Proteomics and phosphoproteomics to study Tuina reverses capsule fibrosis in frozen shoulder: a research report based on rats.

Frozen shoulder (FS) is a common disorder often treated with Tuina, but the mechanisms involved remain unclear. We employed proteomics and phosphoproteomics to investigate the mechanisms associated with the treatment of capsule fibrosis in FS rats. We used a method composed of three weeks of cast immobilization to establish a model of FS. We then administered Tuina once daily for 14 days, evaluated glenohumeral range of motion (ROM), assessed histological changes, and identified differentially expressed proteins (DEPs) using proteomics and phosphoproteomics. This study demonstrated that Tuina could improve glenohumeral ROM and reserve capsule fibrosis in FS rats. Proteomics revealed proteins regulated by Tuina belonging to the PI3K-AKT and ECM receptor interaction signaling pathways. Phosphoproteomics detected differentially phosphorylated proteins regulated by Tuina to be enriched in the MAPK signaling pathway. The combination of proteomics and phosphoproteomics for Protein-Protein Interaction (PPI) network analysis revealed that the phosphorylation of Myh3 and Srsf1 with a node degree larger than the average degree were considered the central regulatory protein modulated by Tuina to reverse capsule fibrosis. Thbs1, Vtn, and Tenascin-W were significantly enriched in PI3K-AKT and ECM receptor interaction signaling pathways and highly expressed in model rats. Tuina resulted in reduced expression of these proteins. Our findings demonstrated some of mechanisms behind the reversal of FS capsule fibrosis following Tuina, a scientific medical therapy for FS patients.

Tuina Intervention in Rabbit Model of Knee Osteoarthritis.

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

Tuina in a Frozen Shoulder Rat Model: An Efficient and Reproducible Protocol.

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS patients in Chinese hospitals, has demonstrated excellent results, but its mechanisms are not fully understood. Building on a previous study, this work aimed to develop a Tuina protocol for an FS rat model. We randomly divided 20 SD rats into control (C; n = 5), FS model (M; n = 5), FS model Tuina treatment (MT; n = 5), and FS model oral treatment (MO; n = 5) groups. This study used the cast immobilization method to establish the FS rat model. The effect of Tuina and oral dexamethasone on the glenohumeral range of motion (ROM) was evaluated, and the histological findings were assessed. Our study showed that Tuina and oral dexamethasone were able to improve shoulder active ROM and preserve the structure of the capsule, with Tuina therapy proving to be more effective than oral dexamethasone. In conclusion, the Tuina protocol established in this study was highly effective for FS.

Molecular Characteristics of Cell Pyroptosis and Its Inhibitors: A Review of Activation, Regulation, and Inhibitors.

Pyroptosis is an active and ordered form of programmed cell death. The signaling pathways of pyroptosis are mainly divided into canonical pathways mediated by caspase-1 and noncanonical pathways mediated by caspase-11. Cell pyroptosis is characterized by the activation of inflammatory caspases (mainly caspase-1, 4, 5, 11) and cleavage of various members of the Gasdermin family to form membrane perforation components, leading to cell membrane rupture, inflammatory mediators release, and cell death. Moderate pyroptosis is an innate immune response that fights against infection and plays an important role in the occurrence and development of the normal function of the immune system. However, excessive pyroptosis occurs and leads to immune disorders in many pathological conditions. Based on canonical pathways, research on pyroptosis regulation has demonstrated several pyroptotic inhibitors, including small-molecule drugs, natural products, and formulations of traditional Chinese medicines. In this paper, we review the characteristics and molecular mechanisms of pyroptosis, summarize inhibitors of pyroptosis, and propound that herbal medicines should be a focus on the research and development for pyroptosis blockers.

