Single-cell transcriptome reveals a novel mechanism of C-Kit+-liver sinusoidal endothelial cells in NASH.

AIM: To understand how liver sinusoidal endothelial cells (LSECs) respond to nonalcoholic steatohepatitis (NASH). METHODS: We profiled single-LSEC from livers of control and MCD-fed mice. The functions of C-Kit+-LSECs were determined using coculture and bone marrow transplantation (BMT) methods. RESULTS: Three special clusters of single-LSEC were differentiated. C-Kit+-LSECs of cluster 0, Msr1+-LSECs of cluster 1 and Bmp4+Selp+-VECs of cluster 2 were revealed, and these cells with diverse ectopic expressions of genes participated in regulation of endothelial, fibrosis and lipid metabolism in NASH. The number of C-Kit+-primary LSECs isolated from MCD mice was lower than control mice. Immunofluorescence co-staining of CD31 and C-KIT showed C-Kit+-LSECs located in hepatic sinusoid were also reduced in NASH patients and MCD mice, compared to AIH patients and control mice respectively. Interestingly, lipotoxic hepatocytes/HSCs cocultured with C-Kit+-LSECs or the livers of MCD mice receipting of C-Kit+-BMCs (bone marrow cells) showed less steatosis, inflammation and fibrosis, higher expression of prolipolytic FXR and PPAR-α, lower expression of TNF-α and α-SMA. Furthermore, coculturing or BMT of C-Kit+-endothelial derived cells could increase the levels of hepatic mitochondrial LC3B, decrease the degree of mitochondrial damage and ROS production through activating Pink1-mediated mitophagy pathway in NASH. CONCLUSIONS: Hence, a novel transcriptomic view of LSECs was revealed to have heterogeneity and complexity in NASH. Importantly, a cluster of C-Kit+-LSECs was confirmed to recovery Pink1-related mitophagy and NASH progression.

[Port-owned Mobile Source Air Pollutant Emission Inventory].

Based on the comprehensive development of the emission inventory of air pollution sources, the emission inventory of self-owned mobile sources of Tianjin coastal ports was researched and formulated. In this study, a gridded emission inventory with a resolution of 3 km×3 km was established for six types of air pollutants from road and non-road mobile sources. The spatial and temporal distribution characteristics of pollutant emissions were analyzed, and the uncertainty of the inventory was analyzed using the Monte Carlo method. The results showed that in 2020, the self-owned mobile sources of coastal ports emitted 148.22 t PM10, 135.34 t PM2.5, 1061.04 t SO2, 4027.16 t NOx, 756.60 t CO, and 237.07 t VOCs, of which the total emissions of road and non-road mobile sources accounted for 6.66% and 93.34% of the mobile source emissions, respectively. The main contributors to motor vehicle pollutant emissions from road mobile sources in the whole port area were small, medium, and large passenger vehicles (gasoline) and heavy trucks (diesel). The main contributors to the pollutants emitted by non-road mobile sources were ships and construction machinery. Uncertainty analysis results showed that the overall uncertainty of mobile sources ranged from -13.3% to 16.53%.

Vsig4+ resident single-Kupffer cells improve hepatic inflammation and fibrosis in NASH.

BACKGROUND: The role of macrophages in the pathogenesis of nonalcoholic steatohepatitis (NASH) is complex and unclear. METHODS: Single-cell RNA sequencing was performed on nonparenchymal cells isolated from NASH and control mice. The expression of Vsig4+ macrophages was verified by qPCR, flow cytometry and immunohistochemistry. Primary hepatic macrophages were cocultured with primary hepatocytes or hepatic stellate cells (LX2) cells by Transwell to detect immunofluorescence and oil red O staining. RESULTS: Two main single macrophage subsets were identified that exhibited a significant change in cell percentage when NASH occurred: resident Kupffer cells (KCs; Cluster 2) and lipid-associated macrophages (LAMs; Cluster 13). Nearly 82% of resident single KCs in Cluster 2 specifically expressed Cd163, and an inhibited subgroup of Cd163+ resident single-KCs was suggested to be protective against NASH. Similar to Cd163, Vsig4 was both enriched in and specific to Cluster 2. The percentage of Vsig4+-KCs was significantly decreased in NASH in vivo and in vitro. Hepatocytes and hepatic stellate cells produced less lipid droplet accumulation, proinflammatory protein (TNF-α) and profibrotic protein (α-SMA) in response to coculture with Vsig4+-KCs than in those cocultured with lipotoxic KCs. CONCLUSIONS: A subgroup of Vsig4+ resident single-KCs was shown to improve hepatic inflammation and fibrosis in NASH.

