[Recent advances in curcumin and its derivatives for treatment of liver diseases].

Curcumin is a principal polyphenolic curcuminoid extracted from turmeric rhizome, which has been used for treating inflammation of joints, ulcers, jaundice and other disorders in Asian traditional medicine. In recent years, many studies have indicated that curcumin plays important roles in treatment of liver diseases. Curcumin attenuates liver injury and non-alcoholic fatty liver disease by lowering the release of inflammation cytokines, minimizing oxidative stress, enhancing the sensitivity of insulin and altering lipid metabolism. Curcumin shows potent anti-fibrosis activity, contributing to inhibit the activation of hepatic stellate cells and reduce the deposition of extracellular matrix by its regulation of PPAR-γ, NF-ΚB and TGF-ß signaling pathways. Moreover, curcumin exhibits anti-cancer effect by inducing G2/M phase cell cycle arrest and apoptosis in several hepatoma cell lines. However, poor water solubility and low bioavailability of curcumin limit its clinical applications. To overcome its limited systemic bioavailability, many new approaches have been explored to deliver curcumin effectively. This article focuses on advances in the effects of curcumin and its derivatives for treatment of liver injury, non-alcoholic fatty liver disease, liver fibrosis and hepatocarcinoma.

A novel approach for transferring oleic acid capped iron oxide nanoparticles to water phase.

A novel and simple method was described to transfer oleic acid stabilized iron oxide nanoparticles from organic solutions to water. The oxidation of OA by sodium periodate in mixed solvents formed a carboxyl group or vicinal diol to make the hydrophobic groups to hydrophilic groups on the surface of the nanoparticles. The characterization of nanoparticles indicated that the phase transfer based on the oxidation of OA was successful performed without change in the size and shape of the iron oxide nanoparticles. The hydrophilic groups on the iron oxide surface stabilized the nanoparticles in aqueous solution and the oxidized nanoparticles can be applied to bimolecular immobilization.

[The effects of curcumin derivative on experimental steatohepatitis].

OBJECTIVE: To observe the effects of curcumin derivative on non-alcoholic steatohepatitis (NASH). METHODS: 60 SD male rats were randomly divided into 5 groups. The NASH model was induced by high fat diet combined with carbon tetrachloride. These rats were then treated with curcumin and curcumin derivative, saline treating group as control. The serum biochemical parameters and liver histological examinations were observed. The TNF alpha, NF-kappa B and HMG-CoA reductase mRNA transcriptions of liver tissue were detected with RT-PCR. The protein expressions of TNF alpha and NF-kappa B were detected by western blot. RESULTS: Compared with the saline group, A remarkable reduction was observed in serum ALT (U/L), AST (U/L) and TC (mmol/L) in rats treated with curcumin derivatives [(69.20 +/- 27.58) vs (102.43 +/- 47.29), (158.00 +/- 39.15) vs (229.50 +/- 105.20) and (2.08 +/- 0.30) vs (2.58 +/- 1.02), P < 0.05]. The degrees of fibrosis were significantly alleviated; Compared with curcumin group, liver index and serum ALT, AST of curcumin derivative group were also significantly decreased [(4.88 +/- 0.62) vs (5.16 +/- 0.61); (69.20 +/- 27.58) vs (82.5 +/- 33.23); (158.00 +/- 39.15) vs (211.75 +/- 106.30), P < 0.05]; The liver steatosis and inflammation grade were also significantly improved .The gene transcriptions of TNF alpha, NF-kappa B and HMG-CoA reductase in curcumin derivative group were significantly lower than those in curcumin and saline group (P < 0.05). CONCLUSION: These results indicate that the water-soluble curcumin derivative displays superior bioavailability to the parent curcumin, which can effectively improve the lipid metabolism and delay the progression of hepatic fibrosis in rats with steatohepatitis.

Cytotoxicity of Fe3O4/Au composite nanoparticles loaded with doxorubicin combined with magnetic field.

