[Methylophiopogonanone a Inhibits LPS/ATP-Induced Macrophage Pyroptosis via ROS/NLRP3 Pathway].

As a byproduct of mitochondrial respiration or metabolism, reactive oxygen species (ROS) can act as a signaling molecule to activate NLR family pyrin domain containing 3 (NLRP3) inflammasome, thereby triggering immune response. NLRP3 inflammasome acts as a sensor of various danger signals and is central to the control of pyroptosis occurrence. Macrophage pyroptosis is closely related to atherosclerosis, arthritis, pulmonary fibrosis and other inflammatory diseases. Methylophiopogonanone A (MO-A) is a main homoisoflavonoid in Chinese herb Ophiopogonis Radix, which has antioxidant effect. However, it is not clear whether MO-A can alleviate macrophage pyroptosis by inhibiting oxidative stress. Here we have shown that MO-A increases the activities of superoxide dismutase (SOD) and catalase (CAT), inhibits the production of ROS, reduces the activation of NLRP3 inflammasome and the release of lactate dehydrogenase (LDH), and inhibits pyroptosis in macrophages induced by lipopolysaccharides (LPS) and adenosine triphosphate (ATP). These effects can be reversed by the ROS promoter H2O2. Therefore, MO-A can inhibit macrophage pyroptosis through the ROS/NLRP3 pathway and may be considered as a candidate drug for the treatment of inflammatory diseases.

Performance evaluation of histone deacetylases in lungs of mice exposed to ovalbumin aerosols.

This study was to investigate expression levels and functional activities of histone deacetylases (HDACs) with potential therapeutic targets selected in animal model of allergic asthma. Mice were sensitized and then challenged with saline (control) or ovalbumin (OVA) for 8 weeks. Airway resistance was determined by increasing concentrations of acetyl-ß-methacholine chloride (0 - 50 mg/ml). The number of cells and cytokine production in bronchoalveolar lavage fluid (BALF) were determined by ELISA. Pathological changes of lung specimens were examined by histochemical staining methods under the light microscope. Expression and quantification of HDACs in lungs were measured using immunohistochemistry and Western blotting analysis. HDAC activity was identified using colorimetric and fluorometric methods. The OVA-treated mice had a significant enhancement in airway resistance with a large number of cells and increased interleukin (IL)-4 and -5 levels in BALF. Morphologically, an infiltration of inflammatory cells into epithelial layer with mucus accumulation and subepithelial fibrosis were seen in the OVA-exposed lungs. The expression levels for HDAC1, HDAC5, HDAC6, and HDAC8 were significantly elevated with weak induction of HDAC 2-4, which was identical with their catalytic activities detected in the lungs. In contrast, HDAC1 and HDAC5 activities were higher than others in the lungs. Individual HDACs are differently regulated in expression levels and functional activities in animal model of allergic asthma. Selective targeting of HDAC1/5 offers an opportunity to improve therapeutic effects of the disease.

Revealing potential functions of VBNC bacteria in polycyclic aromatic hydrocarbons biodegradation.

The bioremediation of polycyclic aromatic hydrocarbon (PAH)-contaminated sites is not running smoothly, because of the lower activity of PAH-degrading bacteria in actual bioremediation applications. The phenomenon of "viable but nonculturable" (VBNC) state may be a main limiting factor for their poor biodegradation capabilities of PAHs. Due to their abilities of entering into the VBNC state, most of bacterial populations with PAH-degradation potential remain unculturable. Resuscitation of VBNC bacteria will enhance the degradation capability of indigenous bacteria which will eventually obtain their better capabilities in environmental bioremediation. Although evidences have been presented indicating that resuscitation of VBNC bacteria in polychlorinated biphenyl (PCB)-contaminated environments not only significantly enhanced PCB degradation, but also obtained novel highly efficient PCB-degrading bacteria, scanty information is available on the VBNC bacteria in PAH-contaminated sites. VBNC bacteria, as a vast majority of potential microbial resource could be the repository of novel highly efficient PAH-biodegraders. Therefore, studies need to be done on resuscitation of VBNC bacteria to overcome key bottlenecks in bioremediation of PAH-contaminated sites. This mini-review provides a new insight into the potential functions of VBNC bacteria in PAHs biodegradation. SIGNIFICANCE AND IMPACT OF THE STUDY: As the vast majority microbial resource, viable but nonculturable (VBNC) bacteria, which showed their potential functions in polycyclic aromatic hydrocarbons (PAHs) biodegradation, can be of great significance in environmental bioremediation. It is therefore important to resuscitate VBNC bacteria for their better capabilities. Meanwhile, preventing the indigenous functional community from entering into the VBNC state will also maintain the high activity of PAH-degrading bacteria in actual bioremediation applications. Undoubtedly, much more work needs to be done to reveal indigenous micro-organisms in the VBNC state from the perspective of environmental functions.

