Bax mRNA Ratio Research Articles

Protective effect of fermented Diospyros lotus L. extracts against the high glucose-induced apoptosis of MIN6 cells.

Date plum persimmon (Diospyros lotus L.) is a fruit crop from the Ebenaceae family. Its microorganism-fermented extract (DPEML) was shown to exhibit a hypoglycemic effect in our previous work. Here, we investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF, and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. In summary, our study suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus. PRACTICAL APPLICATIONS: We investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. We suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus.

Asiaticoside suppresses cell proliferation by inhibiting the NF‑κB signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-associated mortality. Asiaticoside (AC) exhibits antitumor effects; however, to the best of our knowledge, the biological function of AC in CRC cells remains unclear. Therefore, the aim of the present study was to investigate the effect of AC on CRC cells. In the present study, CCK-8 and colony formation assays were performed to assess the effects of AV on human CRC cell lines (HCT116, SW480 and LoVo). Mitochondrial membrane potential was examined by JC-1 staining. Cell apoptosis and cell cycle were monitored by flow cytometry, and the expression of genes was evaluated using RT-qPCR and western blot analysis. Furthermore, the biological effect of AC in vivo was detected using a xenograft mouse model. The findings revealed that 2 µM AC suppressed the proliferation of CRC cells in a time- and dose-dependent manner, but had no adverse effects on normal human intestinal FHC cells at a range of concentrations. AC decreased the mitochondrial membrane potential and increased the apoptosis of CRC cells in a dose-dependent manner. Furthermore, AC induced cell cycle arrest at the G0/G1 phase. AC attenuated IκBα phosphorylation in a dose-dependent manner, thereby preventing P65 from entering the nucleus, and resulting in inhibition of the NF-κB signaling pathway. In addition, AC significantly reduced the expression of CDK4 and Cyclin D1 in a dose-dependent manner, significantly upregulated the activation of caspase-9 and caspase-3, and decreased the Bcl-2/Bax mRNA ratio. Furthermore, treatment with the NF-κB signaling pathway inhibitor JSH-23 significantly increased the cytotoxicity of AC in CRC cells. Findings of the xenograft mice model experiments revealed that AC significantly inhibited colorectal tumor growth in a dose-dependent manner. Overall, AC suppressed activation of the NF-κB signaling pathway by downregulating IκBα phosphorylation. This resulted in inhibition of CRC cell viability and an increase of cell apoptosis, which may form the basis of AC use in the treatment of patients with CRC.

Expression, location and biological effects of four and a half LIM domain protein2 (FHL2) on granulosa cells in ovine.

Previous studies have shown that four and a half LIM domain protein 2(FHL2) plays an essential role in the regulation of follicular development in mammals. Although the FHL2 genes of human and mouse have been well characterized, the expression and location of FHL2 in ovary and the biological functions of FHL2 on granulosa cells (GCs) of ovine are still not clear. In this study, full-length complementary DNA (cDNA) of FHL2 from ovine follicular GCs was amplified by real-time PCR (RT-PCR). The expression and location of FHL2 in ovary and GCs of ovine were studied by immunohistochemistry and immunofluorescence, and the biological effects of FHL2 on the cell proliferation, cell apoptosis, cell cycles and expression level of related genes of ovine GCs were also explored by overexpression or knockdown of FHL2. The results indicated that FHL2 was expressed in ovine follicular GCs and the sequence of the FHL2 cDNA was consistent with that predicted in GenBank, which did not cause an amino acid change. According to the results, FHL2 was expressed in ovine ovary and mainly located in the cytoplasm and nucleus of GCs. In addition, overexpression of FHL2 significantly reduced the cell viability, promoted the cell apoptosis and decreased the percentage of G0/G1 and S phase cells. RT-PCR showed that overexpression of FHL2 significantly increased the mRNA expression level of Bax and decreased the expression of Bcl-2 and the Bcl-2/Bax mRNA ratio compared with the control group. Besides, the knockdown of FHL2 gene in ovine GCs significantly improved the cell viability, suppressed the cell apoptosis, decreased the mRNA expression level of Caspase-3 gene, increased the Bcl-2/Bax mRNA ratio and increased the percentage of S and G2/M phase cells. Our results suggest that FHL2 may play an important role in the biological functions of GCs in ovine.