GoldMag (Fe3O4/Au) nanoparticles have the advantages of both magnetic response in an external magnetic field and the immobilization of molecules on their surface in a single step. The cytotoxicities of GoldMag nanoparticles and GoldMag nanoparticles loaded with doxorubicin (Dox-GoldMag) combined with an external magnetic field were tested in vitro on HepG2 malignant tumor cells. The results showed that cell viability remained above 92% when using GoldMag nanoparticles at a concentration as high as 2.0 mg/ml, suggesting the biocompatibility of the nanoparticles. The IC50 (0.731 microg/ml) of the Dox-GoldMag group was higher than that (0.522 microg/ml) of the Dox group (P < 0. 05). However, the Dox-GoldMag group combined with a magnetic field had an obviously increased inhibition rate for the HepG2 cell line and the IC50 was lower than that of the Dox group (0.421 microg/ml). These results indicated that GoldMag nanoparticles loaded with doxorubicin combined with a permanent magnetic field are more cytotoxic and could be a potential targeted drug delivery system.

[Advance in the study of poly(lactide-co-glycolide) nano/microparticles as gene vector].

Biodegradable nano/microparticles of poly(D, L-lactide-co-glycolide) (PLGA) is a novel non-viral gene vector, which has many advantages, such as safety, non-immunogenicity, easy of large-scale preparation and well load-capability. Therefore, more and more attentions and researches have been paid on its application. Especially, PLGA has shown enormous potential application value and space in the field of plasmid DNA (pDNA) delivery system. On the basis of the current situation of PLGA nano/microparticles for pDNA delivery, this paper focused on summarizing the current preparation approaches and surface modified methods of PLGA particle, furthermore showing its application in gene therapy and genetic vaccine delivery. These showed that PLGA nano/microparticles have extensive prospect in the development of controlled gene delivery system.

Facile preparation of 3,4-diarylpyrroles and hydrogen by a platinum(II) terpyridyl complex.

With visible-light irradiation of the platinum(II) terpyridyl complex 1 (lambda > 450 nm), an effective photocatalytic conversion from readily available 3,4-diaryl-2,5-dihydropyrroles (2a-2e) to 3,4-diarylpyrroles (3a-3e) and hydrogen (H(2)) is achieved with high efficiency and large catalytic turnover in a homogeneous solution.

Magnetic nanoparticles based cancer therapy: current status and applications.

Nanotechnology holds a promising potential for developing biomedical nanoplatforms in cancer therapy. The magnetic nanoparticles, which integrate uniquely appealing features of magnetic manipulation, nanoscale heat generator, localized magnetic field and enzyme-mimics, prompt the development and application of magnetic nanoparticles-based cancer medicine. Considerable success has been achieved in improving the magnetic resonance imaging (MRI) sensitivity, and the therapeutic function of the magnetic nanoparticles should be given adequate attention. This work reviews the current status and applications of magnetic nanoparticles based cancer therapy. The advantages of magnetic nanoparticles that may contribute to improved therapeutics efficacy of clinic cancer treatment are highlighted here.

[Establishing a L02 cell model with whole HBV gene transfection and analyzing its expressions of HLA-A, B, C and MICA/B].

OBJECTIVE: To establish a cell model with HBV secretion by plasmid transfection with whole HBV genes into hepatic L02 cells, and to analyze the effect of HBV on the expression of HLA-A, B, C and MICA/B in L02 cells. METHODS: The mock control plasmid was built by digesting pEcob6 plasmid with EcoR I at the HBV DNA site and ligating the fragment without HBV DNA. The L02 cells were transfected with pEcob6 or mock plasmid and pcDNA3 1-neo by liposome. The expressions of HBsAg, HBcAg/HBeAg and HBV DNA were detected by immunofluorescence assay, Abbott enzymoimmunoassay, or FQ-PCR. The expressions of HLA-A, B, C and MICA/B were determined by FACS and the differences in the two molecules were analyzed. RESULTS: After the transfected cells were selected by G418, the HBsAg and HBeAg in the supernatant of L02-HBV cells were 24.78(S/N) and 4.117(S/N). The quantity of HBV DNA was 9.67 x 10(4) copies/ml, but in L02-mock and in L02 cells they were all negative. Under a confocal microscope HBsAg was brightly shown in the cytoplasm while HBcAg showed dimly in the cytoplasm or in the nuclei. By using FACS, the L02 and L02-mock cells showed some low expressions of HLA-A, B, C and a little expression of MICA/B, while the expression of the two molecules was higher in L02-HBV and the differences were significant (P < 0.05). CONCLUSION: A cell model with the expression of HBV antigens and the secretion of HBV DNA was established by pEcob6 transfection into L02 cells, the transcription or replication of HBV gene might induce stronger expressions of HLA-A, B, C and MICA/B on hepatic cells. They might be related to the immune injuries of hepatic cells.

