OBJECTIVES: Despite all the efforts and increased knowledge of rabies, the exact mechanisms of infection and mortality from the rabies virus are not well understood. To understand the mechanisms underlying the pathogenicity of rabies virus infection, it is crucial to study the tissue that the rabies virus naturally infects in humans. METHODS: Cerebellum brain tissue from 9 human post mortem cases from Iran, who had been infected with rabies virus, were examined histopathologically and immunohistochemically to evaluate the innate immune responses against the rabies virus. RESULTS: Histopathological examination revealed inflammation of the infected cerebellum and immunohistochemical analyses showed an increased immunoreactivity of heat shock protein 70, interleukin-6, interleukin-1, tumor necrosis factor-alpha, caspase-3, caspase-9, toll-like receptor3 and toll-like receptor4 in the infected brain tissue. CONCLUSION: These results indicated the involvement of innate immunity in rabies infected human brain tissue, which may aggravate the progression of this deadly disease.
Brain ; Caspase 3 ; Caspase 9 ; Central Nervous System ; Cerebellum ; HSP70 Heat-Shock Proteins ; Humans ; Immunity, Innate ; Immunohistochemistry ; Inflammation ; Interleukin-1 ; Interleukin-6 ; Iran ; Mortality ; Pathology ; Rabies virus ; Rabies ; Tumor Necrosis Factor-alpha ; Virulence

Rosmarinic acid (RA) is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. The previous studies have revealed that RA had therapeutic effects on hepatocellular carcinoma (HCC) in the H22-xenograft models by inhibiting the inflammatory cytokines and NF-κB p65 pathway in the tumor microenvironment. However, its molecular mechanisms of immunoregulation and pro-apoptotic effect in HCC have not been fully explored. In the present study, RA at 75, 150, and 300 mg/kg was given to H22 tumor-bearing mice via gavage once a day for 10 days. The results showed that RA can effectively inhibit the tumor growth through regulating the ratio of CD4⁺/CD8⁺ and the secretion of interleukin (IL)-2 and interferon-γ, inhibiting the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, thereby up-regulating Bax and Caspase-3 and down-regulating Bcl-2. The underlying mechanisms involved regulation of immune response and induction of HCC cell apoptosis. These results may provide a more comprehensive perspective to clarify the anti-tumor mechanism of RA in HCC.
Animals ; Apoptosis ; Boraginaceae ; Carcinoma, Hepatocellular ; Caspase 3 ; Cytokines ; Interleukin-10 ; Interleukin-6 ; Interleukins ; Lamiaceae ; Mice ; STAT3 Transcription Factor ; Therapeutic Uses ; Tumor Microenvironment

