Identification of biomarkers associated with immune-propionate metabolism in nonalcoholic fatty liver disease.

The mechanisms of the effect of propionate metabolism and immunity on nonalcoholic fatty liver disease (NAFLD) have not been adequately studied. Firstly, differentially expressed-propionate metabolism-related genes (DE-PMRGs) were selected by overlapping PMRGs and differentially expressed genes (DEGs) between the simple steatosis (SS) and health control (HC) groups. Then, common genes were selected by overlapping DE-PMRGs and key module genes obtained from weighted gene co-expression network analysis (WGCNA). Subsequently, the biomarkers were screened out by machine learning algorithms. The expression of the biomarkers was validated by quantitative Real-time PCR. In total, 5 biomarkers (JUN, LDLR, CXCR4, NNMT, and ANXA1) were acquired. The nomogram constructed based on 5 biomarkers had good predictive power for the risk of SS. Next, 5 biomarkers, 11 miRNAs, and 149 lncRNAs were encompassed in the ceRNA regulatory network. The expression of biomarkers was significantly higher in the HC group than in the SS group, which was consistent with the results in the GSE89632 and GSE126848 datasets. In this study, 5 immune and propionate metabolism-related biomarkers (JUN, LDLR, CXCR4, NNMT, and ANXA1) were screened out to provide a basis for exploring the prediction of diagnosis of NAFLD.

Long-term low-dose alcohol intake promotes white adipose tissue browning and reduces obesity in mice.

There are numerous pieces of evidence indicating that moderate alcohol intake has a protective effect on metabolic diseases. Our previous studies revealed that long-term low-dose alcohol intake resists high-fat diet (HFD) induced obesity. A process in which white adipose tissue can be stimulated and turned into heat-producing brown adipose tissue named white adipose browning is associated with energy expenditure and weight loss. In this study we aimed to investigate whether alcohol causes the browning of white adipose tissue and whether the browning of white adipose tissue is involved in the resistance to the occurrence of obesity caused by long-term low-dose alcohol intake. After eight months of alcohol feeding, the body weight of mice had no significant change, but the fat content and lipid deposition in the liver were reduced. Morphological observations revealed that the browning of white adipose tissue occurred. The white adipose tissue browning marker UCP1 gene and protein expression levels were increased and the expression of the PGC1-α/PPAR-α pathway protein and the P38 MAPK/CREB pathway protein was also elevated in the alcohol feeding group. Moderate alcohol drinking increased the secretion of the CXCL14 protein in inguinal subcutaneous adipose tissue, which drove the recruitment of M2 macrophages. Moderate alcohol drinking mice had faster lipid metabolism and slower lipid anabolism. In addition, we found that long-term low-dose alcohol intake prevented the increase of body weight, triglycerides, inflammation and energy expenditure decrease induced by HFD. Moderate alcohol consumption increased the expression of UCP1 and glucose uptake in the adipose tissue of the HFD group. In conclusion, our results show for the first time that alcohol can trigger the browning of white adipose tissue to counteract obesity.

Enhancement of Solubility, Purification, and Inclusion Body Refolding of Active Human Mitochondrial Aldehyde Dehydrogenase 2.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is predominantly linked with acetaldehyde detoxification in the second stage of alcohol metabolism. To intensively study ALDH2 function, a higher purity and uniform composition of the protein is required. An efficient Escherichia coli system for ALDH2 expression was developed by using His and a small ubiquitin-related modifier fusion tag. Most of the recombinant ALDH2s were expressed in the form of inclusion bodies. The ALDH2-enriched inclusion bodies were denatured with 6 M guanidine hydrochloride, and then ALDH2 was ultrafitrated. Finally, ALDH2 was successfully purified through affinity and gel filtration chromatography. The purified ALDH2 was finally preserved by the vacuum freeze-drying method, and its purity was determined to be higher than 95%, with a final media yield of 33.89 mg/L. The specific activity of ALDH2 was 6.1 × 104 U/mg. This work was the first to report pET-SUMO-ALDH2 recombinant plasmid expression in Escherichia coli, and the inclusion bodies were isolated and refolded. Finally, the purified ALDH2 had relatively higher purity, yield, and biological activity.

CPEB3-mediated MTDH mRNA translational suppression restrains hepatocellular carcinoma progression.

Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a sequence-specific RNA-binding protein. We had reported that CPEB3 is involved in hepatocellular carcinoma (HCC) progression. However, the underlying mechanisms of CPEB3 in HCC remain unclear. In this study, we firstly performed RNA immunoprecipitation to uncover the transcriptome-wide CPEB3-bound mRNAs (CPEB3 binder) in HCC. Bioinformatic analysis indicates that CPEB3 binders are closely related to cancer progression, especially HCC metastasis. Further studies confirmed that metadherin (MTDH) is a direct target of CPEB3. CPEB3 can suppress the translation of MTDH mRNA in vivo and in vitro. Besides, luciferase assay demonstrated that CPEB3 interacted with 3'-untranslated region of MTDH mRNA and inhibited its translation. Subsequently, CPEB3 inhibited the epithelial-mesenchymal transition and metastasis of HCC cells through post-transcriptional regulation of MTDH. In addition, cpeb3 knockout mice are more susceptible to carcinogen-induced hepatocarcinogenesis and subsequent lung metastasis. Our results also indicated that CPEB3 was a good prognosis marker, which is downregulated in HCC tissue. In conclusion, our results demonstrated that CPEB3 played an important role in HCC progression and targeting CPEB3-mediated mRNA translation might be a favorable therapeutic approach.

