Antibiotic Resistance Determinants and Virulence Factors of Hypervirulent and Carbapenem Non-Susceptible Pseudomonas aeruginosa.

BACKGROUND: The aim was to investigate the distribution of antibiotic resistance determinants and virulence factors in a group of carbapenem non-susceptible Pseudomonas aeruginosa (P. aeruginosa). METHODS: From March 2018 to May 2019, a total of 98 P. aeruginosa samples were collected from 6 hospitals in Ningbo and Hangzhou, Zhejiang Province, China. Drug susceptibility tests to 13 antimicrobial agents were conducted. The presence of antibiotic resistance determinants and virulence factors were investigated by PCR, including 39 ß-lactamase genes, 14 aminoglycoside modifying enzyme genes, 10 16SrRNA methylase genes, and 11 virulence genes. Phylogenetics of 98 P. aeruginosa was analyzed by sample cluster analysis (UPGMA). RESULTS: PCR revealed the presence of 7 ß-lactamase genes, 5 aminoglycoside modifying enzymes, 1 16S rRNA methylase gene, and 8 virulence genes in total, at least 2 ß-lactamase genes and 4 virulence genes were positive in every isolate. In addition, regional differences in distributions of resistance and virulence genes remained between 2 cities. Sample cluster analysis showed that the strains had obvious aggregation and were divided into several clusters, strains in the same cluster were isolated from different hospitals, even from different cities. CONCLUSIONS: Carrying resistance genes blaPDC and blaOXA-50 group and virulence genes plcH, aprA, and algD were the important epidemiological characteristics of this group of P. aeruginosa. The present findings provide insights into the mechanisms of hypervirulence as well as resistance to ß-lactams and aminoglycosides. To the best of our knowledge, this is the first report of blaPDC, blaOXA-50, and aph(3')-XV in P. aeruginosa in China.

Carvedilol improves liver cirrhosis in rats by inhibiting hepatic stellate cell activation, proliferation, invasion and collagen synthesis.

Portal hypertension (PHT) is one of the most severe consequences of liver cirrhosis. Carvedilol is a first­line pharmacological treatment of PHT. However, the antifibrogenic effects of carvedilol on liver cirrhosis and the intrinsic mechanisms underlying these effects have not been thoroughly investigated. The present study aimed to investigate the antifibrogenic effects of carvedilol on liver cirrhosis in vivo and in vitro. Liver cirrhosis was induced in rats by carbon tetrachloride (CCl4) administration for 9 weeks; carvedilol was administered simultaneously in the experimental group. Blood samples were collected for serum biochemistry. Liver tissues were used for fibrosis evaluation, histological examination, immunohistochemistry and western blot analysis. The human hepatic stellate cell (HSC) line LX­2 was used for in vitro studies. The effects of carvedilol on LX­2 cell proliferation and invasion were evaluated by Cell Counting Kit­8 assay and Transwell invasion assays, respectively. The effect of carvedilol on transforming growth factor ß1 (TGFß1)­induced collagen synthesis in LX­2 cells and the molecular mechanisms were examined by western blot analysis. The results demonstrated that carvedilol improved CCl4­induced structural distortion and fibrosis in the liver. Carvedilol inhibited HSC activation, proliferation and invasion. Carvedilol inhibited HSC collagen synthesis through the TGFß1/SMAD pathway. In conclusion, carvedilol may alleviate liver cirrhosis in rats by inhibiting HSC activation, proliferation, invasion and collagen synthesis. Carvedilol may be a potential treatment of early­stage liver cirrhosis.

Carvedilol Ameliorates Intrahepatic Angiogenesis, Sinusoidal Remodeling and Portal Pressure in Cirrhotic Rats.

BACKGROUND Carvedilol is the first-line drug for the primary prophylaxis of variceal bleeding due to portal hypertension (PHT) in liver cirrhosis. This study aimed to investigate the effects of carvedilol on intrahepatic angiogenesis and sinusoidal remodeling in cirrhotic rats and explore the underlying mechanisms of carvedilol in PHT. MATERIAL AND METHODS For in vivo experiments, carbon tetrachloride was used to induce liver cirrhosis in rats, and carvedilol was simultaneously administered by gavage. The portal pressure was measured in rats, and liver tissues were examined by immunohistochemistry. Sinusoidal remodeling was observed by transmission electron microscopy. For in vitro experiments, the effects of carvedilol on fibronectin (FN) synthesis in human umbilical vein endothelial cells (HUVECs) were explored by quantitative real-time polymerase chain reaction and western blot analysis. RESULTS Portal vein pressure measurements showed that carvedilol reduced portal pressure in cirrhotic rats. Immunohistochemistry assays indicated that carvedilol ameliorated intrahepatic angiogenesis. Transmission electron microscopy examination demonstrated that carvedilol improved sinusoidal remodeling. In the in vitro experiments, carvedilol suppressed transforming growth factor ß1 (TGFß1)-induced FN synthesis in HUVECs by inhibition of the TGFß1/Smads pathway. CONCLUSIONS Carvedilol ameliorated intrahepatic angiogenesis, sinusoidal remodeling and portal pressure in cirrhotic rats. Carvedilol improved sinusoidal remodeling by suppressing FN synthesis in endothelial cells. Carvedilol has potential utility for treating early-stage liver cirrhosis.

