Over-expression of receptor-interacting protein 140 in tumor-associated macrophages suppresses invasion and proliferation of hepatoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of receptor-interacting protein 140 (RIP140) in tumor-associated macrophages (TAMs) in the invasion and proliferation of hepatoma cells in vitro.</p><p><b>METHODS</b>Western blotting, qRT-PCR and flow cytometry were performed to examine the effects of lentivirus-mediated RIP140 over-expression in mouse peritoneal macrophages (PMs). Western blotting, qRT-PCR and immunofluorescence staining were used to detect the expression of RIP140 in TAMs following stimulation of the PMs with hepatocellular carcinoma conditioned medium (HCM) for 24 h. The polarization index and the expression of NF-κB and IL-6 were detected using qRT-PCR in TAMs in HCM-stimulated PMs with or without RIP140 over-expression. Transwell assay and flow cytometry were used to estimate the cell invasion and apoptosis. HE staining and immunohistochemical staining were used to analyze the effects of RIP140-over-expressing macrophages on the growth and tumor formation of H22 cells in BALB/c nude mice.</p><p><b>RESULTS</b>The lentivirus vector efficiently mediated RIP140 over-expression in mouse PMs. HCM stimulation significantly inhibited RIP140 expression in the TAMs and promoted their M2-like polarization. Over-expression of RIP140 in PMs suppressed the invasion and induced apoptosis of HCC cells. RIP140 over-expression inhibited HCM-induced M2 polarization and the activation of NF-κB/IL-6 axis in the TAMs, and RIP140- overexpressing TAMs obviously suppressed the growth of H22 cell xenograft in nude mice.</p><p><b>CONCLUSION</b>Over-expression of RIP140 in TAMs suppresses the growth and proliferation of hepatoma cells possibly by inhibiting M2 polarization of the TAMs.</p>

Effect of ultrasound microbubble carrying herpes simplex virus thymidine kinase on hepatocellular carcinoma in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the effect of ultrasound microbubble carrying herpes simplex virus thymidine kinase hepatocellular carcinoma in mice.</p><p><b>METHODS</b>Kunming mice were inoculated subcutaneously with H22 tumor cells. 40 male mice bearing subcutaneous hepatoma were randomized into 4 groups: PBS (group A), HSV1-TK (group B), HSV1-TK (group C), and microbubble carrying HSV1-TK (group D) were injected into the tail vein every 3 days. Mice in group C and D were exposed to ultrasound. The expression of TK protein was detected by western blot. Ganciclovir (GCV) was intraperitoneally injected at a dose of 100 mg x kg (-1) x d(-1) in group B, group C and group D. The tumor size was measured every 2 days.</p><p><b>RESULTS</b>TK gene could be injected precisely into hepatocellular carcinoma with ultrasound monitor, and the expression of TK protein was found in all 4 groups. Expression in group D was higher than others (P < 0.05). The rate of tumor growth inhibition were 0 in group A, 3.90%+/-1.80% in group B, 22.70%+/-2.86% in group C, 41.25%+/-3.20% in group D (group B vs group C, P < 0.05; group D vs group C, P < 0.05; group D vs group B, P < 0.05).</p><p><b>CONCLUSION</b>Ultrasound microbubble not only improve target gene therapy, but also enhance transfection efficiency.</p>

Effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation (LT).</p><p><b>METHODS</b>Brown-Norway liver allografts were transplanted into Lewis recipients. The transplanted Lewis rats were injected with EK506 (2 mg/kg) and randomly divided into two groups: rats in HBsAg-DCs group (n = 15) were intraperitoneally injected with HBsAg pulsed DCs at 14 d and 28 d after LT, and rats in the HBsAg group (n = 15) were injected with HBsAg (200 mul) once a week for 12 weeks. Rats without any immunosuppressive treatment after LT served as controls (n = 5). IL-2 and IFN-gamma mRNA expression in spleen were analyzed by RT-PCR, serum IL-2, IFN-gamma and anti-HBs were detected by ELISA.</p><p><b>RESULTS</b>High dose of FK506 resulted in the immunosuppressed in LT rats, as evident by low production of IL-2 and IFN-gamma, and without liver rejection compared to rats in the control group. HBsAg-DCs induced high titer of anti-HBs antibody, however, titer of anti-HBs were seldom detectable in the HBsAg group at 1, 2 and 3 mouth after vaccination.</p><p><b>CONCLUSION</b>The capacity of HBsAg-DCs to induce anti-HBs in immunosuppressed rats suggested that DC vaccine may prevent HBV recurrence in liver transplanted patients.</p>

