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.

Network Pharmacology-Based Strategy to Investigate the Mechanisms of Cibotium barometz in Treating Osteoarthritis.

Cibotium barometz is a representative tonifying kidney drug and is widely used for osteoarthritis (OA) in traditional Chinese medicine. However, its regulatory mechanisms in treating OA remain to be sufficiently investigated. The main chemical components of Cibotium barometz were screened through the TCMID database and the corresponding targets were acquired through SwissTargetPrediction. The OA-related targets were obtained from the OMIM, Genecards, Genebank, TTD, and DisGeNET databases. The prediction of key targets and pathways of Cibotium barometz in the treatment of OA was achieved by constructing a compounds-targets network and performing KEGG enrichment analysis. The OA model rats were established by the Hulth method and used to explore the protective effect of Cibotium barometz via cartilage pathological assessment. In vitro models of OA were built by the proinflammatory factor interleukin-1ß (IL-1ß) induced SW1353 cells and used to validate the mechanisms predicted by network pharmacology. Network pharmacology results suggested that the therapeutic effects of Cibotium barometz were closely related to matrix metalloproteinase (MMP)-1, 3, 13 and inflammation-related gene COX2, which are regulated by the NFκB pathway. In vivo experiments revealed that Cibotium barometz could effectively restrain cartilage from degeneration and inhibit the mRNA expression of MMP-1, MMP-3, MMP-13, and COX2 in cartilage. In vitro experiments indicated that Cibotium barometz water extract (CBWE) could significantly inhibit the expression of MMP-1, MMP-3, MMP-13, and PGE2 in IL-1ß-induced SW1353 cells and markedly prevent the translocation of NFκB p65 from the cytoplasm to the nuclei and decrease its phosphorylation level. After small-interfering RNA (siRNA) was used to suppress the synthesis of NFκB p65 to block NFκB signaling pathway, the ability of CBWE to inhibit MMP-1, MMP-3, MMP-13, and PGE2 was greatly reduced. Cibotium barometz has a chondroprotective effect on OA by inhibiting the response to inflammation and substrate degradation, and the related mechanism is associated with the inhibition of the NFκB 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.

Prediction of Rhizoma Drynariae Targets in the Treatment of Osteoarthritis Based on Network Pharmacology and Experimental Verification.

Rhizoma Drynariae has been widely used for the treatment of osteoarthritis (OA), but its potential targets and molecular mechanisms remain to be further explored. Targets of Rhizoma Drynariae and OA were predicted by relevant databases, and a protein-protein interaction (PPI) network was constructed to identify key targets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to obtain related pathways and then select significant pathways associated with OA. The OA chondrocyte model was established by inflammatory factor-induced SW1353 chondrocytes, and molecular docking was conducted to verify the above theoretical prediction. The results showed that a total of 86 Rhizoma Drynariae-OA interaction targets were identified, among which IL-6 and AKT1 were the key targets in the PPI network. Luteolin was the most critical component of Rhizoma Drynariae. KEGG results indicated that the effects of Rhizoma Drynariae on OA are associated with the PI3K/AKT, TNF, IL-17, apoptosis, and HIF-1 signaling pathway. The PI3K/AKT pathway can activate the downstream NF-κB pathway and further regulate the transcription and expression of downstream IL-6, IL-17, HIF-1α, Bax, and TNF, suggesting that the PI3K/AKT/NF-κB pathway is the critical pathway in the treatment of OA with Rhizoma Drynariae. Active components of Rhizoma Drynariae and key proteins of the PI3K/AKT/NF-κB signaling pathway were subjected to molecular docking, whose results showed that luteolin and IKK-α played a critical role. In vitro experiments indicated that both aqueous extracts of Rhizoma Drynariae (AERD) and luteolin inhibited the expression of IL-6 and HIF-1α and suppressed the activation of PI3K/AKT/NF-κB, IL-17, and TNF pathways. The measurement of mitochondrial membrane potential (Δψm) indicated that AERD and luteolin can decrease the LPS-induced early apoptotic cells. Luteolin had a more prominent inhibitory effect than AERD in the abovementioned in vitro experiments. In conclusion, the therapeutic mechanism of Rhizoma Drynariae against OA may be closely related to the inhibition of the PI3K/AKT/NF-κB pathway and downstream pathways, and luteolin plays a vital role in the treatment.

