Network and Experimental Pharmacology to Decode the Action of Wendan Decoction Against Generalized Anxiety Disorder.

Objective: The mechanism of Wendan Decoction (WDD) against Generalized Anxiety Disorder (GAD) was predicted by network pharmacology and validated by in vivo and in vitro experiments. Methods: The targets of WDD for the treatment of GAD were obtained by a search of online databases. Further, PPI network and KEGG enrichment were used to identify the key targets and pathways. Ultimately, these key targets and pathways were validated by in vivo experiments on GAD mice modeled by repeated restraint stress (RRS) and in vitro experiments on inflammatory factor stimulated BV-2 cells. Results: Through searching the databases, the 137 ingredients of WDD that correspond to 938 targets and 4794 targets related to GAD were identified. Among them, 569 overlapping targets were considered as the therapeutic targets of WDD for GAD. PPI analysis showed that the inflammation-related proteins IL-6, TNF, SRC and AKT1 were the key targets, and KEGG enrichment suggested that PI3K/AKT and MAPK signaling pathways were key pathways of WDD in the treatment of GAD. In vivo experiments, RRS mice exhibited abnormality in behavioristics in open field test (OFT) and elevated plus maze (EPM) and increases in serum corticosterone and the percentage of lymphocytes positive for IL-6 in peripheral blood. These abnormal changes can be reversed by WDD and the positive control drug paroxetine. In vitro experiments, WDD can inhibit IL-6 induced activation of PI3K/AKT and MAPK signaling pathways in BV2 cells, and suppress the ensuing release of inflammatory factors TNF-α, IL-1ß and PGE2, and showed a dose-dependent effect. Conclusion: WDD is able to resist GAD by relieving inflammatory response in peripheral and central system.

Chondroprotective Effects of Gubitong Recipe via Inhibiting Excessive Mitophagy of Chondrocytes.

Objective: Osteoarthritis (OA) is the most common degenerative joint disorder and a leading cause of disability. A previous randomized controlled trial has shown that Gubitong (GBT) recipe can improve OA-related symptoms and articular function without noticeable side effects. However, the underlying mechanisms remain unclear. This study aims to explore the therapeutic mechanisms of the GBT recipe for OA through in vivo and in vitro experiments. Methods: Rats of the OA model were established by Hulth surgery and intervened with the GBT recipe and then were subjected to pathological assessment of the cartilage. Matrix metalloproteinase 13 (MMP-13) expression in cartilage tissues was assessed by immunohistochemical staining. Chondrocytes were isolated from sucking rats and stimulated with LPS to establish an in vitro model. After intervened by water extraction of the GBT recipe, the fluorescent signal of Mtphagy Dye and mitochondrial membrane potential (Δψm) were detected to determine the states of mitophagy and mitochondrial dynamics of chondrocytes in vitro, respectively. Western blot test was used to detect levels of proteins related to catabolism of the cartilage matrix, mitophagy, and PI3K/AKT pathway. Results: In in vivo experiments, the GBT recipe can effectively inhibit the cartilage degeneration of chondrocytes in OA rats, as well as markedly suppress the expression of MMP-13. In vitro experiments on LPS-induced chondrocytes exhibited increase in mitochondrial depolarization and excessive mitophagy, and the GBT recipe can alleviate these changes. LPS-stimulated chondrocytes showed increases in MMP-13, PINK1, and Parkin in cell lysates and LC3II/LC3I ratio in the mitochondrial fraction, and the GBT recipe can inhibit these increases in a dose-dependent manner. Moreover, the GBT recipe can attenuate the abnormal activation of PI3K/AKT pathway induced by LPS. Conclusion: The GBT recipe exhibits chondroprotective effects through inhibiting excessive mitophagy of chondrocytes, which may be associated with its inhibitory effect on the abnormal activation of PI3K/AKT pathway.

Integrating Network Pharmacology and Experimental Validation to Explore the Key Mechanism of Gubitong Recipe in the Treatment of Osteoarthritis.

