylserine translocation, and mitochondrial membrane depolarization. Additionally, upregulation of Bax, downregulation of Bcl-2, and apoptotic fragmented DNA were also observed. Thymol induced ROS by reducing the SOD level which was confirmed via in vitro and in silico analysis. Furthermore, the levels of lipid peroxides and protein carbonyl content were elevated in thymol-treated groups. Notably, N-acetyl cysteine pretreatment reversed the efficacy of thymol on A549 cells. Moreover, thymol-treated human PBMC cells did not show any significant cytotoxicity. CONCLUSION: Overall, our results confirmed that thymol can act as a safe and potent therapeutic agent to treat NSCLC.

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma.

OBJECTIVES: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. METHODS: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. RESULTS: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. CONCLUSION: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2643-2649, 2020.

Protective Role of Hydrogen Gas on Oxidative Damage and Apoptosis in Intestinal Porcine Epithelial Cells (IPEC-J2) Induced by Deoxynivalenol: A Preliminary Study.

To explore the protective role of hydrogen gas (H2) on oxidative damage and apoptosis in intestinal porcine epithelial cells (IPEC-J2) induced by deoxynivalenol (DON), cells were assigned to four treatment groups, including control, 5 µM DON, H2-saturated medium, and 5 µM DON + H2-saturated medium treatments. After 12 h of different treatments, the cell viability, biomarkers of cell redox states, and gene expression of antioxidant enzymes and apoptosis were observed and detected. Furthermore, caspase-3 and Bax protein expressions were measured by Western blot analysis. Our results demonstrated that the 5 µM DON significantly caused cytotoxicity to IPEC-J2 cells by reducing cell viability and increasing lactate dehydrogenase release in culture supernatants. Moreover, DON treatments significantly increased levels of 8-hydroxy-2'-deoxyguanosine, 3-nitrotyrosine, and malonaldehyde; however, they decreased total superoxide dismutase and catalase activities and downregulated messenger RNA (mRNA) expression related to antioxidant enzymes in cells. The 5 µM DON treatment also downregulated Bcl-2 expression and upregulated caspase-3 and Bax expression. However, the H2-saturated medium significantly improved cell growth status and reversed the change of redox states and expression of genes and proteins related to apoptosis induced by DON in IPEC-J2 cells. In conclusion, H2 could protect IPEC-J2 cells from DON-induced oxidative damage and apoptosis in vitro.

In vitro photodynamic therapy of endothelial cells using hematoporphyrin monomethyl ether (Hemoporfin): Relevance to treatment of port wine stains.

Hemoporfin (hematoporphyrin monomethyl ether, HMME) is a relatively new photosensitizer that has achieved success in mediating photodynamic therapy (PDT) of port wine stains in China. However, the exact mechanism of Hemoporfin PDT on endothelial cell proliferation and apoptosis is unclear. The present study investigated the mechanism of action of HMME-PDT on endothelial cells in vitro. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. HMME-PDT treated the cells and detected the phototoxicity by cell counting kit-8 (CCK-8) assay, apoptosis by Flow cytometry assay and quantification of the secreted VEGF-A levels using ELISA and different proteins expression by quantitative real-time PCR and Western blotting. Phototoxicity was caused in an HMME and light dose-dependent manner. Apoptosis was induced as shown by Annexin-V/propidium iodide staining and morphological changes. The Bax/Bcl-2 ratio was increased as shown by Western blot for protein and RT-qPCR for mRNA. VEGF-A expression was reduced and signaling molecules in the Akt/mTOR pathway were inhibited as shown by ELISA and immunofluorescence. Hemoporfin (hematoporphyrin monomethyl ether, HMME) has achieved success in mediating photodynamic therapy (PDT) of port wine stains. The clinical success of HMME-PDT with low recurrence rates can be explained by inhibition of endothelial cell proliferation through VEGF/Akt /mTOR pathway.

Renal effects of exendin-4 in an animal model of brain death.