Low-dose ethanol intake prevents high-fat diet-induced adverse cardiovascular events in mice.

A high-fat diet is recognized as an important factor in the development of cardiovascular diseases including cardiomyopathy. Besides high-fat diets, large quantities of ethanol also induce cardiomyopathy in both animals and humans. Emerging evidence suggests that low ethanol intake may have a protective effect on the cardiovascular system. This study aimed to clarify whether low-dose ethanol intake could prevent high-fat diet-induced adverse effects on cardiomyocytes in mice. After 6-8 weeks of feeding, the heart weight significantly decreased in ethanol + HFD mice compared to HFD mice. In addition, cardiac triglycerides and lipid droplets also decreased, but no statistically significant difference in cholesterol level was found between the two groups. Expression of the fatty acid transporters Cd36, Slc27a1 and Got2 was downregulated in the ethanol + HFD group. According to echocardiography, the mass and volume of the left ventricle were reduced, and the ejection fraction (EF) and fractional shortening (FS) were increased in mice fed with alcohol. Low doses of ethanol reduced the cardiomyocytes' cross-sectional area and the expression of the hypertrophic markers ANP and BNP. Moreover, Col1a1, the main collagen type expressed in the heart, was also reduced by low-dose ethanol consumption. Also, the expression of Rgs5, a crucial component of the signaling pathway involved in cardiac remodeling and heart failure, was upregulated in response to ethanol intake. The data suggest that low ethanol intake prevents adverse effects induced by a high-fat diet, such as lipid accumulation, cardiac dysfunction, hypertrophy and fibrosis. Furthermore, low ethanol intake upregulates Rgs5, which suggests it plays a role in cardiac remodeling and heart failure.

Niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells.

Our previous study found that the anthelmintic drug niclosamide relaxed the constricted arteries and inhibited proliferation and migration of vascular smooth muscle cells. Here, we investigated the effect of niclosamide ethanolamine (NEN) on trachea function and the proliferation and migration of trachea smooth muscle cells. Isometric tension of trachea was recorded by multi-channel myograph system. The cell proliferation was detected by using BrdU cell proliferation assay. The cell migration ability was evaluated by using scratch assay. The protein level was measured by using western blot technique. Acute treatment with NEN dose-dependently relaxed acetylcholine chloride (Ach)- and High K+ physiological salt solution (KPSS)-induced constriction of mice trachea. Pre-treatment with NEN inhibited Ach- and KPSS-induced constriction of mice trachea. NEN treatment inhibited proliferation of human bronchial smooth muscle cells (HBSMCs), inhibited migration of HBSMCs and rat primary trachea smooth muscle cells. NEN treatment activated adenosine monophosphate activated protein kinase (AMPK) activity and inhibited signal transducer and activator of transcription 3 (STAT3) activity in HBSMCs. In conclusion, niclosamide ethanolamine induces trachea relaxation and inhibits proliferation and migration of trachea smooth muscle cells, indicating that niclosamide might be a potential drug for chronic asthma treatment.

Characterizations of mitochondrial uncoupling induced by chemical mitochondrial uncouplers in cardiomyocytes.

Induction of mild mitochondrial uncoupling is protective in a variety of disorders; however, it is unclear how to recognize the mild mitochondrial uncoupling induced by chemical mitochondrial uncouplers. The aim of the present study is to identify the pharmacological properties of mitochondrial uncoupling induced by mitochondrial uncouplers in cardiomyocytes. Neonatal rat cardiomyocytes were cultured. Protein levels were measured by using western blot technique. The whole cell respiratory function was determined by using high-resolution respirometry. The typical types of chemical mitochondrial uncouplers, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), niclosamide, and BAM15, induced biphasic change of STAT3 activity in cardiomyocytes, activating STAT3 at low dose and inhibiting STAT3 at high dose, though the dose range of these drugs was distinct. Low-dose uncouplers induced STAT3 activation through the mild increase of mitochondrial ROS (mitoROS) generation and the subsequent JAK/STAT3 activation in cardiomyocytes. However, high-dose uncouplers induced inhibition of STAT3, decrease of ATP production, and cardiomyocyte death. High-dose uncouplers induced STAT3 inhibition through the excessive mitoROS generation and the decreased ATP -induced AMPK activation. Low-dose mitochondrial uncouplers attenuated doxorubicin (DOX)-induced STAT3 inhibition and cardiomyocyte death, and activated STAT3 contributed to the cardioprotection of low-dose mitochondrial uncouplers. Uncoupler-induced mild mitochondrial uncoupling in cardiomyocytes is characterized by STAT3 activation and ATP increase whereas excessive mitochondrial uncoupling is characterized by STAT3 inhibition, ATP decrease and cell injury. Development of mitochondrial uncoupler with optimal dose window of inducing mild uncoupling is a promising strategy for heart protection.