Hepatocyte-derived exosomal miR-27a activateshepatic stellate cells through the inhibitionof PINK1-mediated mitophagy in MAFLD.

The role of exosome-mediated mitophagy in the crosstalk between hepatocytes (HCs) and hepatic stellate cells (HSCs) in metabolic-associated fatty liver disease (MAFLD) remains unknown. Serum exosomal miR-27a levels were markedly increased and positively correlated with liver fibrosis in MAFLD patients and mice. Exosomal miR-27a was released from lipotoxic HCs and specifically transmitted to recipient-activated HSCs. PINK1, the key target of miR-27a, primarily mediates mitophagy. Overexpression of miR-27a or knockdown of PINK1 or lipotoxic HC-exosomal miR-27a impaired mitochondria (inhibiting mitophagy, respiration, membrane potential, and transcription while promoting reactive oxygen species production) in activated HSCs and stimulated HSC-derived fibroblasts (promoting activation and proliferation while inhibiting autophagy). High exosomal miR-27a serum levels and a lack of hepatic PINK1-mediated mitophagy were directly related to liver fibrosis in MAFLD mice. Lipotoxic HC exosome transplantation aggravated the degree of PINK1-mediated mitophagy suppression, steatohepatitis, lipidosis, and fibrosis in the livers of MAFLD mice with cirrhosis. Both in vitro and in vivo, exosomes derived from miR-27a-knockdown HCs could not facilitate the abovementioned deteriorating effects. In conclusion, lipotoxic HC-exosomal miR-27a plays a pivotal role in inhibiting mitophagy and in promoting MAFLD-related liver fibrosis by negatively regulating PINK1 expression.

Zinc-α2-glycoprotein 1 attenuates non-alcoholic fatty liver disease by negatively regulating tumour necrosis factor-α.

BACKGROUND: Zinc-α2-glycoprotein 1 (AZGP1) plays important roles in metabolism-related diseases. The underlying molecular mechanisms and therapeutic effects of AZGP1 remain unknown in non-alcoholic fatty liver disease (NAFLD). AIM: To explore the effects and potential mechanism of AZGP1 on NAFLD in vivo and in vitro. METHODS: The expression of AZGP1 and its effects on hepatocytes were examined in NAFLD patients, CCl4-treated mice fed a high fat diet (HFD), and human LO2 cells. RESULTS: AZGP1 levels were significantly decreased in liver tissues of NAFLD patients and mice. AZGP1 knockdown was found to activate inflammation; enhance steatogenesis, including promoting lipogenesis [sterol regulatory element-binding protein (SREBP)-1c, liver X receptor (LXR), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and stearoyl CoA desaturase 1 (SCD)-1], increasing lipid transport and accumulation [fatty acid transport protein (FATP), carnitine palmitoyl transferase (CPT)-1A, and adiponectin], and reducing fatty acid ß-oxidation [farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)-α]; accelerate proliferation; and reverse apoptosis in LO2 cells. AZGP1 overexpression (OV-AZGP1) had the opposite effects. Furthermore, AZGP1 alleviated NAFLD by blocking TNF-α-mediated inflammation and intracellular lipid deposition, promoting proliferation, and inhibiting apoptosis in LO2 cells. Finally, treatment with OV-AZGP1 plasmid dramatically improved liver injury and eliminated liver fat in NAFLD mice. CONCLUSION: AZGP1 attenuates NAFLD with regard to ameliorating inflammation, accelerating lipolysis, promoting proliferation, and reducing apoptosis by negatively regulating TNF-α. AZGP1 is suggested to be a novel promising therapeutic target for NAFLD.

MicroRNA-194 inactivates hepatic stellate cells and alleviates liver fibrosis by inhibiting AKT2.

BACKGROUND: Activation of hepatic stellate cells (HSCs) is a pivotal event in the onset and progression of liver fibrosis. Loss of microRNA-194 (miR-194) has been reported in activated HSCs, but the actual role of miR-194 in liver fibrosis remains uncertain. AIM: To explore the role and potential mechanism of miR-194-mediated regulation of liver fibrosis in vitro and in vivo. METHODS: The expression of miR-194 was examined in human fibrotic liver tissues, activated HSCs, and a carbon tetrachloride (CCl4) mouse model by qPCR. The effects of AKT2 regulation by miR-194 on the activation and proliferation of HSCs were assessed in vitro. For in vivo experiments, we reintroduced miR-194 in mice using a miR-194 agomir to investigate the functions of miR-194 in liver fibrosis. RESULTS: MiR-194 expression was notably lacking in activated HSCs from both humans and mice. Overexpression of miR-194 (OV-miR-194) inhibited α-smooth muscle actin (α-SMA) and type I collagen (Col I) expression and suppressed cell proliferation in HSCs by causing cell cycle arrest in G0/G1 phase. AKT2 was predicted to be a target of miR-194. Notably, the effects of miR-194 knockdown in HSCs were almost blocked by AKT2 deletion, indicating that miR-194 plays a role in HSCs via regulation of AKT2. Finally, miR-194 agomir treatment dramatically ameliorated liver fibrosis in CCl4-treated mice. CONCLUSION: We revealed that miR-194 plays a protective role by inhibiting the activation and proliferation of HSCs via AKT2 suppression. Our results further propose miR-194 as a potential therapeutic target for liver fibrosis.

