[MIG7 Regulates the Vasculogenic Mimicry Formation in Hepatocellular Carcinoma then Effects the Metastasis Potential of HCC].

OBJECTIVES: To investigate the expression of migration-inducing gene 7 (MIG7) in different HCC lines and its relationship with vasculogenic mimicry (VM) formation and metastatic potentiality. METHODS: Employing immunostaining to detect MIG7 protein expression and VM formation in 40 matched pairs of primary and metastatic HCC specimens from 40 patients, and investigating the correlation of VM formation with MIG7 protein expression. Detecting VM formation in HCC lines with different metastatic ability (MHCC-97H, MHCC-97L, Huh-7) and human normal hepatocyte line (L-02) through three-dimensional culture, and detecting MIG7 mRNA expression with RT-PCR, investigating the correlation of MIG7 protein expression with VM formation and HCC metastatic potentiality with Western blot assay; screening the HCC cell line with high MIG7 expression. RESULTS: In 40 matched pairs of HCC tissue, there was a significant positive correlation between MIG7 protein expression and VM formation ( rs=0.595, P<0.01). The capability of VM formation of MHCC-97H with high metastatic potentiality was stronger than that of MHCC-97L with low metastatic potentiality and Huh-7 with non-metastatic potentiality, and there was no VM formation in L-02. The result of RT-PCR and Western blot assay indicated the same. CONCLUSIONS: MIG7 expression in HCC tissue is high and correlated positively with VM formation. MIG7 expression in different HCC cell lines is coincident with theirs VM formation, invasion and metastasis. MIG7 is a potential target for inhibiting the invasion and metastasis of HCC.

Berbamine ameliorates ethanol-induced liver injury by inhibition of hepatic inflammation in mice.

Alcoholic liver disease (ALD) has become one of the leading causes of death in the world. Berbamine (BM), a natural product mainly derived from Berberis vulgaris L, possesses multiple bioactivities as a traditional medicine. However, the protective effect of BM on ALD remains unknown. In this study, we investigated the effect of BM on ethanol-induced hepatic injury in mice and its underlying mechanism. It was shown that BM at 0.3125-40 µmol·L-1had no effect on macrophages and hepatocytes proliferation. BM at 5-20 µmol·L-1 significantly inhibited lipopolysaccharide (LPS) or acetate-induced IL-1ß and IL-6 mRNA expression in RAW264.7 cells. Moreover, BM treatment significantly inhibited LPS-induced p65 and STAT3 phosphorylation in RAW264.7 cells. Hepatic histopathology analysis showed that inflammatory cells infiltration and lipid accumulation were suppressed by 25 and 50 mg·kg-1 BM administration in ethanol-induced hepatic injury mouse model. Meanwhile, BM treatment significantly inhibited serum ALT and AST levels in ethanol-fed mice. Oil red O staining results showed that BM administration ameliorated hepatic lipid accumulation in ethanol-fed mice. Preventions of ethanol-induced hepatic injury by BM were reflected by markedly decreased serum and hepatic triglyceride (TG) and total cholesterol (TC) contents. Real-time PCR results showed that BM treatment significantly inhibited pro-inflammatory cytokines mRNA expression in ethanol-fed mouse liver. Remarkably, the mechanism of action of BM was related to the reduction of ethanol-induced NF-κB and STAT3 phosphorylation levels in liver. In addition, BM treatment significantly inhibited ERK phosphorylation but not JNK and p38 of MAPK pathway. Taken together, our results demonstrate a beneficial effect of BM on ethanol-induced liver injury via a mechanism associated with inactivation of NF-κB, STAT3 and ERK pathway, which gives insight into the further evaluation of the therapeutic potential of BM for ALD.

[Fluorescence spectra and imaging of Platymonas subcordiformis via LSCM].

Primary investigation of Platymonas subcordiformis was performed with laser scanning microscopic technology. Both autofluorescence spectra and autofluorescence imaging of Platymonas subcordiformis were achieved by one photon excitation of 488 nm Ar+ laser. Autofluorescence images revealed cup-shaped object inside the Platymonas subcordiformis cell, and the corresponding fluorescence peak located at 682 nm was attributed to chloroplast. Moreover, in single channel mode, there existed a round-shaped object in chloroplast center, showing strong fluorescence by 800 nm femtosecond laser excitation. Also, the authors obtained both the individual cup-shaped chloroplast and round-shaped object image and the overlaid images by using dual-channel mode. Meanwhile, six main fluorescence peaks were found. Single photon excitation can be used to obtain autofluorescence image and autofluorescence spectra of Platymonas subcordiformis, while two photon excitation combined with multitrack mode and Lambda mode can be used not only to observe internal structure, but also for the analysis of existence of biological and chemical substances with higher sensitivity than single photon excitation. LSCM technique can be a promising tool for rapid, convenient, real-time and effective investigation in ocean algae.

[Two-photon excitation fluorescence of 5-ALA induced PpIX in DHL cells].

