Ming-Shi Shiao

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis

MicroRNA-122 (miR-122), which accounts for 70% of the livers total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-α-treated human aortic endothelial cells

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water‐soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial‐leukocyte adhesion molecules by tumor necrosis factor‐α (TNF‐α)‐treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 μg/ml), the TNF‐α‐induced expression of vascular adhesion molecule‐1 (VCAM‐1) was notably attenuated (77.2 ± 3.2% and 80.0 ± 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 μg/ml), 84.5 ± 1.9%, 78.8 ± 1.2%, 58.9 ± 0.4%, 58.7 ± 0.9%, and 57.4 ± 0.3%, respectively. Dose‐dependent lowering of expression of intercellular cell adhesion molecule‐1 (ICAM‐1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E‐selectin) was not affected. SME (50 μg/ml) or Sal B (5 μg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF‐α‐stimulated HAECs (45.7 ± 2.5% and 55.8 ± 1.2%, respectively). SME or Sal B significantly inhibited TNF‐α‐induced activation of nuclear factor kappa B (NF‐κB) in HAECs (0.36‐ and 0.48‐fold, respectively). These results demonstrate that SME and Sal B have anti‐inflammatory properties and may explain their anti‐atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 82:512–521, 2001.

Increase of Vitamin E Content in LDL and Reduction of Atherosclerosis in Cholesterol-Fed Rabbits by a Water-Soluble Antioxidant-Rich Fraction of Salvia miltiorrhiza

Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. Salvia miltiorrhiza Bunge is a Chinese herb widely used for the treatment of atherosclerosis-related disorders. Salvianolic acid B (Sal B), a water-soluble polyphenolic antioxidant isolated from the roots of this plant, was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit LDL oxidation more effectively than probucol. In order to evaluate the antiatherogenic potential, New Zealand White rabbits were fed for 12 weeks a normal diet, a high cholesterol diet, a high cholesterol diet containing 1% probucol, or a high cholesterol diet containing a 5% water-soluble extract of S miltiorrhiza (SM). Both SM and probucol feeding reduced plasma cholesterol. LDLs from the SM-treated group were more resistant to Cu2+-induced oxidation and contained more vitamin E (21.7+/-2.1 mmol/micromol LDL cholesterol) than did LDLs from the high cholesterol diet group (9.6+/-1.8 nmnol/micromol LDL cholesterol) (P<.005). Endothelial damage, determined at week 6, was reduced by 53% in the SM group (P<.01). SM treatment reduced the atherosclerotic area in the abdominal aorta by 56% (P<.005) and cholesterol deposition in the thoracic aorta by 50% (P<.005). The severity of atherosclerosis in the SM group was significantly reduced after adjustment by using cholesterol exposure as an index of the cholesterol-lowering effect. This study concludes that the reduction of atherosclerosis by SM relies not only on its cholesterol-lowering effect but more heavily on its antioxidant potential to prevent endothelial damage and inhibit LDL oxidative modification in hypercholesterolemic animals.

Cordyceps sinensis as an immunomodulatory agent

Effects of various fractions of methanol extracts from fruiting bodies of Cordyceps sinensis on the lymphoproliferative response, natural killer (NK) cell activity, and phytohemagglutinin (PHA) stimulated interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) production on human mononuclear cells (HMNC) were studied. Two of the 15 column fractions (CS-36-39 and CS-48-51) significantly inhibited the blastogenesis response (IC50 = 71.0 +/- 3.0 and 21.7 +/- 2.0 micrograms/ml, respectively), NK cell activity (IC50 = 25.0 +/- 2.5 and 12.9 +/- 5.8 micrograms/ml, respectively) and IL-2 production of HMNC stimulated by PHA (IC50 = 9.6 +/- 2.3 and 5.5 +/- 1.6 micrograms/ml, respectively). TNF-alpha production in HMNC cultures was also blocked by CS-36-39 and CS-48-51 (IC50 = 2.7 +/- 1.0 and 12.5 +/- 3.8 micrograms/ml, respectively). These results indicated that neither CS-36-39 nor CS-48-51 was cytotoxic on HMNC, and that immunosuppressive ingredients are contained in Cordyceps sinensis.

Growth Inhibitors Against Tumor Cells in Cordyceps sinensis Other than Cordycepin and Polysaccharides

Cordyceps sinensis is a parasitic fungus that has been used as a Chinese medicine for a long time. In the present study, inhibitory effects of crude methanolic extracts of C. sinensis fruiting bodies on various tumor cell lines were demonstrated. The crude methanolic extracts were fractionated into 15 fractions by silica gel column chromatography. Two of the 15 fractions (CS-36-39 and CS-48-51) significantly inhibited the growth of K562, Vero, Wish, Calu-1, and Raji tumor cell lines. The inhibitory activities were not due to the polysaccharides, which have been removed in the extracting process. The polarities of these two fractions indicated that they were different from that of cordycepin. Therefore, it is suggested that tumor cell growth inhibitors, other than cordycepin and polysaccharides, are contained in C. sinensis.

Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis.

