Resveratrol inhibits LPS-induced MAPKs activation via activation of the phosphatidylinositol 3-kinase pathway in murine RAW 264.7 macrophage cells.

BACKGROUND: Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells. METHODOLOGY/PRINCIPAL FINDINGS: Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol's action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1. CONCLUSION AND IMPLICATIONS: This investigation demonstrates that PI3-K/Akt activation is an important signaling in resveratrol-mediated activation of AMPK phosphorylation and SIRT1 expression, and inhibition of phosphorylation of CREB and MAPKs activation, proinflammatory mediators and cytokines production in response to LPS in RAW 264.7 cells.

[The effects of p38MAPK and HBxAg on cell proliferation and apoptosis in human hepatocarcinogenesis].

OBJECTIVE: To investigate the effects of host-derived p38 mitogen-activated protein kinase subunit 38 (p38MAPK) and the hepatitis B virus X antigen (HbxAg) on cell proliferation and apoptosis in human hepatocellular carcinoma (HCC), and to study the mechanism underlying hepatocarcinogenesis. METHODS: Liver tissues were biopsied from healthy individuals and patients with chronic hepatitis B (CHB), liver cirrhosis, paratumor cirrhosis, and HCC. Immunohistochemical staining was used to detect expressions of HBxAg, p38MAPK, cell cycle G2/M phase-related factors (cdc25B, p34cdc2, cyclin B1), and cell proliferation factor ki-67.The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method (known as TUNEL) was used to detect apoptosis. RESULTS: The highest rates of HBxAg were detected in CHB (65.0%) and HCC (44.4%) liver samples, and the antigen was mainly expressed in nuclei. Increasingly higher rates of p38MAPK, cdc25B, cyclin B1, and p34cdc2 expression were detected with increases in disease severity: normal liver (40.0%, 20.0%, 20.0%, and 30.0%, respectively), chronic hepatitis B (60.0%, 65.0%, 40.0%, and 50.0%, respectively), liver cirrhosis (65.0%, 75.0%, 70.0%, and 55.0%, respectively), paratumor cirrhosis (66.7%, 75.0%, 75.0%, and 63.9%, respectively), and HCC (77.8%, 80.6%, 80.6%, and 72.2%, respectively). In addition, the intracellular location of p38MAPK expression was different under different disease conditions, showing nuclear expression in CHB and liver cirrhosis samples and cytoplasmic expression in paratumor cirrhosis and HCC samples (x2 = 1.11, P more than 0.05). The proliferation index (PI) and the apoptosis index (AI) were both increased along with disease severity (normal more than CHB more than paratumor cirrhosis more than HCC) (PI: 0.0000+/-0.000, 0.0502+/-0.011, 0.0411+/-0.009, 0.0762+/-0.017; AI: 0.0351+/-0.024, 0.0607+/-0.022, 0.0562+/-0.013, 0.0716+/-0.011), with the notable exception for liver cirrhosis (PI: 0.1810+/-0.036 and AI: 0.1200+/-0.018). PI in poorly-differentiated HCC (0.2285+/-0.062) was significantly higher than in well-differentiated HCC (0.1216+/-0.032, t = 2.082, P = 0.044). AI in well-differentiated HCC (0.152+/-0.026) was significantly higher than in poorly-differentiated HCC (0.081+/-0.022, t = 2.129, P = 0.041). CONCLUSIONS: In the process of hepatocarcinogenesis, HBxAg may cause a series of abnormal changes in cell cycle, proliferation and apoptosis by affecting the expression of p38MAPK.

Expression and hypermethylation of p27 kip1 in hepatocarcinogenesis.

AIM: To study the expressions of p27 kip1 protein and p27mRNA, the hypermethylation of p27 kip1 and the relation between them in various stages of hepatocarcinogenesis. METHODS: p27 protein and p27mRNA were detected by immunohistochemical staining and in situ hybridization respectively in 68 cases of normal liver, liver cirrhosis, pericancerous cirrhosis and hepatocellular carcinoma (HCC). The hypermethylation of p27 kip1 was detected by methylation-specific PCR (MSP) in 44 cases of normal liver, liver cirrhosis, and HCC. RESULTS: The positive rate of p27 protein was 66.7% (4/6) in normal liver, 60.0% (6/10) in liver cirrhosis, 50.0% (12/24) in pericancerous cirrhosis and 21.4% (6/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 protein significantly decreased in HCC compared to that in the other groups (P = 0.006, chi2 = 7.664). The positive rate of p27 kip1 mRNA was 83.3% (5/6) in normal liver, 70.0% (7/10) in liver cirrhosis, 75.0% (18/24) in pericancerous cirrhosis and 25.0% (7/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 kip1 mRNA also significantly decreased in HCC compared to that in the other groups (P = 0.000, chi2 = 16.600). In addition, there was a significant correlation between the expression of p27 protein and p27mRNA in the integrated group of normal liver and liver cirrhosis. However, no significant correlation was found between pericancerous cirrhosis and HCC. Using MSP, we found that 1 HCC in 44 cases (including 6 cases of normal liver, 10 cases of liver cirrhosis and 28 cases of HCC) was methylated, whose p27 protein and p27mRNA were negative. CONCLUSION: The reduction or loss of p27 protein and p27mRNA are potentially involved in hepatocarcinogenesis. The hypermethylation of p27 might lead to the loss of p27mRNA transcription.

[Expression of transforming growth factor-beta1 and c-fos and p27 in human middle ear cholesteatoma].