Total Flavonoids of Rhizoma Drynariae Restore the MMP/TIMP Balance in Models of Osteoarthritis by Inhibiting the Activation of the NF-κB and PI3K/AKT Pathways.

Total flavonoids of Rhizoma Drynariae (TFRD) have been shown to have beneficial effects on osteoarthritis (OA) clinically, but the mechanisms have not been elucidated. In this study, we investigated the effect of TFRD on articular cartilage in an OA rat model established by the Hulth method and in SW1353 chondrocytes induced by the proinflammatory factor interleukin-1ß (IL-1ß). The results showed that TFRD could alleviate the pathological changes in knee cartilage in OA model rats. In vivo, the qPCR analysis indicated that the mRNA levels of matrix metalloproteinases, MMP-1, MMP-3, and MMP-13, were decreased, while tissue inhibitor of matrix metalloproteinases- (TIMP-) 4 was increased in cartilage, and these changes could be partially prevented by TFRD. In vitro experiments showed that IL-1ß could significantly increase the expression of MMP-1, MMP-3, and MMP-13 and decrease the expression of TIMP-4 in SW1353 cells at the mRNA and protein levels. TFRD could increase the expression of MMP-3 and MMP-13 and decrease the expression of TIMP-4. Transfection of siRNA and addition of pathway inhibitors were used to clarify that inhibition of NF-κB and PI3K/AKT pathway decreased MMP-1, MMP-3, and MMP-13 and increased TIMP-4 expression. We also found that in IL-1ß-induced SW1353 cells, TFRD pretreatment had a modest inhibitory effect on p-AKT (Ser473) and reversed the increase of nuclear factor kappa-B (NF-κB) p65 in nuclear fraction and the decrease of inhibitor of NF-κB(IκB)-α in the cytosolic fraction. Further immunofluorescence confirmed that TFRD can inhibit IL-1ß-induced NF-κB p65 translocation to the nucleus to some extent. In conclusion, TFRD showed chondroprotective effects by restoring the MMP/TIMP balance in OA models by suppressing the activation of the NF-κB and PI3K/AKT pathways.

Benefits and Safety of Tripterygium Glycosides and Total Glucosides of Paeony for Rheumatoid Arthritis: An Overview of Systematic Reviews.

OBJECTIVE: To summarize the evidence from systematic reviews (SRs) on the benefits and safety of Tripterygium glycosides (TG) and total glucosides of paeony (TGP), commonly used to treat rheumatoid arthritis (RA) in China, for patients with RA. METHODS: SRs of randomized controlled trials (RCTs) on TG or TGP in treating RA were included, by searching 8 databases from their inception until December 2017. Two authors extracted data independently. We assessed the quality of SRs using AMSTAR and graded the quality of evidence according to the GRADE approach. RESULTS: Eleven SRs containing an average of 7.6 RCTs, involving a total of 7,012 participants were included in this overview. On the basis of included SRs, TG and TGP could improve the following indexes for RA patients: American College of Rheumatology (ACR) 20 response rate, ACR50 response rate and ACR70 response rate, swollen joint count, tender joint count, erythrocyte sedimentation rate and C-reactive protein. Moreover, TGP could reduce incidence of hepatotoxicity. The most common adverse effects of TG were gastrointestinal discomfort and gonad toxicity, while for TGP was mild to moderate diarrhea. The overall quality of evidence for these findings ranged from "low" to "moderate". CONCLUSIONS: TG and TGP might be 2 potentially effective complementary and alternative drugs for patients with RA. Nevertheless, due to gonad toxicity, TG should only be considered in elderly patients or patients without reproductive needs. More evidence from high quality RCTs and SRs is warranted to support the use of TG and TGP for RA patients.

[Research,evaluation,industry status and development strategy of traditional Chinese medicine functional food].