[Immune response to HSP70-HBcAg(18-27) complex in HBV transgenic mice].

OBJECTIVES: To study the cellular immune response to HSP70-HBcAg(18-27) complex in HBV transgenic mice. METHOD: HSP70-HBcAg(18-27) complex was reconstituted in vitro, then it was injected into HBV transgenic mice to observe the cellular immune response. At the same time, we investigated whether HSP70-HBcAg(18-27) complex could generate antigen specific cytotoxic T lymphocyte responses in spleen cells. RESULTS: Our results demonstrated that HSP70-HBcAg(18-27) complex increased levels of CD4+ and CD8+ T cells in the spleens and peripheral blood of HBV transgenic mice, and the complex also activated dendritic and natural killer cells. CONCLUSION: HSP70-HBcAg(18-27) complex has an immunological antigenicity in raising the immunoresponse to chronic HBV infection in HBV transgenic mice. HSP70-HBcAg(18-27) complex might be considered as a candidate for further studies on its role as a therapeutic vaccine against chronic HBV infection in humans.

Ultrasmall Ferrite Nanoparticles Synthesized via Dynamic Simultaneous Thermal Decomposition for High-Performance and Multifunctional T1 Magnetic Resonance Imaging Contrast Agent.

Large-scale synthesis of monodisperse ultrasmall metal ferrite nanoparticles as well as understanding the correlations between chemical composition and MR signal enhancement is critical for developing next-generation, ultrasensitive T1 magnetic resonance imaging (MRI) nanoprobes. Herein, taking ultrasmall MnFe2O4 nanoparticles (UMFNPs) as a model system, we report a general dynamic simultaneous thermal decomposition (DSTD) strategy for controllable synthesis of monodisperse ultrasmall metal ferrite nanoparticles with sizes smaller than 4 nm. The comparison study revealed that the DSTD using the iron-eruciate paired with a metal-oleate precursor enabled a nucleation-doping process, which is crucial for particle size and distribution control of ultrasmall metal ferrite nanoparticles. The principle of DSTD synthesis has been further confirmed by synthesizing NiFe2O4 and CoFe2O4 nanoparticles with well-controlled sizes of ∼3 nm. More significantly, the success in DSTD synthesis allows us to tune both MR and biochemical properties of magnetic iron oxide nanoprobes by adjusting their chemical composition. Beneficial from the Mn2+ dopant, the synthesized UMFNPs exhibited the highest r1 relaxivity (up to 8.43 mM-1 s-1) among the ferrite nanoparticles with similar sizes reported so far and demonstrated a multifunctional T1 MR nanoprobe for in vivo high-resolution blood pool and liver-specific MRI simultaneously. Our study provides a general strategy to synthesize ultrasmall multicomponent magnetic nanoparticles, which offers possibilities for the chemical design of a highly sensitive ultrasmall magnetic nanoparticle based T1 MRI probe for various clinical diagnosis applications.

Switch between charge transfer and local excited states in 4-aminophenyl-substituted Hantzsch 1,4-dihydropyridine induced by pH change and transition metal ions.

