Construction of lentiviral vector for late endosomal/lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 interference and its role on inflammatory factor secretion of macrophages / 中华传染病杂志

Objective@#To construct lentiviral vector of late endosomal/lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 (lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.@*Methods@#Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage. There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control. The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α secreted by the cells were detected by RT-qPCR. T test was used for comparison between groups.@*Results@#The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully. The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000±0.000, 0.596±0.125 and 0.120±0.080, respectively. The expression of lamtor2 in the sh2 group was significantly lower than that in the sh1 group (t=3.399, P=0.015), and they were both significantly lower than the control group (t=3.333 and 9.734, respectively, both P< 0.05). After infection with Klebsiella pneumoniae, expression levels of IL-1β (t=15.20), IL-6 (t=43.30) and TNF-α (t=12.67) were significantly higher than those in the control group (all P<0.01).@*Conclusion@#The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism, which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

Construction of lentiviral vector for late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 interference and its role on inflammatory factor secretion of macrophages / 中华传染病杂志

Objective To construct lentiviral vector of late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 ( lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.Methods Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage.There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control.The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction ( RT-qPCR ) and Western blot.The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-αsecreted by the cells were detected by RT-qPCR.T test was used for comparison between groups.Results The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully.The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000 ±0.000, 0.596 ±0.125 and 0.120 ±0.080, respectively.The expression of lamtor2 in the sh2 group was significantly lower than that in the sh 1 group (t＝3.399, P＝0.015), and they were both significantly lower than the control group ( t ＝3.333 and 9.734, respectively, both P ＜0.05).After infection with Klebsiella pneumoniae, expression levels of IL-1β( t ＝15.20), IL-6 (t＝43.30) and TNF-α(t＝12.67) were significantly higher than those in the control group (all P＜0.01).Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism , which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

Triterpenoids from the roots of Rubus parvifolius / 中国天然药物

Two new oleanane-type triterpenoids, parvifolactone A (1) and rubuside P (2), together with 11 known triterpenoids, fupenzic acid (3), 18,19-seco,2α,3α-dihydroxyl-19-oxo-urs-11,13(18)-dien-28-oic acid (4), euscaphic acid (5), maslinic acid (6), 1β- hydroxyeuscaphic acid (7), 2α,3α,19α,23-tetrahydroxyolean-12-en-28-oic acid (8), 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (9), glucosyl pinfaensate (10), rubuside J (11), 2α,3α,19α,23-tetrahydroxyurs-12-en-24,28-dioic acid (12), and 2α,3β,19α- trihydroxyurs-12-en-23,28-dioic acid (13), were isolated from the roots of Rubus parvifolius.

Flavonoids from Urena lobata / 中草药

Objective: To study the chemical constituents of Urena lobata. Methods: Compounds were isolated and purified using various column chromatographies such as silica gel, Sephadex LH-20, and prep HPLC. Their structures were identified by physicochemical properties and various spectroscopic experiments, including HRESIMS, 1H-NMR, 13C-NMR, HSQC, and HMBC. Results: Fourteen flavonoids were obtained from the ethyl acetate extract of U. lobata, including apigenin-6-C-(6″-O-trans- caffeoyl)-β-D-glucopyranoside (1), tiliroside (2), kaempferol-3-O-(6″-O-cis-p-coumaroyl)-β-D-glucopyranoside (3), kaempferol-3- O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside (4), rutin (5), astragalin (6), baicalin (7), myricetrin (8), isoquercitrin (9), baicalein (10), luteolin (11), apigenin (12), kaempferol (13), and quercetin (14). Conclusion: Compound 1 is a new compound named urenalobside A, compounds 3, 4, 7, and 8 are firstly obtained from the family Malvaceae, and compound 10 is firstly isolated from genus Urena Linn.