ABSTRACT
Paris saponin VII (PSVII), a bioactive constituent extracted from Trillium tschonoskii Maxim., is cytotoxic to several cancer types. This study was designed to explore whether PSVII prevents non-small-cell lung cancer (NSCLC) proliferation and to investigate its molecular target. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. In cultured human NSCLC cell lines, PSVII induces autophagy by activating AMPK and inhibiting mTOR signaling. Furthermore, PSVII-induced autophagy activation was reversed by the AMPK inhibitor compound C. Computational docking analysis showed that PSVII directly interacted with the allosteric drug and metabolite site of AMPK to stabilize its activation. Microscale thermophoresis assay and drug affinity responsive target stability assay further confirmed the high affinity between PSVII and AMPK. In summary, PSVII acts as a direct AMPK activator to induce cell autophagy, which inhibits the growth of NSCLC cells. In the future, PSVII therapy should be applied to treat patients with NSCLC.
ABSTRACT
Objective: To evaluate the preventive effect of dihydrocumin (DHC) on an in vitro model of nonalcoholic fatty liver disease (NAFLD) and investigate the signal transduction pathways underlying DHC treatment. Methods: Oleic acid (OA, 0.5 mmol/L) induced hepatic steatosis was established in HepG2 cells as in vitro model of NAFLD. After cells were co-treated by OA and DHC (0, 5, 10, and 20 μmol/L) for 24 h, the cellular contents of triglyceride (TG), reactive oxygen species (ROS) and NO were determined by cellular biochemical assays. Signaling pathways involved in glucolipid metabolism and oxidative stress including SREBP-1C, PNPLA3, PPARα, PI3K, the phosphorylation of AKT (pAKT), AKT, and Nrf2 on the mRNA and protein levels were determined by RT-qPCR and Western blotting. The glucose uptake was determined by fluorospectrophotometry using 2-NBDG as a fluorescence probe. Results: Compared with the control group, the content of TG, ROS and NO was significantly increased, the uptake of 2-NBDG was decreased. and the expression of SREBP-1C and PNPLA3on the mRNA and protein levels were up-regulated and the protein expression levels for PPARα, PI3K and Nrf2, as well as the ratio of pAKT to AKT were down-regulated in OA-induced cells. Compared with OA treated group, DHC decreased the levels of cellular TG and NO, as well as the mRNA and protein expression levels of SREBP-1C and PNPLA3, and increased the uptake of 2-NBDG, while at the same time increasing the cellular glucose uptake and the protein expression levels of PPARα, PI3K, pAKT/AKT, and Nrf2 in OA-induced HepG2 cells. Conclusion: DHC protected OA-induced hepatic steatosis by inhibiting lipid accumulation and oxidative/nitrative stress and increasing hepatic insulin sensitivity. Furthermore, the effect of DHC is likely associated with its role in the regulation of SREBP-1C, PNPLA3, PPARα, Nrf2, PI3K and AKT signaling pathways. DHC may have the effects on relieving the resistance of insulin and promoting the uptake of glucose in the hepatocytes by upregulating PI3K expression and pAKT/AKT level. Through increasing the expression of Nrf2 and reducing the content of NO in the cells, DHC could alleviate the inflammatory reaction and oxidative damage of liver cells.
ABSTRACT
Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by a grinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary (1.0 mm,ouside diametre)was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter -15 μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a "suitable"opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.
ABSTRACT
Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by a grinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary (1.0 mm,ouside diametre)was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter -15 μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a "suitable"opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.