Degree and distribution of substitution of hydroxypropyl-ß-cyclodextrin in enantioselective liquid-liquid extraction and countercurrent chromatographic enantioseparation.

Nine types of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) with different degrees and distributions of substitution were synthesised, and nine racemates were selected to investigate the effect of different degrees and distributions of substitution of HP-ß-CD on the enantioseparation factor. 1H NMR and GC/MS were used to characterise the synthesised HP-ß-CD. The degree and distribution of substitution had a significant influence on enantioselective liquid-liquid extraction and enantioseparation by countercurrent chromatography. For most of the tested racemates, increasing both the degree of substitution and distribution of substitution at the C-2 position for HP-ß-CD would lead to an increasing enantioseparation factor; the optimal enantioseparation factor of 2-phenylbutyric acid, tropic acid, 2,3-diphenylpropionic acid, 2-(4-hydroxylphenyl) propanoic acid, and naproxen was increased to 1.77, 1.53, 1.67, 1.61, and 1.75, respectively. The enantioseparation of racemic naproxen, 2-(4-hydroxylphenyl) propanoic acid, and 2,3-diphenylpropionic acid by countercurrent chromatography was optimised using HP-ß-CD with a degree of substitution of 16.5, and peak resolution was significantly improved to 1.03, 1.35, and 1.01, respectively.

High-performance liquid chromatography micro-fraction bioactive evaluation combined with countercurrent chromatographic separation of antioxidants from Citrus peel and their tyrosinase inhibition activities.

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.

Analytical enantioseparation of N-alkyl drugs by reversed-phase liquid chromatography with carboxymethyl-ß-cyclodextrin as mobile phase additive.

Carboxymethyl-ß-cyclodextrins (CM-ß-CDs) with five kinds of degrees of substitution were synthesized and characterized. Analytical enantioseparation of six basic drugs containing N-alkyl groups, including pheniramine, chlorpheniramine, labetalol, propranolol, venlafaxine, and trans-paroxol, was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) using the synthesized CM-ß-CD as chiral mobile phase additives. Key influence factors were optimized, including organic modifier, pH value, CM-ß-CD with different degrees of substitution, and concentration of CM-ß-CD. The mobile phase was composed of methanol and 10 mmol L-1 of phosphate buffer pH 4.0 containing 10 mmol L-1 of CM-ß-CD. Peak resolution for six racemic drugs was gradually increased with an increasing degree of substitution of the synthesized CM-ß-CD. The stoichiometric ratio and binding constants for the inclusion complex formed by CM-ß-CD and enantiomer were determined, which showed that the stoichiometric ratio for each inclusion complex was 1:1.

Solvent strength of organic phase for two biphasic solvent systems in high speed countercurrent chromatography.

In this work, relationships between solvent strength of organic phase (ψ) for two biphasic solvent systems in high speed countercurrent chromatography, hexane-ethyl-acetate-methanol-water (HEMWat) and ethyl acetate-n-butanol-water (EBuWat), and partition coefficient (K) were investigated using four retention models, including Jandera's model (ABM), Neue-Kuss model (NK), linear-solvent-strength model (LSS) and quadratic-solvent-strength model (QSS). Experimental results showed that ABM model had the best fitting results for HEMWat system while NK model and QSS model had good fitting results in EBuWat system. Thus, a mathematical relationship between partition coefficient (K) and solvent strength of organic phase (ψ) could be obtained by measurement of partition coefficients of the target compounds with three different volume ratios of organic phase. At the same time, a functional map was proposed to construct to get a maneuverable region so that an optimal two-phase solvent system for separation of a target compound could be selected easily, which saved a lot of manpower for high speed countercurrent chromatographic separation. The application of this new method was declared by successful separation of two components, apigenin-6-C-ß-D-xylopyranosyl-8-C-α-L-arabinopyranoside and vicenin-3, from dried leaves of Dendrobium officinale Kimura et Migo using high speed countercurrent chromatography.

Enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography using anionic ß-cyclodextrin as chiral selector.