Niclosamide inhibits vascular smooth muscle cell proliferation and migration and attenuates neointimal hyperplasia in injured rat carotid arteries.

BACKGROUND AND PURPOSE: The anti-helminthic drug niclosamide regulates multiple cellular signals including STAT3, AMP-activated protein kinase (AMPK), Akt, Wnt/ß-catenin and mitochondrial uncoupling which are involved in neointimal hyperplasia. Here we have examined the effects of niclosamide on vascular smooth muscle cell proliferation, migration and neointimal hyperplasia and assessed the potential mechanisms. EXPERIMENTAL APPROACH: Cell migration was measured by using wound-induced migration assay and Boyden chamber assay. Protein levels were measured by using Western blot technique. Neointimal hyperplasia in vivo was induced in rats by balloon injury to the carotid artery. KEY RESULTS: Niclosamide treatment inhibited serum-induced (15% FBS) and PDGF-BB-induced proliferation and migration of vascular smooth muscle cells (A10 cells). Niclosamide showed no cytotoxicity at anti-proliferative concentrations, but induced cell apoptosis at higher concentrations. Niclosamide treatment inhibited serum-induced (15% FBS) and PDGF-BB-induced STAT3 activation (increased protein levels of p-STAT3 at Tyr705 ) but activated AMPK, in A10 cells. Niclosamide exerted no significant effects on ß-catenin expression and the activities of ERK1/2 and Akt in A10 cells. Injection (i.p.) of soluble pegylated niclosamide (PEG5000-niclosamide) (equivalent to niclosamide 25 mg·kg-1 ) attenuated neointimal hyperplasia following balloon-injury in rat carotid arteries in vivo. CONCLUSIONS AND IMPLICATIONS: Niclosamide inhibited vascular smooth muscle cell proliferation and migration and attenuated neointimal hyperplasia in balloon-injured rat carotid arteries through a mechanism involving inhibition of STAT3.

Mitochondrial Fission Inhibitors Suppress Endothelin-1-Induced Artery Constriction.

BACKGROUND/AIMS: Endothelin-1 is implicated in the pathogenesis of hypertension, but the underlying mechanisms remained elusive. Our previous study found that inhibition of mitochondrial fission of smooth muscle cells suppressed phenylephrine- and high K+-induced artery constriction. Here, we studied the effects of mitochondrial fission inhibitors on endothelin-1-induced vasoconstriction. METHODS: The tension of rat mesenteric arteries and thoracic aorta was measured by using a multi-wire myograph system. Mitochondrial morphology of aortic smooth muscle cells was observed by using transmission electron microscopy. RESULTS: Dynamin-related protein-1 selective inhibitor mdivi-1 relaxed endothelin-1-induced constriction, and mdivi-1 pre-treatment prevented endothelin-1-induced constriction of rat mesenteric arteries with intact and denuded endothelium. Mdivi-1 had a similar inhibitory effect on rat thoracic aorta. Another mitochondrial fission inhibitor dynasore showed similar effects as mdivi-1 in rat mesenteric arteries. Mdivi-1 inhibited endothelin-1-induced increase of mitochondrial fission in smooth muscle cells of rat aorta. Rho-associated protein kinase inhibitor Y-27632 which relaxed endothelin-1-induced vasoconstriction inhibited endothelin-1-induced mitochondrial fission in smooth muscle cells of rat aorta. CONCLUSION: Endothelin-1 increases mitochondrial fission in vascular smooth muscle cells, and mitochondrial fission inhibitors suppress endothelin-1-induced vasoconstriction.

Arterial relaxation is coupled to inhibition of mitochondrial fission in arterial smooth muscle cells: comparison of vasorelaxant effects of verapamil and phentolamine.

Mitochondria are morphologically dynamic organelles which undergo fission and fusion processes. Our previous study found that arterial constriction was always accompanied by increased mitochondrial fission in smooth muscle cells, whereas inhibition of mitochondrial fission in smooth muscle cells was associated with arterial relaxation. Here, we used the typical vasorelaxants, verapamil and phentolamine, to further confirm the coupling between arterial constriction and mitochondrial fission in rat aorta. Results showed that phentolamine but not verapamil induced vasorelaxation in phenylephrine (PE)-induced rat thoracic aorta constriction. Verapamil, but not phentolamine, induced vasorelaxation in high K+ (KPSS)-induced rat thoracic aorta constriction. Pre-treatment with phentolamine prevented PE- but not KPSS-induced aorta constriction and pre-treatment with verapamil prevented both PE- and KPSS-induced aorta constriction. Transmission electron microscopy (TEM) results showed that verapamil but not phentolamine inhibited KPSS-induced excessive mitochondrial fission in aortic smooth muscle cells, and verapamil prevented both PE- and KPSS-induced excessive mitochondrial fission in aortic smooth muscle cells. Verapamil inhibited KPSS-induced excessive mitochondrial fission in cultured vascular smooth muscle cells (A10). These results further demonstrate that arterial relaxation is coupled to inhibition of mitochondrial fission in arterial smooth muscle cells.