Carvedilol attenuates liver fibrosis by suppressing autophagy and promoting apoptosis in hepatic stellate cells.

Carvedilol has been identified as a promising agent for the treatment of liver fibrosis. Meanwhile, autophagy and apoptosis have been reported to play key roles in the activation of hepatic stellate cells (HSCs), which can contribute to the progression of liver fibrosis. However, the effects of carvedilol on autophagy and apoptosis in HSCs remain unclear. Our study aimed to detect these effects and identify the underlying mechanisms by which carvedilol mediates HSC autophagy and apoptosis. For this purpose, the LX-2 cell line was used in this study, and the cells were exposed to various concentrations of carvedilol for specific times. First, we found that carvedilol increased autophagic marker levels, the number of GFP-LC3-containing puncta and LC3B-II levels in LX-2 cells. Interestingly, the addition of chloroquine (CQ) failed to enhance the effects on GFP-LC3 puncta and LC3B-II levels, and carvedilol treatment resulted in a significant increase in p62 protein levels. Moreover, carvedilol treatment led to the accumulation of yellow dots only in GFP-RFP-LC3-LX-2 cells, similar to the results following CQ treatment, indicating that carvedilol inhibited autophagic flux. Next, we found evidence that carvedilol inhibited autophagic flux by increasing lysosomal pH and not by impairing the fusion of autophagosomes with lysosomes. Moreover, carvedilol substantially reduced the viability of LX-2 cells and noticeably induced cell apoptosis, as observed by flow cytometry. In addition, increased levels of cleaved caspase-3, cleaved caspase-8 and cleaved PARP, increased Bax activity and decreased Bcl-2 expression were detected in LX-2 cells. Finally, the carvedilol treatment inhibited autophagy and subsequently induced apoptosis in vitro. In conclusion, carvedilol suppresses autophagy and promotes apoptosis in HSCs and the late-stage inhibition of autophagy preceded the induction of apoptosis.

Propranolol prevents liver cirrhosis by inhibiting hepatic stellate cell activation mediated by the PDGFR/Akt pathway.

Propranolol is known to reduce portal pressure by decreasing blood flow to the splanchnic circulation and the liver. However, it is unknown if propranolol improves fibrogenesis and sinusoidal remodeling in the cirrhotic liver. The aim of this study was to investigate the therapeutic effects of propranolol on carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and the intrinsic mechanisms underlying those effects. In this study, a hepatic cirrhosis mouse model was induced by CCl4 administration for 6 weeks. Propranolol was simultaneously administered orally in the experimental group. Liver tissue and blood samples were collected for histological and molecular analyses. LX-2 cells induced by platelet-derived growth factor-BB (PDGF-BB) were used to evaluate the anti-fibrogenic effect of propranolol in vitro. The results showed that treatment of mice with CCl4 induced hepatic fibrosis, as evidenced by inflammatory cell infiltration, collagen deposition and abnormal vascular formation in the liver tissue. All these changes were significantly attenuated by propranolol treatment. Furthermore, we also found that propranolol inhibited PDGF-BB-induced hepatic stellate cell migration, fibrogenesis, and PDGFR/Akt phosphorylation. Taken together, propranolol might prevent CCl4-induced liver injury and fibrosis at least partially through inhibiting the PDGF-BB-induced PDGFR/Akt pathway. The anti-fibrogenic effect of propranolol may support its status as a first-line treatment in patients with chronic liver disease.

Metformin attenuates motility, contraction, and fibrogenic response of hepatic stellate cells in vivo and in vitro by activating AMP-activated protein kinase.

AIM: To investigate the effect of metformin on activated hepatic stellate cells (HSCs) and the possible signaling pathways involved. METHODS: A fibrotic mouse model was generated by intraperitoneal injection of carbon tetrachloride (CCl4) and subsequent treatment with or without metformin. The level of fibrosis was detected by hematoxylin-eosin staining, Sirius Red staining, and immunohistochemistry. The HSC cell line LX-2 was used for in vitro studies. The effect of metformin on cell proliferation (CCK8 assay), motility (scratch test and Transwell assay), contraction (collagen gel contraction assay), extracellular matrix (ECM) secretion (Western blot), and angiogenesis (ELISA and tube formation assay) was investigated. We also analyzed the possible signaling pathways involved by Western blot analysis. RESULTS: Mice developed marked liver fibrosis after intraperitoneal injection with CCl4 for 6 wk. Metformin decreased the activation of HSCs, reduced the deposition of ECM, and inhibited angiogenesis in CCl4-treated mice. Platelet-derived growth factor (PDGF) promoted the fibrogenic response of HSCs in vitro, while metformin inhibited the activation, proliferation, migration, and contraction of HSCs, and reduced the secretion of ECM. Metformin decreased the expression of vascular endothelial growth factor (VEGF) in HSCs through inhibition of hypoxia inducible factor (HIF)-1α in both PDGF-BB treatment and hypoxic conditions, and it down-regulated VEGF secretion by HSCs and inhibited HSC-based angiogenesis in hypoxic conditions in vitro. The inhibitory effects of metformin on activated HSCs were mediated by inhibiting the Akt/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) pathways via the activation of adenosine monophosphate-activated protein kinase (AMPK). CONCLUSION: Metformin attenuates the fibrogenic response of HSCs in vivo and in vitro, and may therefore be useful for the treatment of chronic liver diseases.