| SECTION IThe construction of eukaryotic expression vector for short hairpin RNA targeting gene of rat a7nAchR and production of recombinant adenovirusObjectiveTo construct eukaryotic expression vector for shRNA targeting gene of rat a7nAchR, and select recombinant adenovirus that displayed the best effect of a7nAchR gene silencing.MethodsWe searched sequence of rat a7nAchR in genebank, and designed and synthesized DNA templates (AY318, AY857, AY444+559, AY318+857) which would encode shRNA of a7nAchR. To construct plasmid vector which would encode one or two shRNA of a7nAchR, DNA templates listed were connected with linearized plasmid vector (pGenesil-1.1, pGenesil-1.4, pGenesil-1.2, pGenesil-1.3). The recombinant plasmid vectors infected DH5a cells and cultured in LB media with kanamycin. In order to identify the recombinant plasmid vectors, plasmids from DH5a were extracted and electrophoresed in 1% agarose gel. To construct recombinant adenovirus plasmid, a LR homologous recombination method in vitro was used to transfer expression box of shRNA from pGenesil to pGSadeno from adenovirus plasmids. Then digested and extracted plasmids from infected DH5a cells in similar style, and electrophoresed in 1% agarose gel to identify the recombinant adenovirus plasmid of pGSadeno. To obtain specific titer of recombinant adenovirus, we extracted DNA from the recombinant adenovirus and infected HEK293 cells increasingly. To identify the effect of ?7nAchR gene silencing, we infected GH3 cells with the recombinant adenovirus and PCR was implied to detect the expression of a7nAchR mRNA in different groups (pGSadeno+AY318 shRNA, pGSadeno +AY857 shRNA, pGSadeno+AY444+559 shRNA, pGSadeno+HK shRNA and Normal group).ResultsThe recombinant plasmids of pGenesil-1.1-AY318 and pGenesil-1.4-AY857 were digested to two DNA fragments of 1000bp and 700bp separately with Sac?. This implied the success of shRNA combined with pGenesil. Similarly, the recombinant plasmids of pGenesil-1.3-AY318+857 and pGenesil-1.2-AY444+559 were digested with Hind? and EcoRI, and showed a 700bp fragment. Moreover, pGSadeno-AY444+559, AY318, AY857 shRNA were digested with Xba? and showed a 2.5Kb fragment. These suggested the success of recombinant adenovirus. The GH3 cell expressed green fluorescent protein under infection of recombinant adenovirus implied the success of infectious recombinant adenovirus construction. The down-regulated of a7nAchR mRNA expression in infected GH3 cells suggested the recombinant adenovirus displayed gene silencing effect targeting a7nAchR, and pGSadeno-AY857 showed the best effect.ConclusionWe successfully constructed the recombinant adenovirus of pGSadeno-?7nAchR-shRNA, and pGSadeno-AY857 shRNA showed the best effect.SECTION ? The moleculer mechanism of berberine on improving insulin resistance of type 2 diabetic rats through a7n AchR related cholinergic anti-inflammatory pathwayObjectiveTo explore the mechanism of berberine (BBR) on improving insulin resistance of type 2 diabetic rats via a7nAchR related cholinergic anti-inflammatory pathway (CAP).MethodsThe rat model of T2DM was established by high fat and high sugar feeding with a low dose injection of STZ. All rats were randomly divided into control, model, BBR, nicotine, metformin groups. The rat model of T2DM with ?7nAchR gene silencing was established with the recombinant adenovirus, and the rats were divided into control, diabetic, Ad-HK, Ad-?7nAchR-siRNA and Ad-?7nAchR-siRNA-BBR groups. After six weeks treatment, all rats were sacrificed. The plasma TCHE activity, Ach and FPG levels were examined. The expression of ?7nAchR protein in the liver tissue was detected by immunochemistry method, and the mean optical density (MOD) was applied to measure the a7nAchR protein level. The protein expression of NF-?B p65, PI-3K, IRS-1, IRS-1 ser307, Glut2 in liver tissue, and Glut4 in adipose tissue were measured by western-blot assay. Meanwhile, the mRNA expression of IRS-1, IR, ?7nAchR in tissues of