A network pharmacology strategy to investigate the anti-osteoarthritis mechanism of main lignans components of Schisandrae Fructus.

Osteoarthritis (OA) is a chronic age-related progressive joint disorder. Degradation of the cartilage extracellular matrix (ECM) is considered a hallmark of OA and may be a target for new therapeutic methods. Schisandrae Fructus (SF) has been shown to be effective in treating OA. The major active components of SF are lignans. However, the targets of SF and the pharmacological mechanisms underlying the effects of SF lignans in the treatment of OA have not been elucidated. Therefore, based on network pharmacology, this research predicted the treatment targets of six lignans in SF, constructed a protein-protein interaction network and identified 15 hub genes in the OA-target protein-protein interaction network. Through Gene Ontology function and pathway analyses, the gene functions of lignans in the treatment of OA were determined. Finally, the anti-OA effects of lignans and underlying mechanisms identified in the network pharmacology analysis were verified by molecular docking, real-time PCR and western blotting in vitro. The biological processes of the genes and proteins targeted by lignans in the treatment of OA included the immune response, inflammatory response, cell signal transduction and phospholipid metabolism. Moreover, 20 metabolic pathways were enriched. Network pharmacology, molecular docking and in vitro and in vivo experimental results revealed that SF, schisanhenol and gamma-schisandrin inhibited EGFR and MAPK14 gene expression by inhibiting SRC gene expression and activity and then decreased MMP 13 and collagen II protein and gene expression. This research provides a basis for further study of the anti-OA effects and mechanisms of SF, schisanhenol and gamma-schisandrin.

The 1st step initiation essential for allergen-specific IgE antibody production upon the 2nd step: Induction of non-specific IgE+ small B cells containing secondly-sensitized allergen-specific ones in mice firstly-sensitized with an allergen.

There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.

Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa.

Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.

Antitumor effects in vitro and in vivo and mechanisms of protection against melanoma B16F10-Nex2 cells by fastuosain, a cysteine proteinase from Bromelia fastuosa.

In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57Bl/6 mice, fastuosain and bromelain injected intraperitoneally were protective, and very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, and became round and detached, forming strongly bound cell clusters in suspension. Peritoneal cells recruited and activated by fastuosain treatment (mainly monocytic cells and lymphocytes) migrated to the lung, where pulmonary melanoma metastases grew. Adoptive transference of peritoneal cells recruited by fastuosain had no protective effect against lung metastases in recipient mice. Treatment of green fluorescent protein-chimeric animals with fastuosain did not change the number of cells that migrated to the lung, compared to PBS-injected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, and cathepsins B and L cross-reacted in ELISA and recognized surface and cytoplasmic components expressed on B16F10-Nex2 cells. Anti-fastuosain antibodies were cytotoxic/lytic to B16F10-Nex2 cells. Antitumor effects of fastuosain involve mainly the direct effect of the enzyme and elicitation of protective antibodies.

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.

Carica papaya pollen allergy.

BACKGROUND: Carica papaya (CP) trees are widely cultivated in tropical and subtropical areas; however, CP pollen allergy has not been previously described. OBJECTIVE: To study patients with CP pollen hypersensitivity. METHODS: A CP pollen extract was elaborated. Skin prick tests (SPTs) with this extract, as well as with commercial papaya fruit and papain extracts, were performed. Specific IgE levels to CP pollen, papaya fruit, and papain were determined. Specific conjunctival challenge tests to the CP pollen extract were also performed. RAST inhibition studies among CP pollen, papaya fruit, and papain were carried out. Twenty atopic patients were used as a control group for in vivo and in vitro tests. RESULTS: Six patients with clinical histories of seasonal rhinoconjunctivitis or bronchial asthma in relation to CP trees exposure, suggestive of IgE-mediated respiratory allergy, were studied. Commercial SPT and specific serum IgE to papaya fruit and papain were positive in our patients. An IgE-mediated hypersensitivity to a CP pollen extract was demonstrated in all patients, by means of SPTs, specific serum IgE determinations, and conjunctival challenge tests. Control atopic subjects showed negative SPTs, specific IgE, and conjunctival challenge tests to the CP pollen extract. On RAST inhibition studies using CP pollen extract in solid phase, a significant crossreactivity was found among CP pollen, papaya fruit, and papain. CONCLUSIONS: Our study suggests that papaya flower pollen is able to induce respiratory IgE-mediated allergy. The existence of common allergens among papaya flower pollen, papaya fruit, and papain has been demonstrated by RAST inhibition.

Asthma caused by Ficus benjamina latex: evidence of cross-reactivity with fig fruit and papain.