PURPOSE: To investigate whether dexmedetomidine (Dex) can reduce the production of inflammatory factor IL-1β by inhibiting the activation of NLRP3 inflammasome in hippocampal microglia, thereby alleviating the inflammatory response of the central nervous system induced by surgical injury. METHODS: Exploratory laparotomy was used in experimental models in this study. Totally 48 Sprague Dawley male rats were randomly divided into 4 groups (n = 12 for each), respectively sham control (group A), laparotomy only (group B); and Dex treatment with different doses of 5 μg/kg (group D1) or 10 μg/kg (group D2). Rats in groups D1 and D2 were intraperitoneally injected with corresponding doses of Dex every 6 h. The rats were sacrificed 12 h after operation; the hippocampus tissues were isolated, and frozen sections were made. The microglia activation was estimated by immunohistochemistry. The protein expression of NLRP3, caspase-1, ASC and IL-1β were detected by immunoblotting. All data were presented as mean ± standard deviation, and independent sample t test was used to analyze the statistical difference between groups. RESULTS: The activated microglia in the hippocampus of the rats significantly increased after laparotomy (group B vs. sham control, p < 0.01). After Dex treatment, the number was decreased in a dose-dependent way (group D1 vs. D2, p < 0.05), however the activated microglia in both groups were still higher than that of sham controls (both p < 0.05). Further Western blot analysis showed that the protein expression levels of NLRP3, caspase-1, ASC and downstream cytokine IL-1β in the hippocampus from the laparotomy group were significantly higher than those of the sham control group (all p < 0.01). The elevated expression of these proteins was relieved after Dex treatment, also in a dose-dependent way (D2 vs. D1 group, p < 0.05). CONCLUSION Dex can inhibit the activation of microglia and NLRP3 inflammasome in the hippocampus of rats after operation, and the synthesis and secretion of IL-1β are also reduced in a dose-dependent manner by using Dex. Hence, Dex can alleviate inflammation activation on the central nervous system induced by surgical injury.
Animals ; Dexmedetomidine ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Hippocampus ; metabolism ; Immunohistochemistry ; Inflammasomes ; metabolism ; Inflammation Mediators ; metabolism ; Injections, Intraperitoneal ; Interleukin-1beta ; metabolism ; Laparotomy ; adverse effects ; Male ; Microglia ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVE: Behcet's disease (BD) is an autoimmune disorder that causes most commonly mouth and genital ulcerations and erythema nodules of the skin and currently has limited options of therapeutic medicines. Metformin is recently reported to suppress immune reaction, and we hypothesized that metformin could be an option for treatment of BD. METHODS: Thirty patients with BD were enrolled in this perspective single-blinded, before-after study. We recorded the changes in the mucocutaneous activity index for BD (MAIBD), relapse frequency, C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) after metformin treatment to assess the changes in the disease activity. We also analyzed the changes in the protein and mRNA expression levels of Foxp3, interleukin-35 (IL-35), transforming growth factor-β (TGF-β), Ror-γt, IL-17, and tumor necrosis factor- (TNF-) in these patients using ELISA and qRT-PCR. RESULTS: Of the 30 patients enrolled, 26 completed the trial. After the treatment, favorable responses were achieved in 88.46% (23/26) of the patients, and partial remission was obtained in 11.54% (4/26) of them. During the treatment, 8 patients complained of gastrointestinal side effects, for which 4 chose to withdraw from the study in the first week. Our results showed that metformin treatment decreased MAIBD and relapse frequency in the patients, and significantly lowered the clinical inflammatory indexes including CRP and ESR. The results of ELISA and qRT-PCR revealed that metformin treatment obviously increased Foxp3 and TGF-β expressions at both the protein and mRNA levels and significantly decreased the levels of ROR-γt, IL-17 and TNF- as well as IL-35 level in these patients. CONCLUSIONS Metformin treatment relieves the clinical symptoms, reduces the inflammatory reaction indexes and regulates the Treg/Th17 axis in patients with BD, suggesting the potential of metformin as a candidate medicine for treatment of BD.
Behcet Syndrome ; drug therapy ; metabolism ; Controlled Before-After Studies ; Forkhead Transcription Factors ; metabolism ; Humans ; Immunosuppressive Agents ; adverse effects ; therapeutic use ; Interleukin-17 ; metabolism ; Interleukins ; metabolism ; Metformin ; adverse effects ; therapeutic use ; Neoplasm Recurrence, Local ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; metabolism ; Recurrence ; Single-Blind Method ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To investigate the expression of cytokine signal suppressor 3 (SOCS3) in colon cancer tissue and the mechanism by which SOCS3 regulates the proliferation and invasion of colon cancer. METHODS: We collected the specimens of tumor tissues and paired adjacent tissues from 80 patients with colon cancer undergoing radical resection in our hospital between July, 2014 and May, 2017, and the expression of SOCS3 in the tissue samples was analyzed using Western blotting. We also transfected colon cancer cell line SW480 with a SOCS3-overexpressing plasmid or a small interference RNA (siRNA) for SOCS3 knockdown, and the changes in the cell proliferation and invasion capacity were evaluated using CCK-8 assay and Transwell assay, respectively. The effect of demethylation and IL-6 treatment on SOCS3 expression and the proliferation and invasion of SW480 cells were observed. RESULTS: Colon cancer tissues showed a lowered expression of SOCS3 compared with the adjacent tissues. Over-expression of SOCS3 significantly inhibited while SOCS3 knockdown obviously promoted the proliferation and invasion of SW480 cells . Demethylation treatment up-regulated SOCS3 expression and inhibited the proliferation and invasion capacity of SW480 cells; IL-6 treatment of the cells caused the reverse changes. CONCLUSIONS SOCS3 participates in the development and progression of colon cancer and serves as a potential target for colon cancer treatment. In patients with colon cancer, the low expression of SOCS3 possibly as a result of methylation may promote the proliferation and invasion of the cancer cells.
Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; etiology ; pathology ; Cytokines ; Demethylation ; Disease Progression ; Humans ; Interleukin-6 ; pharmacology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein ; genetics ; metabolism ; Transfection

OBJECTIVE: To study the association of Nod-like receptor protein 3 (NLRP3) inflammasome with inflammatory response in the acute stage and coronary artery lesion (CAL) in children with Kawasaki disease (KD). METHODS: A total of 42 children with KD who were hospitalized from January to October 2017 were enrolled as the KD group, among whom 9 had CAL (CAL group) and 33 had no CAL (NCAL group). Fifteen age- and gender-matched children with pneumonia and pyrexia were enrolled as the pneumonia-pyrexia group. Fifteen healthy children were enrolled as the healthy control group. Real-time PCR was used to measure the mRNA expression of NLRP3 inflammasome (NLRP3, ASC and caspase-1) in peripheral blood mononuclear cells. The Spearman rank correlation test was used to investigate the correlation of NLRP3 mRNA expression with serum levels of C-reactive protein, erythrocyte sedimentation rate, interleukin-6, interleukin-1β, procalcitonin, albumin and prealbumin. RESULTS: The KD group had significantly higher mRNA expression of NLRP3, ASC and caspase-1 in the acute stage than the pneumonia-pyrexia and healthy control groups (P<0.05). The CAL group had significantly higher mRNA expression of NLRP3 than the NCAL group (P<0.05). NLRP3 mRNA expression was correlated with C-reactive protein, interleukin-6, interleukin-1β, and prealbumin levels in children with KD in the acute stage (r=0.449, 0.376, 0.427, and -0.416 respectively; P<0.05). CONCLUSIONS NLRP3 inflammasome may participate in inflammatory response in the acute stage and the development of CAL in children with KD.
Child ; Humans ; Inflammasomes ; Interleukin-1beta ; Leukocytes, Mononuclear ; Mucocutaneous Lymph Node Syndrome ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(HO),calcium-sensitive receptor agonist gadolinium trichloride(GdCl),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of HO.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without HO.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with HO,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the HO group and HO+GdCl group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of HO concentration were similar.HO-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of HO.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the HO +siRNA treatment group and the HO treatment group.Conclusion CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.
Animals ; Cathepsin B ; antagonists & inhibitors ; metabolism ; Cell Line ; Gene Silencing ; Hydrogen Peroxide ; Inflammasomes ; metabolism ; Interleukin-1beta ; Mice ; Microglia ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Oxidative Stress ; Pyrin Domain ; Transient Receptor Potential Channels ; metabolism