Impact of combined ischemic preconditioning and remote ischemic perconditioning on ischemia-reperfusion injury after liver transplantation

Ischemic preconditioning (IPC) and remote ischemic perconditioning (RIPer) confer protective effects against liver ischemia-reperfusion injury (IRI), but data about RIPer applying in liver transplantation is lacking. The study aimed to evaluate whether the combination of IPC and RIPer provides reinforced protective effects. C57BL/6 mice (160 pairs) were allocated into four groups: control, subjected to liver transplantation only; IPC, donor hilar was clamped for 10 min followed by 15 min of reperfusion; RIPer, three cycles of occlusion (5 min) and opening (5 min) of femoral vascular bundle were performed before reperfusion; IPC + RIPer, donors and recipients were subjected to IPC and RIPer respectively. Liver tissues were obtained for histological evaluation, TUNEL staining, malondialdehyde assays, GSH-Px assays, and NF-κB p65 protein and Bcl-2/Bax mRNA analyses. Blood samples were used to evaluate ALT, AST, TNF-α, NOx levels and flow cytometry. We found that protective efficacy of RIPer is less than IPC in terms of ALT, TNF-α, GSH-Px and NOx at 2 h postoperation, but almost equivalent at 24 h and 72 h postoperation. Except for Suzuki scores, ALT, Bcl-2/Bax mRNA ratio, other indices showed that combined treatment brought enhanced attenuation in IRI, compared with single treatment, through additive effects on antioxidation, anti-apoptosis, modulation of microcirculation disturbance, and inhibition of innate immune response. This study suggested a combined strategy that could enhance protection against IRI in clinical liver transplantation, otherwise, provided a hint that RIPer’s mechanism might be partly or totally different from IPC in humoral pathway.

Melatonin protects against paraquat-induced damage during in vitro maturation of bovine oocytes.

Paraquat (PQ), a broad-spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ-induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro-produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic-related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ-exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ-induced p38 mitogen-activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl-histone H3 lysine 4 (H3K4me3) and trimethyl-histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ-induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.

Modulations of DNMT1 and HDAC1 are involved in the OTA-induced cytotoxicity and apoptosis in vitro

Ochratoxin A (OTA) as a fungal metabolite is reported to induce cytotoxicity and apoptosis through the mechanism of oxidative stress. Oxidative stress could induce the epigenetic enzymes modifications. However, whether epigenetic enzymes modifications are involved in OTA-induced cytotoxicity and apoptosis has not been reported until now. Therefore, the objectives of this study were to verify OTA-induced cytotoxicity and apoptosis and to investigate the potential role of epigenetic enzymes in OTA-induced cytotoxicity and apoptosis in PK15 cells. The results demonstrated that OTA at 4 μg/ml treatment for 12 h and 24 h induced cytotoxicity and apoptosis as demonstrated by decreasing cell viability, increasing LDH release, Annexin V/PI staining, Bcl-2/Bax mRNA ratio and apoptotic nuclei in PK15 cells. OTA treatment up-regulated ROS production and down-regulated GSH levels. In addition, OTA treatment activated the epigenetics related enzymes DNA methyltransferase 1 (DNMT1) and Histone deacetylase 1 (HDAC1). Adding DNMT1 inhibitor (5-Aza-2dc) or HDAC1 inhibitor (LBH589) depressed the up-regulation of DNMT1 or HDAC1 expression, the decreases of GSH levels and increases of ROS production induced by OTA, respectively. Furthermore, inhibition of DNMT1 or HDAC1 by their inhibitor reversed the decreases of cell viability and increases of LDH activity and apoptosis induced by OTA, respectively. In conclusion, the observed effects indicate that the critical modulation of DNMT1 and HDAC1 is related to OTA-induced cytotoxicity and apoptosis.

