Xiaobo Cai

Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation

Proliferating hepatic stellate cells (HSCs) respond to liver damage by secreting collagens that form fibrous scar tissue, which can lead to cirrhosis if in appropriately regulated. Advancement of microRNA (miRNA) hepatic therapies has been hampered by difficulties in delivering miRNA to damaged tissue. However, exosomes secreted by adipose‐derived mesenchymal stem cells (ADSCs) can be exploited to deliver miRNAs to HSCs. ADSCs were engineered to overexpress miRNA‐181‐5p (miR‐181‐5p‐ADSCs) to selectively home exosomes to mouse hepatic stellate (HST‐T6) cells or a CCl4‐induced liver fibrosis murine model and compared with non‐targeting control Caenorhabditis elegans miR‐67 (cel‐miR‐67)‐ADSCs. In vitro analysis confirmed that the transfer of miR‐181‐5p from miR‐181‐5p‐ADSCs occurred via secreted exosomal uptake. Exosomes were visualized in HST‐T6 cells using cyc3‐labelled pre‐miRNA‐transfected ADSCs with/without the exosomal inhibitor, GW4869. The effects of miRNA‐181‐5p overexpression on the fibrosis associated STAT3/Bcl‐2/Beclin 1 pathway and components of the extracellular matrix were assessed. Exosomes from miR181‐5p‐ADSCs down‐regulated Stat3 and Bcl‐2 and activated autophagy in the HST‐T6 cells. Furthermore, the up‐regulated expression of fibrotic genes in HST‐T6 cells induced by TGF‐β1 was repressed following the addition of isolated miR181‐5p‐ADSC exosomes compared with miR‐67‐ADSCexosomes. Exosome therapy attenuated liver injury and significantly down‐regulated collagen I, vimentin, α‐SMA and fibronectin in liver, compared with controls. Taken together, the effective anti‐fibrotic function of engineered ADSCs is able to selectively transfer miR‐181‐5p to damaged liver cells and will pave the way for the use of exosome‐ADSCs for therapeutic delivery of miRNA targeting liver disease.

Analysis of the differential expression of circulating microRNAs during the progression of hepatic fibrosis in patients with chronic hepatitis B virus infection

Considering the limitations of liver biopsy, reliable non-invasive serum biomarkers of liver fibrosis are required for early diagnosis. The present study analyzed the expression profile of circulating micro (mi)RNAs during the development and progression of hepatic fibrosis in patients with chronic hepatitis B virus (HBV) infection, aiming to identify novel earlier diagnostic biomarkers. Fresh plasma samples were collected from 50 patients diagnosed with chronic HBV infection and hepatic fibrosis. These patients were classified into five groups (S0, S1, S2, S3 and S4; n=10 per group) based on Scheuers staging criteria. The differential expression of the circulating miRNAs was determined by performing miRNA microarray hybridization. Finally, the target genes of the miRNAs were predicted and classified using gene ontology analysis. A total of 140 miRNAs were detected in the S1–S4 patient groups, and their expression levels were >2-fold higher compared with those in the S0 group. The numbers of miRNAs differentially expressed in the S1–S4 patient groups were 48, 97, 84 and 56, respectively, with 12 miRNAs differentially expressed at all stages, 10 of which were upregulated and two of which were downregulated. The target genes of the miRNAs identified were found to be involved in 100 signal transduction pathways, the majority of which affected hepatic fibrosis via the TGF-/Smad, Wnt, MAPK, Jak/STAT and VEGF pathways. The differential expression levels of miRNAs were closely associated with the staging of hepatic fibrosis. The results of the present study provide evidence to facilitate the development and application of non-invasive biomarkers for earlier diagnosis of hepatic fibrosis.

Isolation and Culture of Single Cell Types from Rat Liver

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

CXCL6 promotes human hepatocyte proliferation through the CXCR1–NFκB pathway and inhibits collagen I secretion by hepatic stellate cells

Hepatocyte proliferation and collagen I (COLI) secretion are important processes during liver regeneration. This study aimed to investigate the role of CXCL6 in hepatocyte proliferation and COLI secretion. Serum CXCL6 levels in patients with chronic hepatitis B (CHB) were examined and the effects of CXCL6 on the proliferation of L02 hepatocytes and the secretion of COLI from LX2 human hepatic stellate cells were evaluated. We found that serum CXCL6 levels increased gradually with disease progression of CHB, and there was positive correlation between serum CXCL6 level and alanine transaminase (ALT) and aspartate transaminase (AST). In vitro, CXCL6 promoted L02 proliferation but this was blocked upon CXCR1 knockdown. The level of phospho-IκBα was upregulated by CXCL6 but downregulated by CXCR1 siRNA in L02 cells. CXCL6 inhibited the secretion of COLI by LX2 cells, dependent on CXCR1 and CXCR2. Taken together, these data suggest that increased expression of CXCL6 during CHB could promote hepatocyte proliferation through the CXCR1-NFκB pathway and inhibit the secretion of COLI by hepatic stellate cells.

CXCL6-EGFR-induced Kupffer cells secrete TGF-β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C-MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP‐2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF‐β in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC‐T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF‐β showed increased expression of α‐SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C‐MYC/EZH2 pathway by enhancing the SMAD3‐BRD4 interaction and promoting direct binding of BRD4 to the C‐MYC promoter and CMY‐C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl4‐induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF‐β in serum and the expression of α‐SMA, SMAD3, BRD4, C‐MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF‐β by KCs and thereby activating HSCs.