The absorption and fluorescence spectra of a Hantzsch 1,4-dihydropyridine derivative bearing a N,N-dimethylaminophenyl group at 4-position (H(2)Py-PhN(CH(3))(2)) in aprotic solvents have been examined and compared to model compounds 4-phenyl- and 4-methyl-substituted Hantzsch 1,4-dihydropyridines (H(2)Py-Ph and H(2)Py-Me). While H(2)Py-Ph and H(2)Py-Me show fluorescence around 420 nm from the local excited state of the dihydropyridine chromophore, H(2)Py-PhN(CH(3))(2) exhibits fluorescence around 520 nm from the intramolecular charge transfer (ICT) state involving the aniline and dihydropyridine groups as donor and acceptor, respectively. Upon addition of an acid to the solution of H(2)Py-PhN(CH(3))(2), the amino group in the aniline is protonated. Thus, the photoinduced intramolecular charge transfer is prevented, and only the fluorescence from the local excited state of the dihydropyridine chromophore can be detected. These changes in the fluorescence behavior are fully reversible: subsequent addition of a base to the acidic solution leads to the recovery of the ICT fluorescence and the quenching of the local fluorescence. Transition metal ions also can switch the fluorescence of H(2)Py-PhN(CH(3))(2). Evidence for the interaction between transition metal ions and the amino group in the dimethylaniline have been provided by absorption and emission spectrum as well as NMR studies.

[Immune adjuvant effect of TB.HSP70 to its accompanying cytotoxic T lymphocytes epitope elicits HBV specific immune response].

OBJECTIVE: To investigate whether Mycobacterium tuberculosis heat shock protein 70 (TB.HSP70) can be used as an adjuvant carrier to stimulate immune response to an accompanying cytotoxic T lymphocytes (CTL) epitope peptide from hepatitis B virus (HBV) core antigen. METHODS: In vitro, peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B volunteers were stimulated with TB.HSP70-CTL fusion protein and TB.HSP70/CTL complex, and then HBV specific CTL activity was assessed. In vivo, the CD4+ T, CD8+ T and natural killer cells (NKs) in the peripheral blood and in spleens of the immunized mice were measured by flow cytometry and the protective HBV specific immune responses of the mice were also evaluated. RESULTS: The results revealed that both of them could induce HBV specific CTL response in human PBMCs and in the immunized mice. In the mice they activated CD4+ T, CD8+ T and NKs. Furthermore, the immunocompetence of the TB.HSP70-CTL fusion protein was stronger than that of TB.HSP70/CTL complex. The HBV specific killing rate was 28.9%, the CD8+ T cell population was 43.9% and the NKs was 13.6% in splenocytes of immunized mice with TB.HSP70-CTL fusion protein. The CTL peptide alone was capable of generating weak CTL lysis. The TB.HSP70 showed almost no target cell killing. CONCLUSION: The results demonstrate that TB.HSP70 may be used as a new adjuvant carrier to improve the immunogenicity of short CTL epitope and produce effective CTL response.

Application of a Designed Synthetic Ligand for the Separation and Purification of Alkaline Phosphatase.

An artificial affinity ligand comprising a p-aminobenzyl phosphoric acid as inhibitor was designed and coupled to an agarose support by chlorotriazine ring to separate and purify alkaline phosphatase from calf intestine. The optimal density of the ligand is 6 &mgr;mol/g adsorbent. After elution with sodium chloride and inorganic phosphoric acid a 300-fold purified enzymatic preparation was obtained in one step with a yield of 90%.

Relationship between the different replication status of HBV and mutations in the core promoter in mothers and their children infected via mother-to-infant transmission.

OBJECTIVE: To study the relationship between the different replication status of hepatitis B virus (HBV) and mutations in the core promoter (CP) in mother and her child infected by mother-to-infant transmission. METHODS: The core promoter was amplified by PCR and cloned into pGEM-T vector with the T-A cloning technique. The recombinant plasmid pGEM-CP was confirmed by digestion with restriction enzyme Apa I and Sac I. Two clones were selected to be sequenced in each patient. RESULTS: Every pair of mother and child had same serotype and genotype and the homology of nucleotides encoding "a" determinant was 98%-100%. The number of mutations in the core promoter of patients with a high replication status was less than that in those with a low replication status. Mutations were mainly distributed in basia core promoter (BCP) and the inhibitor region of Kunitz-type serine protease. This difference was not associated with mother or child. CONCLUSION: The different replication status of HBV is caused by mutations in the core promoter in mother and child infected by mother-to-infant transmission and appears to be not associated with the status of development of the infection.

