Role of PCSK9 in sulforaphane attenuating palmitic acid-induced autophagic flux in hepatic cells

Objective To determine the effect and underlying mechanism of sulforaphane (SFN) on hepatocyte injury and autophagy. Methods HHL5 cells were treated with 200 μmol/L palmitic acid (PA) for 24 h to establish a hepatocyte injury model. Then the model was treated with 5 μmol/L SFN and co-cultured with 200 μmol/L PA for 24 h. So there were 3 groups of cells, that is, control group, PA group, and PA+SFN group. Cell viability, malonaldehyde (MDA) content, and production of reactive oxygen species (ROS) were measured with CCk-8 assay, MDA reagent kit, and CellROXTM Deep Red Reagent, respectively. qPCR was used to detect the transcription levels of IL-1β and TNF-α, and Western blotting was employed to measure the protein expression of SQSTM1 and LC3II. The total RNA of the cell was extracted by TRIzol reagent to sequence the whole gene transcriptome, and the RNA sequence was analyzed to figure out differentially expressed genes. And the results were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, HHL5 cells were pretreated with PCSK9 siRNA for 24 h, then the mRNA level of PCSK9 was measured by qPCR, and the expression of SQSTM1 and LC3 II were measured by Western blotting. Results Compared with the PA group, the cell survival rate was increased, and the levels of MDA and ROS were decreased significantly in the PA+SFN group (P < 0.05). The transcriptional levels of IL-1β and TNF-α were reduced obviously in the PA+SFN group (P < 0.05). The expression of SQSTM1 was decreased and that of LC3II was increased in the PA+SFN group. KEGG enrichment analysis suggested that differential expressed genes were enriched on the autophagy pathway. PCSK9 was selected as our candidate gene. Compared with the control group, the transcriptional level of PCSK9 was increased in the PA group, while was decreased significantly in the PA+SFN group (P < 0.05). The level was elevated in the PA group but decreased after SFN intervention. After PCSK9 knockdown, the ROS level in the PA group was decreased compared with that in the PA group without siPCSK9 treatment, which was consistent with the trend of SFN-induced ROS reduction. After PCSK9 knockdown, the expression level of LC3II was increased, which was consistent with the trend of SFN-induced autophagy flux recovery. Conclusion SFN can attenuate hepatocyte injury induced by PA, which may be related to the inhibition of PCSK9 at transcriptional level, thus regulating autophagic flux

Long and repeat-rich intronic sequences favor circular rna formation under conditions of reduced spliceosome activity

Circular RNAs (circRNAs), an important class of regulatory RNAs, have been shown to be the most prevalent in the brain compared with other tissues. However the processes governing their biogenesis in neurons are still elusive. Moreover, little is known about whether and how different biogenesis factors work in synchrony to generate neuronal circRNAs. To address this question, we pharmacologically inhibited the spliceosome and profiled rat neuronal circRNAs using RNA sequencing. We identified over 100 circRNAs that were up-regulated and a few circRNAs that were down-regulated upon spliceosome inhibition. Bioinformatic analysis revealed that up-regulated circRNAs possess significantly longer flanking introns compared with the un-changed circRNA population. Moreover, the flanking introns of up-regulated circRNAs harbor a higher number of distinct repeat sequences and more reverse complementary motifs compared with the unchanged circRNAs. Taken together, our data demonstrate that the biogenesis of circRNAs containing distinct intronic features becomes favored under conditions of limited spliceosome activity

Serum lipoprotein(a) positively correlates with coronary artery calcification in low-risk chinese han patients: a study from a single center.