Two anionic ß-cyclodextrins as chiral selectors were successfully applied in the enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography. Sulfobutyl ether-ß-cyclodextrin and carboxymethyl-ß-cyclodextrin showed opposite enantioselectivity for both duloxetine and N-methyl duloxetine enantiomers. Two biphasic solvent systems, n-hexane: 0.1 mol/L phosphate buffer pH 7.6 with 50 mmol/L of sulfobutyl ether-ß-cyclodextrin (1:1, v/v) and n-hexane: 0.1 mol/L phosphate buffer pH 7.2 with 50 mmol/L of carboxymethyl-ß-cyclodextrin (1:1, v/v), were selected for N-methyl duloxetine. Enantioseparation of duloxetine was achieved by recycling countercurrent chromatography using a solvent system composed of n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.2 with 20 mmol/L of sulfobutyl ether-ß-cyclodextrin or carboxymethyl-ß-cyclodextrin (1:1, v/v). A solvent system composed of n-hexane: n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.6 containing 20 mmol/L of sulfobutyl ether-ß-cyclodextrin (6:4:10, v/v) was selected for enantioseparation of fluoxetine.

Enantioseparation of five racemic N-alkyl drugs by reverse phase HPLC using sulfobutylether-ß-cyclodextrin as a chiral mobile phase additive.

Analytical enantioseparations of five N-alkyl drugs, fluoxetine hydrochloride, labetalol, venlafaxine hydrochloride, trans-paroxol, and atropine sulfate, were investigated by reverse phase high-performance liquid chromatography with sulfobutylether-ß-cyclodextrin as chiral mobile phase additive. Effects of various factors such as composition of mobile phase, concentration of cyclodextrins, and column temperature on retention and enantioselectivity were studied. Apparent formation constant between methanol, acetonitrile, and sulfobutylether-ß-cyclodextrin were determined to be 2.90 × 10-3 and 1.00 × 10-4 L mmol-1 under 25°C using UV-spectrophotometry. Van't Hoff plots were used to investigate thermodynamic parameters for enantiomers-stationary phase interaction and formation of inclusion complex. Two retention models were employed individually for evaluation of inclusion complexation between five racemates and sulfobutylether-ß-cyclodextrin. The second model with complex adsorption was more accord with the retention behavior of fluoxetine hydrochloride, labetalol, and venlafaxine hydrochloride enantiomers, while the first model was more consistent with the retention behaviors of trans-paroxol and atropine sulfate. In the selected mobile phase, stoichiometric ratio for both of inclusion complex was found to be 1:1.

Solvent strength of aqueous phase for two typical biphasic solvent systems in high-speed countercurrent chromatography.

In the present work, influence of solvent strength of aqueous phase for two frequently-used biphasic solvent system in partition coefficient (K) of selected solutes were mainly studied, and a new method for selection of biphasic solvent system was proposed for high-speed countercurrent chromatographic separations. Solvent strength was referred to the typical theory that was deeply investigated in conventional reversed-phase liquid chromatography. Experimental results showed that a linear relationship between log(K) of solutes and apparent content of methanol in biphasic solvent system was found for the biphasic solvent system hexane-ethyl acetate-methanol-water (HEMWat), which was consistent with the relationship between real content and apparent content of methanol in this system. Meanwhile, a quadratic relationship was found between log(K) of solutes and apparent content of methanol in biphasic solvent system chloroform-methanol-water (ChMWat), in which it was found that the relationship between real content and apparent content of methanol in this system was also quadratic. In addition, a visual and simple method was proposed to select a suitable biphasic solvent system for separation of target compounds by high-speed countercurrent chromatography with isocratic elution, which saves a lot of manpower and material resources in order to find a suitable two-solvent system. An optimal biphasic solvent system for isolation of several tested compounds by high-speed countercurrent chromatography was easily obtained using our proposed method.

Cannulation Selection of Portal Venous and Splenic Venous Catheterization in Venovenous Bypass of Swine Orthotopic Liver Transplantation.

BACKGROUND The aim of this study was to compare the hemodynamic changes in 2 different cannulations in portal system (portal venous catheterization and splenic venous catheterization) during venovenous bypass (VVB) of swine orthotopic liver transplantation (OLT) MATERIAL AND METHODS Thirty pairs (a total of 60) of healthy Duroc pigs were selected for OLT. According to the difference of cannulation in portal venous system during VVB, these pigs were divided into 2 groups: the PVC group (pigs with portal venous catheterization, n=15) and the SVC group (pigs with splenic venous catheterization, n=15). Intraoperative hemodynamic parameters were monitored continuously. RESULTS Two recipients in the PVC group died: 1 died of unsmooth bypass during the operation and 1 died of disseminated intravascular coagulation (DIC). There was only 1 death in the SVC group, due to hemorrhagic shock. The duration of anhepatic phase (AP) in the SVC group was significantly shorter than in the PVC group (P<0.05). Moreover, hemodynamic parameters in phase III (5 min after start of portal vein suturing) and phase IV (5 min after graft reperfusion) were remarkably different between the SVC group and the PVC group (P<0.05). CONCLUSIONS Our results show that VVB via splenic venous catheterization in swine OLT: 1) shortens the AP time; 2) keeps the hemodynamics stable; and 3) reduces the occurrence of postoperative complications. Thus, SVC appears to be superior to PVC.