[A study of the characteristics of pre S/S gene mutation of hepatitis B virus (HBV) in mothers and HBV-infected children via mother-to-infant transmission].

OBJECTIVE: To investigate the characteristics of mutations in pre S/S gene of HBV in asymptomatic carrier (AsC) children infected through mother-to-infant transmission and their AsC mothers with different degree of viremia. METHODS: According to the levels of viremia in every pair of mother and child, 15 pairs of child and mother were divided into three groups 5 pairs in each group in this study: group I (both children and mothers had high viremia), group II (children had high and mothers had low viremia) and group III (children had low and mothers had high viremia). pre S/S gene was amplified by PCR and cloned into pGEM-T vector with T-A cloning technique. The recombinant plasmid pGEM- preS/S was confirmed by digestion with restriction enzyme ApaI and SacI. Tow clones were selected to be sequenced in each patient. The mutations of preS/S were compared with HBV DNA consensus sequence of Chongqing. RESULTS: In each group the subtypes of HBV were B/adw2 in 4 pairs and C/adrq+ in one pair. The preS/S clones in patients infected with subtype B/adw2 HBV were analyzed. It was shown that there was no difference among the four high viremic groups or between the two low viremic groups in the number of mutation and the mutational position. However, there was significant difference between the high viremic group and low viremic group. The mutation was not related to age. In the two low viremic groups (the mothers of group II and the children of group III), there were 86-94 mutational positions in 13/16 clones. There were 86 same mutational positions causing 37 amino acid changes in 11/13 clones and 90 same mutational positions causing 38 amino acid changes in 2/13 clones. Most of the changed amino acids were located within T and B cell epitopes of the envelope protein or/and the surrounding regions. Sixty-two mutational positions that resulted in 28 amino acid changes were same in these two mutational sequences. CONCLUSIONS: The mutation of HBV is not associated with the duration of infection. There are many differences of mutation when HBV comes from a same strain in hosts with different degrees of viremia. There are some regular patterns in the mutation of HBV after occurrence of HBeAg seroconversion. The mutation could be related to the escape of the attack of host's immunity.

[The primary study on the anti-HBV effect of whole recombinant yeast].

OBJECTIVES: Based on the immunologic character of Pichia pastoris yeast, a new therapeutic vaccine, whole recombinant yeast, was used to explore a new way to activate cell-mediated anti-viral immunity. METHODS: The recombinant plasmids, pPIC9K/S and PIC9K/hsp(1-370)-S, were constructed by inserting the gene encoding HBsAg, HSP70 (1-370) -HBsAg into vector pPIC9K and then the recombinants were transfected into Pichia pastoris yeast,GS115, respectively. Then that recombinant yeast immunized BALB/C mice were detected for humoral and cellular immunity to HBsAg. RESULTS: Recombinant yeast successfully activated the humoral immunity to HBsAg in mice, but failed to activate the cellular immunity. CONCLUSION: The whole recombinant yeast can be used as vaccine, but need further study for optimal way of immunization.

[Specific immune responses in Balb/c mice induced by plasmid coexpressing hepatitis B surface antigen and granulocyte-macrophage colony stimulating factor].

OBJECTIVES: To investigate the improvement of specific immune responses induced by plasmid coexpressing hepatitis B surface antigen (HBsAg) and granulocyte-macrophage colony stimulating factor (GM-CSF). METHODS: All Balb/c (H-2d) mice were immunized with pGM-CSF/S, pS/GM-CSF, pS or control plasmids. 4 weeks later, anti-HBs titer and the levels of IL-2, IL-4 and IFN-gamma in the supernatant of splenocytes were detected using enzyme- linked immunosorbent assay (ELISA), and HBsAg-specific cytotoxic T lymphocytes (CTL) activity was measured with a 51Cr release assay, using P815/S transfectants as target cells. RESULTS: The anti-HBs antibody titers in the serum, the levels of IL-2 and IFN-gamma, and the CTL activity in pcDNA3.1-GM-CSF-S immuned mice were higher than those in PcDNA3.1-S immunized mice (F=4.176, P<0.01; F=31.188, P<0.01; F=31.796, P<0.01; F

[Heat shock protein 70-HBsAg complex inducing antigen-specific cytotoxic T lymphocyte immune response].