Expression of human triggering receptor expressed on myeloid cells 1 in peripheral blood mononuclear cells of patients with acute obstructive suppurative cholangitis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression pattern of human triggering receptor expressed on myeloid cells 1 (TREM-1) mRNA in peripheral blood mononuclear cells and its clinical significance in acute obstructive suppurative cholangitis (AOSC).</p><p><b>METHODS</b>Peripheral blood mononuclear cells were collected from 36 patients with AOSC and 40 healthy adults. TREM-1 mRNA was determined by semi-quantitative RT-PCR, and TREM-1 protein by immunocytochemistry. Enzyme-linked immunosorbent assay (TNF-alpha) was used to detect the level of tumor necrosis factor-alpha (TNF-alpha), and immunoturbidimetry employed to detect C reactive protein.</p><p><b>RESULTS</b>The expression of TREM-1 mRNA relative to beta-actin was 1.007-/+0.252 in patients with AOSC, significantly higher than that in the healthy adults (0.457-/+0.053, P<0.05). The two groups also showed significantly different TREM protein expression (P<0.01). The AOSC patients exhibited significantly higher levels of TNF-alpha and C reactive protein than the healthy adults (P<0.01).</p><p><b>CONCLUSION</b>The expression of human TREM-1 in peripheral blood mononuclear cells is up-regulated obviously in early stage of AOSC, probably suggesting an important role of TREM-1 in the development of AOSC.</p>

Intercellular transfer of P-glycoprotein in hepatocellular cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether there is intercellular transfer of functional P-glycoprotein(P-gp) from P-gp-positive cells to P-gp-negative cells in vitro.</p><p><b>METHODS</b>HepG2/GFP cells, a HepG2 cell line stably expressing GFP, were co-cultured with HepG2/ADM cells, an adriamycin-resistant cell line derived from HepG2 cells. The distribution of P-gp in hepatocellular carcinoma cell was observed under laser scanning confocal microscope (LSCM). Immunomagnetic beads were used to separate HepG2/GFP cells from the mixed culture. The abundance of P-gp was analyzed by western blot, and the expression of mdr1 mRNA was detected by qRT-PCR.</p><p><b>RESULTS</b>Yellow fluorescence was detected in HepG2/aqMDR cells, green fluorescence was detected in HepG2/GFP cells, red fluorescence was detected in HepG2/ADM cells by LSCM. The level of P-gp protein in HepG2/aqMDR cells was lower than that in HepG2/ADM cells, but higher than that in HepG2/GFP cells (q = 35.07, P < 0.05) and HepG2 cells (q = 36.87, P < 0.05). The expression of mdr1 mRNA in HepG2/ADM cells was higher than that in HepG2/aqMDR, HepG2 and HepG2/GFP cells, but there was no significant difference in mdr1 mRNA among HepG2/aqMDR, HepG2 and HepG2/GFP cells (F = 2.30, P > 0.05).</p><p><b>CONCLUSIONS</b>P-gp can transfer from drug resistant hepatocellular cells to sensitive hepatocellular carcinoma cells. This study suggests a novel mechanism of multidrug resistance in hepatocellular carcinoma.</p>

Influence of continuous high-volume hemofiltration on IRAK-4 protein expression in severe acute pancreatitis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the influence of continuous high-volume hemofiltration (CHVHF) on interleukin 1 receptor-associated kinase-4 (IRAK-4) and tumor necrosis factor-alpha (TNF-alpha) levels in patients with severe acute pancreatitis (SAP).</p><p><b>METHODS</b>Forty-one patients with SAP were randomly divided into two groups to receive treatment with CHVHF plus conventional therapy (21 patients) and conventional therapy only (20 patients). Venous blood samples were taken before and 12, 24, and 72 h after the treatment for evaluation of APACHE II scores. The mRNA and protein levels of IRAK-4 in the monocytes were determined by real-time PCR and Western blotting, respectively, and serum TNF-alpha levels was detected using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Among the 21 patients receiving CHVHF, 18 survived and 3 died, and in the conventional therapy group, death occurred in 5 cases. In the surviving patients of CHVHF group, the APACHE II scores, IRAK-4 mRNA and protein levels and TNF-alpha levels were all significantly lowered after the treatment, and these indices were also significantly lower than those in the conventional group after treatment (P<0.05).</p><p><b>CONCLUSION</b>CHVHF is effective in reducing monocyte IRAK-4 levels and serum TNF-alpha level in SPA patients, and thus alleviates the symptoms and improves the prognosis of SAP, possibly by reducing the level of the activators that induce monocyte activation via the Toll-like receptor.</p>

The protection mechanisms of glycine against liver injury induced by lipopolysaccharides / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the protective mechanisms of glycine (Gly) on lipopolysaccharides (LPS) induced liver injury.</p><p><b>METHODS</b>BABL/c mice were randomly divided into a LPS group, in which the animals were intraperitoneally injected with 10 mg/kg LPS, and a Gly group, in which the mice were pretreated with a 5% Gly-containing diet for 3 days before receiving the same dose of LPS. The livers of the mice were examined for histopathological changes. The TNF alpha and interleukin-10 (IL-10) levels in the blood plasma were measured using ELISA analysis. The mRNA expression of TNF alpha, IL-10 and Toll-like receptor 4 (TLR4) in hepatic tissues were detected using RT-PCR analysis. Protein expression of TLR4 in livers was detected using immunohistochemistry.</p><p><b>RESULTS</b>The Gly group mice had an improved survival rate and attenuated LPS-induced pathological changes in the liver tissues in comparison with those of the LPS group animals. The TNF alpha levels [(1,852.80+/-126.64) pg/ml vs (708.83+/-51.29) pg/ml, P<0.05] in plasma, as well as the expression of TNF alpha (A 1.59+/-0.14 vs. 0.91+/-0.11, P<0.05) and TLR4 (A 0.97+/-0.12 vs. 0.53+/-0.11, P<0.05) mRNA in liver tissues were decreased. However, the levels of plasma interleukin-10 [(344.09+/-31.70) pg/ml vs (418.64+/-38.86) pg/ml, P<0.05] were significantly increased and the peaking time left, shifted.</p><p><b>CONCLUSIONS</b>Gly pretreatment could attenuate LPS -induced liver injury in mice, which may be associated with its role in down-regulating TLR4 expression and up-regulating IL-10 production.</p>

