A Strategy for Quality Control of Vespa magnifica (Smith) Venom Based on HPLC Fingerprint Analysis and Multi-Component Separation Combined with Quantitative Analysis.

As a folk medicine of the Jingpo minority in Yunnan province, the venom of Vespa magnifica has been commonly used for the treatment of rheumatoid arthritis. Quality standardization of the wasp venom is a necessary step for its pharmaceutical research and development. To control the quality of the wasp venom, a method based on high-performance liquid chromatography (HPLC) was developed for chemical fingerprint analysis. In the chromatographic fingerprinting, chemometrics procedures, including similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA), were applied to classify 134 batches (S1-S134) of wasp venom from different origins. The HPLC fingerprint method displayed good precision (Relative standard deviation, RSD < 0.27%), stability (in 16 h, RSD < 0.34%), and repeatability (RSD < 1.00%). Simultaneously, four compounds (VMS1, VMS2, VMS3, and VMS4) in the wasp venom were purified and identified. VMS1 was 5-hydroxytryptamine, and the other compounds were three peptides that were sequenced as follows: Gly-Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg-Ile-Asp-NH2 (VMS2), Ile-Asn-Leu-Lys-Ala-Ile-Ala-Ala-Leu-Ala-Lys-Lys-Leu-Leu-NH2 (VMS3), and Phe-Leu-Pro-Ile-Ile-Gly-Lys-Leu-Leu-Ser-Gly-Leu-Leu-NH2 (VMS4). The quantifications for these components were 110.2 mg/g, 26.9 mg/g, 216.3 mg/g, and 58.0 mg/g, respectively. The results of this work indicated that the combination of the chemical fingerprint and quantitative analysis offers a reasonable way to evaluate the quality of wasp venom.

Kangfuxin alleviates ulcerative colitis in rats by inhibiting NF-κB p65 activation and regulating T lymphocyte subsets.

Objectives: Ulcerative colitis (UC) remains an enduring, idiopathic inflammatory bowel disease marked by persistent mucosal inflammation initiating from the rectum and extending in a proximal direction. An ethanol extract of Periplaneta americana L., namely Kangfuxin (KFX), has a significant historical presence in Traditional Chinese Medicine and has been broadly utilized in clinical practice for the treatment of injury. Here, we aimed to determine the effect of KFX on 2,4,6-trinitro'benzene sulfonic acid (TNBS)-induced UC in Sprague-Dawley rats. Materials and Methods: We established the UC model by TNBS/ethanol method. Then, the rats were subject to KFX (50, 100, 200 mg/kg/day) for 2 weeks by intragastric gavage. The body weight, disease activity index (DAI), colonic mucosal injury index (CMDI), and histopathological score were evaluated. The colonic tissue interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), IL-10, transforming growth factor-1 (TGF-ß1), and epidermal growth factor (EGF) were determined by Elisa. To study T-lymphocyte subsets, flow cytometry was performed. In addition, the expression level of NF-κB p65 was evaluated by immunohistochemistry and western blot analysis. Results: Compared with the TNBS-triggered colitis rats, the treatment of rats with KFX significantly increased the body weight, and decreased DAI, CMDI, and histopathological score. Also, KFX elicited a reduction in the secretion of colonic pro-inflammatory cytokines, namely IL-1ß, IL-6, and TNF-α, concomitant with up-regulation of IL-10, TGF-ß1, and EGF levels. Upon KFX treatment, the CD3+CD4+/CD3+CD8+ ratio in the spleen decreased, while the CD3+CD8+ subset and the CD3+CD4+CD25+/CD3+CD4+ ratio demonstrated an increase. In addition, the expression of NF-κB p65 in the colon was decreased. Conclusion: KFX effectively suppresses TNBS-induced colitis by inhibiting the activation of NF-κB p65 and regulating the ratio of CD4+/CD8+.

