Matrine suppresses NLRP3 inflammasome activation via regulating PTPN2/JNK/SREBP2 pathway in sepsis.

BACKGROUND: Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Abnormal activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome plays a vital role in the pathogenesis of sepsis. Matrine is proved to show good anti-inflammatory properties, whereas its effect and the underlying molecular machinery on sepsis remains unclear. PURPOSE: The aim of this study is to evaluate the effect and mechanism of Matrine on sepsis. STUDY DESIGN: THP-1 cells and J774A.1 cells were stimulated by lipopolysaccharide (LPS) with nigericin or adenosine triphosphate (ATP) to establish an in vitro model. Cecal ligation and puncture (CLP)-induced sepsis mouse model was used. Matrine was given by gavage. METHODS: To investigate the NLRP3 inflammasome activation, phorbol myristate acetate (PMA)-induced THP-1 cells were first primed with LPS and then stimulated by matrine, followed by treatment with nigericin or ATP. The concentration of interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) in the cell culture supernatant was detected. The mechanism was explored by cell death assay, immunoblots and immunofluorescence in vitro. C57BL/6 mice were intragastrically administered with matrine for 5 days before CLP. The therapeutic effect of matrine was evaluated by symptoms, pathological analysis, ELISA and RT-qPCR. RESULTS: Our results revealed that matrine inhibited IL-1ß and IL-18 secretion, suppressed caspase-1 activation, reduced cell death, and blocked ASC speck formation upon NLRP3 inflammasome activation. Furthermore, matrine restrains NLRP3 inflammasome activation as well as pyroptosis through regulating the protein tyrosine phosphatase non-receptor type 2 (PTPN2)/JNK/SREBP2 signaling. Matrine also prominently improved the symptoms and pathological changes with reduced levels of TNF-α, IL-1ß, and IL-6 in the lung tissues and serum in a dose-dependent manner. CONCLUSION: Matrine effectively alleviates the symptoms of CLP-induced sepsis in mice, restrains NLRP3 inflammasome activation by regulating PTPN2/JNK/SREBP2 signaling pathway, and may become a promising therapeutic agent for sepsis treatment.

Immunization with HBsAg-Fc fusion protein induces a predominant production of Th1 cytokines and reduces HBsAg level in transgenic mice.

BACKGROUND: The Fc receptor associated pathway might improve the immune responses against hepatitis B virus (HBV) as previously described by us. In addition, the Flt3 ligand (FL) has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses. In this study, the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice. METHODS: The fusion proteins composed of HBsAg and the Fc domain of murine IgG1 (HBsAg-Fc) and/or the Flt3 ligand, and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice, respectively. Serum and liver HBsAg levels, serum anti-HBsAg and cytokine profile, and the activities of alanine aminotransferase (ALT)/AST were investigated after immunization. RESULTS: After six injections, the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group. In addition, serum Th1 cytokines and ALT/AST activities were highest in this group, indicating an effective induction of a favorable cellular immune response. Interestingly, the fusion protein containing HBsAg-Fc and the Flt3 ligand stimulated an alternative Th1-type immune response featured with high level productions of tumor necrosis factor α (TNF-α) and monocyte chemoabstractant protein 1 (MCP-1), causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level. CONCLUSION: HBsAg-Fc is effective in eliciting both the humoral and cellular immune responses against HBsAg in HBsAg transgenic mice, which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.

[Development and identification of polyclonal antibodies against HIV-1 Vpr-derived polypeptides].

To develop polyclonal antibodies against predicted B cell epitopes in HIV-1 accessory protein Vpr, the prepared consensus Vpr amino acid sequence was used to predict potential B cell epitopes by online softwares (ABCpred and Bcepred), the synthesized polypeptides of B-cell epitopes were subsequently conjugated with keyhole limpet hemocyanin (KLH) and then used to immunize rabbits. The antibody titers were determined by ELISA, and antibody specifity was analyzed by Western-Blotting and immunoprecipitation, respectively. Amino acid residues 3-19 (N) and 82-95 (C) of Vpr were predicted as the potential B cell epitopes. After inoculation of the conjugation of synthesized peptide to KLH, the antibody titers in rabbit sera against N and C peptides reached more than 1:100000 by ELISA. Western-Blotting analysis showed that the polyclonal antibodies reacted with both wild Vpr and fusion protein of GFP with Vpr, no matter Vpr was derived from HIV-1 B subtype or CRF07_BC recombinant form; Immunoprecipitation analysis showed similar reactions to Western-Blotting results. Two B cell epitopes of Vpr were successfully predicted by Bio-informatics methods and polyclonal antibodies against those peptides could be successfully prepared.