BACKGROUND: Ficus benjamina or weeping fig is a plant used increasingly for indoor decoration that can cause allergic rhinitis and asthma. OBJECTIVE: We report a clinical and immunologic study in a patient with perennial asthma caused by F. benjamina latex in whom several episodes of angioedema of the oropharyngeal tract and tongue followed ingestion of figs and kiwi. METHODS: Hypersensitivity to latex from F. benjamina and from Hevea brasiliensis, fig fruit, kiwi, papain, and bromelain was investigated by means of skin prick test, specific IgE determination by CAP, histamine release test, and bronchial provocation test to F. benjamina latex. CAP-inhibition assays were carried out to study possible cross-reactivity among these antigens. RESULTS: Hypersensitivity to F. benjamina latex, fig, kiwi, and proteases was demonstrated by means of skin prick test, determination of specific IgE and histamine release test. Bronchial provocation test with F. benjamina latex resulted in a dual asthmatic reaction, confirming the etiologic role of this plant. A rise of eosinophil cationic protein in patient's serum was observed 21 hours after bronchial challenge, suggesting activation of eosinophils. Inhibition assays showed that F. benjamina latex as liquid-phase inhibited up to 95% the CAP to fig and up to 57% the CAP to papain. Neither sensitization nor cross-allergenicity with H. brasiliensis latex was found. CONCLUSIONS: Hypersensitivity to F. benjamina latex may cause IgE-mediated respiratory allergy. The association with allergy to fig and papain is likely due to the existence of cross-reactive allergen structures.

Monoclonal antibodies against Dermatophagoides group I allergens as pseudo-cystatins blocking the catalytic site of cysteine proteinases.

The enzyme allergens Der p I and Der f I produced by the house dust mites Dermatophagoides pteronyssinus and D. farinae display partial sequence homology with other members of the cysteine proteinase superfamily. We report that certain widely used mouse mAbs against these Group I allergens indeed crossreact with the plant enzymes papain, bromelain and ficin. The recognition sites of these anti Group I mAbs comprise conformational and thermolabile epitopes involved in molding the catalytic center of the proteinases. Thus, the mAbs inhibit the enzymatic hydrolysis of specific chromogenic substrates by the Group I allergens, while specific cysteine proteinase inhibitors abolish the recognition of the enzymes by the mAbs. Similarly, activation of the thiol-proteases with L-cysteine abrogates their binding in the two-site mAb system, indicating that the mAbs recognize a proenzyme conformational peptide epitope. It follows that mAb-based assays for mite Group I components can neither detect the allergens after inactivation, nor in their fully activated forms.

Pulmonary disease in workers exposed to papain: clinico-physiological and immunological studies.

Of the twenty-three employees at a pharmaceutical plant manufacturing a new product containing papain, twelve had respiratory symptoms of cough, wheezing, dyspnoea, or chest paint. Most were studied with in-depth interviews by a doctor, extensive pulmonary function tests, and immunoserological tests for IgE and precipitating antibodies specific for papain, as well as total IgE antibodies to common natural allergens. There were significant correlates (all P values < 0.05) between the presence of specific IgE antibodies to papain and decreases of FEV1, FEF75--85, TLC, RV, and response to bronchodilators as percentage change from baseline for all spirographic flow rates. Atopic workers developed pulmonary symptoms and antipapain antibodies significantly sooner after papain exposure than did the others. Duration of exposure had no effect on symptomatology, pulmonary function, or immunological response. However, those judged to have the greatest amount of dust exposure per work-day had significantly more pulmonary symptoms (P < 0.005). Papain produced lung diseases by acting as an inhalant allergen rather than a proteolytic enzyme. Papain is a potent sensitizer in humans for the production of respiratory disease. The pulmonary reactions, based on physiological data, seem to involve small airways, alveolar, and interstitial lung tissue in an inflammatory rather than destructive manner, and thus resemble bronchitis and interstitial lung disease rather than pulmonary emphysema or typical bronchial asthma.

Studies on the specificity of human IgE-antibodies to the plant proteases papain and bromelain.

The sera of seven patients clinically hypersensitive to papain--in one case also to baromelain--and the sera of sixty asthmatic patients with allergies to other inhalant and food allergens were investigated for IgE antibody activity to the plant proteases papain and bromelain and to common allergens by RAST, confirmed in some sera by RAST inhibition. There seems to be a relation between the antibody reactions to papain and bromelain, in several cases also between the reactions to these proteases and to grass pollen and flour. Studies by RAST inhibition showed that papain, bromelain, wheat flour, rye flour, grass pollen and birch pollen mutually inhibit IgE antibody to each antigen; but the degree of inhibition varies among the different sera and allergens. Our results suggest that these allergens from various plants, besides having specific antigenic determinants, also possess similar or even identical antigenically active regions, leading to immunological cross-reactivity.