The aim of the present study was to investigate the effect of salidroside (Sal) on inflammatory activation induced by lipopolysaccharide (LPS) in the co-culture of rat alveolar macrophages (AM) NR 8383 and type II alveolar epithelial cells (AEC II) RLE-6TN. CCK-8 colorimetric method was used to detect cell proliferation percentage. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-10 (IL-10) in the supernatant. Western blot was used to examine the expression levels of phosphorylated AKT (p-AKT) and total AKT protein. The results showed that pretreatment of RLE-6TN cells or co-culture of RLE-6TN and NR 8383 cells with 32 and 128 µg/mL Sal for 1 h, followed by continuous culture for 24 h, significantly increased the cell proliferation (P < 0.05). Compared with control group, 32 and 128 µg/mL Sal pretreatment significantly increased the ratio of p-AKT/AKT in RLE-6TN cells (P < 0.05). Pretreatment of 32 µg/mL Sal not only inhibited the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05), but also enhanced the inhibitory effect of RLE-6TN and NR 8383 cells co-culture on the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05). In addition, 32 µg/mL Sal pretreatment promoted LPS-induced IL-10 secretion by NR 8383 cells (P < 0.05), and enhanced the promoting effect of co-culture of RLE-6TN and NR 8383 cells on the IL-10 secretion by LPS-induced NR 8383 cells (P < 0.05). In conclusion, Sal may directly inhibit LPS-induced inflammatory activation of AM (NR 8383), promote the proliferation of AEC II (RLE-6TN) through PI3K/AKT signaling pathway, and enhance the regulatory effect of AEC II on LPS-induced inflammatory activation of AM.
Alveolar Epithelial Cells ; drug effects ; metabolism ; Animals ; Cell Line ; Chemokine CXCL2 ; metabolism ; Coculture Techniques ; Glucosides ; pharmacology ; Interleukin-10 ; metabolism ; Lipopolysaccharides ; Macrophages, Alveolar ; drug effects ; metabolism ; Phenols ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

The aim of this paper was to study the effect and mechanism of alcohol extract from Polygonum cuspidatum(PCE) on acute gouty arthritis in C57 BL/6 mice through NLRP3/ASC/caspase-1 axis. The model mice which injected with ankle joint injection of sodium urate crystals(MSU) were orally administrated with three different concentration of PCE, with colchicine as positive control. HE staining was used for observing the morphological changes of synovial tissue; concentration of IL-1β, IL-6 and TNF-α secreted by synovial tissue of the ankle joint were detected by ELISA; mRNA and protein expression of NLRP3, ASC and caspase-1 in synovial tissue were detected by RT-PCR and Western blot respectively. The results showed that the swelling degree of ankle joint in model mice were significantly elevated; expression of IL-1β, IL-6 and TNF-α were significantly increased; mRNA and protein expression of NLRP3, ASC and caspase-1 also significant increase, compared with normal control group. The swelling degree of ankle joint significantly relief; expression of IL-1β, IL-6 and TNF-α in joint synovium significantly decrease; mRNA and protein expression of NLRP3, ASC and caspase-1 were significantly decrease in PCE treatment group compared with model group. Our research implied that alcohol extract from P. cuspidatum had positive effect on acute gouty arthritis in mice, and the regulation of NLRP3/ASC/caspase-1 axis may be its mechanism.
Animals ; Ankle Joint ; physiopathology ; Arthritis, Gouty ; drug therapy ; CARD Signaling Adaptor Proteins ; metabolism ; Caspase 1 ; metabolism ; Fallopia japonica ; chemistry ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Plant Extracts ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism ; Uric Acid

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 μmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1β (IL-1β) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1β and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1β and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.
Caspase 1 ; Gene Expression Regulation ; drug effects ; Interleukin-1beta ; genetics ; Macrophages ; drug effects ; NLR Family, Pyrin Domain-Containing 3 Protein ; genetics ; Pyroptosis ; drug effects ; Serum Albumin ; pharmacology