BACKGROUND: Elevated plasma levels of lipoprotein(a) (Lp(a)) and a higher degree of coronary artery calcification (CAC) are both considered to be risk factors for atherosclerosis. However, previous studies have demonstrated that the relationship between Lp(a) levels and the degree of CAC indicates significant heterogeneity that may be due to varying ethnicities. The purpose of this study was to examine the predictive power of Lp(a) for CAC as measured by multidetector computed tomography (MDCT) in the Han ethnic group of China. METHODS: A total of 1082 subjects were recruited in this study. The patients were divided into four groups: patients without hypertension or diabetes were group 1, patients with hypertension were group 2, patients with diabetes were group 3 and patients with both hypertension and diabetes were group 4. CAC score (CACs), lipid profiles (Lp(a), LDL, HDL, TG, TC), HbA1C, glucose, personal health history and body morphology were measured in all participants. The predictive power of Lp(a) for calcified atherosclerotic plaque was determined by correlations and ordinal logistic regression. RESULTS: There was no significant difference in the CACs between group 2 and group 3 (z = 1.790, p = 0.736), and there were significant differences among the other groups. However, there was no significant difference in the total Lp(a) among the 4 groups (χ(2) = 0.649, p = 0.885). Only In group 1, Lp(a) was a statistically significant predictor of the presence of calcified coronary plaque using ordinal logistic regression. CONCLUSIONS: Levels of Lp(a) positively correlate with CACs among Chinese Han people who are without diabetes and hypertension, suggesting that Lp(a) may be an important risk factor for the presence of calcified atheromas

3,6-Dihydroxyflavone regulates microRNA-34a through DNA methylation

Abstract Background Breast cancer is the common cancer in China. In previous study, we determined that 3,6-dihydroxyflavone (3,6-DHF) increases miR-34a significantly in breast carcinogenesis, but the mechanism remains unclear. Methods We used qRT-PCR to analyze miR-34a and ten-eleven translocation (TET)1, TET2, TET3 levels in breast cancer cells. With a cellular breast carcinogenesis model and an experimental model of carcinogenesis in rats, TET1 levels were evaluated by western blot analysis and immunofluorescence. TET1 and 5hmC (5-hydroxymethylcytosine) levels were evaluated by immunofluorescence in nude mouse xenografts of MDA-MB-231 cells. Chromatin immunoprecipitation(ChIP) assayed for TET1 on the TET1 promoter, and dot blot analysis of DNA 5hmC was performed in MDA-MB-231 cells. We evaluated the mechanism of 3,6-DHF on the expression of tumor suppressor miR-34a by transfecting them with DNA methyltransferase (DNMT)1 plasmid and TET1 siRNA in breast cancer cells. Methylation-specific PCR detected methylation of the miR-34a promoter. Results First, we found that 3,6-DHF promotes the expression of TET1 during carcinogen-induced breast carcinogenesis in MCF10A cells and in rats. 3,6-DHF also increased TET1 and 5hmC levels in MDA-MB-231 cells. Further study indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibited the 3,6-DHF reactivation effect on expression of miR-34a in breast cancer cells. Methylation-specific PCR assays indicated that TET1 siRNA and pcDNA3/Myc-DNMT1 inhibit the effect of 3,6-DHF on the demethylation of the miR-34a promoter. Conclusions Our study showed that 3,6-DHF effectively increases TET1 expression by inhibiting DNMT1 and DNA hypermethylation, and consequently up-regulates miR-34a in breast carcinogenesis

Towards Online Estimation of Human Joint Muscular Torque with a Lower Limb Exoskeleton Robot

Exoskeleton robots demonstrate promise in their application in assisting or enhancing human physical capacity. Joint muscular torques (JMT) reflect human effort, which can be applied on an exoskeleton robot to realize an active power-assist function. The estimation of human JMT with a wearable exoskeleton is challenging. This paper proposed a novel human lower limb JMT estimation method based on the inverse dynamics of the human body. The method has two main parts: the inverse dynamic approach (IDA) and the sensing system. We solve the inverse dynamics of each human leg separately to shorten the serial chain and reduce computational complexity, and divide the JMT into the mass-induced one and the foot-contact-force (FCF)-induced one to avoid switching the dynamic equation due to different contact states of the feet. An exoskeleton embedded sensing system is designed to obtain the user&rsquo;s motion data and FCF required by the IDA by mapping motion information from the exoskeleton to the human body. Compared with the popular electromyography (EMG) and wearable sensor based solutions, electrodes, sensors, and complex wiring on the human body are eliminated to improve wearing convenience. A comparison experiment shows that this method produces close output to a motion analysis system with different subjects in different motion

Effect of fruit juice on cholesterol and blood pressure in adults: a meta-analysis of 19 randomized controlled trials.