[The role of the AMPK-mTOR pathway in paraquat-induce autophagy in PC12 cells].

Objective: To investigate the regulation of AMPK-mTOR signal transduction pathway in paraquat-induced autophagy of pheochromocytoma cells (PC12) . Methods: The PC12 cell were treated with terminal concentrations of 0, 25, 50, 100, 200, 300 and 400 µmol/L PQ for 24 hours, and the cells were induced by 300 µmol/L PQ for different time (6, 12, 24, 48 h) . MTT was used to detect the relative survival rate of cells, and the dose/time-effect relationship was determined respectively. The cells were treated with PQ at concentrations of 0, 100, 200 and 300 µmol/L PQ for 24 hours, the lactate dehydrogenase (LDH) activity in the culture supernatant was detected by spectrophotometry. The expression and distribution of autophagic lysosomes were observed by MDC staining. The intracellular reactive oxygen species (ROS) was detected by dichlorofluorescein diacetate (DCFH-DA) . The expression of microtubule-related protein 1 light chain 3 (LC3) was measured by immunofluorescence. The protein level of LC3Ⅱ, p62, Beclin1 and p-AMPK, p-mTOR were detected by Western blot. Results: Compared with the control group, the cell survival rate of the 100, 200, 300, 400 µmol/L PQ group decreased significantly, and showed a dose-dependent pattern (P<0.05) . The survival rate of cells treated with 300 µmol/L PQ decreased significantly with the prolongation of exposure time (12, 24, 48 h) (P<0.05) . Compared with the control group, the activity of LDH in 100, 200, 300 µmol/L PQ-treated group were significantly higher while The fluorescence intensity of ROS was significantly increased (P<0.05) . MDC staining showed the density of autophagic lysosomes and fluorescence intensity in PQ-treated group significantly decreased (P<0.05) . Immunofluorescence results showed the LC3 fluorescence intensity of PQ-treated group decreased which was consistent with MDC staining results. Western blot showed that compared with the control group, the expression levels of autophagy related proteins LC3Ⅱand Beclin1 in PQ-treated group were significantly lower, while the expression level of p62 protein was higher (P<0.05) . p-AMPK protein level decreased and p-mTOR protein expression increased in 200 and 300µmol/L PQ-treatd groups, with statistically significant difference (P<0.05) . Conclusion: AMPK-mTOR signaling pathway played a regulatory role in PQ-induced decreased autophagy of PC12 cell.

[Therapeutic effects of Tubastatin A Hcl on airway inflammation in acute mice asthma model].

Objective: To investigated the therapeutic effects of Tubastatin A Hcl, a selective HDAC6 inhibitor, on airway inflammation in acute mice bronchial asthma (asthma) model. Methods: A total of 48 BALB/C mice were randomly divided into control group, asthma group, dexamethasone group and Tubastatin A Hcl group with 12 mice in each group. Then the airway hyperresponsiveness was assessed in each group; total cell number, different cell number, levels of Interleukin (IL)-4, IL-5 and Interferon-γ (IFN-γ) in the bronchoalveolar lavage fluid (BALF) were detected; the lung tissues of each group were stained with HE to observe the inflammatory cells infiltration. AB-PAS was used to observe the goblet cell metaplasia in tracheal epithelium. Masson staining was used to observe the collagen deposition in lung tissue. Results: The airway reactivity in Tubastatin A Hcl group was significantly lower than that in asthma group [(4.18±0.94) vs (6.02±0.47), P<0.05]; in the Tubastatin A Hcl group, the total inflammatory cells [(57.0±5.7)×10(4)/ml vs (87.0±5.6)×10(4)/ml], eosinophil cells [(6.8±1.7)×10(4)/ml vs (12.3±3.5)×10(4)/ml] and levels of IL-4 [(19.3±2.7) vs (26.2±3.2)ng/ml] in BALF were obviously lower than those of asthma group (all P<0.05); IL-5 in Tubastatin A Hcl group was lower and IFN-γ was higher than that of asthma group, while there were no significant differences (both P>0.05). The degree of inflammatory cells infiltrations around the airway and vascular, number of inflammatory cells [(9.80±2.42) vs (20.67±7.53)], score of inflammation [(2.20±0.70) vs (3.60±0.68) points, ], and percentage of goblet cell metaplasia [(50.46±5.03)% vs (71.06±5.38)%] in the lung tissue of Tubastatin A Hcl group were lower than that of asthma group (all P<0.05). Although collagen deposition in the lung tissue of Tubastatin A Hcl group was lower than asthma group, there were no significant differences (P>0.05). However, all of the above results in the dexamethasone group were slightly better than Tubastatin A Hcl group, which had no significant differences (P>0.05). Conclusion: Tubastatin A Hcl can effectively alleviate the level of airway inflammation in acute asthma, but its anti-inflammatory effect is limited, which is not as significant as dexamethasone.