In vitro immune toxicity of ochratoxin A in porcine alveolar macrophages: A role for the ROS-relative TLR4/MyD88 signaling pathway

Ochratoxin A (OTA) frequently contaminates a wide variety of food and feeds, inducing immune toxicity of animals. However, the underlying mechanism remains unclear. In the present study, porcine alveolar macrophages (PAMs) cell line 3D4/21 were chosen as a model, and treated by various concentrations of OTA. The results showed that OTA induced immune toxicity of PAMs in a dose-dependent manner, as demonstrated by cell viability, LDH release, Annexin V/PI staining, Bcl-2/Bax mRNA ratio, and pro-inflammatory cytokines expression. OTA increased intracellular ROS levels of PAMs by flow cytometry and confocal microscopy. NAC, a commonly used antioxidant, alleviated the OTA-induced increase of ROS production, apoptosis, and LDH release and decrease of cell viability. Furthermore, OTA enhanced the mRNA and protein expression of TLR4 and MyD88 and the phosphorylation of ERK1/2, p38, and NF-κB p65. Knockdown of TLR4 by using a TLR4-specific siRNA alleviated the OTA-induced immune toxicity. These data indicate that OTA induced immune toxicity via ROS-relative TLR4/MyD88 signaling pathway in PAMs.

Amelioration of CCl4-induced liver injury in rats by selenizing Astragalus polysaccharides: Role of proinflammatory cytokines, oxidative stress and hepatic stellate cells

Selenizing Astragalus polysaccharides (sAPS) were prepared by nitric acid–sodium selenite method. Effect of sAPS on carbon tetrachloride (CCl4)-induced liver injury and the underlying mechanisms were investigated in the rat. Forty male Wistar rats were divided into five equal groups as follows: control group; CCl4 group; CCl4+Astragalus polysaccharides group; CCl4+sodium selenite group and CCl4+selenizing Astragalus polysaccharides group. The results showed that sAPS significantly decreased the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase in the serum, malondialdehyde and hydroxyproline content in liver (P<0.01), and increased the levels of total protein, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase in liver of rats induced by CCl4. In addition, expression levels of antioxidant-related genes (GPX1, SOD1, and Nrf2) were significantly increased following supplementation of the sAPS (P<0.01). Furthermore, sAPS effectively ameliorated CCl4 induced hepatic necrosis and inflammation, and it also reduced the expression levels of proinflammatory cytokines including TNF-α, IL-6, COX-2 and NFκB (P<0.01). Moreover, sAPS significantly decreased the expression levels of α-smooth muscle actin, collagen 1, TGF-β1, but increased the Bcl-2/Bax mRNA ratio in rats administered CCl4 (P<0.01). Taken together, it could be concluded that sAPS could increase the activities of Astragalus polysaccharides and sodium selenite to protect the liver from damage by attenuating hepatic inflammation, oxidative stress, fibrogenesis, and induces apoptosis and cell cycle arrest in hepatic stellate cells.

Effects of cadmium on Bcl-2/ Bax expression ratio in rat cortex brain and hippocampus.

To investigate the underlying mechanism of neurotoxicity of cadmium, we examined the effects of intraperitoneal injection of cadmium on messenger RNA (mRNA) expression of Bcl-2 (B-cell lymphoma 2) and Bax (Bcl2-associated x) genes and caspase-3/7 activation in rat hippocampus and frontal cortex. Twenty-eight male Wistar rats weighing 200-250 g were randomly divided into four groups. Control group received saline and three other groups received cadmium at doses of 1, 2 and 4 mg/kg (body weight) for 15 successive days. One day after the last injection, the hippocampus and frontal cortex were dissected and removed and then the expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction and apoptotic studies was done using caspase-3/7 activation assay. Cadmium reduced the mRNA level of Bcl-2 in the control group at doses of 1 ( p < 0.01), 2 and 4 mg/kg ( p < 0.001) in rat hippocampus and cortex cells. The mRNA level of Bax increased significantly compared to the control group at the doses of 1 ( p < 0.05), 2 and 4 mg/kg ( p < 0.001) in rat hippocampus. The mRNA level of Bax was increased significantly compared to the control group at the doses of 2 and 4 mg/kg ( p < 0.001) in rat cortex cells. Cadmium increased caspase-3/7 activity at doses of 1, 2 and 4 mg/kg in rat hippocampus. Caspase-3/7 activity was increased significantly at dose of 4 mg/kg in rat cortex. This decreased Bcl-2/Bax mRNA ratio induces cell apoptosis. Apoptotic effect of cadmium may be through the mitochondrial pathway by the activation of caspase-3/7.