The effects of Twist-2 on liver endotoxin tolerance induced by a low dose of lipopolysaccharide.

Endotoxin tolerance is an important mechanism for preventing uncontrolled inflammatory cytokine production in bacterial sepsis. However, its molecular mechanisms remain largely unknown. It was reported that Twist-2 protein was a negative regulator for cytokine signaling by repressing the nuclear factor (NF)-κB-dependent cytokine pathway. However, the relationship between Twist-2 and endotoxin tolerance is unclear. Endotoxin tolerance models of BABL/c mice and isolated Kupffer cells (KCs) were established to observe the changes of Twist-2 during endotoxin tolerance. Then, Twist-2 shRNA was used to specifically inhibit Twist-2 gene in KCs to further explore the role of Twist-2 in endotoxin tolerance. The expression of Twist-2 was analyzed by immunohistochemistry, reverse transcription polymerase chain reaction, and Western blotting, respectively. The responses to lipopolysaccharide were assessed by the activation of nuclear factor-κB and the production of tumor necrosis factor-α. The histopathologic changes in the liver of the non-endotoxin tolerance group were more serious than those of the endotoxin tolerance group. Endotoxin tolerance also led to less activation of nuclear factor-κB, lower expression levels of tumor necrosis factor-α mRNA, and more expression of Twist-2 than those of non-endotoxin tolerance group in liver and KCs. Moreover, the inhibitive effects partly weaken in KCs transfected with Twist-2 shRNA. Twist-2 was involved in endotoxin tolerance through inhibiting NF-κB trans-activation and cytokines transcriptional activities. It may be a new target for the clinical treatment of sepsis and other inflammatory diseases.

Taurine attenuates liver injury by downregulating phosphorylated p38 MAPK of Kupffer cells in rats with severe acute pancreatitis.

This study was undertaken to clarify the effects of taurine on liver injury in rats with severe acute pancreatitis (SAP). Rats were randomly assigned to three groups: a sham operation (SO), a SAP (established by infusion of 5% taurocholate), and a SAP given taurine (Taur). At 12 and 24 h post-operation, taurine pretreatment significantly attenuated hepatic tissue injury induced by SAP, and concurrently, serum alanine aminotransferase, aspartate transaminase, and amylase levels were significantly reduced by taurine pretreatment. Compared with the SO group, the total and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) expression and nuclear factor-κB (NF-κB) activity of Kupffer cells (KCs) were significantly higher in the SAP group, but taurine pretreatment inhibited the total and phosphorylated p38 MAPK expression and NF-κB activity of KCs in the SAP group. The increase of tumor necrosis factor-α and interleukin-lß in cultured supernate of the SAP rat-derived KCs was also significantly inhibited by taurine pretreatment. These results suggest that taurine pretreatment ameliorated liver injury in rats with SAP mainly by inhibiting phosphorylated p38 MAPK and NF-κB activity in KCs, which may play an important role in liver injury.

[Role of glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL) on lipopolysaccharide indu-ced Kupffer cells apoptosis].

AIM: To study the role of glucocorticoid-induced tumor necrosis factor-related protein ligand (GITRL) on apoptosis of mouse Kupffer cells (KCs) induced by lipopolysaccharide (LPS). METHODS: The KCs were isolated from BALB/c mice and transfected with Control siRNA or GITRL siRNA for 24 h. The KCs were randomly divided into four groups including control group: cultured in media alone, dexamethasone (Dex) group: media with Dex 10 µmol/L, LPS group: media with LPS 1 mg/L, and LPS+Dex group: media with LPS 1 mg/L and Dex 10 µmol/L. At 24 h after treatment, the expression of GITRL was detected by immunocytochemistry. The apoptosis of KCs was measured by Annexin V-FITC/PI double staining and FCM. RESULTS: The GITRL expression of KCs was increased by LPS challenge (P < 0.05), whereas Dex treatment attenuated the increase. LPS challenge induced KCs apoptosis, but the LPS induced apoptosis was inhibited by GITRL siRNA transfection or Dex treatment (P < 0.05, respectively). CONCLUSION: LPS could induce mouse KCs apoptosis, which may be depend on GITRL signal transduction.