Y-box Protein-1 Regulates the Expression of Collagen I in Hepatic Progenitor Cells via PDGFR-β/ERK/p90RSK Signalling

Y-box protein-1 (YB-1) is a highly conserved transcription factor that is involved in multiple biological processes via transcriptional regulation of several genes, including p53, cyclin D1, and EGFR. YB-1 has been reported to be overexpressed in injured livers. This study aims to explore the functions of YB-1 in hepatic progenitor cells (HPCs). Herein, chromatin immunoprecipitation sequencing (ChIP-sequencing) and RNA-sequencing assays identified that YB-1 participated in the biological adhesion process and ECM-receptor interactions in HPCs. Further study demonstrated that YB-1 modulated the expression of extracellular matrix components in HPCs. ChIP-sequencing assays established that PDGFR-β was a target gene of YB-1, and luciferase reporter assays confirmed that YB-1 negatively regulated PDGFR-β promoter activity in HPCs. In addition, PDGFR-β can regulate the expression of collagen I through ERK/p90RSK signalling, and disruption of the signalling pathway with a PDGFR-β inhibitor or ERK1/2 inhibitor abolished the regulatory effect of PDGFR-β on collagen I expression in HPCs. Conclusively, YB-1 can modulate the expression of collagen I in HPCs via direct binding to the PDGFR-β promoter, negatively regulating its expression. In addition, the ERK/p90RSK axis serves as the downstream signalling pathway of PDGFR-β.

Cytokeratin19 positive hepatocellular carcinoma is associated with increased peritumoral ductular reaction.

BACKGROUND AND AIMS Cytokeratin19 positive (CK19+) hepatocellular carcinoma (HCC) is thought to derive from liver progenitor cells (LPC). However, whether peritumoralductular reaction (DR) differs between CK19+ and CK19 negative (CK19-) HCC patients remains unclear. MATERIAL AND METHODS One hundred and twenty HBV-related HCC patients were enrolled in this study. Clinicopathological variables were collected, and immunohistochemistry staining for CK19, proliferating cell nuclear antigen (PCNA), interleukin-6 (IL-6) and β-catenin were performed in tumor and peritumor liver tissues. RESULTS CK19+ HCC patients had higher grade of peritumoral DR and proportion of proliferative DR than the CK19- group. The mean number or the proportion of cytoplasmic β-catenin+ DR was higher in the CK19+ group than in the CK19- group. Furthermore, there were more patients with nuclear β-catenin+ peritumoral DR in the CK19+ group as compared to the CK19- group. CONCLUSION Peritumoral DR was more abundant and proliferative in CK19+ HCC patients, with higher level of nuclear translocation of β-catenin. However, it is unclear whether peritumoral DR is the cause or result of poor prognosis in these patients.BACKGROUND AND AIMS Cytokeratin19 positive (CK19+) hepatocellular carcinoma (HCC) is thought to derive from liver progenitor cells (LPC). However, whether peritumoralductular reaction (DR) differs between CK19+ and CK19 negative (CK19-) HCC patients remains unclear. MATERIAL AND METHODS One hundred and twenty HBV-related HCC patients were enrolled in this study. Clini-copathological variables were collected, and immunohistochemistry staining for CK19, proliferating cell nuclear antigen (PCNA), inter-leukin-6 (IL-6) and β-catenin were performed in tumor and peritumor liver tissues. RESULTS CK19+ HCC patients had higher grade of peritumoral DR and proportion of proliferative DR than the CK19- group. The mean number or the proportion of cytoplasmic β-catenin+ DR was higher in the CK19+ group than in the CK19- group. Furthermore, there were more patients with nuclear β-catenin+ peritumoral DR in the CK19+ group as compared to the CK19- group. CONCLUSION Peritumoral DR was more abundant and proliferative in CK19+ HCC patients, with higher level of nuclear translocation of β-catenin. However, it is unclear whether peritumoral DR is the cause or result of poor prognosis in these patients.

Peritumoral ductular reaction is related to nuclear translocation of β-catenin in hepatocellular carcinoma.

Increased peritumoral ductular reaction (DR) is related to poor prognosis in hepatocellular carcinoma (HCC) but the mechanism is unclear. Nuclear translocation of β-catenin is correlated with HCC metastasis and recurrence. Thus, we aim to explore whether there is a relationship between peritumoral DR and tumoral nuclear translocation of β-catenin in HCC. Hepatitis B virus (HBV)-related HCC patients (n=120) were enrolled into this study from January 2003 to December 2007. Clinicopathological variables were collected and immunohistochemistry staining for cytokeratin 19 (CK19), proliferating cell nuclear antigen (PCNA), β-catenin, phosphorylated-Smad2 (PSmad2) and transforming growth factor-β1 (TGF-β1) were performed in tumor and/or peritumor liver tissues. Peritumoral DR is significantly correlated with local inflammation (P<0.001), fibrosis (P<0.001), tumor size (P=0.006) and CK19 expression in the tumor (P=0.005). More patients with peritumoral DR had nuclear accumulation of β-catenin than patients with mild peritumoral DR (37.50% vs. 14. 58%, P=0.011). HCCs in the obvious DR group had stronger expression of PSmad2 than that in the mild DR group, and patients with nuclear translocation of β-catenin also had higher PSmad2 expression. In conclusion, increased peritumoral DR is related to tumoral nuclear translocation of β-catenin in HCC and enhanced action of TGF-β1 signaling may be involved in this relationship.

Computer-assisted dielectric control in curing process of armature windings of DC motors

In view of the fact that the dielectric characteristics of armature windings are similar for different norms of the motors, the dielectric response can be used to control the curing process. Appropriate physical properties, such as ultimate tensile strength and the percentage of elongation at break, are required. Since the extent of the cure is the cause of all events, the dielectric features corresponding to the appropriate physical properties can be used as criteria. The optimum curing point is determined by analyzing the dielectric response of armature windings with a dielectrometer interfaced with a microcomputer. A preliminary evaluation of the approach is presented.<<ETX>>