OBJECTIVE: To explore the possibility of cell-medicated immune response induced with heat shock protein 70 (HSP70)-HBsAg protein complex in vitro. METHODS: HSP70-HBsAg complex was reconstituted in vitro which was injected into mice in order to observe that whether HSP70-HBsAg would stimulate humoral and cellular immune responses. HSP70, HSP70-HBsAg complex and HBsAg were used to activate the dentritic cell (DC) individually, which would initiate homogeneic T lymphocyte to transform to cytotoxic T lymphocyte (CTL). The cytotoxicity of CTL was detected with MTT assay. RESULTS: HSP70-HBsAg complex elicited both humoral and cellular immune responses against HBsAg in mice. Specific CD8+ CTL response was readily induced by HSP70-HBsAg complex and HBsAg, especially the former. CONCLUSIONS: HSP70-HBsAg complex is immunogenic and HSP70 can lead to great efficient CTL response. And HSP70-HBsAg complex may be used as a protein vaccine for immunotherapy for chronic hepatitis B.

[PreS/S gene mutations in HBV in children infected through mother-to-infant transmission and in their mothers].

OBJECTIVES: To investigate the characteristics of mutations in PreS/S gene of HBV in children infected through mother-to-infant transmission and in their mothers with different degree of viremia. METHODS: There were 15 pairs of child and mother in this study. Mothers of all children were chronic asymptomatic HBsAg carrier (ASC) before pregnancy and the children were not inoculated against HBV after birth. Anti-HBV medicine was never administrated to all subjects. The serological markers of hepatitis A, B, C, D and E virus were tested and the titers of serum HBV DNA were quantitated. PreS/S gene was amplified by PCR and cloned into pGEM-T vector with T-A cloning technique. The recombinant plasmid pGEM-PreS/S was confirmed by digestion with restriction enzyme ApaI and SacI. Two clones were selected to be sequenced from each patient. RESULTS: According to the degree of viremia in every pair of mother and child, 15 pairs of child and mother were divided into three groups: group A (both children and mothers had high viremia with HBeAg-positive), group B (high in children and low in mothers with anti-HBe positive), and group C (low in children and high in mothers), and there were 5 pairs in each group. The subtype of each pair was the same. There were 4/5 pairs of HBV with B/adw2 and 1/5 pair of HBV with C/adrq+ in each group. It was shown that there were no difference among the four high viremia groups or between the two low viremia groups in the number of mutations and the number of mutational positions. However, there was significant difference between high viremia group and low viremia group. The mutation was not related to age. There were 56 mutational positions and there was no mutational hotspot in high viremia patients. In two low viremia groups (the mothers in group B and the children in group C), there were 113 mutational positions and 85 mutational positions were hotspots (owned by 5/8 clones in each) which could make 37 amino acids changed. Most of mutational amino acids were located within T and B cell epitopes of envelope protein or/and located in the surrounding regions. CONCLUSIONS: There are many differences in HBV with different degree of viremia, even if it comes from the same strain. There are some regular patterns in the mutations of HBV after HBeAg seroconversion happened.

[Prokaryocytic expression of the shortened hepatitis B surface antigen and antigenic analysis].

OBJECTIVE: To study the expression of the shortened hepatitis B surface antigen in prokaryocyte and detect the antigenic characters. METHODS: Firstly, the gene fragments coding the 152 and 124 amino acids of the carboxyl terminus of hepatitis B surface antigen (HBsAg) were amplified by polymerase chain reaction (PCR). Secondly, they were cloned to plasmid pBKS+, and the accuracy of those constructions were confirmed by restriction enzyme digestion and DNA sequencing. Then, they were cloned to prokaryocytic expression vector-plasmid pET32a(+). The recombinant plasmids were transfected into E.coli BL21 and induced to express with IPTG. RESULTS: The recombinant plasmids were successfully constructed. In E.coli BL21, the protein was expressed in a fusion fashion and could be recognized by monoclonal antibody against HBsAg with ELISA and Western blot. CONCLUSION: The shortened HBsAg can be expressed in prokaryocyte.