[Therapeutic effects of histone deacetylase inhibitor givinostat on air inflammation and high airway resistance in a murine asthma model].

Objective: To investigate the therapeutic effects of givinostat, a histone deacetylase inhibitor (HDACI), on the development of chronic asthma with airway inflammation, airway remodeling and airway hyperresponsiveness (AHR). Methods: BALB/C mice were randomly divided into control group, asthma group, dexamethasone group and givinostat group (n=12 per group). AHR was assessed. Total cell numbers and differential counts, interleukin-4(IL-4), interleukin-5(IL-5) and interferon-γ (IFNγ) levels in the bronchoalveolar lavage fluid (BALF) were measured in the above 4 groups. The pathology of lung tissue was evaluated. Immunohistochemical (IHC) staining and Western blot were used to detect α smooth muscle actin(α-SMA) and transforming growth factor-ß1(TGFß1). Results: Compared with the asthma only group, givinostat treatment relieved airway resistance (2.96±1.01 vs 6.50±0.79, P<0.05). Total inflammatory cells [(33.04±5.62)×10(4)/ml vs (98.04±9.27)×10(4)/ml, P<0.01], eosinophil cells [(9.17±2.33)×10(4)/ml vs(37.64±6.98)×10(4)/ml, P<0.01], IL-4 [(10.12±2.98)ng/ml vs (16.88±2.78)ng/ml, P<0.05] and IL-5 [(27.09±3.62)ng/ml vs (37.86±7.34)ng/ml, P<0.05] levels were all reduced in givinostat group, while IFNγ [(91.86±23.73)pg/ml vs (60.49±11.88)pg/ml, P>0.05] was enhanced in BALF. Inflammatory cell infiltration around the airway was reduced, with decreased inflammatory cell score[(1.60±0.69)points vs (3.40±0.68) points, P<0.01] and inflammatory cell number (111.65±31.41 vs 601.25±186.85, P<0.01). The goblet cell metaplasia [(26.36±2.33)% vs (57.21±11.56)%] and collagen deposition area [(52.77±7.58)µm(2)/µm vs (111.81±12.40)µm(2)/µm] were obviously reduced (P<0.01). The expressions of α-SMA and TGFß1 in the lung tissue were both significantly decreased (P<0.01). Conclusion: Givinostat treatment can reduce airway inflammation, airway remodeling and airway hyperresponsiveness in chronic asthma. Its effect is comparable to that of glucocorticoid hormone treatment.

TLR4 gene expression in pig populations and its association with resistance to Escherichia coli F18.

TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.

Age-dependent expression of the BPI gene in Sutai piglets.

We compared and analyzed the expression of the BPI gene of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 by the real-time PCR method, in order to determine if it is involved in protection against disease caused by ETEC F18. There was a significant difference between 18 and 35-day expression in the jejunum. There were also significant differences between 35-day expression and expression at the other development stages in the duodenum. There were no significant differences in expression at 8, 18, and 30 days in the jejunum. We conclude that the porcine BPI gene may be the direct factor that resisted the ETEC F18 in weaning piglets, and that the resistance to ETEC F18 in weaning piglets is related to up-regulation of mRNA expression of BPI gene to a certain extent.

Peptide binding to MHC class I is determined by individual pockets in the binding groove.

