| Chronic low-grade inflammation state is one of the important pathogenesis in obesity and type2diabetes, That the secretion and expression of much inflammatory adipocytokines and inflammatory markers was significantly increased in serumb of obese type2diabetic patients, for example, interleukin6, tumor necrosis factor alph, monocyte chemoattractant protein1, plasminogen activator inhibitor1and resisitin etc. These inflammatory cytokines caused insulin resistance. They plays an important role in regulate glucose and lipid metabolism, vascular biology and treatment of obesity and related diseases such as type2diabetes, obesity, hypertension, atherosclerosis, cardiovascular disease. Anti-inflammatory factor of adiponectin improve insulin sensitivity through activate AMPK pathway. Therefore, against the inflammatory state or enhance the anti-inflammatory factor of adipose tissue helps to improve treatment of insulin resistance and type2diabetes.Rhizoma Coptidisand Ginseng are commonly used in treat of type2diabetes in clinica. Compatible use of both, can reduce the levels of blood sugar and insulin in diabetic rats, while reducing the free fatty acids in serum and protecting the morphology and function of islet β cells. Shows the significant effects in improve insulin resistance. Berberine and ginsenoside are the main active ingredients of Rhizoma Coptidis and Ginseng. They belonged to Qi replenishing heRbs and heat evacuating heRbs respectively, However, both compounds have the same and significant regulatory benefits on glucose and lipid metabolism.Objectives:1) To Investigate the effects of combination of berberine and ginsenosides Rbi on the mRNA expression of TNF-a, IL-6, MCP-1, Resisitin and adiponectin in3T3-L1adipocytes.2) To explore the effects of combination of berberine and ginsenosides Rbi on the protein expression of IKK-0, JNK and the phosphorylation levels of IKK-β(ser181), JNK in3T3-L1adipocytes.3) To observe the effects of combination of berberine and ginsenosides Rb1on the transposition of NF-KB(p65)into nucleus in3T3-L1adipocytes. Methods:Part One Effect of combination of berberine and ginsenosides Rb1on the expression of inflammatory adipocytokines in3T3-L1adipocytesCytokine expression (TNF-α,resistin, IL-6, MCP-1,adiponeetin) in fully differented3T3-L1adipocytes were measured by quantitative real-time PCR with or without TNF-α for24hour. respectively, give medicine to nondiabetic mice and mice homozygous for the diabetes spontaneous mutation, After14days, fasting10hours, measurement of fasting plasma glucose and serum insulin, with pentobarbital sodium anesthesia (30mg/kg). Collect tissue and serum samples.By the methods of enzyme-linked immunosorbent (ELISA) to detect the contents of TNF alpha, adiponectin.Part Tow Effect of combination of berberine and ginsenosides Rb1on inflammatory signaling pathways in3T3-L1adipocytesThe levels of IκB kinaseβ(IKK-β), IKK-β(ser181), JNK and the phosphorylation of JNK protein were detected in fully differented3T3-L1adipocytes by Western blotting with or without TNF-α for24hour.Part Three Effect of combination of berberine and ginsenosides Rb1on the translocation of NF-κB P65into nucleus in3T3-L1adipocytesThe distribution of NF-κB p65protein inside the adipocytes was observed in fully differented3T3-L1adipocytes through confocal laser scanning micrscopy (CLSM) with or with TNF-α for24hour.Results:Part One Effect of combination of berberine and ginsenosides Rb1on the expression of inflammatory adipocytokines in3T3-L1adipocytes1) Effect of combination of berberine and ginsenosides Rb1on the expression of inflammatory adipocytokines in3T3-L1adipocytes in normal.Compare with control group the expression of TNF-α、IL-6、Resistin、MCP-1are reduced on the mRNA with the berberine, but adiponectin doesn’t have obvious change. Adiponectin is increased in group of ginsenosides Rb1, but the expression of TNF-α、IL-6、Resistin、MCP-1are not effected, while rosiglitazone used as positive control drug. When combined with ginsenoside Rb1, the inhibitory effects to3T3-L1adipocytes of berberine are weakened.2) Effect of combination of berberine and ginsenosides Rb1on the expression of inflammatory adipocytokines in3T3-L1adipocytes with TNF-α the expression of TNF-a, IL-6. Resistin, MCP-1are reduced on the mRNA with the berberine and adiponectin is increased with TNF-a. the expression of TNF-a and MCP-1are inhibited in group of ginsenosides Rb1. they are not effected but the expression of TNF-a and MCP-1are inhibited in group of combination.3) Effect of combination of berberine and ginsenosides Rbl on the expression of insulin and factor in mice。After fasting for10hours, measurement of fasting plasma glucose and serum insulin, The Ber and Rb1+Ber group had significant difference (P<0.01).Compared with normal rats, the db/db mice in14days after delivery, The food intake and body weight of the group of Ber and Ber+Rb1were reduced in mice, the proportion of epididymis and the liver also decreased obviously. Significantly reduced fasting blood glucose and serum insulin. As well as can significantly restored the mRNA expression of adiponectin (P<0.01); after treatment of Rb1, the mRNA expression of the TNF-a has reduced, as well as recovered the mRNA expression of Adiponectin partially (P <0.05).Part Tow Effect of combination of berberine and ginsenosides Rb1on inflammatory signaling pathways in3T3-L1adipocytesThe expression of total IKK-β, JNK were not changed by berberine and ginsenoside Rbl treatment, but berberine can suppress the degrees of phosphorylation of IKK-β(ser181) and JNK inTNF-a indued insulin rsistanee state, but ginsenoside Rbl and combine had no effect on signaling protein, while rosiglitazone used as positive control drug.Part Three Effect of combination of berberine and ginsenosides Rbi on the translocation of NF-κB P65into nucleus in3T3-L1adipocytesAfter the intervention of TNF-a for24hours, the nuclear translocation of NF-KB(p65) protein were increased, but it can be reversed by berberine treatment, however, we did not observed the effect of ginsenoside Rbl and combine on the nuclear translocation of NF-KB(p65) protein, while rosiglitazone used as positive control drug.Conclusion:1) in vitro, the expression of TNF-α, IL-6, Resistin, MCP-1are reduced with the berberine, The food intake and body weight of the group of Ber and Ber+Rb1were reduced in mice, the proportion of epididymis and the liver also decreased obviously, Significantly reduced fasting blood glucose and serum insulin, at the time The expression of total IKK-(3, JNK are inhibited and the nuclear translocation of NF-κB(p65) protein is increased. Berberine may regulate the expression and secretion of inflammatory cytokines through targeting IKK/NF-κB signaling.2) The expression of TNF-a, MCP-1, adiponectin were changed, but have no effect on IKK-β, JNK and NF-κB(p65) in ginsenosides Rb1. These results help us to conclude that berberine and ginsenoside Rbl may regulate cytokine expression and secretion through different molecular mechanism.3) There no apparent effect of combination of berberine and ginsenoside Rb1on expression of cytokine and secretion of adipocytes and protein. But diabetes is improved by ginsenosides Rb1and berberine, There are obvious differences in the molecular mechanism, and further illustrates the role of both the inflammatory state are not the same. |
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