Canagliflozin attenuates hypertension induced myocardial hypertrophy and fibrosis via RAS and TGF-β1/Smad pathway

Objective:To investigate the effects of cagliazin,a sodium-glucose cotransporter 2 inhibitor(SGLT-2I),on ventricular remodeling in spontaneously hypertensive rats(SHR)through renin angiotensin system(RAS)and transforming growth factor-β1(TGF-β1).Methods:The experiment was divided into 4 groups:normal blood pressure control group,SHR group,cagliet net low-dose group(30mg/kg),cagliet net high-dose group(60mg/kg),once a day for 8 weeks.Normal blood pressure rats(WKY)were used as the control group to measure blood pressure with tail sleeve sphygmomanometer(BP)and blood glucose level was measured with glucose meter Cardiac function was evaluated by echocardiography,cell area of left ventricle was evaluated by histomorphology,real-time quantitative polymerase chain reaction and protein imprinting hybridization were used to detect TGF-β1 Smad4 renin from type I collagen(Col1a)type III collagen(Col3a)matrix metalloproteinase 2(MMP-2)Expression results of angiotensin II1 type receptor 1(AGTR1)and Angiotensin II2 type receptor 2(AGTR2).Results:After 8 weeks of administration,the cardiac weight/body weight ratio(HW/BW)of left ventricular weight/heart weight ratio(LVW/HW)of kaglinet low-dose group and high-dose group was statistically significant compared with that of spontaneous hypertensive rats(P&lt);Compared with SHRs,the expression of Col1a,Col3a,MMP2,TGF-β1,Smad4,Renin AGTR1 was significantly down-regulated and the expression of AGTR2 was up-regulated in cagliet net low-dose and high-dose groups Conclusions:Cagliazin can improve hypertension-induced cardiac remodeling by regulating RAS and TGF-β1/Smad signaling pathways.Conclusion:From the results,canaglifozin was found to ameliorate pressure overload-induced cardiac remodeling by regulating the RAS and TGF-β1/Smad signaling pathway.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

Objective: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus(rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based r AAV2 vector delivery strategy.Methods: The melittin peptide was inserted into the rAAV2 capsid either in the loop Ⅷ of all viral proteins(VPs) or at the N terminus of VP2. Various r AAV2-gfp or-fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.Results: rAAV2 vectors with melittin peptide inserted in the loop Ⅷ of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of r AAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.Conclusion: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.