Bai-Hu-Tang regulates endothelin-1 and its signalling pathway in vascular endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Bai-Hu-Tang (BHT) is traditionally used to treat human and animal fever syndrome with four symptoms: large and vigorous pulse, large thirst, high sweat, and high heat. AIM OF THE STUDY: To investigate the mechanism of vasodilation regulation of Bai-Hu-Tang in primary vascular endothelial cells stimulated by lipopolysaccharide (LPS). MATERIALS AND METHODS: A hydrophilic concentrate of BHT was prepared, and the main components of mangiferin and timosaponin BⅡ were determined by HLPC analysis. The rabbit fever model was constructed by intravenous injection of LPS (15 µg/kg body weight), and BHT was gavaged to treat febrile rabbits. After treatment for 6 h, animal peripheral blood was collected, and serum was isolated for endothelin-1 (ET-1) and nitric oxide (NO) assays. Rabbit vascular endothelial cells (RVECs) were isolated and stimulated with 1 µg/mL LPS, and then inflammatory cells were treated with 125 or 250 µg/mL BHT for 24 h. The supernatant cytokines TNF-ɑ, IL-1ß, IL-6, and ET-1 were detected by ELISA kits. Gene expression levels of endothelin receptor type B (ETB receptor) were analysed by real-time polymerase chain reaction (RT-PCR), and protein expression levels of PI3K and Akt were detected by Western blot. A nitrite assay was used to measure intracellular nitric oxide (NO) production, and nitric oxide synthase (NOS) was measured by the T-NOS colorimetric method. RESULTS: Animal experiments demonstrated that BHT significantly restored ET-1 and NO in animal peripheral blood, which were disordered in LPS-induced fever rabbits. Moreover, a cytotoxicity assay demonstrated that BHT ≤700 µg/mL is innoxious to RVECs. BHT significantly repressed cellular TNF-α, IL-1ß, and ET-1, which were originally elevated by LPS in RVECs. Meanwhile, BHT elevated the gene expression level of the ETB receptor and promoted NOS and NO production in RVECs induced by LPS. CONCLUSION: BHT can inhibit excessive ET-1 secretion induced by LPS in vascular endothelial cells and activate the classic ET-1 signalling pathway to promote NO production, which may facilitate vasodilation of smooth muscle cells.

iTRAQ-based quantitative proteomic analysis reveals Bai-Hu-Tang enhances phagocytosis and cross-presentation against LPS fever in rabbit.

ETHNOPHARMACOLOGICAL RELEVANCE: Bai-Hu-Tang (BHT), a classical anti-febrile Chinese formula comprising of liquorice, anemarrhena rhizome, gypsum and rice, has been traditionally used to anti-febrile treatment and promote the production of body fluid to relieve thirst. In this paper, we aim to explore anti-febrile mechanism of BHT at protein level through analyzing alteration of differentially expressed proteins (DEPs) both lipopolysaccharide (LPS) fever syndrome and that was treated with BHT in rabbits. MATERIALS AND METHODS: Febrile model was induced by LPS injection (i.v.) in rabbits, and BHT (750mg dry extract/kg body weight) was gavaged to another group of LPS fever rabbits. After sacrifice of animals, total protein of liver tissue was isolated, and two-dimensional liquid chromatography (LC) - tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling analysis was employed to quantitatively identify differentially expressed proteins in two group animals, which were compared with control group. Then bioinformatic analysis of DEPs was conducted through hierarchical Clustering, Venn analysis, gene ontology (GO) annotation enrichment, and kyoto encyclopedia of genes and genomes (KEGG) pathways enrichment. RESULT: The results demonstrated there were 63 and 109 DEPs in LPS fever group and BHT-treated group, respectively. Enrichment analysis of GO annotations indicated that BHT mainly regulated expression of some extracellular structural proteins for response to stimulus and stress. KEGG analysis showed that ribosome and phagosome were the most significant pathways. Thereinto, several proteins in phagosome pathway were significantly up-regulated by BHT, including F-actin, coronin, Rac, and major histocompatibility complex class I (MHC I), which work in phagocytosis and cross-presentation CONCLUSION: BHT may contribute to pyrogen clearance by boosting antigenic phagocytosis, degradation, and cross presentation in the liver.

Differentially expressed genes of LPS febrile symptom in rabbits and that treated with Bai-Hu-tang, a classical anti-febrile Chinese herb formula.

