OBJECTIVETo assess the response in THP-1 treated with Rv3671c protein in Mycobacterium tuberculosis (M.tuberculosis).

METHODSThe gene encoding Rv3671c protein of M.tuberculosis was cloned into pET-28a vector and then expressed in Escherichia coli. The Rv3671c was purified with Ni-NTA affinity and ion exchange chromatography. The detection of protein concentration was by Lowry method.THP-1 cell was stimulated with Rv3671c protein and cells were analyzed by Hochest staining under fluorescence microscopy to assay cell death (apoptosis and necrosis). TNF-α and IL-1β were detected by ELISA at each stimulating time.

RESULTSThe Rv3671c protein of M.tuberculosis was successfully expressed in Escherichia coli. The purity of recombinant Rv3671c protein was 95%, and the protein concentration was up to 0.4 mg/ml. The nucleus of THP-1 was isolated and necrosis-like under fluorescence when cells were stimulated by Rv3671c protein. The levels of TNF-α and IL-1β in supernatant were 19 000 and 16 500 pg/ml respectively, and were significantly higher than control cells with the levels of 2100 and 3800 pg/ml separately.

CONCLUSIONThe necrosis of THP-1 cells could be stimulated by Rv3671c protein of M.tuberculosis and it was probably associated with high cytokines TNF-α and IL-1β levels.

Bacterial Proteins ; pharmacology ; Cell Death ; Cell Line ; Humans ; Interleukin-1beta ; metabolism ; Macrophages ; cytology ; metabolism ; Mycobacterium tuberculosis ; genetics ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo establish a rabbit model of intervertebral disc degeneration by puncturing the anulus fibrosus through an approach between the longissimus dorsi muscle and obliquus externus abdominis.

METHODSThe L(4/5) and L(5/6) intervetebral discs of 6 New Zealand white rabbits were punctured by an 18-gauge pin in the anterolateral annular fibrosus through an approach between the longissimus dorsi muscle and the obliquus externus abdominis with the right transverse processes of L(5) and L(6) resected; the L(2/3) discs were used as the control without exposure or needle stab, and the L(3/4) discs were subjected to sham operation with the discs exposed but not punctured after resecting the right transverse process of L(4). X-ray and magnetic resonance imaging (MRI) were performed preoperatively and at the 4th week after puncture. At 4 weeks after the operation, histological and immunohistochemical analyses of the discs were carried out.

RESULTSX-ray of the punctured discs at 4 weeks after the operation presented a significant decrease of disc height, osteophytosis formation, and end-plate stiffness; an obvious decrease of signal intensity on T(2)-weighted images was found in the puncture group but not in the control or sham-operated groups. Gross morphological inspection showed atrophy of the nucleus pulposus, which became loose, soft, and fragile with a light yellow color. Histological and immunohistochemical analyses showed a significant decrease of notochordal cells and type II collagen in the nucleus pulposus in the puncture group as compared to the control and sham-operated groups.

CONCLUSIONPuncture through the approach between the longissimus dorsi muscle and the obliquus externus abdominis allows the establishment of a reliable animal model for studying intervertebral disc degeneration.

Animals ; Disease Models, Animal ; Female ; Intervertebral Disc Degeneration ; Lumbar Vertebrae ; physiopathology ; Male ; Rabbits

Evodiamine(EVO)is an indoloquinazoline alkaloid isolated from the fruits of Euodia rutaecarpa,which has long been used in traditional Chinese medicine to treat various diseases.Modern medical research shows that evodiamine has various pharmacological activities and extremely high medicinal value such as anti-inflammation,anti-tumor,losing weight,treating Alzheimer's,and antibacterium.Evodiamine can interact with a variety of proteins.For example,the interaction between evodiamine and TRPV1 induces its activation to exert anti-inflammatory and losing weight; the interaction with DNA topoisomerase I inhibits its function and thus inhibit bacterial proliferation; the interaction with tubulin promotes NLRP3 inflammasome activation,and exerts anti-tumor and anti-inflammatory functions.In recent years,with the in-depth study of evodiamine,pharmacological research and mechanism of evodiamine has significant improvement.Recent progress of pharmacological effect of evodiamine is highlighted in this review.

Pyroptosis is a novel process of programmed inflammatory necrosis, which is closely related to microbial infections and autoimmune diseases. In recent years it has been proved that Gasdermin D is an executive protein of pyroptosis. Cleaved Gasdermin D leads to the formation of pores in cell membrane to induce pyroptosis, and the release of inflammatory factors such as IL-1β and IL-18 aggravates the occurrence of inflammatory responses. With the deepening of Gasdermin D research, it is gradually clear its protein structure and activation sites, which has other activation pathways apart from inducing inflammasome activation. Activation of Gasdermin D does not necessarily mean cell death, even if it could induce the formation of pores in cell membrane and regulation of inflammatory cytokine secretion. These findings offer insight into Gasdermin D function and change our understanding of pyroptosis and programmed necrosis. The recent progress of Gasdermin D is highlighted in this review.