Calycosin mitigates chondrocyte inflammation and apoptosis by inhibiting the PI3K/AKT and NF-κB pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Shaoyao Gancao Decoction (SG-Tang), originated from the Treatise on Febrile Diseases, is often used to treat OA pain symptoms. Whereas its efficacy has been verified by several clinical studies, the underlying mechanism remained unclear. Network pharmacology and UPLC-QTOF-MS analysis found that calycosin could be regarded as the active components of SG-Tang in treating OA. However, the effect of calycosin on cartilage destruction and the pathogenesis of OA are not known. Therefore, we evaluated the benefits of calycosin for OA and revealed the underlying mechanisms. AIM OF STUDY: Using network pharmacology, UPLC-QTOF-MS analysis and experiments, the active components of SG-Tang were analyzed to explore their potential therapeutic mechanism in OA. MATERIALS AND METHODS: The components of SG-Tang were detected by UPLC-QTOF-MS, and the possible active components and mechanism of SG-Tang in the treatment of OA were screened by network pharmacology. The OA mouse model was constructed by DMM. In total, 30 mice were randomly divided into three groups: Sham, DMM, and DMM + Calycosin. H&E, safranin O/fast green staining and the OARSI scores were used to evaluate joint injury in mice. In addition, OA models were established using chondrocytes treated with 10 ng/mL IL-1ß. Treatment groups were treated with 100, 200 or 400 µM calycosin. CCK-8 assay was used for assessing the cytotoxic effects of calycosin. TUNEL staining and Western blotting were used to detect chondrocyte apoptosis. In addition, PI3K/Akt and NF-κB signaling pathway-related markers and cartilage matrix-related indicators were also detected. RESULTS: In vivo studies showed that calycosin inhibited IL-1ß-induced IL-6 and TNF-α production, as well as iNOS and COX-2 expression. Meanwhile, calycosin could inhibit IL-1ß-induced degradation of cartilage matrix, including downregulation of MMP3, MMP-13, collagen II and aggrecan. NF-κB and PI3K/AKT were also inhibited by calycosin in OA chondrocytes. Furthermore, calycosin inhibited IL-1ß-induced apoptosis in mouse chondrocytes. In a mouse model of OA, our results suggest that calycosin has a chondroprotective effect. CONCLUSIONS: According to this study, calycosin may act as a protective agent against OA by inhibiting the PI3K/AKT and NF-κB pathways. Furthermore, this study suggested that calycosin is a potential candidate for the treatment of OA.

Sanse Powder Essential Oil Nanoemulsion Negatively Regulates TRPA1 by AMPK/mTOR Signaling in Synovitis: Knee Osteoarthritis Rat Model and Fibroblast-Like Synoviocyte Isolates.

Synovitis is the primary driving factor for the occurrence and development of knee osteoarthritis (KOA) and fibroblast-like synoviocytes (FLSs) and plays a crucial role during this process. Our previous works revealed that transient receptor potential ankyrin 1 (TRPA1) ion channels mediate the amplification of KOA synovitis. In recent years, essential oils have been proved to have blocking effect on transient receptor potential channels. Meanwhile, the therapeutic effect of Sanse Powder on KOA synovitis has been confirmed in clinical trials and basic studies; although, the mechanism remains unclear. In the present study, Sanse Powder essential oil nanoemulsion (SP-NEs) was prepared, and then chemical composition, physicochemical properties, and stability were investigated. Besides, both in MIA-induced KOA rats and in LPS-stimulated FLSs, we investigated whether SP-NES could alleviate KOA synovitis by interfering with AMP-activated protein kinase- (AMPK-) mammalian target of rapamycin (mTOR), an energy sensing pathway proved to negatively regulate the TRPA1. Our research shows that the top three substances in SP-NEs were tumerone, delta-cadinene, and Ar-tumerone, which accounted for 51.62% of the total, and should be considered as the main pharmacodynamic ingredient. Less inflammatory cell infiltration and type I collagen deposition were found in the synovial tissue of KOA rats treated with SP-NEs, as well as the downregulated expressions of interleukin (IL)-1ß, IL-18, and TRPA1. Besides, SP-NEs increased the phosphorylation level of AMPK and decreased the phosphorylation level of mTOR in the KOA model, and SP-NEs also upregulated expressions of peroxisome proliferator-activated receptor-gamma (PPARγ) and PPARγ coactivator-1α and downstream signaling molecules of AMPK-mTOR in vivo and in vitro. To conclude, a kind of Chinese herbal medicine for external use which is effective in treating synovitis of KOA was extracted and prepared into essential oil nanoemulsion with stable properties in the present study. It may alleviate synovitis in experimental KOA through the negative regulation of TRPA1 by AMPK-mTOR signaling.