Acupuncture with reinforcing and reducing twirling manipulation inhibits hippocampal neuronal apoptosis in spontaneously hypertensive rats.

To observe the effects of different acupuncture manipulations on blood pressure and target organ damage in spontaneously hypertensive rats (SHRs), this study used the reinforcing twirling method (1.5-2-mm depth; rotating needle clockwise for 360° and then counter clockwise for 360°, with the thumb moving heavily forward and gently backward, 60 times per minute for 1 minute, and retaining needle for 9 minutes), the reducing twirling method (1.5-2-mm depth; rotating needle counter clockwise for 360° and then clockwise for 360°, with the thumb moving heavily backward and gently forward, 60 times per minute for 1 minute, and retaining needle for 9 minutes), and the needle retaining method (1.5-2-mm depth and retaining the needle for 10 minutes). Bilateral Taichong (LR3) was treated by acupuncture using different manipulations and manual stimulation. Reinforcing twirling, reducing twirling, and needle retaining resulted in a decreased number of apoptotic cells, reduced Bax mRNA and protein expression, and an increased Bcl-2/Bax ratio in the hippocampus compared with the SHR group. Among these groups, the Bcl-2/Bax protein ratio was highest in the reducing twirling group, and the Bcl-2/Bax mRNA ratio was highest in the needle retaining group. These results suggest that reinforcing twirling, reducing twirling, and needle retaining methods all improve blood pressure and prevent target organ damage by increasing the hippocampal Bcl-2/Bax ratio and inhibiting cell apoptosis in the hippocampus in SHR.

Neuroprotective Effects of Engineered Polymeric Nasal Microspheres Containing Hydroxypropyl-β-cyclodextrin on β-Amyloid (1-42)–Induced Toxicity

β-Amyloid (Aβ) plaques are the key neurotoxic assemblies in Alzheimer disease. It has been suggested that an interaction occurs between membrane cholesterol and Aβ aggregation in the brain. Cyclodextrins can remove cholesterol from cell membranes and change receptor function. This study aimed to investigate the effect of hydroxypropyl-β-cyclodextrin (HP-CD) polymeric microspheres, based on chitosan or sodium alginate, on the levels of lipid peroxidation, reactive oxygen species production, and mitochondrial function in brain synaptosomes. The effect of microspheres on DNA fragmentation, the expression of Bcl-2, Bax, and Apex1 mRNAs in rat hippocampus after Aβ(1-42) peptide-induced neurotoxicity was also evaluated. Comparison with HP-CD raw material was performed. Aβ(1-42) treatment significantly decreased the mitochondrial activity of Apex1 and Bcl-2 mRNAs, induced DNA fragmentation, and increased mRNA levels of Bax. Treatment with HP-CD microspheres against Aβ(1-42) significantly reduced DNA fragmentation and increased the Bcl-2/Bax mRNA ratio and mitochondrial function. In addition, HP-CD microspheres used against Aβ(1-42) decreased the levels of lipid peroxidation and reactive oxygen species production. These results indicate that nasally administered spray-dried HP-CD microspheres are able to provide protection against Aβ(1-42)-induced neurotoxicity, due to the suppressed levels of oxidative stress and apoptotic signals in the rat hippocampus.