Cordyceps sinensis (C. sinensis) is one of the well known fungi used in traditional Chinese medicine for treatment asthma and bronchial and lung inflammation. In this study, effects of C. sinensis methanolic extracts on bronchoalveolar lavage fluids (BALF) cells proliferation, inflammatory cytokines production, and genes expression were evaluated. The proliferative response of BALF cells to lipopolysaccharide (LPS) was determined by the tritiated thymidine uptake method. The cell-free supernatants were harvested then tested for interlukin-1beta (IL-1beta), interlukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), and interferon-gamma (IFN-gamma) by the enzyme immunoassay. The results indicated that the CS-19-22 fraction dose dependently suppressed BALF cells proliferation activated by LPS. The CS-19-22 fraction also reduced IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha production in LPS activated BALF cell cultures. Furthermore, the IL-12 and IFN-gamma production in activated BALF cells were enhanced by CS-19-22 treatment. The CS-19-22 fraction did not affect IL-1beta, IL-6, TNF-alpha, and IL-8 mRNAs expression in BALF cells detected by reverse transcription-polymerase chain reaction (RT-PCR). By contrast, the CS-19-22 fraction increased IL-12 and IFN-gamma mRNAs expression and decreased IL-10 mRNA expression in the BALF cells activated with LPS. These results indicated the CS-19-22 fraction suppressed IL-1beta, IL-6, TNF-alpha, and IL-8 cytokines production in BALF cells through other than inhibition of mRNAs expression pathway. These results also demonstrate that the therapeutic activity of C. sinensis in Chinese medicine may be related to modulation of TH1 and TH2 cells functions in bronchial airway.

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells.

Salvianolic acid B (Sal B), a water‐soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo‐E‐deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP‐2 and MMP‐9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo‐E‐deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP‐2 and MMP‐9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP‐2 and barely detectable levels for MMP‐9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS‐induced cell migration through the inactivation of MMP‐2 and MMP‐9 protein synthesis as well as the downregulation of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun NH2‐terminal kinase (JNK). These results demonstrate that Sal B has anti‐migration properties on smooth muscle cells and may explain its anti‐atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 100: 372–384, 2007.

Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aorta.

Inflammation plays an essential role in atherosclerosis and post‐angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water‐soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)‐treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol‐fed apoE deficient mice were investigated. In unstimulated HASMCs, COX‐2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX‐1 mRNA and protein. The activation of COX‐2 protein synthesis in LPS‐stimulated HASMCs was shown to involve the activation of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2), c‐Jun NH2‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX‐2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E2 production and NADPH oxidase activity in LPS‐treated HASMCs. When apoE‐deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 ± 0.009%) compared to the apoE‐deficient group (0.114 ± 0.043%) and there was a significant reduction in COX‐2 protein expression in the thickened intima. These results demonstrate that Sal B has anti‐inflammatory properties and may explain its anti‐atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 98: 618–631, 2006.

Reduced expression of endothelial connexin37 and connexin40 in hyperlipidemic mice: recovery of connexin37 after 7-day simvastatin treatment.

Objective—We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. Methods and Results—Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both P <0.01). After simvastatin treatment, Cx40 remained depressed, but Cx37 recovered to 94% of the level found in non–cholesterol-fed animals (P <0.01). Electron microscopy demonstrated that gap junctions were smaller in animals fed the CED compared with those given simvastatin and with controls fed NC (P <0.01). Endothelial connexins were rare in the atherosclerotic plaques of apoE-deficient mice. Conclusions—Mouse aortic endothelial gap junctions and connexins are downregulated during long-term hyperlipidemia. Short-term treatment with simvastatin leads to recovery of Cx37 expression but not Cx40 expression.

Resveratrol ameliorates metabolic disorders and muscle wasting in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is characterized by dysregulated energy metabolism. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in diabetic animals. However, its overall in vivo effects on energy metabolism and the underlying mechanism require further investigation. In the present study, electrospray ionization-tandem mass spectrometry was employed to characterize the urine and plasma metabolomes of control, streptozotocin-induced DM and RSV-treated DM rats. Using principal component analysis (PCA) and heat map analysis, we discovered significant differences among control and experimental groups. RSV treatment significantly reduced the metabolic abnormalities in DM rats. Compared with the age-matched control rats, the level of carnitine was lower, and the levels of acetylcarnitine and butyrylcarnitine were higher in the urine and plasma of DM rats. RSV treatment ameliorated the deranged carnitine metabolism in DM rats. In addition, RSV treatment attenuated the diabetic ketoacidosis and muscle protein degradation, as evidenced from the attenuation of elevated urinary methyl-histidine and plasma branched-chain amino acids levels in DM rats. The beneficial effects of RSV in DM rats were correlated with activation of hepatic AMP-activated protein kinase and SIRT1 expression, increase of hepatic and muscular mitochondrial biogenesis and inhibition of muscle NF-κB activities. We concluded that RSV possesses multiple beneficial metabolic effects in insulin-deficient DM rats, particularly in improving energy metabolism and reducing protein wasting.