H-2Kb and HLA-A2 are MHC4 class I molecules with a similar overall structure. Important differences between these two class I molecules reside in the structure of the individual pockets in the antigenic-peptide-binding groove. H-2Kb, which has a deep C pocket, binds specifically peptides with a tyrosine or a phenylalanine at position 5. In contrast, HLA-A2 has a shallow C pocket, which cannot accommodate large side chains at position 5. Site-directed mutagenesis was used to generate a chimera between the murine H-2Kb and the human HLA-A2 [H-2Kb/HLA-A2(C')]. The structure of this chimera is similar to H-2Kb except for the region around the deep C pocket, where residues at positions 9, 97 and 99 were substituted with those bulkier residues from HLA-A2. Peptide binding between this chimera and H-2Kb-binding peptides [VSV (52-59), OVA (257-264), and MCMV pp89 (168-176)], revealed that the deep C pocket of H-2Kb was crucial for high-affinity binding. While a peptide, VSV (52-59), was found to bind with severalfold lower 'affinity' to H-2Kb/HLA-A2(C') than to the wild-type H-2Kb, a VSV analogue with the tyrosine in position 5 (Tyr5) substituted with an alanine was found to bind with a similar 'affinity' to both MHC class I molecules. Computer-aided modelling of the H-2Kb/HLA-A2(C') complex indicates that the VSV (52-59) peptide probably binds to the chimeric MHC molecule with the peptide side chain of anchor residue Tyr5 pointing away from the groove. These results confirm a role of the individual pockets in determining peptide-binding affinity and specificity and suggest that this may be accomplished by changes in side-chain orientation.

Analysis of the structure of empty and peptide-loaded major histocompatibility complex molecules at the cell surface.

We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.

Analysis of TCR V beta gene usage and encephalitogenicity of myelin basic protein peptide p91-103 reactive T cell clones in SJL mice: lack of evidence for V gene hypothesis.

We have analyzed the epitope specificity and encephalitogenicity of peptides that span the C terminus of MBP, p84-103. Our studies show that multiple antigenic epitopes with disease-inducing capacity exist in SJL mice. Three peptides that span this region were examined and found to be immunogenic. However, the mode of immunization (active or passive) determined the incidence and severity of EAE. In our experiments adoptive transfer of p91-103-reactive T cell lines was most consistent in the development of disease. Interestingly, the response to peptides p89-101, p91-103, and p84-102 was absent following immunization with MBP. This suggests that although p91-103 and p89-101 were encephalitogenic they were not the major immunogenic epitopes following immunization with MBP. Analysis of a panel of eight p91-103-reactive T cell clones showed significant heterogeneity in the fine specificity, the TCR V beta gene usage, and in their ability of transfer EAE. These studies suggest that in SJL mice the epitopes involved in the pathogenesis of disease are multiple and there is no clear correlation between encephalitogenicity and TCR V beta gene usage. These observations argue against the presence of a dominant TCR V beta gene in the pathogenesis of EAE in SJL mice.

Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex-peptide complexes.

Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) class II gene-linked autoimmune demyelinating disease of the central nervous system. To develop therapies that will specifically inactivate only the autoantigen-reactive T cells, mice were treated with soluble MHC class II molecules that had been complexed with encephalitogenic peptides. Intravenous injections of 300 micrograms of complexes consisting of encephalitogenic peptide 91-103 of myelin basic protein plus I-As protein on day 0, 4, and 7 were effective in preventing experimental allergic encephalomyelitis. Similarly, administration of 45 micrograms of I-As protein complexed to peptide 139-151 from proteolipoprotein on day 1, 4, and 7 prevented mortality and significantly reduced paralysis induced by immunization with the encephalitogenic proteolipoprotein peptide. Histological examination of sections of animal brains revealed that treatment with I-As protein plus myelin basic protein 91-103 peptide prevents the development of inflammatory lesions characteristic of experimental allergic encephalomyelitis. Thus, treatment with MHC-self-peptide complexes could serve as a highly specific therapeutic modality in treating autoimmune disease when the putative autoantigen and the MHC restricting elements are known.

Treatment of chronic relapsing experimental allergic encephalomyelitis with the intravenous administration of splenocytes coupled to encephalitogenic peptide 91-103 of myelin basic protein.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) is an autoimmune demyelinating disease of the central nervous system and serves as an experimental model of human multiple sclerosis. Amino acid residues p91-103 of myelin basic protein are encephalitogenic in SJL mice and transfer of T cell lines that recognize this epitope results in CR-EAE. We show here that coculture of T cells in the presence of p91-103 that has been chemically cross-linked to the antigen presenting cells renders the T cell lines tolerant to the antigen. Injection of p91-103 coupled splenocytes into animals that had received encephalitogenic p91-103 reactive T cells significantly reduced the incidence and severity of EAE. Furthermore, treatment of mice with a single injection of antigen coupled splenocytes after they had recovered from their initial paralytic attack prevented the development of subsequent clinical relapses in all animals. These studies indicate that this effect is long lasting and can be successfully accomplished in an established autoimmune disease. Hence this form of immunotherapy may be considered as a therapeutic modality in the treatment of autoimmune diseases when the autoantigens are known.