ABSTRACT
The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.
Subject(s)
Humans , Adenoviridae , Genetics , Glucose , Metabolism , Glycogen Synthase Kinase 3 beta , Metabolism , Hep G2 Cells , MicroRNAs , Genetics , Metabolism , Signal Transduction , Genetics , TransfectionABSTRACT
We constructed recombinant adenoviruses expressing miR-29b2c (Ad-miR29b2c), and analyzed their effects on the proliferation and migration of HGC-27 and MGC-803 cells. miR-29b2c gene was amplified by PCR from genomic DNA and cloned into the pAdTrack-CMV vector to create the shuttle plasmid pAdT-29b2c. The recombinant plasmid was verified by restriction enzyme digestion and sequencing. The linearized shuttle vector was mixed with an adenoviral backbone plasmid (pAdEasy-1), followed by cotransformation into competent BJ5183 cells to generate the recombinant plasmid pAd-miR-29b2c. Finally, recombinant adenoviral vectors were generated by transfecting the recombinant plasmid into 293A packaging cell line. HGC-27 and MGC-803 cells were infected with the recombinant adenoviruses expressing pAd-miR-29b2c, then MTT and wound-healing assay were used to analyze the effects of pAd-miR-29b2c on the proliferation and migration of HGC-27 and MGC-803 cells. The miR-29b and miR-29c levels were significantly increased in HGC-27 cells after infected with pAd-miR-29b2c. MTT and wound-healing analysis also revealed a significant decrease in proliferation and migration of HGC-27 and MGC-803 cells compared to the control Ad-GFP-infected cells. Furthermore, western blotting results demonstrated that the protein expression level of δ-catenin was reduced in pAd-miR-29b2c transfected HGC-27 and MGC-803 cells. Taken together, the recombinant adenoviral vector was generated, and it can significantly inhibit the proliferation and migration of HGC-27 and MGC-803 cells.
ABSTRACT
The isoflavone calycosin-7-O-β-d-glucopyranoside (CG) is a principal constituent of Astragalus membranaceus (AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin (Ocal) mRNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2 (BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family (WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.
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The effects of magnolol (Mag) on hyperglycemia and hyperlipemia, hepatic oxidative stress and cytochrome P4502E1 (CYP2E1) activity of diabetic rats induced by high-fat diet (HFD) and streptozotocin (STZ) were studied. After oral administration of Mag (25, 50 and 100 mg x kg(-1) x d(-1)) for continuous 10 weeks, the blood glucose and lipids (TC, TG and LDL-C) levels, as well as the hepatic CYP2E1 activity and MDA content of diabetic rats, decreased significantly (P < 0.05 or P < 0.01), whereas the oral glucose tolerance and hepatic antioxidant enzymatic activities (CAT and GSH-Px) of diabetic rats, increased significantly (P < 0.05 or P < 0.01). The results indicated that Mag was effective against the hepatic oxidative damage, hyperglycemia and hyperlipemia of diabetic rats induced by HFD and STZ, and the inhibition of Mag on hepatic CYP2E1 activity could be an important mechanism of Mag against hepatic insulin resistance and oxidative damage.
ABSTRACT
This study sought to determine the impact of dental fluorosis severity on demineralization and remineralization of human fluorosed teeth in vitro. Surface enamel microhardness was measured on the enamel blocks before and after demineralization and after remineralization. The results showed that after demineralization, the sequence of % Surface microhardness demineralization (% SMHD) was TFI4 (18.92 +/- 1.31) < TFI3 (20.50 +/- 1.32) < TFI2 (25.08 +/- 1.69) < TFI1 (27.77 +/- 1.79) < TFI0 (30.70 +/- 1.35) (P < 0.05), and there was no statistically significant differences between TFI1 (27.77 +/- 1.79) and the normal group TFI0 (30.70 +/- 1.35). After remineralization, the sequence of % Surface microhardness remineralization (% SMHR) was TFI1 (55.17 +/- 1.23) > TFI0 (53.97 +/- 3.05) > TFI2 (49.17 +/- 1.81) > TFI3 (44.85 +/- 1.89) > TFI4 (36.51 +/- 2.95) (P < 0.05). Moderately fluorosed enamel showed a significatnt resistance to caries, but mildly fluorosed enamel could get better remineralization. These facts and figures deserve clinicians' attention.
Subject(s)
Humans , Dental Enamel , Chemistry , Pathology , Fluorosis, Dental , Metabolism , In Vitro Techniques , Tooth Demineralization , Tooth RemineralizationABSTRACT
Objective:To investigate the effects of Maijun'an Tablets on expression of endothelin-1(ET-1) and nitricoxide synthase(NOS) in tissues and plasma of spontaneous hypertensive rats (SHR). Methods:12 spontaneous hypertensive rats (SHR) were divided randomly into normal control group and drug administration group. Rats in drug administration group were orally given with Maijun'an Tablets (hydrochlorothiazide 10 mg?kg-1.d-1,puerarin 36 mg?kg-1.d-1 and total alkaloid of rhynchophylla 122 mg?kg-1.d-1) once a day for 4 weeks,and rats in control group were orally dosed with equal amount of physiological saline. All animals were sacrificed after 4 weeks. The mRNA and protein contents of ET-1 and NOS in plasma,liver,kidnay and blood vessel were determined. Results:Compared with the control group,Maijun'an Tablets can effectively reduce blood pressure of SHRs,regulate down the content of ET-1 and regulate up the content of NOS at mRNA and protein levels. Conclusion:The up-regulation of NOS and down-regulation of ET-1 at mRNA and protein levels were the major antihypertensive machnisam of Maijun'an Tablets for SHRs.
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Objective:To study the effects of ginsenoside Rb1 on cell proliferation,differentiation and OPG/RANKL mRNA expression of rat osteoblast cultured in vitro.Methods:Rat osteoblast cultured in vitro was treated by a series of different concentration of ginsenoside Rb1 for different time respectively.The proliferation of osteoblast was detected by MTT method.The differentiation of osteoblast was detected by the activity of ALP.mRNA expression of osteoprotegerin (OPG) and RANKL(receptor activator nuclear factor-kappa B ligand,RANK) were semi-quantified by RT-PCR,and the ratio of OPG/RANKL mRNA was used to analyze the differentiation of ginsenoside Rb1 to osteoclast.Results:Ginsenoside Rb1 at a concentration of 1.12?10-9mol/L to 1.12?10-5mol/L can promote the proliferation of osteoblast after 72h of treatment (P
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Objective:To study the effects of diosgenin on cell proliferation, differentiation and OPG/RANKL mRNA expression of rat osteoblasts cultured in vitro for analyzing the prevention and treatment mechanism of diosgenin. Methods: Rat osteoblast cultured in vitro was treated by a series of different concentrations of diosgenin for different time respectively. The proliferation of osteoblast was detected by MTT method. The differentiation of osteoblast was detected by the activity of ALP mRNA expression of osteoprotegerin(OPG) and RANKL(receptor activator nuclear factor-kappa B ligand,RANK) were semi-quantified by RT-PCR, and the ratio of OPG/RANKL mRNA was used to evaluated the effect of diosgenin on osteoclast signal transduction pathway of OPG/RANKL/RANK system. Results: Diosgen at the concentration of 3.64?10-8 and 3.64?10-7mol/L can promote the proliferation, enhance the activity of ALP and raise the ratio of OPG/RANKL mRNA of rat osteoblast cultured in vitro(P