linc-SCRG1 accelerates liver fibrosis by decreasing RNA-binding protein tristetraprolin.

The biologic roles of long noncoding RNAs (lncRNAs) in liver fibrosis remained unknown. Through microarray analysis, linc-SCRG1 (a lncRNA with transcript length 3118 bp) was found up-regulated 13.62-fold in human cirrhotic tissues. Quantitative PCR verified that linc-SCRG1 increased along with liver fibrosis progression in human tissues and in activated LX2 cells induced by TGF-ß1. Knockdown of linc-SCRG1 significantly reversed the effects of TGF-ß1 on LX2, including inhibiting activation, promoting apoptosis, reducing proliferation, lessening invasion, and down-regulating genes [fibrosis-related mRNA: α-smooth muscle actin ( α-SMA), type I collagen, and B-cell lymphoma-2; invasion-related mRNA: matrix metallopeptidase-2 ( MMP-2), MMP-9, and MMP-13; inflammation-related mRNA: TNF-α, IL-6, and IL-10]. linc-SCRG1 had binding sites with tristetraprolin (TTP), a kind of RNA-binding protein, and specifically combined to TTP proteins. Overexpression of linc-SCRG1 would cause TTP mRNA unstably and proteins decreasing. TTP mRNA was proved having negative relevance with linc-SCRG1 and was gradually reduced during human liver fibrosis progression. Overexpressing TTP resulted in knockdown of lincSCRG1 and degraded downstream target genes ( MMP-2 and TNF-α) in activated LX2. Overexpressing TTP had the same effects as small interfering RNA-lincSCRG1 (si- lincSCRG1), whereas knockdown of TTP had reversal effects on si- lincSCRG1 in activated LX2. In summary, linc-SCRG1 reduced TTP and restricted its degradation of target genes TNF-α and MMP-2. Therefore, linc-SCRG1 had a repressing TTP-elicited inactivation effect on hepatic stellate cell (HSC) phenotypes. Inhibition of linc-SCRG1 may be a novel therapeutic approach to inactivate HSCs and extenuate human liver fibrosis.-Wu, J.-C., Luo, S.-Z., Liu, T., Lu, L.-G., Xu, M.-Y. linc-SCRG1 accelerates liver fibrosis by decreasing RNA-binding protein tristetraprolin.

STAT3 aggravates TGF-ß1-induced hepatic epithelial-to-mesenchymal transition and migration.

Signal transducer and activator of transcription 3 (STAT3) has been shown to affect epithelial-to-mesenchymal transition (EMT) in cancers. We investigated the underlying molecular mechanisms of STAT3 crosstalk with Snail-Smad3/transforming growth factor (TGF)-ß1 signaling pathways during the EMT in hepatocellular carcinoma (HCC). STAT3 and TGF-ß1 expressions are examined in liver tissues of HCC patients and rats. The effect of IL-6/ STAT3 crosstalk with Snail-Smad3/TGF-ß1 on EMT, carcinogenesis, migration and invasion are tested in vitro and in vivo. Phosphorylation of STAT3 and TGF-ß1 proteins are universally high and positively co-expressed in HCC tissues from human and rats. Hepatic lower p-STAT3 proteins are related to earlier tumor stages in HCC patients. AG490 (a JAK2-specific inhibitor) treatment could reduce tumor numbers and sizes depending on suppression of STAT3 signaling in HCC rats. TGF-ß1 could induce EMT along with an E-cadherin decrease, while vimentin, Snail, p-Smad2/3, and p-STAT3/STAT3 increase in HepG2. SIS3 (a specific inhibitor of Smad3) could markedly inhibit Snail, Vim and p-STAT3 along with blocking phosphorylation of Smad3, but E-cadherin could be activated in HepG2. IL-6 activates STAT3 signaling and then has cascading consequences for activating Snail-Smad3/TGF-ß1 and vimentin as well as migration and invasion in liver cancer cells. In contrast, AG490 has an effect that inhibits phosphorylation of STAT3, lowers Snail-p-Smad3 protein levels, decreases TGF-ß1-related PAI-1 promoter activation and then reduces migration or invasion of liver cancer cells. STAT3 functions as a positive regulator to activate TGF-ß1-induced EMT and metastasis of HCC. STAT3 and the Snail-Smad3/TGF-ß1 signaling pathways synergistically augment EMT and migration in HCC.