Differentiated impact of procyanidins from evening primrose on human breast cancer cells

Abstract This study examines some of the biological activities of an evening primrose flavanol preparation (EPFP) against non-invasive human breast cancer cells (MCF-7). The results are compared with those obtained for highly invasive human breast cancer cells (MDAMB-231). The results show, for the first time, that EPFP reduces MCF-7 cell number, IC50 = 75 µM gallic acid equivalents/GAE for 72 h incubation, and reduces migration to 52% of the control value at 100 µM L−1 GAE. EPFP caused favorable changes in Bcl-2/Bax mRNA ratio, which rendered MCF-7 cells more sensitive to apoptosis: the number of apoptotic cells increased 2.2-fold vs. control at 100 µM GAE. Furthermore, 100 µ M L−1 GAE EPFP caused a 1.8-fold reduction in the activity of metalloproteinase-9 (MMP-9) secreted to the culture medium by MCF-7 cells. Moreover, EPFP suppressed the expression of selected genes of matrix metalloproteinases (MMPs), a proliferation marker (Ki67), and vascular endothelial growth factor (VEGF). In conclusion, the results of this study suggest that EPFP may exhibit proapoptotic, antiproliferative, antimigratory, and antimetastatic potential towards both selected human breast cancer cell lines, which is more pronounced in the case of the highly invasive MDA-MB-231 cells.

Flavanols from Evening Primrose (Oenothera paradoxa) Defatted Seeds Inhibit Prostate Cells Invasiveness and Cause Changes in Bcl-2/Bax mRNA Ratio

In this study, we assessed the influence of an evening primrose flavanol preparation (EPFP) on proliferation and invasiveness of human prostate cancer cells (DU 145) and immortalized prostate epithelial cells (PNT1A). We report for the first time that EPFP reduces DU 145 cell proliferation (IC50 = 97 μM GAE for 72 h incubation) and invasiveness (by 24% versus control at 75 μM GAE). EPFP strongly inhibited PNT1A invasiveness in a concentration-dependent manner (by 67% versus control at 75 μM GAE) and did not cause a reduction in their proliferation. Furthermore, EPFP inhibited the activities of MMP-2 and MMP-9 secreted to culture medium by PNT1A cells by 84% and 34% versus control at 100 μM GAE, respectively. In the case of DU 145, MMP-9 activity at 100 μM GAE was reduced by 37% versus control. Moreover, the evening primrose seed flavanols suppressed the expression of selected genes (MMP-1, MMP-9, MMP-14, c-Fos, c-Jun, and VEGF) and also caused favorable changes in Bcl-2/Bax mRNA ratio which render DU 145 cells more sensitive to apoptosis-triggering agents. An additional confirmation of the proapoptotic activity of EPFP toward DU 145 was visualization of characteristic apoptotic bodies by DAPI staining. In conclusion, this study suggests that EPFP may increase apoptosis and reduce angiogenesis of prostate cancer cells.

Anti-Stress Effects of Carnosine on Restraint-Evoked Immunocompromise in Mice through Spleen Lymphocyte Number Maintenance

Carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, has been characterized as a putative neurotransmitter and serves as a reservoir for brain histamine, which could act on histaminergic neurons system to relieve stress-induced damages. However, understanding of the role of carnosine in stress-evoked immunocompromise is limited. In this study, results showed that when mice were subjected to restraint stress, spleen index and the number of spleen lymphocytes including Natural Killer (NK) cells were obviously decreased. Results also demonstrated that restraint stress decreased the cytotoxic activity of NK cells per spleen (LU10/spleen) while the activity of a single NK cell (LU10/106 cells) was not changed. However, oral administration of carnosine (150 and 300 mg/kg) increased spleen index and number of spleen lymphocytes (including NK cells), and elevated the cytotoxic activity of NK cells per spleen in restraint-stressed mice. These results indicated that carnosine ameliorated stress-evoked immunocompromise through spleen lymphocyte number maintenance. Carnosine was further found to reduce stress-induced elevation of plasma corticosterone level. On the other hand, results showed that carnosine and RU486 (a glucocorticoids receptor antagonist) treatment prevented the reduction in mitochondrion membrane potential and the release of mitochondrial cytochrome c into cytoplasm, increased Bcl-2/Bax mRNA ratio, as well as decreased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in spleen lymphocytes of stressed mice. The results above suggested that the maintenance of spleen lymphocyte number by carnosine was related with the inhibition of lymphocytes apoptosis caused by glucocorticoids overflow. The stimulation of lymphocyte proliferation by carnosine also contributed to the maintenance of spleen lymphocyte number in stressed mice. In view of the elevated histamine level, the anti-stress effects of carnosine on restraint-evoked immunocompromise might be via carnosine-histamine metabolic pathway. Taken together, carnosine maintained spleen lymphocyte number by inhibiting lymphocyte apoptosis and stimulating lymphocyte proliferation, thus prevented immunocompromise in restraint-stressed mice.