liver and muscle and Glut4 of muscle tissue were measured by real-time PCR.ResultsCompared with the control group, the FPG, FINS, HOMA-IR levels and TCHE activity were significantly increased in model group, while the weight and plasma Ach level were obviously decreased. The plasma levels of inflammatory cytokines IL-1? and TNF-? were elevated. The protein expressions of NF-?B p65 and a7nAchR, and the ratio of pIRS-1 ser307/IRS-1 in liver tissue were increased. However, the protein expressions of PI-3K p85 and GLUT2 in liver tissue and GLUT4 in adipose tissue were decreased. The mRNA expressions of IR and IRS-1 in liver and muscle tissues and GLUT4 mRNA expression in muscle tissue were down-regulated. The intervention of berberine can reverse the above changes. It manifested as the decrease in FPG, FINS and HOMA-IR levels and TCHE activity, and the increase in Ach level. Moreover, the plasma levels of IL-1? and TNF-? were decreased and the protein expression of NF-?B p65 in liver tissue was down-regulated. The protein expressions of a7nAchR, PI-3K p85 and GLUT2 in liver tissue and GLUT4 protein expression in adipose tissue were elevated. The mRNA expressions of a7nAchR, IR and IRS-1 in liver tissue, and a7nAchR, IRS-1 and GLUT4 mRNA expressions in muscle tissue were up-regulated.The effect of berberine in the rats with a7nAchR gene silencing showed a different result. Compared with the control group, the rats in other groups displayed the increase in FPG, FINS, HOMA-IR levels. Except the Ad-a7nAchR-siRNA-BBR group, the TCHE activity was increased and Ach level the decreased. The plasma levels of IL-1? and TNF-? were elevated. The protein expressions of NF-?B p65 and the ratio of pIRS-1 ser307/IRS-1 in liver tissue were up-regulated. However, the protein expressions of PI-3K and GLUT2 in liver tissue and GLUT4 protein expression in adipose tissue were down-regulated. The mRNA expressions of IR, IRS-1 in liver and muscle tissues and GLUT4 mRNA expression in muscle tissue were down-regulated. The protein expression of a7nAchR in liver tissue was up-regulated in model and Ad-HK groups, but was down-regulated in Ad-a7nAchR-siRNA and Ad-a7nAchR-siRNA-BBR groups. The mRNA expression of a7nAchR in liver and muscle tissues were up-regulated in model and Ad-HK groups. Compared with the model and Ad-HK groups, the mRNA and protein expressions of a7nAchR in liver tissue were down-regulated in groups of Ad-a7nAchR-siRNA and Ad-a7nAchR-siRNA-BBR groups, and the mRNA expression of a7nAchR in muscle tissue was decreased. However, the decline of TCHE activity and the arising of Ach level were only displayed in Ad-?7nAchR-siRNA-BBR group. Compared with the Ad-?7nAchR-siRNA group, BBR just displayed the inhibition of TCHE activity and the increase of Ach level in Ad-?7nAchR-siRNA-BBR group.Conclusion1. We successfully established the rat models of T2DM with insulin resistance, and T2DM with ?7nAchR gene silencing.2. BBR showed good effect on inhibiting TCHE activity and increasing Ach level in the rats of T2DM and T2DM with a7nAchR gene silencing. However, BBR decreased the levels of plasma inflammatory factors and liver NF-?B p65 protein expression only in T2DM rats. These results implied that BBR could attenuate inflammatory response in T2DM rats by activating ?7nAchR related CAP (Ach-?7nAchR-JAK/STAT-NF-?B).3. BBR can improve insulin resistance by regulating the gene expressions of insulin signaling pathway (IR/IRS-1-PI3K/AKT-GLUT) in T2DM rats rather than T2DM with a7nAchR gene silencing rats. These results inferred the importance of ?7nAchR in inducing insulin resistance.4. BBR improved the glucose metabolism disorder and insulin resistance by activating CAP and inhibiting inflammatory response in T2DM rats, but not in rats of T2DM with ?7nAchR gene silencing. Therefore, we concluded that BBR may improve insulin resistance of T2DM by activating ?7nAchR related CAP (Ach-?7nAchR-JAK/STAT-NF-?B). |
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