Berberrubine and its analog, hydroxypropyl-berberrubine, regulate LDLR and PCSK9 expression via the ERK signal pathway to exert cholesterol-lowering effects in human hepatoma HepG2 cells.

Berberine (BBR), the major isoquinoline alkaloid in Chinese herb Rhizoma coptidis, has significant lipid-lowering effect by upregulating hepatic low-density lipoprotein receptor (LDLR) expression. In a previous study, we have indicated that berberrubine (M3), a major metabolite of BBR in vivo, displays the most potential hypolipidemic effects via upregulating LDLR expression in human hepatoma (HepG2) cells compared with BBR and 3 other metabolites. Accordingly, 9 M3 analogs (A1-A9) were modified at the C9 position. We aimed to find a new promising agent by evaluating the cholesterol-lowering effect and clarifying the related molecular mechanism. In the current study, the cellular cholesterol content was assayed with a commercial cholesterol assay kit. Real-time polymerase chain reaction and Western blot assay were used to explore the molecular mechanism of M3 and its analogs on the hypolipidemic effect. Among M3 and its analogs, hydroxypropyl-berberrubine (A8) exhibited the highest potential effects on the upregulation of LDLR expression, which was accompanied by a steady decline of proprotein convertase subtilisin/kexin type 9 (PCSK9) messenger RNA and protein levels. Furthermore, inhibition of extracellular signal-regulated kinase (ERK) activity with PD98059 prevented the upregulation of LDLR and downregulation of PCSK9 induced by A8. The current study revealed that M3 and its structurally modified analog, A8, could regulate hepatic LDLR and PCSK9 expression to exert lipid-lowering effects via the ERK signal pathway, while A8 showed a stronger effect and might be a promising drug candidate against hyperlipidemia.

Phased peristaltic micropumping for continuous sampling and hardcoded droplet generation.

Droplet microfluidics has recently emerged as a new engineering tool for biochemical analysis of small sample volumes. Droplet generation is most commonly achieved by introducing aqueous and oil phases into a T-junction or a flow focusing channel geometry. This method produces droplets that are sensitive to changes in flow conditions and fluid composition. Here, we present an alternative approach using a simple peristaltic micropump to deliver the aqueous and oil phases in antiphase pulses resulting in a robust "chopping"-like method of droplet generation. This method offers controllable droplet dynamics, with droplet volumes solely determined by the pump design, and is insensitive to liquid properties and flow rates. Importantly, sequences of droplets with controlled composition can be hardcoded into the pump, allowing chemical operations such as titrations and dilutions to be easily achieved. The push-pull pump is compact and can continuously collect samples, generating droplets close to the sampling site and with short stabilisation time. We envisage that this robust droplet generation method is highly suited for continuous in situ sampling and chemical measurement, allowing droplet microfluidics to step out of the lab and into field-deployable applications.

Akt/AMPK/mTOR pathway was involved in the autophagy induced by vitamin E succinate in human gastric cancer SGC-7901 cells.

Vitamin E succinate (VES), a derivative of vitamin E, is a promising cancer chemopreventive agent that inhibits tumor promotion by inducing apoptotic cell death. The effects of VES on autophagy, an intricate programmed process which helps cells survive in some stressed situations by degrading some cytoplasmic material, are unclear. When human gastric cancer cells SCG-7901 were exposed to VES, both the level of microtubule-associated protein 1 light chain 3 and the yeast ATG6 homolog Beclin-1 increased, and related autophagy genes were activated, thereby suggesting that autophagy was induced by VES. We also observed that VES-induced autophagy was accompanied by the activation of AMP-activated protein kinases (AMPK). VES-induced autophagy decreased when AMPK was inhibited by using small interfering RNA (siRNA), thereby suggesting that VES-induced autophagy is mediated by AMPK. Moreover, further studies revealed that the decreased activity of mammalian target of rapamycin (mTOR) and its downstream targets P70S6K and 4EBP-1 were involved in VES-activated autophagy associated with AMPK activation. The experiments also showed that the activity of protein kinases B (Akt)-mTOR axis was inhibited by VES. VES-induced AMPK activation could be attenuated by Akt activation. Overall, our studies demonstrated that AMPK was involved in the VES-induced autophagy. Crosstalk exists between AMPK and the Akt/mTOR axis. The results elucidated the mechanism of VES-induced autophagy in human gastric cancer cells.

Effect of vitamin E succinate on the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor in gastric cancer cells and CD4(+) T cells.