Bone marrow mesenchymal stem cells protect against folic acid- induced acute kidney injury

Acute kidney injury constitutes a syndrome responsible by a major percentage of acute kidney failures and it continues being associated to high mortality rates. Induced mainly by ischemia-reperfusion injury and nephrotoxic drugs, this condition is marked by a decrease in organ function and histopathological pattern of acute tubular necrosis. In search of more efficient therapies, a great deal of attention has been given to the therapeutic use of stem cells to treat kidney injuries. Bone marrow stem cells in particular have received a great attention due to its immunomodulatory properties, and its therapeutic mechanisms are intensely being studied in the literature. Purpose: In view of recent findings, the aim of our research was to get a better understanding on the potential role of bone marrow mesenchymal stem cells in a murine model of acute nephotoxicity induced by folic acid. Methods: C57Bl/6j mice (8 weeks) were submitted to acute kidney injury by folic acid (200mg/kg) administered intraperitoneally. After 24 hours, mice received mesenchymal stem cells (5.105 cells per animal) through retro-orbital intravenous injection. Mice were sacrificed after 24 hours and blood and kidneys were harvested for analysis. Results: Stem cell treatment conferred functional improvement seen through lower creatinine and urea serum levels in 8 week old C57Bl/6j mice in comparison to mice treated only with folic acid (200mg/kg body weight). This amelioration are also correlated to down regulation of kidney pro-inflammatory cytokine mRNA levels as TNF-a, IL-6 and IL-1b in stem cell treated mice. In addition, treated mice demonstrated higher levels of immunostaining for proliferating cell nuclear antigen and a tendency towards a higher Bcl-2/Bax mRNA ratio, indicating higher tissue regeneration and protection against injury-induced apoptosis. Conclusion: These results indicate bone marrow stem cells as an efficient tool in nephrotoxic kidney injury treatment.&#x0D;

Effects of BNP preconditioning on myocardial cell apoptosis and expressions of bcl-2 and Bax during myocardial ischemia-reperfusion injury in rats

To study the effects of B-type natriuretic peptide (BNP) preconditioning on the apoptosis and expressions of Bcl-2 and Bax in rat cardiomyocytes during myocardial ischemia-reperfusion. Twenty-one male Sprague-Dawley rats weighing (250 ± 50) g were randomly divided into 3 groups of sham operation (SHAM), ischemia-reperfusion (I/R) and B-type natriuretic peptide (BNP). A rat model of in vivo myocardial ischemia-reperfusion injury was established by ligating the left anterior descending coronary artery for 35 minutes and then reperfusing for 240 minutes. The apoptosis of myocardial cell was determined by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end-labeling (TUNEL) method. Real-time polymerase chain reaction and Western blot were used to detect the expression changes of Bcl-2 and Bax in rat ischemia myocardium. The apoptotic indices of SHAM, BNP and I/R groups were 5.4% ± 4.2%, 22.5% ± 9.5% and 45.2% ± 13.0% respectively (P < 0.05). The Bcl-2 protein expression of SHAM, BNP and I/R groups were 0.87 ± 0.09, 0.70 ± 0.07 and 0.38 ± 0.09 respectively (P < 0.05). The Bax protein expression of SHAM, BNP and I/R groups were 0.08 ± 0.04, 0.39 ± 0.09 and 0.71 ± 0.18 respectively (P < 0.01). The Bcl-2/Bax mRNA ratio of SHAN, BNP and I/R groups were 0.763 ± 0.154, 0.099 ± 0.025 and 0.022 ± 0.024 respectively (P < 0.05). The BNP preconditioning can decrease the myocardial apoptosis induced by ischemia-reperfusion injury. The mechanisms may be associated with an elevated expression of Bcl-2, an increased ratio of Bcl-2/Bax and a lowered expression of Bax.