The therapeutic effect of wasp venom (Vespa magnifica, Smith) and its effective part on rheumatoid arthritis fibroblast-like synoviocytes through modulating inflammation, redox homeostasis and ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a chronic inflammatory disease that is related to the aberrant proliferation of fibroblast-like synoviocytes (FLS). Wasp venom (WV, Vespa magnifica, Smith), an insect secretion, has been used to treat RA in Chinese Jingpo national minority's ancient prescription. However, the potential mechanisms haven't been clarified. AIM OF THE STUDY: The purposes of this paper were two-fold. First, to investigate which was the best anti-RA effective part of WV-I (molecular weight less than 3 kDa), WV-II (molecular weight 3-10 kDa) and WV-III (molecular weight more than 10 kDa) that were separated from WV. Second, to explore the underlying molecular mechanism of WV and WV-II that was best effective part in RA. MATERIALS AND METHODS: The wasps were electrically stimulated and the secretions were collected. WV-I, WV-II and WV-III were acquired by ultracentrifuge method according to molecular weight. Next, WV, WV-I, WV-II and WV-III were identified by HPLC. Functional annotation and pathway analysis of WV used to bioinformatics analysis. RNA-seq analyses were constructed to identify differentially expressed genes (DEGs). GO and KEGG pathway analyses were performed by Metascape database. STRING was used to analyze the PPI network from DEGs. Next, PPI network was visualized using Cytoscape that based on MCODE. The pivotal genes of PPI network and MCODE analysis were verified by qRT-PCR. Subsequently, MH7A cells were performed by MTT assay to evaluate the ability of inhibiting cell proliferation. Luciferase activity assay was conducted in HepG2/STAT1 or HepG2/STAT3 cells to assess STAT1/3 sensitivity of WV, WV-I, WV-II and WV-III. Additionally, interleukin (IL)-1ß and IL-6 expression levels were detected by ELISA kits. Intracellular thioredoxin reductase (TrxR) enzyme was evaluated by TrxR activity assay kit. ROS levels, lipid ROS levels and Mitochondrial membrane potential (MMP) were assessed by fluorescence probe. Cell apoptosis and MMP were measured by using flow cytometry. Furthermore, the key proteins of JAK/STAT signaling pathway, protein levels of TrxR and glutathione peroxidase 4 axis (GPX4) were examined by Western blotting assay. RESULTS: RNA-sequencing analysis of WV displayed be related to oxidation-reduction, inflammation and apoptosis. The data displayed that WV, WV-II and WV-III inhibited significantly cells proliferation in human MH7A cell line compared to WV-I treatment group, but WV-III had no significant suppressive effect on luciferase activity of STAT3 compared with IL-6-induced group. Combined with earlier reports that WV-III contained major allergens, we selected WV and WV-II further to study the mechanism of anti-RA. In addition, WV and WV-II decreased the level of IL-1ß and IL-6 in TNF-α-induced MH7A cells via inactivating of JAK/STAT signaling pathway. On the other hand, WV and WV-II down-regulated the TrxR activity to produce ROS and induce cell apoptosis. Furthermore, WV and WV-II could accumulate lipid ROS to induce GPX4-mediated ferroptosis. CONCLUSIONS: Taken together, the experimental results revealed that WV and WV-II were potential therapeutic agents for RA through modulating JAK/STAT signaling pathways, redox homeostasis and ferroptosis in MH7A cells. Of note, WV-II was an effective part and the predominant active monomer in WV-II will be further explored in the future.

Isolation and identification of the components in Cybister chinensis Motschulsky against inflammation and their mechanisms of action based on network pharmacology and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Cybister chinensis Motschulsky belongs to the family Dytiscidae. As a traditional Chinese medicine, the insect is called Longshi in the folk and is commonly used to treat enuresis in children and frequent urination in the elderly. AIM OF THE STUDY: Inflammation is involved in chronic kidney disease. The previous study proved ethanol extract of C. chinensis exhibited anti-inflammation effects in the Doxorubicin-induced kidney disease. However, the material basis and their possible mechanism of the insect were still unclear. Thus, we aimed to separate the active compounds of the ethanol extract from C. chinensis and to investigate their possible mechanism of anti-inflammation by network pharmacology and molecular docking. MATERIALS AND METHODS: The insect was extracted with 75% ethanol to produce ethanol extracts and then were extracted by petroleum ether, ethyl acetate and n-butanol respectively. Silica gel column chromatography and preparative HPLC were applied to separate the compounds of the extract. The compounds were characterized and identified by NMR and mass. The compound associated genes were collected by BATMAN-TCM database and the inflammation associated genes were obtained through DigSee database. The protein-protein interaction (PPI) network was carried out via Search Tool for the Retrieval of Interacting Genes/Protein (STRING) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) target pathway analysis was performed in Database for Annotation, Visualization and Integrated Discovery (DAVID). The possible mechanism of compounds against inflammation was investigated by molecular docking. Finally, the anti-inflammatory effect of the representative compound was verified by the LPS-induced Raw 264.7 cell inflammatory model. TNF-α, IL-1ß and IL-6 of the cell supernatants were analyzed via using ELISA kits and the key proteins in JAK2/STAT3 signaling pathway were verified via the Western blot assays. RESULTS: Among crude extracts from C. chinensis, ethyl acetate extract showed the obvious anti-inflammatory effects. Nine compounds were isolated from ethyl acetate extract of Cybister chinensis for the first time, including benzoic acid (1), hydroxytyrosol (2), protocatechualdehyde (3), N-[2-(4-hydroxyphenyl)ethyl]acetamide (4), (2E)-3-phenylprop-2-enoic acid (5), 3-phenylpropionic acid (6), methyl 3,4-dihydroxybenzoate (7), 1,4-diphenyl butane-2,3-diol (8) and p-N,N-dimethylaminobenzaldehyde (9). After searching in the database, 1079 compound associated genes and 467 inflammation associated genes were found. The 137 common targets covered 77 signaling pathways, in which HIF-1 signaling pathway, TNF signaling pathway, influenza A, PI3K/Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway and Jak-STAT signaling pathway were important for inflammation. Molecular docking studies showed compound 1, 4, 5, 6, 7 and 8 were the potential inhibitors of JAK2 protein. In addition, the in vitro test showed compound 5 reduced the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner. Furthermore, it was found that compound 5 inhibited the expression of p-JAK2 and p-STAT3 in LPS-induced RAW264.7 cells in a dose-dependent manner. CONCLUSIONS: Based on the network pharmacology and molecular docking, the study suggested that C. chinensis could relieve the inflammation based on the multi-compounds and multi-pathways, which provided the foundation for the medicinal application of C. chinensis.