MicroRNA profile analysis in the liver fibrotic tissues of chronic hepatitis B patients.

OBJECTIVE: We aimed to identify the features of microRNA (miRNA) at different fibrotic stages in patients with hepatitis B virus (HBV)-related liver fibrosis. METHODS: Liver tissues were collected from 40 chronic hepatitis B (CHB) patients at fibrotic stages S0-4. Microarrays of miRNAs and genomic informatics analysis were performed. RESULTS: In total, 105 miRNAs were differentially expressed in fibrotic tissues (S1-4 groups) compared with no fibrotic tissues (S0 group; P < 0.05). Combined with three classifications, 17 differential miRNAs were found to be closely related to fibrotic stages (over twofold change and P < 0.05). Five miRNAs had a signature that correlated with serum biochemical parameters and liver inflammatory grades. The receiver operating characteristic (ROC) curve showed that six miRNAs performed excellently in the diagnosis of liver fibrosis, with the area under the ROC curve (AUROC) over 0.8; among them hsa-miR-214-3p had the highest AUROC (0.867). Gene ontology functions of differential miRNAs mainly involved in the cellular and developmental processes, localization, biological regulation, binding, transcriptional regulator and organelle. We also found that 23 novel signaling pathways were dysregulated in the liver fibrosis. CONCLUSIONS: MiRNA profile signature, including 17 differential miRNAs and 23 dysregulated signaling pathways, was associated with liver fibrosis. Hepatic inflammatory grades were correlated with the differential miRNA. Some miRNAs can be used for the diagnosis of liver fibrosis.

Effects of social isolation, re-socialization and age on cognitive and aggressive behaviors of Kunming mice and BALB/c mice.

Both Kunming (KM) mice and BALB/c mice have been widely used as rodent models to investigate stress-associated mental diseases. However, little is known about the different behaviors of KM mice and BALB/c mice after social isolation, particularly cognitive and aggressive behaviors. In this study, the behaviors of KM and BALB/c mice isolated for 2, 4 and 8 weeks and age-matched controls were evaluated using object recognition, object location and resident-intruder tests. The recovery of behavioral deficits by re-socialization was also examined for the isolated mice in adolescence. Our study showed that isolation for 2, 4 and 8 weeks led to cognitive deficits and increased aggressiveness for both KM and BALB/c mice. An important finding is that re-socialization could completely recover spatial/non-spatial cognitive deficits resulted from social isolation for both KM and BALB/c mice. In addition, age only impacted aggressiveness of KM mice. Moreover, isolation duration showed different impacts on cognitive and aggressive behaviors for both KM and BALB/c mice. Furthermore, BALB/c mice showed weak spatial/non-spatial memory and low aggressiveness when they were at the same age and isolation duration, compared to KM mice. In conclusion, KM mice and BALB/c mice behaved characteristically under physiology and isolation conditions.

[The clinical classification method research of keloid].

OBJECTIVE: To explore the clinical classification method of keloids and providing a thread for the treatment of keloids. METHODS: To summarize the 600 cases of keloid patients we accepted and diagnosed from November 2004 to October 2012, and filling in keloid patients information sheet, recording the keloids form by photographs, analyzing the treatment, putting forward the classification method of keloids in clinic. RESULTS: According to the position and quantity that keloids grow, the keloid patients are divided into four major categories:one in single site, one in each site, more than one in single site and more than one in each site; According to the area and thickness of keloids, the keloid single lesion is divided into four subclasses: type of small area and thin, type of small area and thick, type of large areas and thin,type of large areas and thick; According to the number of lesions, keloid multiple lesions is divided into two subgenera: isolated multiple and dispersion multiple, different kinds of keloids suit different methods of treatment. CONCLUSION: The clinical classification method of keloids can be used to provide thought for the treatment of keloids, and have a good application value.

Maggot therapy for repairing serious infective wound in a severely burned patient.

The larvae of musca domestica were put in use to discard the dead tissue of a case of severe burn. A total of 50 000 aseptic maggots were put onto the infective wound surface, and aseptic dressings overlaid the surface. Three days later, another 20 000 maggots were put onto the wound for the second therapy. After twice maggot debridement, most necrotic muscle tissues of the wound were cleaned up, and eventually fresh granulation tissue grew and later the wound was covered and healed by 3 times of skin grafting. The result demonstrates that maggot therapy is safe and effective with no adverse complications except pain.