Peony glycosides protect against corticosterone-induced neurotoxicity in PC12 cells.

Preclinical and clinical investigations have shown the involvement of dysregulation of hypothalamic-pituitary-adrenal (HPA) axis in the pathogenesis of depression. Hypercortisolemia and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Previous studies in our laboratory have demonstrated the antidepressant-like activity of total glycosides of peony (TGP) in mice. This study aimed to examine the effect of TGP treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Treating the cells with corticosterone at 200 muM for 48 h caused apoptotic cell death. The cytotoxicity was associated with the activation of caspase-3 activity and the decrease in the mRNA ratio of bcl-2 to bax. TPG treatment at increasing doses (1-10 mg/l) protected against the corticosterone-induced toxicity in PC12 cells in a dose-dependent manner. The cytoprotection afforded by TGP treatment was associated with the inhibition of caspase-3 activity and the up-regulation of bcl-2/bax mRNA ratio. The anti-apoptotic effect of TGP is therefore likely mediated by the suppression of the mitochondrial pathway leading to apoptosis.

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50μM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7μg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7μg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.

Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan™) from Norway spruce ( Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells

Lignans are plant polyphenols which may possess anticancer, antioxidant, antimicrobial, anti-inflammatory and immunomodulatory activities. In particular, the lignan 7-hydroxymatairesinol (HMR/lignan™, HMR) is a novel precursor of the mammalian lignan enterolactone (EL). In the present study, we investigated the estrogenicity of HMR and of EL in comparison to estradiol (E2), by measuring their effects on growth and apoptotic markers in the human estrogen-sensitive cell line MCF-7. HMR, EL and E2 concentration-dependently increased the percentage of MCF-7 cells in the S phase of the cell cycle, with the following relative potencies: E2 ≅ EL ≫ HMR, and efficacies: E2 > HMR ≫ EL. Treatment of MCF-7 cells with either HMR, EL or E2 also increased the Bcl-2/Bax mRNA ratio. The effects of HMR and EL were reduced in the presence of the estrogen receptor (ER) antagonist tamoxifene. We conclude that both HMR and its metabolite EL are endowed with estrogenic activity, which is likely to be exerted through the contribution of ER-dependent pathways and to target the same intracellular mechanisms acted upon by E2. The estrogenicity of HMR and EL is however milder than that of E2, as indicated by the lower potencies and efficacies of both lignans. The present results support the notion that dietary supplementation with HMR may result in a mild estrogenic activity, both directly and by providing a suitable source for endogenous EL.

Influence of age or circadian time on bcl-2 and bax mRNA expression in the rat hippocampus after corticosterone exposure

A rapid elevation in the level of endogenous corticosterone (CORT) functions in the stress response associated with the hypothalamus–pituitary–adrenal axis, and it has been well documented that high levels of CORT play neurotoxic roles in the hippocampus. Both aging and the circadian rhythm possibly affect the sensitivity to CORT, although their endogenous modifications in the CORT-mediated events remain unclear. To explore the influence of age or circadian time on hippocampal vulnerability to excess CORT, we examined the relative mRNA expression of bcl-2 and bax in the dentate gyrus (DG) and the CA1 subfield, compared with the CA3 as an internal standard, after acute CORT administration using in situ RT-PCR. Male rats aged 10 weeks (young) or 6 months (adult) were treated with CORT at 0800 or 2000 h. The bcl-2 to bax mRNA ratio in the dentate gyrus (DG) was significantly decreased 2 h after CORT exposure in the young rats treated at 0800 or 2000 h. In the adult rats, the treatment with CORT at 0800 h significantly decreased the bcl-2 to bax ratio, whereas the treatment at 2000 h was ineffective; the discrepancy between the treatment time points was apparent in adult rats, but not in young rats. Our results emphasize the importance of circadian time as well as age as a factor influencing the stress paradigm.