BACKGROUND: The effect of fruit juice on serum cholesterol and blood pressure in humans has generated inconsistent results. We aimed to quantitatively evaluate the effect of fruit juice on serum cholesterol and blood pressure in adults. METHODS: We performed a strategic literature search of PubMed, EMBASE, and the Cochrane Library (updated to October, 2012) for randomized controlled trials that evaluated the effects of fruit juice on serum cholesterol and blood pressure. Study quality was assessed by using the Jadad scale. Weighted mean differences were calculated for net changes in cholesterol and blood pressure by using fixed-effects model. Prespecified subgroup and sensitivity analyses were conducted to explore the potential heterogeneity. RESULTS: Nineteen trials comprising a total of 618 subjects were included in this meta-analysis. Fruit juice consumption borderlinely reduced the diastolic blood pressure (DBP) by 2.07 mm Hg (95% CI: -3.75, -0.39 mm Hg; p = 0.02), but did not show significant effects on total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) concentrations or systolic blood pressure (SBP) values. A significant reduction of TC concentration was observed in low-median intake of total polyphenols group. Subgroup analyses for HDL-C and LDL-C concentrations did not show statistically significant results. No significant heterogeneity was detected for all the measures. CONCLUSION: This meta-analysis suggested that fruit juice had a borderline significant effect on reducing DBP, but had no effect on TC, HDL-C, LDL-C concentrations or SBP

Estrogen receptor and PI3K/Akt signaling pathway involvement in S-(-)equol-induced activation of Nrf2/ARE in endothelial cells.

S-(-)equol, a natural product of the isoflavone daidzein, has been reported to offer cytoprotective effects with respect to the cardiovascular system, but how this occurs is unclear. Interestingly, S-(-)equol is produced by the human gut, suggesting a role in physiological processes. We report that treatment of human umbilical vein endothelial cells and EA.hy926 cells with S-(-)equol induces ARE-luciferase reporter gene activity that is dose and time dependent. S-(-)equol (10-250 nM) increases nuclear factor-erythroid 2-related factor 2 (Nrf2) as well as gene products of Nrf2 target genes heme oxygenase-1 (HO-1) and NAD(P)H (nicotinamide-adenine-dinucleotide-phosphate) quinone oxidoreductase 1 (NQO1). Endothelial cells transfected with an HA-Nrf2 expression plasmid had elevated HA-Nrf2, HO-1, and NQO1 in response to S-(-)equol exposure. S-(-)equol treatment affected Nrf2 mRNA only slightly but significantly increased HO-1 and NQO1 mRNA. The pretreatment of cells with specific ER inhibitors or PI3K/Akt (ICI182,780 and LY294002) increased Nrf2, HO-1, and NQO1 protein, impaired nuclear translocation of HA-Nrf2, and decreased ARE-luciferase activity. Identical experiments were conducted with daidzein, which had effects similar to S-(-)equol. In addition, DPN treatment (an ERβ agonist) induced the ARE-luciferase reporter gene, promoting Nrf2 nuclear translocation. Cell pretreatment with an ERβ antagonist (PHTPP) impaired S-(-)equol-induced Nrf2 activation. Pre-incubation of cells followed by co-treatment with S-(-)equol significantly improved cell survival in response to H2O2 or tBHP and reduced apoptotic and TUNEL-positively-stained cells. Notably, the ability of S-(-)equol to protect against H2O2-induced cell apoptosis was attenuated in cells transfected with an siRNA against Nrf2. Thus, beneficial effects of S-(-)equol with respect to cytoprotective antioxidant gene activation may represent a novel strategy to prevent and treat cardiovascular diseases