ETHNOPHARMACOLOGICAL RELEVANCE: Bai-Hu-Tang (BHT) has been traditionally used to clear heat and engender fluids. AIM OF THE STUDY: To reveal the alteration of differentially expressed genes (DEGs) between lipopolysaccharide (LPS) febrile syndrome in rabbits and treatment with BHT which is a classical anti-febrile formula in traditional Chinese medicine. MATERIALS AND METHODS: Febrile model was induced by LPS injection (i.v.) in rabbits, and BHT was gavaged to another group of febrile rabbits. After sacrifice of animals, total RNA of liver tissue was isolated, processed, and hybridized to rabbit cDNA microarrays obtained from Agilent Co. The data of DEGs were obtained by lazer scanning and analyzed with Cluster program 3.0. Then bioinformatic analysis of DEGs was conducted through gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, expression levels of four relative genes were detected by quantitative real time ployenzyme chain reaction (qRT-PCR) to validate the accuracy of microarrays. RESULT: The results demonstrated that genes expression pattern could be clustered into three groups significantly, and there were 606 up-regulated genes and 859 down-regulated genes in the model group, and 106 up-regulated genes and 429 down-regulated genes in BHT treated group. There were 286 DEGs existed as the common in two experimental groups. Enrichment analysis of GO annotations indicated that DEGs in model and BHT treated animals mainly referred catalytic activity and oxidoreductase activity for metabolic processes located in the membrane system at intracellular part, and binding activities increased significantly in treatment with BHT. Enrichment of KEGG analysis showed that the pathways of phagosome and protein processing in endoplasmic reticulum contained the most altered genes in the LPS group, but the percentage of phagosome pathway almost doubled in BHT group. Most DEGs involved in the LPS signal recognition system was up-regulated in LPS group, but partly decreased in BHT group. RT-PCR results of eight relative genes were consistent with the results of microarrays. CONCLUSION: DEGs of LPS febrile syndrome mainly involved oxidoreductase and catalytic activity of the metabolic processes, and pathways of processing protein for pyrotoxin recognition; BHT mostly regulated the DEGs in the phagosome pathway to clear LPS in the liver, and partly interfered with gene expression in LPS recognition system. The study provided an important pioneering result on gene expression profiling research, and will facilitate the clinical care or further studies of the formula.

Aqueous extract of Bai-Hu-Tang, a classical Chinese herb formula, prevents excessive immune response and liver injury induced by LPS in rabbits.

ETHNOPHARMACOLOGICAL RELEVANCE: Bai-Hu-Tang (BHT) was traditionally used to reduce fever heat and promote generation of body fluids. AIM OF THE STUDY: To investigate the effect and mechanism of BHT in the prevention of lipopolysaccharide (LPS) fever in manners of immune modulation. MATERIALS AND METHODS: The model of fever syndrome of Chinese medicine pattern was imitated by LPS injection i.v. in rabbits, and BHT was gavaged. The serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL-6, 10) and immunoglobulin (IgG, IgA, and IgM) were determined by enzyme-linked immunosorbent assay (ELISA); alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested by biochemical methods. Liver tissue damage was detected by hematoxylin-eosin (H&E) stain. Subpopulation of T cells was detected by Fluorescence Activated Cell Sorter (FACS). Genes expression of Toll-like receptor 4 (TLR4) and lipopolysaccharide binding protein (LBP) in liver tissue were assayed by real-time polymerase chain reaction (RT-PCR). RESULT: The results demonstrated that BHT prevented sudden increase of IL-10, TNF-α, ALT and AST, and liver damage induced by LPS. BHT also prevented significant decrease of the percentage of CD(8+) T cells since LPS injection. At the same time, BHT did not affect the gene expression of TLR4 and serum concentration of three immunoglobulins, which were increased by LPS, but made gene expression of LBP higher. CONCLUSION: The results of this study indicated that BHT played an important role in immunity protection and anti-injury through preventing immunoinflammatory damage by LPS. The achievement thereby scientifically provided mechanism of BHT in the prevention of febrile disease, and supported its traditional use.

Cytotoxicity, apoptosis-inducing effects and structure-activity relationships of four natural xanthones from Gentianopsis paludosa Ma in HepG2 and HL-60 cells.

Four xanthones were isolated from Gentianopsis paludosa Ma and were identified by modern spectroscopic methods. Cytotoxicity of the four xanthones was tested on HepG2 cells and HL-60 cells by sulphorhodamine B (SRB) assay. Clonogenic survival assay, trypan blue exclusion method, AO/EB staining and DNA fragmentation assay were conducted to investigate the effect on growth inhibition and apoptosis in the two cell lines in vitro. At the same time, structure-activity relationships (SARs) of the xanthones were investigated. The results showed that the xanthones had significant cytotoxicity and inhibition of proliferation in both HepG2 cells and HL-60 cells, and could induce apoptosis in these two cell lines. SARs indicated that the methoxy group had more cytotoxic contribution than the hydroxyl group at site C-8 in the structural scaffold of xanthone. The glycosidea at site C-1 may aggravate the stereospecific blockade of compound 4 and reduced its cytotoxic activity.