Background: Gubitong Recipe (GBT) is a prescription based on the Traditional Chinese Medicine (TCM) theory of tonifying the kidney yang and strengthening the bone. A previous multicentral randomized clinical trial has shown that GBT can effectively relieve joint pain and improve quality of life with a high safety in treating osteoarthritis (OA). This study is aimed at elucidating the active compounds, potential targets, and mechanisms of GBT for treating OA. Method: The network pharmacology method was used to predict the key active compounds, targets, and mechanisms of GBT in treating OA. An OA rat model was established with Hulth surgery, and the pathological changes of articular cartilage were observed to evaluate the effects of GBT. Chondrocytes were stimulated with LPS to establish in vitro models, and key targets and mechanisms predicted by network pharmacology were verified via qRT-PCR, ELISA, western blot, and immunofluorescence. The Contribution Index Model and molecular docking were used to determine the key active compounds of GBT and the major nodes affecting predicted pathways. Result: A total of 500 compounds were acquired from related databases, where 87 active compounds and their 254 corresponding targets were identified. 2979 OA-related genes were collected from three databases, 150 of which were GBT-regulating OA genes. The compound-target network weight analysis and PPI results showed that IL-6 and PGE2 are key targets of GBT in treating OA. KEGG results showed that PI3K/AKT, Toll-like receptor, NFκB, TNF, and HIF-1 are the key signaling pathways. An in vivo experiment showed that GBT could effectively suppress cartilage degradation of OA rats. In vitro experiments demonstrated that GBT can inhibit the key targets of KEGG-related pathways. Molecular-docking results suggested that luteolin, licochalcone A, and ß-carotene were key targets of GBT, and the mechanisms may be associated with the NFκB signaling pathway. Blockage experiments showed that the NFκB pathway is the key pathway of GBT in treating OA. Conclusion: This study verified that GBT can effectively protect articular cartilage through multitarget and multipathway, and its inhibitory effect on the NFκB pathway is the most key mechanism in treating OA.

Network Pharmacology Analysis and Experimental Validation to Investigate the Mechanism of Total Flavonoids of Rhizoma Drynariae in Treating Rheumatoid Arthritis.

Objective: The study aimed to explore the mechanism of total flavonoids of Rhizoma Drynariae (TFRD) in the treatment of rheumatoid arthritis (RA) based on network pharmacology and experimental validation. Methods: The active components of TFRD were identified from TCMSP and TCMID databases. Relevant targets of the active compounds of TFRD and RA-related targets were predicted by public databases online. A component-target (C-T) regulatory network was constructed by Cytoscape. The genes of TFRD regulating RA were imported into STRING database to construct a protein-protein interaction (PPI) network in order to predict the key targets. KEGG enrichment analysis was performed to predict the crucial mechanism of TFRD against RA. The active components of TFRD underwent molecular docking with the key proteins. Collagen-induced arthritis (CIA) model of rats and inflammatory factors-stimulated fibroblast-like synoviocytes were used in vivo and in vitro to validate the efficacy and predicted critical mechanisms of TFRD. Results: Network Pharmacology analysis revealed that TFRD had 14 active compounds, corresponding to 213 targets, and RA related to 2814 genes. There were 137 intersection genes between TFRD and RA. KEGG indicated that therapeutic effects of TFRD on RA involves T cell receptor signaling pathway, Th17 cell differentiation, IL-17 signaling pathway, TNF signaling pathway, MAPK signaling pathway and PI3K/AKT signaling pathway. In vivo experiments suggested TFRD can alleviate the inflammatory response, joint swelling and synovial abnormality of CIA rats. TFRD contributed to the decrease of Th17 cells and the down-regulated secretion of IL-17A and TNF-α of activated lymphocyte in CIA model. In vitro experiments confirmed TFRD can effectively inhibit the inflammatory response of fibroblast-like synoviocytes and suppress the abnormal activation of MAPK, PI3K/AKT and NFκB signaling pathways. Conclusion: The treatment of RA with TFRD is closely related to inhibiting Th17 differentiation and inflammatory response of synoviocytes.