OBJECTIVES: To analyze the expression of transforming growth factor-beta1 (TGF-beta1), cyclin dependent kinase inhibitor (p27) and c-fos in human middle ear cholesteatomas and to investigate the correlation between their expression and the ability of erosion of cholesteatoma. METHODS: Immunohistochemical staining (SP method) of 31 cholesteatomas and 11 external ear canal skin samples from patients and 10 external ear canal skin samples from healthful men which were taken intraoperatively, was performed for c-fos, TGF-beta1 and p27 positivity. The signals representing the expression of c-fos, TGF-beta1 and p27 were observed under microscope and scanned into a computer by an image scanner. The gray-scale of positive signals were quantitated by image processing computer. RESULTS: The percentage of positive expression of TGF-beta1 and c-fos in cholesteatoma were 87.1% and 83.9%, respectively. Their expression tended to be increased greatly compared with which in the skins of the control groups. Positive p27 signals were not observed in cholesteatomas and external ear skin tissues. It showed statistically significant correlation between expression of c-fos and the ability of erosion of cholesteatoma. There was correlation between the express ion of TGF-beta1 and the ability of erosion of cholesteatoma too. But there was no correlation between the expression of c-fos and TGF-beta1. CONCLUSION: The expression of c-fos in cholesteatoma was significally higher compared with which in the skin of external auditory of cholesteatoma patients and healthful men, which indicate that c-fos plays an important role in the hyperproliferative of cholesteatoma. The expression of TGF-beta1 was significant higher in cholesteatoma, which indicate that cytokines such as TGF-beta1 play a great role in the etiology of cholesteatoma.

Detection of HBV, PCNA and GST-pi in hepatocellular carcinoma and chronic liver diseases.

AIM: To investigate the change of HBV DNA, PCNA and GST-pi in chronic liver disease and hepatocellular carcinoma (HCC). METHODS: Hepatitis B surface antigen (HBsAg), proliferating cell nuclear antigen (PCNA) and glutathione S-transferases (GST-pi) were detected by immunohistochemical staining and HBV DNA was detected by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded sections with a total of 111 specimens of chronic hepatitis, liver cirrhosis, paratumorous tissue, HCC and normal liver tissue. RESULTS: The positive rates of HBsAg and HBVDNA were 62.5 %(15/24) and 75.0 %(12/16) in chronic hepatitis, 64.0 %(16/25) and 83.3 %(15/18) in liver cirrhosis, 72.7 %(16/22) and 85.7 %(12/14) in the paratumorous tissu and 45.0 %(14/31) and 64.3 %(9/14) in HCC. The positive HBVDNA granules in chronic hepatitis, liver cirrhosis and the paratumorous tissue were more intense than that in HCC. The positive rates of PCNA and GST-pi were 34.8 %(8/23) and 25.0 %(4/16) in chronic hepatitis, 73.7 %(14/19) and 17.6 %(3/17) in liver cirrhosis, 86.7 %(13/15) and 53.3 %(8/15) in the paratumorous tissue, 100 %(15/15) and 60.0 %(9/15) in HCC, respectively, and the positive rate of GST-pi in the paratumorous tissue was significantly higher than that in the liver cirrhosis without tumor (P<0.05), but same as that in HCC (P>0.05). CONCLUSION: The HBV infection may increase expression of PCNA and GST-pi. The paratumor cirrhosis may be a sequential lesion of precancerous cirrhosis around HCC.

[Expression of GST-pi and HBV infection in hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: The expression of glutathione S-transferases(GST-pi) might abnormally increase in many carcinogenesis, and the alteration of GST-pi preceded than that alteration of cell morphology. This study was designed to investigate the expression of glutathione S-transferases (GST-pi) and its relationship with hepatitis B virus (HBV) infection in hepatocellular carcinoma (HCC). METHODS: Hepatitis B surface antigen (HBsAg), Hepatitis B core antigen (HBcAg), and GST-pi were detected by immunohistochemical staining and HBV DNA was detected by in situ hybridization (ISH) in total 86 samples of chronic hepatitis, liver cirrhosis, paratumor cirrhosis, HCC, and normal liver tissue. RESULTS: The positive expression rates of HBsAg, HBcAg, and HBV DNA were 61.9% (13/21), 42.9% (9/21), and 75.0% (12/16) in chronic hepatitis; 64.0% (16/25), 36.0% (9/25), and 83.3% (15/18) in liver cirrhosis; 72.7% (16/22), 61.1% (11/18), and 85.7% (12/14) in the paratumor cirrhosis, as well as 45.0% (14/31), 50.0% (14/28) and 64.3% (9/14) in HCC. HBV DNA positive granules in chronic hepatitis, liver cirrhosis, and the paratumor cirrhosis were more and stronger than that in HCC, respectively. The positive expression rates of GST-pi were 25.0% (4/16), 17.6% (3/17), 53.3% (8/15), and 60.0% (9/15) in chronic hepatitis, liver cirrhosis, the Paratumor cirrhosis, and HCC, respectively. The positive rate of GST-pi in the paratumor cirrhosis was significantly higher than that in the liver cirrhosis without tumor (P < 0.05), but the same as in HCC (P > 0.05). CONCLUSIONS: Most of the HCC cases were closely associated with chronic hepatitis and liver cirrhosis of HBV infection. The HBV infection may increase expression of GST-pi. The paratumor cirrhosis may be a sequential lesion of precancerous cirrhosis around HCC.