Two-photon fluorescence microscopy is a novel imaging technique, which is primarily sensitive to a specimen's response coming from an in-focus plane, thus has low photo-bleaching and photo-damage to biological samples. 5-ALA induced production of PpIX in DHL cells was excited by 820 nm femtosecond laser; two-photon excitation fluorescence of single cell was obtained in Lambda mode of laser scanning confocal microscope. The specific fluorescence intensity of PpIX which accumulated in DHL cells was measured at 2, 4 and 10 mmol x L(-1) concentration of 5-ALA with different incubation time, which reflected the kinetics of 5-ALA accumulated in DHL cells. Accumulation of PpIX in DHL cells was a dynamic change process. Biphasic alterations of PpIX accumulation were noted: PpIX content enhanced with the increasing time and reached the maximal value around 3 h, however PpIX content decreased in the subsequent incubation time. Results indicate that two-photon fluorescence based on laser scanning microscope can be a useful technology for studying the kinetics of 5-ALA induced PpIX production in DHL cells and other leukemia cells.

[Inhibitory effect of Mig-7 silencing by retrovirus-mediated shRNA on vasculogenic mimicry, invasion and metastasis of human hepatocellular carcinoma cells in vitro].

OBJECTIVE: To explore the inhibitory effect of migration-inducing gene 7 (Mig-7) gene silencing induced by retroviral-mediated small hairpin RNA (shRNA) on vasculogenic mimicry (VM), invasion and metastasis of human hepatocellular carcinoma (HCC) cells in vitro. METHODS: Two target sequences (Mig-7 shRNA-1 and Mig-7 shRNA-2) and one negative control sequence (Mig-7 shRNA-N) were synthesized. The recombinant retroviral vectors carrying Mig-7 shRNA were constructed, and HCC cell line MHCC-97H were transfected with Mig-7 shRNA-1, Mig-7 shRNA-2, Mig-7 shRNA-N, or the empty vector, or treated with 125 µg/mL recombinant human endostatin (ES). Mig-7 expression in the treated cells was detected using semi-quantitative PCR and Western blotting. The inhibitory effect of Mig-7 silencing on VM formation was investigated in a 3-dimensional cell culture system; the changes in cell adhesion, invasion and migration were assessed with intercellular adhesion assay, Transwell invasion assay and Transwell migration assay, respectively. RESULTS: The expression of Mig-7 at both mRNA and protein levels decreased significantly, VM formation, invasion and metastasis were suppressed, while intercellular adhesion increased significantly in MHCC-97H cells in Mig-7 shRNA-1 and Mig-7 shRNA-2 groups (P<0.05); such changes were not observed in cells transfected with Mig-7 shRNA-N or the empty vector, nor in cells treated with ES. CONCLUSIONS: Mig-7 silencing by retroviral-mediated shRNA significantly inhibits VM formation, invasion and metastasis and increases the intercellular adhesion of the HCC cells, while ES does not have such inhibitory effects.

Inhibition of hepatic cells pyroptosis attenuates CLP-induced acute liver injury.

Pyroptosis is a programmed cell death associated with caspase-1 and accompanied by the secretion of a large number of pro-inflammatory cytokines. In the acute stage of sepsis, the release of several pro-inflammatory cytokines aggravates hepatic cell death, and acute liver injury is aggravated with the progress of the disease, resulting in acute liver failure with a very high mortality rate. The present study investigated the effect of inhibiting hepatic cell pyroptosis on the septic acute liver injury. Septic acute liver injury mice model was established by cecal ligation and puncture (CLP model). The liver tissues were assessed for inflammatory infiltration by HE, serum concentrations of ALT, AST, IL-1ß, and IL-18 were examined by ELISA, hepatic cell pyroptosis was determined by flow cytometry, and expressions of caspase-1 and NLRP3 were assessed by Western blot. CLP-induced acute liver injury was distinct at 24 h post-operation, with the highest hepatic cell pyroptosis rate. The pyroptosis rate and liver injury indexes were positively correlated. Western blot showed that the expressions of pyroptosis-related proteins, caspase-1, and NLRP3, were increased. Normal mouse hepatic cells were cultured in vitro and LPS+ATP introduced to establish the cell model of septic acute liver injury. The expressions of caspase-1, NLRP3, IL-1ß, and IL-18 in LPS+ATP group were significantly higher than the control group by Western blot and ELISA. The inhibitors of NLRP3 (Glyburide) and caspase-1 (AC-YVAD-CMK) alone or in combination were used to pre-treat the hepatic cells, which revealed that the pyroptosis rate was decreased and the cell damage alleviated. The in vivo assay in rats showed that post inhibitor treatment, the 10-days survival was significantly improved and the liver damage reduced. Therefore, inhibiting the hepatic cell pyroptosis could alleviate CLP-induced acute liver injury, providing a novel treatment target for septic acute liver injury.

Evaluation of collagen alteration after topical photodynamic therapy (PDT) using second harmonic generation (SHG) microscopy--in vivo study in a mouse model.

Topical photodynamic therapy (PDT) mediated by 5-aminolevulinic acid (ALA) has been used for the treatment of age-related skin lesions for therapeutic or cosmetic purposes. The modulation of collagen component and structure might play a significant role in influencing treatment outcomes of PDT. In this study, the effect of multi-session low dose ALA PDT on skin rejuvenation was examined in a hairless mouse model. Changes in collagen and skin texture were investigated by histological examination and in vivo second harmonic generation (SHG) microscopy. Results indicated that multi-session PDT could improve the collagen density and arrangement of skin tissue. SHG microscopy combined with quantitative collagen analysis could provide a useful tool for the evaluation of collagen alteration induced by ALA PDT.