Diagnosis and management of choledochoduodenal fistula.

Recent advances in hepatobiliary imaging techniques have led to the increased detection of choledochoduodenal fistula. However, the diagnosis and treatment of choledochoduodenal fistula is still a challenge. In this study, we summarize how patients were diagnosed and treated for choledochoduodenal fistula at our institution. Sixty-six patients with choledochoduodenal fistula were diagnosed and treated in our department from January 2000 to June 2009. Sixty-one patients were treated operatively, whereas five patients were treated with medicine. Patients with choledochoduodenal fistula were confirmed by endoscopic retrograde cholangiography. Of the 61 patients needing surgical intervention, clinical outcomes were excellent in 57 patients, and five patients underwent successful laparoscopic surgery for repairing the choledochoduodenal fistula. Follow-up of these patients for 6 months to 10 years showed they did not suffer from further cholangitis. A patients' past history of biliary disease, upper abdominal pain, fever, and jaundice may lead to choledochoduodenal fistula. Operative therapy, including laparoscopic surgery, was the primary treatment for most patients, regardless of the preoperative diagnosis.

Up-regulation of IRAK-M is essential for endotoxin tolerance induced by a low dose of lipopolysaccharide in Kupffer cells.

BACKGROUND: Endotoxin tolerance (ET) is an important mechanism to maintain the homeostasis of Kupffer cells (KCs), because KCs are continually exposed to various pathogen-associated molecular patterns including lipopolysaccharide (LPS). ET involves multiple changes in cell signal transduction pathways; however, not all signaling pathways are down-regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including interleukin-1 receptor-associated kinase M (IRAK-M) expression. The aim of this study is to clarify weather or not IRAK-M is involved in the mechanisms of ET in KCs through dampening nuclear factor-kappa B (NF-kappaB) mediated pathway. MATERIALS AND METHODS: KCs isolated from male C57BL/6J mice were seeded in 24-well plates at 1 x 10(6) cells/well and cultured overnight prior to transfection, were randomly divided into two groups: the pIRAK-M-short hairpin RNA (shRNA) group (transfected with IRAK-M shRNA) and the control group (transfected with control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group (incubation in Dulbecco's modified Eagle's medium [Invitrogen, Carlsbad, CA] with 10% fetal bovine serum) and endotoxin pretreatment group (incubation in the same medium containing 10 ng/mL LPS), named pIRAK-M-EP, pIRAK-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h. The expression of IRAK-M gene and protein level were determined by reverse transcription-polymerase chain reaction and Western blot, the activities of NF-kappaB were estimated by electrophoretic mobility shift assay and enzyme-linked immunosorbent assay, and the supernatant tumor necrosis factor-alpha levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: The recombinant plasmid of pIRAK-M-shRNA specifically inhibited IRAK-M expression after it was transfected into KCs. At 3 h after 100 ng/mL LPS was added to the medium, IRAK-M expression was significantly induced in pCV-EP than that in pCV-NEP; however, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, accompanied with lowest level of NF-kappaB activation and tumor necrosis factor-alpha levels in pCV-EP, and a dramatic enhancement in the other three groups (P < 0.01). CONCLUSIONS: Although a primary low dose of LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted in pIRAK-M-EP than in pCV-EP, indicating that the absence of IRAK-M caused abnormal enhancement of inflammatory effects. IRAK-M negatively regulates toll-like receptors signaling and involves in the mechanisms of ET in KCs through dampening NF-kappaB mediated pathway; therefore it may be a key component of this important control system, and a new target for the clinical treatment of sepsis.

Glycine blunts transplantative liver ischemia-reperfusion injury by downregulating interleukin 1 receptor associated kinase-4.