Organ transplantation is the gold standard therapy for the majority of patients with terminal organ failure. However, it is still a limited treatment especially due to the low number of brain death (BD) donors in relation to the number of waiting list recipients. Strategies to increase the quantity and quality of donor organs have been studied, and the administration of exendin-4 (Ex-4) to the donor may be a promising approach. Male Wistar rats were randomized into 3 groups: (1) control, without central nervous system injury; (2) BD induced experimentally, and (3) BD induced experimentally + Ex-4 administered immediately after BD induction. After BD induction, animals were monitored for 6 h before blood collection and kidney biopsy. Kidney function was assessed by biochemical quantification of plasma kidney markers. Gene and protein expressions of inflammation- and stress-related genes were evaluated by RT-qPCR and immunoblot analysis. Animals treated with Ex-4 had lower creatinine and urea levels compared with controls. BD induced oxidative stress in kidney tissue through increased expression of Ucp2, Sod2 and Inos, and Ex-4 administration reduced the expression of these genes. Ex-4 also induced increased expression of the anti-apoptotic Bcl2 gene. Nlrp3 and Tnf expressions were up-regulated in the BD group compared with controls, but Ex-4 treatment had no effect on these genes. Our findings suggest that Ex-4 administration in BD rats reduces BD-induced kidney damage by decreasing the expression of oxidative stress genes and increasing the expression of Bcl2.

Rhazyaminine from Rhazya stricta Inhibits Metastasis and Induces Apoptosis by Downregulating Bcl-2 Gene in MCF7 Cell Line.

BACKGROUND: The role of alkaloids isolated from Rhazya stricta Decne (Apocynaceae family) (RS) in targeting genes involved in cancer and metastasis remains to be elucidated. OBJECTIVE: Identify and characterize new compounds from RS, which inhibit gene(s) involved in the survival, invasion, self-renewal, and metastatic processes of cancer cells. METHODS: Bioinformatics study was performed using HISAT2, stringtie, and ballgown pipeline to understand expressional differences between a normal epithelial cell line-MCF10A and MCF7. NMR and ATR-FTIR were performed to elucidate the structure of rhazyaminine (R.A), isolated from R stricta. Cell viability assay was performed using 0, 25, and 50 µg/mL of total extract of R stricta (TERS) and R.A, respectively, for 0, 24, and 48 hours, followed by scratch assay. In addition, total RNA was isolated for RNA- seq analysis of MCF7 cell line treated with R.A followed by qRT-PCR analysis of Bcl-2 gene. RESULTS: Deptor, which is upregulated in MCF7 compared with MCF10A as found in our bioinformatics study was downregulated by R.A. Furthermore, R.A effectively reduced cell viability to around 50% ( P < .05) and restricted cell migration in scratch assay. Thirteen genes, related to metastasis and cancer stem cells, were downregulated by R.A according to RNA- seq analysis. Additionally, qRT-PCR validated the downregulation of Bcl-2 gene in R.A-treated cells by less than 0.5 folds ( P < .05). CONCLUSION: R.A successfully downregulated key genes involved in apoptosis, cell survival, epithelial-mesenchymal transition, cancer stem cell proliferation, and Wnt signal transduction pathway making it an excellent "lead candidate" molecule for in vivo proof-of-concept studies.

Puerarin attenuates neurological deficits via Bcl-2/Bax/cleaved caspase-3 and Sirt3/SOD2 apoptotic pathways in subarachnoid hemorrhage mice.

BACKGROUND: Subarachnoid hemorrhage (SAH) results in many brain dysfunctions and the neuroprotective function of puerarin after brain damage has been demonstrated in several studies. But whether puerarin can reduce brain nerve damage after SAH is not clear.In this study, we hypothesized that puerarin had the neuroprotective effect after SAH, and this protection could be mediated by bothBcl-2/Bax/Cleaved caspase-3 and SIRT3/SOD2 apoptotic signaling pathways. METHODS: First, we used neurological score, brain water content and so on to detect the neurological deficits after SAH. Then apoptosis neuron rate was detected by TUNEL staining. Western blot was carried out to explore the alteration of Blc-2, Bax, cleaved caspase-3 and Sirt3.Also, ROS acitivity and As-lysine level of SOD2 should be detected with assays. RESULTS: We demonstrate that puerarin attenuated the neurological deficits, effectively relieves cerebral edema, and reduce BBB disruption in SAH mice.And we revealed that a reduced rate of apoptosis neuron has been found out in puerarin treatment after SAH. In addition, obviously higher ratio of Blc-2/Bax and decreased expression of cleaved caspase-3 in puerarin-treated SAH micecomparing with vehicle-treated SAH animals had been found. Furthermore, puerarin effectively reversed these alterations in expression and inhibits ROSproduction induced by SAH. Also, puerarin can increase SOD activation after SAH and protect the expression of Sirt3 after SAH. CONCLUSIONS: In coclusion, puerarin can provide potential neuroprotection from the SAH damages, and can be act as a novel therapy for SAH.