With the development of social economy,people's demand for health services is growing rapidly. As health resource with Chinese characteristics,health food containing Chinese materia medica have broad prospect and great market space for development.However,at present,there are still many problems of health food containing Chinese materia media in the research,development,evaluation and market application. In addition,due to lack of theoretical support of traditional Chinese medicine(TCM) in the research and development of health food containing Chinese materia media,blurred boundaries between health food containing Chinese materia media and other health products as well as TCM are present,lacking of TCM characteristics. In the evaluation process of health food containing Chinese materia media,the construction of functional food laws,regulations and evaluation norms is relatively lagging behind,which can't meet the needs of health food containing Chinese materia media research and development,severely restricting the development of health food containing Chinese materia media. Based on the research and evaluation of health food containing Chinese materia media,the existing problems were reviewed and the reasons for the deficiencies were analyzed in this paper. Guided by the theory of TCM,based on the constitution identification in TCM,and combined with modern scientific and technological means,a new research and development mode of functional food was put forward in this paper to distinguish health food containing Chinese materia media from TCM as well as general health products. Nevertheless,we should ensure the vitality of Chinese medicine health products with original thinking and scientific and technological connotations,and accelerate the harmonious,rapid and sustainable development of Chinese medicine health industry.

[Some thoughts on health food formulated with traditional Chinese medicine].

The health food industry is an important support for the big health industry and the strategy of healthy China. The Chinese medicine prescription health food has exceeded 60% of the total declared health food. However,the main basis for its function evaluation,the Technical Specification for Inspection and Evaluation of Health Food,was abolished in 2018,and 27 of them were based on modern medical and nutritional theories. Quantitative efficacy evaluation methods in western pharmacology are short of function claims and function evaluation methods reflecting the characteristics of traditional Chinese medicine,which could affect the health food industry to a certain extent. Therefore,the establishment of the evaluation mechanism of Chinese medicine prescription health food which conforms to the positioning of health food and the theory of traditional Chinese medicine is helpful for the healthy development of health food industry. In this paper,this problem was explained from five aspects. First,how to differentiate the positioning of Chinese medicine prescription health food from ordinary food and medicine,and how to embody the characteristics of Chinese medicine. Secondly,the relationship between traditional Chinese medicine prescription health food and Chinese patent medicine. Thirdly,how to scientifically and reasonably determine the raw materials of traditional Chinese medicine prescription health food. Fourthly,how to explain the function claim of traditional Chinese medicine prescription health food,and how to evaluate its function scientifically and reasonably. Fifthly,the functional evaluation of Chinese herbal medicine prescription health food is connected with other national scientific and technological strategies. In this paper,a preliminary analysis of the Chinese medicine prescription health food was conducted from the above five aspects,and some personal views and suggestions were put forward,hoping to provide reference for the competent authorities and researchers.

[Current situation analysis and supervision suggestions of traditional Chinese medicine health food claiming to enhance immune function].

Number of products claiming to enhance immunity function ranks first among all the approved health food products,and number of products whose formula contains Chinese medicine accounts for two thirds of all the products claiming to enhance immunity function. Chinese medicine allowed to be used in health food has a specified range,and the safety of raw materials is generally higher.The usage amount of raw materials shall not exceed the upper limit stipulated in the literature or regulations. The claim of enhancement of immunity function is put forward by western medicine based on modern pharmacology and nutriology. However,immunity is wide in scope and complex in mechanism. The health food that contains Chinese medicine plays an active effect in enhancement of immunity under the guidance of Chinese medicine theory in many ways such as immune organs,immune cells and immune molecules. In this article,the author first analyzed the current use of raw materials for health food,then summarized the approved health food claiming to enhance immunity function,and conducted statistical analysis on the enhancement of immunity for traditional Chinese medicine(TCM)health food,use of raw materials,compatibility of raw materials,conducted in-depth analysis on health food formula,safety,health function,quality control and production process from the perspectives of technical review,supervision and management. Finally,some suggestions on registration and supervision of TCM health food claiming to enhance immunity function were put forward from the perspectives of problems found in the supervision and from the demand of TCM health food in the future.