[HIV-1 infection affects the expression of host cell factor TSG101 and Alix].

To investigate the effects of HIV-1 infection on the expression of host factors TSG101 (Tumor Susceptibility Gene 101) and Alix (ALG-2-interacting protein X). HIV-1 infectious clone pNL4-3 was used to infect TZM-bl, PM1, Jurkat cell lines and human peripheral blood mononuclear cells (PBMC). Twenty-four hours post-infection, the infected or uninfected cells were harvested respectively for extraction of total RNAs and total cellular proteins, which were subsequently used in RT-PCR and Western-blotting respectively to quantify TSG101 and Alix, respectively. Our data showed that HIV-1 infection resulted in various influences on the expression of TSG101 and Alix in the cell lines and the primary PBMC. A down-regulation was mainly observed in the cell lines, whereas an up-regulation of TSG101 was identified in primary PBMC. Three patterns were observed for down-regulation, including dual down-regulation of TSG101 and Alix for Jurkat cells, single down-regulation of Alix for TZM-bl cells and marginal or no influence on PM1 cells. The dual down-regulation of Alix and TSG101 in Jurkat cells coincided with less expression of HIV-1 p24 protein. This is the first-line evidence that HIV-1 infection affects the expression of host factors TSG101 and Alix, the down-regulation of these molecules may influence the HIV-1 replication. The underlying mechanism remains to be addressed.

[Expression of CD66c (CEACM6) in adult acute leukemia and its significance].

OBJECTIVE: To explore the expression of CD66c (CEACM6) in adult acute leukemia and its significance. METHODS: Acute leukemia cell lines HL-60, K562, LCL721.221 and Jurkat were cultured in vitro. RT-PCR and multi-parameter flow cytometry were applied to analysis of CD66c mRNA and protein expression respectively in the cell lines and patient' s bone marrow leukemic cells. Cytogenetic analysis for 199 bone marrow samples from leukemia patients and Minimal Residual Disease (MRD) detection for 25 CD66c positive B lineage ALL were performed. RESULTS: (1) CD66c expression both on cell surface and in plasma were negative in all the cell lines. (2) Four of 127 AML (3.15%) (mainly of M2 and M4), and 28 of 79 ALL (35.44%) (all of B linage ALL) were CD66c positive the subtypes of the ALL being common B-ALL (20/54) and pre B-ALL (8/11) including 8 Ph + B-linage ALL. (3) Six-month relapse rate was significantly different between the MRD positive and negative patients. (4) CD66c mRNA was strongly expressed in B-linage ALL. For the cell lines, only the HL60 cells weakly expressed CD66c mRNA. CONCLUSION: CD66c expression could be a useful bio-marker for the MRD analysis in ALL, and is closely associated with its transcription level.

[Eukaryotic expression and tumor-targeting modification of human mutated-IL-18 fusion gene].

Interleukin-18 (IL-18) is a proinflammatory cytokinin. This protein has a role in regulating immune responses and exhibits significant anti- tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL18 mature peptide via a linker peptide of (Gly4ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-gamma to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF Receptor competitive test in human epithelial cancer A431 cell line showed that EGF- IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. The results suggested that EGF-IL18 fusion protein could specifically be targeted on tumor cells. The work may provide a new insight into the cytokinin for the tumor treatment.

Rational design of an EGF-IL18 fusion protein: implication for developing tumor therapeutics.

Interleukin-18 (IL-18) is a proinflammatory cytokine. This protein has a role in regulating immune responses and exhibits significant anti-tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL-18 mature peptide via a linker peptide of (Gly(4)Ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-gamma to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF receptor competitive test in human epithelial cancer A431 cell line showed that EGF-IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. These suggest that this rationally designed protein can be further developed as novel tumor therapeutics.