Effects of cyclosporin, nifedipine and phenytoin on gingival myofibroblast transdifferentiation in monkeys

Abstract Objective: Myofibroblasts have been associated with the development of several pathologic fibrotic conditions. This longitudinal study aims to assess the proliferative and antiapoptotic effects of cyclosporin, nifedipine and phenytoin on gingival connective tissue cells of nonhuman primate, as well as to analyze a possible role of myofibroblasts in gingival overgrowth. Materials and Methods: Gingival samples from the right superior canine area were obtained from 12 male monkeys ( Sapajus spp ) to comprise the control group. After one week, the animals were randomly assigned to three groups, which received daily oral doses of cyclosporin, nifedipine or phenytoin for 120 days. Gingival samples were collected from the left superior canine area of two animals of each group at 52 and 120 days. Histological sections were stained with hematoxylin and eosin, and immunoreacted against α-SMA, Ki- 67 and bcl-2. Results: α-SMA immunoreaction was negative in the control and experimental groups. Similarly, no difference between groups concerning immunostaining against Ki-67 and bcl-2 was observed in connective tissue cells. Conclusion: Based on this methodology, it may be concluded that gingival overgrowths induced by cyclosporin, nifedipine and phenytoin are not associated with neither myofibroblast transdifferentiation, proliferation nor apoptosis of gingival connective cells in monkeys.

Effects of cyclosporin, nifedipine and phenytoin on gingival myofibroblast transdifferentiation in monkeys.

OBJECTIVE: Myofibroblasts have been associated with the development of several pathologic fibrotic conditions. This longitudinal study aims to assess the proliferative and antiapoptotic effects of cyclosporin, nifedipine and phenytoin on gingival connective tissue cells of nonhuman primate, as well as to analyze a possible role of myofibroblasts in gingival overgrowth. MATERIALS AND METHODS: Gingival samples from the right superior canine area were obtained from 12 male monkeys ( Sapajus spp ) to comprise the control group. After one week, the animals were randomly assigned to three groups, which received daily oral doses of cyclosporin, nifedipine or phenytoin for 120 days. Gingival samples were collected from the left superior canine area of two animals of each group at 52 and 120 days. Histological sections were stained with hematoxylin and eosin, and immunoreacted against α-SMA, Ki- 67 and bcl-2. RESULTS: α-SMA immunoreaction was negative in the control and experimental groups. Similarly, no difference between groups concerning immunostaining against Ki-67 and bcl-2 was observed in connective tissue cells. CONCLUSION: Based on this methodology, it may be concluded that gingival overgrowths induced by cyclosporin, nifedipine and phenytoin are not associated with neither myofibroblast transdifferentiation, proliferation nor apoptosis of gingival connective cells in monkeys.

[Mechanism of angiotensin II (Ang II) on the proliferation of human hepatoma cell line HepG2 cells].

Objective: To study the effect and mechanism of angiotensin (Ang II) on the proliferation of human hepatocellular carcinoma HepG2 cells. Methods: The effects of different concentrations of Ang II's (10(-8)-10(-4) mol/L) on proliferated hepatocellular carcinoma HepG2 cells were detected by CCK-8 assay. The expression of angiotensin II type 1 receptor (AT1) protein and activation of ERK1/2 protein in hepatocellular carcinoma HepG2 cells after processing with Ang II were assayed by Western blot. The cells were pretreated with candesartan (AT1 receptor antagonist), sorafenib (Raf kinase inhibitor) and PD98059 (ERK1/2 inhibitor) for 1.5 h and then Ang II (10(-6) mol/L) was added. CCK-8 assay was used to determine whether it could reverse the proliferation of Ang II, and ERK phosphorylation levels were detected by Western blot. The changes in Bcl-2 and c-myc gene expression before and after Ang II processing were detected by Rt-PCR. According to different data, t-test, one-way analysis of variance or SNK method were used for statistical analysis. Results: HepG2 cells treated with different concentrations of Ang II promoted cell proliferation after 24h and 48h. After 24 h, cell vitality was strongest with Ang II concentration 10(-5) mol/L and the absorbance value was 0.990 8±0.097 8; and again after 48 h, the cell viability was strongest with Ang II concentration 10(-6) mol/L and the absorbance value was 1.302 7 ± 0.030 9. Moreover, the pro-proliferation effect of Ang II on HepG2 cells blocked candesartan, sorafenib and ERK1/2 isolated inhibitors. After treatment with 10(-6) mol/L Ang II, Western blot showed that Ang II significantly promoted AT1 receptor expression and phosphorylation of ERK1/2 protein confirmed that Ang II activated the AT1/RAF/ERK1/2 signaling pathway. In addition, Rt-PCR detection showed that the downstream of Bcl-2 and c-myc genes expressions rose significantly when the concentration of Ang II ranged from 10(-8) to 10(-6) mol/L. Conclusion: Ang II can promote the proliferation of HepG2 cells by activating AT1/Raf /ERK1/2 signaling pathway and enhance the downstream of Bcl-2 and c-myc gene expression.