Network Pharmacology Approach to Uncover the Mechanism Governing the Effect of Simiao Powder on Knee Osteoarthritis.

OBJECTIVE: To explore the molecular mechanism of Simiao powder in the treatment of knee osteoarthritis. METHODS: Based on oral bioavailability and drug-likeness, the main active components of Simiao powder were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). GeneCard, OMIM, DisGeNET, DrugBank, PharmGkb, and the Therapeutic Target Database were used to establish target databases for knee osteoarthritis. Cytoscape software was used to construct a visual interactive network diagram of "active ingredient - action target - disease." The STRING database was used to construct a protein interaction network and analyze related protein interaction relationships. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) biological process enrichment analysis were performed on the core targets. Additionally, Discovery Studio software was used for molecular docking verification of active pharmaceutical ingredients and disease targets. RESULTS: Thirty-seven active components of Simiao powder were screened, including 106 common targets. The results of network analysis showed that the targets were mainly involved in regulating biological processes such as cell metabolism and apoptosis. Simiao powder components were predicted to exert their therapeutic effect on the AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, and HIF-1 signaling pathway. The molecular docking results showed that the active components of Simiao powder had a good match with the targets of IL1B, MMP9, CXCL8, MAPK8, JUN, IL6, MAPK1, EGF, VEGFA, AKT1, and PTGS2. CONCLUSION: Simiao powder has multisystem, multicomponent, and multitarget characteristics in treating knee osteoarthritis. Its possible mechanism of action includes inhibiting the inflammatory response, regulating immune function, and resisting oxidative stress to control the occurrence and development of the disease. Quercetin, wogonin, kaempferol, beta-sitosterol, and other active ingredients may be the material basis for the treatment of knee osteoarthritis.

Total flavone of Abelmoschl manihot L. medic exhibits protective effect against hind-limb ischemia in rat model.

Total flavones of Abelmoschus manihot L. Medic (TFA) is the major active component isolated from Chinese herb Abelmoschus manihot L. Medic. TFA has shown neuroprotective effect against cerebral ischemia injury in rats and rabbits. However, the effects of TFA on hind-limb ischemia and the underlying mechanisms remain unclear. Therefore, in the present study, we used a rat hind-limb ischemia model to investigate protective effect of TFA against limb ischemia injury. The rat model of hind-limb ischemia was established. Treatment groups received TFA at two different doses (160 and 40mg/kg) daily for 10 days. Sham operated control group and model group received saline. At the end the rats were sacrificed, hindlimb tissues were stained with Haematoxylin-Eosin and Masson's trichrome. RNA and protein were extracted from tissues for PCR and Western blot analysis. The results showed that TFA reduced lower limb ischemic injury, recovered tissue volume and diminished fibrosis and muscle degeneration. Mechanistically, we showed that TFA increased the expression of anti-apoptotic factor such as Bcl-2 and survivin, decreased the expression of pro-apoptotic factor such as Caspase 3, Bax and Bak and inhibited the activation of caspase 3 and 9. In summary, this study proves new evidence that TFA protects hind-limb against ischemia injury by inhibiting apoptosis and could be a promising therapeutic agent for acute lower extremity ischemia.

Effect of Ultrasound-Enhanced Transdermal Drug Delivery Efficiency of Nanoparticles and Brucine.

Brucine is the active component in traditional Chinese medicine "Ma-Qian-Zi" (Strychnos nux-vomica Linn), with capabilities of analgesic, anti-inflammatory, anti-tumor and so on. It is crucial how to break through the impact of cuticle skin which reduces the penetration of drugs to improve drug transmission rate. The aim of this study is to improve the local drug concentration by using ultrasound. We used fresh porcine skin to study the effects of ultrasound on the transdermal absorption of brucine under the influence of various acoustic parameters, including frequency, amplitude and irradiation time. The transdermal conditions of yellow-green fluorescent nanoparticles and brucine in skin samples were observed by laser confocal microscopy and ultraviolet spectrophotometry. The results show that under ultrasonic conditions, the permeability of the skin to the fluorescent label and brucine (e.g., the depth and concentration of penetration) is increased compared to its passive diffusion permeability. The best ultrasound penetration can make the penetration depth of more than 110 microns, fluorescent nanoparticles and brucine concentration increased to 2-3 times. This work will provide supportive data on how the brucine is better used for transdermal drug delivery (TDD).