AIM: To determine whether glycine could downregulate interleukin 1 receptor associated kinase-4 (IRAK-4) expression to interfere with lipopolysaccharides (LPS) signal transduction and blunt transplantative liver ischemia-reperfusion injury (I/RI). METHODS: SD rats were randomly divided into two groups: donor animals of the glycine group (n=40) were given glycine (1.5 mL; 300 mmol/L, iv) 1 h before harvest, and the control group were treated with 1.5 mL physiological saline (n= 40). Orthotopic liver transplantation was then performed according to the Kamada technique. Ten animals in each group were followed up for 7 d after surgery to assess survival. The remaining animals in each group were divided into 3 subgroups (n=10) at 1h, 2 h and 6 h after portal vein reperfusion. Levels of LPS, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin in portal circulation, as well as IRAK-4 and TNF-alpha expression, NF-kappaB transcriptional activity and morphological study of liver tissues were analyzed. RESULTS: Reperfusion resulted in a significant elevation of LPS concentrations in each group persisting to the end of our study. However, glycine, which led to improved survival rate and liver function, significantly alleviated liver parenchyma cell damage by downregulating IRAK-4, TNF-alpha expression and NF-kappaB transcriptional activity compared with the control group. CONCLUSION: Glycine can attenuate hepatic I/RI by downregulating IRAK-4 to interfere with LPS signal transduction.

[The influence with block the endotoxin signal transduction for ischemia/reperfusion injury of graft liver in rats].

OBJECTIVE: To explore the feasibility of interleukin 1 receptor associated kinase-4 (IRAK-4) as gene therapy target for liver ischemia/reperfusion injury (I/RI) and effective approach in vivo for short hairpin RNA (shRNA) interference used to gene therapy in liver graft hqappened. METHODS: Sprague-Dawley rats were randomly divided into three groups: the control group, the in vivo transfection group (IVT) and the cold ischemia transfection group (CIT). Experiments of orthotopic liver transplantation were performed by two-cuff method. CIT were perfused with IRAK-4-shRNA plasmid (pSIIRAK-4) during cold ischemia phase, IVT received the equivalent volumes (2 mL) of pSIIRAK-4 after portal vein inosculated, and the control group leaved without any treatment. At 0 min, 60 min and 180 min after reperfusion, the expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The serum TNF-alpha level was detected by ELISA. Liver histopathological changes and cell apoptosis were observed by electron microscope and TUNEL. RESULTS: After reperfusion, the expression of IRAK-4 were largely depressed in CIT than that of IVT and the control group (P < 0.01), and furthermore, the serum TNF-alpha level, proportion of hepatocyte apoptosis and severity of hepatocyte injury were also lower than the latter. CONCLUSION: These results indicate that depression IRAK-4 expression with IRAK-4-shRNA through portal vein perfusion during cold ischemia phase could effectively blunt graft hepatic I/RI.

[The protection mechanisms of glycine against liver injury induced by lipopolysaccharides].

OBJECTIVE: To explore the protective mechanisms of glycine (Gly) on lipopolysaccharides (LPS) induced liver injury. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[The mechanism and treatment phases chosen of glycine for inhibition lipopolysaccharide induced Kupffer cells activation].

OBJECTIVE: To explore the possible mechanism and optimal treatment phase of glycine for inhibition lipopolysaccharide (LPS) induced Kupffer cells (KCs) activation. METHODS: 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). RESULTS: 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). CONCLUSION: The results indicated that prophylactic or simultaneous treatment with glycine could effectively inhibit LPS-induced KCs activation by inhibiting IRAK-4 expression.

[Expression changes of interleukin-1 receptor associated kinase-4 during endotoxin tolerance development in kupffer cells].

OBJECTIVE: 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). METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[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].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.

[Expression of CD14 protein in U937 cells induced by vitamin D3].

AIM: To observe the 1, 25-dihydroxy vitamin D3 (Vit D3) induced CD14 expression in human U937 cell line and the reaction of the cells to LPS stimulation following the induction. METHODS: U937 cells were cocultured with 0.1 mumol/L Vit D3 for 24 hours to induce the expression of CD14 gene and the sensitiveness of U937 cells to stimulation of LPS at various concentrations and for different times were observed. RESULTS: Vit D3 stably induced U937 cells to express CD14 mRNA and CD14 protein. The sensitivity of U937 cells to LPS stimulation increased notably after Vit D3 induction. It was demonstrated that low concentration of LPS stimulated the activation of NF-kappaB in U937/CD14 cells, and enhanced the transcription and expression of TNF-alpha gene, and even release of TNF-alpha into the culture supernatant. CONCLUSION: Vit D3 can induce U937 cells to express CD14 gene and CD14 protein and enhance the reactivity of U937/CD14 cells to LPS stimulation.