Immunoexpression of vascular endothelial growth factor and B-cell lymphoma 2 in the uterine tissue of rats treated with melatonin in the estrus phase1.

PURPOSE: To investigate the effect of melatonin on uterine tissue in the ovariectomized rat model. METHODS: Fourty Wistar albino rats were divided into four groups for histologic and immunohistochemical examination. The rats were first numbered randomly and then randomly divided into 4 equal groups: control (group 1), torsion (group 2), torsion+detorsion (group 3) and torsion+detorsion+melatonin (group 4) groups. In addition, four Wistar albino rats were used for western blot analysis in each group. And also, malondialdehyde (MDA) levels were measured biochemically in all rats. RESULTS: The histopathological examination of the uterine tissue in rats ovarectomized showed a degeneration in uterine glands, dilation of blood vessels in the internal layer with a thrombosis and bleeding, abnormal nucleuses and vacuolated cytoplasm above and below the nucleus. In torsion group, the apoptotic cells increased in luminal epithelium and gland cells. In the melatonin group showed that the Bcl2 negative effect on the uterine epithelium and did not lead to apoptotic cells. CONCLUSION: The increase in vascular endothelial growth factor expression resulted in the rearrangement of endothelial cell growth and the induction of angiogenesis.

Immunoexpression of vascular endothelial growth factor and B-cell lymphoma 2 in the uterine tissue of rats treated with melatonin in the estrus phase

Abstract Purpose: To investigate the effect of melatonin on uterine tissue in the ovariectomized rat model. Methods: Fourty Wistar albino rats were divided into four groups for histologic and immunohistochemical examination. The rats were first numbered randomly and then randomly divided into 4 equal groups: control (group 1), torsion (group 2), torsion+detorsion (group 3) and torsion+detorsion+melatonin (group 4) groups. In addition, four Wistar albino rats were used for western blot analysis in each group. And also, malondialdehyde (MDA) levels were measured biochemically in all rats. Results: The histopathological examination of the uterine tissue in rats ovarectomized showed a degeneration in uterine glands, dilation of blood vessels in the internal layer with a thrombosis and bleeding, abnormal nucleuses and vacuolated cytoplasm above and below the nucleus. In torsion group, the apoptotic cells increased in luminal epithelium and gland cells. In the melatonin group showed that the Bcl2 negative effect on the uterine epithelium and did not lead to apoptotic cells. Conclusion: The increase in vascular endothelial growth factor expression resulted in the rearrangement of endothelial cell growth and the induction of angiogenesis.

Hydrogen protects against hyperoxia-induced apoptosis in type II alveolar epithelial cells via activation of PI3K/Akt/Foxo3a signaling pathway.

Oxidative stress is regarded as a key regulator in the pathogenesis of prolonged hyperoxia-induced lung injury, which causes injury to alveolar epithelial cells and eventually leads to development of bronchopulmonary dysplasia (BPD). Many studies have shown that hydrogen has a protective effect in a variety of cells. However, the mechanisms by which hydrogen rescues cells from damage due to oxidative stress in BPD remains to be fully elucidated. This study sought to evaluate the effects of hydrogen on hyperoxia-induced lung injury and to investigate the underlying mechanism. Primary type II alveolar epithelial cells (AECIIs) were divided into four groups: control (21% oxygen), hyperoxia (95% oxygen), hyperoxia + hydrogen, and hyperoxia + hydrogen + LY294002 (a PI3K/Akt inhibitor). Proliferation and apoptosis of AECIIs were assessed using MTS assay and flow cytometry (FCM), respectively. Gene and protein expression were detected by quantitative polymerase chain reaction (q-PCR) and western blot analysis. Stimulation with hyperoxia decreased the expression of P-Akt, P- FoxO3a, cyclinD1 and Bcl-2. Hyperoxic conditions increased levels of Bim, Bax, and Foxo3a, which induced proliferation restriction and apoptosis of AECIIs. These effects of hyperoxia were reversed with hydrogen pretreatment. Furthermore, the protective effects of hydrogen were abrogated by PI3K/Akt inhibitor LY294002. The results indicate that hydrogen protects AECIIs from hyperoxia-induced apoptosis by inhibiting apoptosis factors and promoting the expression of anti-apoptosis factors. These effects were associated with activation of the PI3K/Akt/FoxO3a pathway.