Kangfuxin Liquid Ameliorates Dextran Sulfate Sodium (DSS)-Induced Acute Ulcerative Colitis in Mice by Modulating Immune Response and Suppressing Inflammation.

BACKGROUND The aim of this study was to determine the effect of kangfuxin liquid (KFXL) on inflammatory response, and its underlying mechanism in treating acute ulcerative colitis (UC) in mice induced by dextran sulfate sodium (DSS). MATERIAL AND METHODS Mice were provided drinking water containing DSS (3%) for 7 days to induce acute enteritis. The mice were divided into 6 groups: a control group, a DSS-induced (vehicle) group, a sulfasalazine (SASP) group, and low-, medium-, and high-dose kangfuxin liquid groups. Disease activity index (DAI), colon mucosa damage index (CMDI), histopathological score (HS), and organ index were monitored daily. The levels of interleukin-1ß (IL-1ß), interleukin-10 (IL-10) in serum and interleukin-17 (IL-17) and epidermal growth factor (EGF) in colon tissue were assessed by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to assess the changes of T lymphocyte subsets in spleens of mice to evaluate the therapeutic effect of drugs on acute UC in mice. RESULTS Different doses of kangfuxin liquid reduced the DAI, CMDI, and HS scores (P<0.01 or P<0.05) of acute UC mice, reduced the level of IL-1ß and IL-17 in serum, increased the expression of IL-10 in serum and EGF in colon tissue, increased the number of CD3⁺ T cells, and decreased the level of CD4⁺ T cells and the ratio of CD4⁺/CD8⁺. CONCLUSIONS Kangfuxin liquid has a therapeutic effect on DSS-induced acute UC in mice, and its mechanism of action may be associated with regulating immune function and reducing intestinal inflammatory response.

Wasp Venom Possesses Potential Therapeutic Effect in Experimental Models of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease. Wasp venom (WV), which is considered as a traditional folk medicine in Jingpo nationality in Yunnan, China, relieves rheumatoid arthritis. The current study aimed to investigate the effect of wasp venom ameliorating rheumatoid arthritis symptoms in experimental rats. We established a model of type II collagen- (CII-) induced arthritis (CIA) in SD rats and examined the inhibition of inflammation and autoimmune response. The antiarthritic effects of WV were evaluated through the paw swelling, and histopathological score and histopathology changes of the affected paw were assessed. The anti-inflammation effects were assayed by the level of IL-6, TNF-α, IL-1ß, and the number of inflammatory cells in peripheral blood. The alteration of the T cell subset ratio in the spleen of rats was detected by flow cytometry, and at the same time, the viscera index and immune serum globulin levels were evaluated. The results suggested that various doses of WV (0.125, 0.25, and 0.5 mg/kg) significantly alleviated paw swelling and arthritis score in CIA rats with the untreated control (P < 0.05). WV (0.25 and 0.5 mg/kg) relieved synovial tissue lesions of ankle joints and histopathology scores of synoviocyte hyperplasia and inflammatory cell infiltration with vehicle group (P < 0.05). Regarding immunological regulation, 0.5 mg/kg WV lowered the immune serum globulin levels (P < 0.05), and we further found that WV (0.5 mg/kg) suppressed the immune response of Th cells, while enhancing the functions of Tc cells and Treg cells in spleen cells markedly (P < 0.05). The immunosuppressive action of WV displayed was analogous to its inhibitory effect on IL-1ß, TNF-α, IL-8, IL-6, COX-2, and PGE2 levels in rat serum. In conclusion, these findings demonstrated that WV exhibited antiarthritic activity, which might be associated with their inhibitory effects on immunoregulation and anti-inflammatory action.