Gastric malignancy, which shows poor prognosis, is one of the most frequent causes of cancer-associated deaths. Vitamin E succinate (VES) inhibits cell proliferation and induces apoptosis in a concentration- and time-dependent manner. We explored the effect of VES on the expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor in gastric cancer cells and CD4(+) T cells. On one hand, VES dose-dependently regulated the expression of the TRAIL receptor in gastric cancer cells. Moreover, the activation of the TRAIL receptor, death receptor 4 (DR4), and death receptor 5 (DR5) in gastric cancer cells increased for up to 12 h. On the other hand, the expression of TRAIL protein in human CD4(+) T cells was obviously upregulated in the presence of VES. On the basis of these findings, we combined VES and human CD4(+) T cells to induce apoptosis of MKN28 human gastric cancer cells. The results showed that VES induced higher gastric cancer cell apoptosis when combined with human CD4(+) T cells than when applied alone. We conclude that VES can induce the expression of TRAIL receptor in gastric cancer cells, as well as the expression of TRAIL in CD4(+) T cells. Overall, our results provide a theoretical basis for future immunotherapy studies.

Berberine metabolites could induce low density lipoprotein receptor up-regulation to exert lipid-lowering effects in human hepatoma cells.

Berberine (BBR) is an isoquinoline alkaloid isolated from several Chinese herbal medicines, such as Coptis chinensis, Berberis aristata, and Coptis japonica. It exhibits a lipid-lowering effect by up-regulating the hepatic low density lipoprotein receptor (LDLR) expression. However, the plasma concentration of BBR is very low after oral administration for the reason that BBR is poorly absorbed and rapidly metabolized. Therefore, it is hard to explain the pharmacological effects of BBR in vivo. Here, RT-PCR, Western blotting and Oil Red O staining were used to investigate the effects of four BBR metabolites on LDLR expression and lipid accumulation in human hepatoma Hep G2 cells. Our results suggested that BBR increased the LDLR mRNA and protein levels in a time- and dose-dependent manner. Four metabolites of BBR, jatrorrhizine, columbamine, berberrubine and demethyleneberberine, were found to be able to up-regulate LDLR mRNA and protein expression. Moreover, almost all the metabolites had potent effects on inhibiting cellular lipid accumulation. These results suggest that both BBR and its metabolites exhibit lipid-lowering effects by up-regulating LDLR expression, and BBR and its metabolites might be the in vivo active forms of BBR produced after oral administration. This study provides information to help us understand the mechanisms underlying the hypolipidemic effects of BBR in vivo.

Berberine metabolites exhibit triglyceride-lowering effects via activation of AMP-activated protein kinase in Hep G2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma coptidis (Huanglian in Chinese) is commonly used in Chinese folk medicine to treat diarrhea, diabetes, hypertension, hyperlipidemia and tumors. This herb has increasingly gained attention because of its use as a hypolipidemic herb. Berberine (BBR) is the most important constituent of R. coptidis that contribute to the pharmacological efficacy of the herb. AIM OF THE STUDY: Pharmacokinetic studies have indicated that BBR has poor oral bioavailability. Interestingly, several reports show that absorbed BBR is extensively metabolized in rats and humans. We speculate that the BBR metabolites might be responsible for the pharmacological effects. The aim of this study is to examine BBR metabolites for their triglyceride (TG)-lowering activities and the molecular mechanism to clarify BBR genuine effective forms in vivo. MATERIALS AND METHODS: Four BBR metabolites were examined their TG-lowering effects with a commercial triglyceride assay kit. Real-time PCR and Western blotting were used to confirm genes and proteins of interest, respectively. RESULTS: Among those BBR metabolites, M2 exhibited the more potential effects on TG-lowering and AMP-activated protein kinase (AMPK) activation in Hep G2 cells as compared with BBR. Moreover, BBR and M2 inhibited gene expressions of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), glycerol-3-phosphate acyltransferase (GPAT) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), but motivated gene expression of medium chain acyl-CoA dehydrogenase (mCAD) significantly. CONCLUSIONS: The results suggested that the TG-lowering effects of BBR and M2 might be partially mediated by the up-regulation of lipolysis gene expressions and down-regulation of lipogenesis gene expressions through activation of the AMPK signaling pathway. BBR and its metabolites might be in vivo active forms of oral doses of BBR, and M2 might be a promising drug candidate against hyperlipidemia.