The mechanism and treatment phases chosen of glycine for inhibition lipopolysaccharide induced Kupffer cells activation / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the possible mechanism and optimal treatment phase of glycine for inhibition lipopolysaccharide (LPS) induced Kupffer cells (KCs) activation.</p><p><b>METHODS</b>The KCs were isolated from 40 BALB/c mice and divided into four groups: the endotoxin group, the prevention group, the early treatment group and the later treatment group (n = 10). The endotoxin group was treated with 10 mg/L LPS, and in the other three groups, glycine (1 mmol/L) was given 24 h before, or at 0 h or 4 h respectively after LPS stimulation. At 0 h, 1 h, 2 h, 6 h and 12 h after LPS stimulation, the mRNA levels and protein expression of interleukin-1 receptor associated kinase-4 (IRAK-4) were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively, and nuclear factor-kappaB (NF-kappaB) activities as well as tumor necrosis factor alpha (TNF-alpha) levels were also detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The climax values of IRAK-4, NF-kappaB and TNF-alpha were significantly higher in the endotoxin group and the later treatment group than that in the other two groups (t = 3.17, 4.33, 2.47, 126.73, P < 0.01).</p><p><b>CONCLUSION</b>The results indicated that prophylactic or simultaneous treatment with glycine could effectively inhibit LPS-induced KCs activation by inhibiting IRAK-4 expression.</p>

Expression changes of interleukin-1 receptor associated kinase-4 during endotoxin tolerance development in kupffer cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs).</p><p><b>METHODS</b>Isolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation.</p><p><b>RESULTS</b>The ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01).</p><p><b>CONCLUSIONS</b>Pretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.</p>

An experimental study of the inhibitory effects on the activation of endotoxin-induced Kupffer cells through short hairpin RNA targeting interleukin-1 receptor associated kinase-4 gene / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells (KCs) through short hairpin RNA (shRNA) targeting interleukin-1 receptor associated kinase-4 (IRAK-4) gene.</p><p><b>METHODS</b>Two effective transfection shRNA plasmid (pSIIRAK-4-A, pSIIRAK-4-B) and one invalidated plasmids (pSIIRAK-4-C) targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups: the normal control group, the RNAi control group (pSIIRAK-4-C) and the RNAi effective group (pSIIRAK-4-A, pSIIRAK-4-B). Then KCs were stimulated with 0.1 microg/ml lipopolysaccharide (LPS) after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF-kappaB in KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h.</p><p><b>RESULTS</b>The level of IRAK-4, the activities of NF-kappaB and the TNF-alpha level in the RNAi effective group were evidently lower than those in normal and RNAi control groups (P < 0.01) at 1 h, 3 h, and 6 h. Especially, the pSIIRAK-4-A group in which the changes of the above indices were of no difference (P > 0.05), had better inhibited effects than that of the pSIIRAK-4-B group (P < 0.01).</p><p><b>CONCLUSION</b>The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.</p>

Expression of CD14 and Toll-like receptor 4 on Kupffer cells and its role in ischemia-reperfusion injury on rat liver graft / 中华外科杂志

<p><b>OBJECTIVE</b>To study the expression of lipopolysaccharide (LPS) receptor CD14 and Toll-like receptor 4 (TLR4) on Kupffer cells and its role in ischemia-reperfusion injury (IRI) on rat liver graft.</p><p><b>METHODS</b>The Kupffer cells following IRI were isolated and divided into control, ischemia-reperfusion (IR), and anti-CD14 antibody group. The mRNA and protein expression of CD14 and TLR4, nuclear factor kappa B (NF-kappaB) activity and TNF-alpha level in supernatant were measured.</p><p><b>RESULTS</b>The mRNA and protein expression of CD14 and TLR4 in IR group were significantly higher than those in control group (P < 0.01). The NF-kappaB activity and TNF-alpha level in IR group were significantly higher than those in control group (P < 0.01), and they greatly decreased after anti CD14 antibody treatment (compared with IR group, P < 0.05), but were still significantly higher than those in control group (P < 0.01).</p><p><b>CONCLUSIONS</b>LPS following IRI could up-regulate CD14 and TLR4 gene and protein expression on Kupffer cells, and subsequently activate NF-kappaB to produce cytokines, but other signal transduction pathways might also participate in the IRI.</p>