Study on the mechanism of Tong-Qiao-Huo-Xue decoction regulating apoptosis via ASK1/MKK4/JNK pathway in MCAO/R rats.

BACKGROUND: Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear. PURPOSE: Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury. METHODS: Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively. RESULTS: HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax. CONCLUSION: By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.

Tong-Qiao-Huo-Xue decoction activates PI3K/Akt/mTOR pathway to reduce BMECs autophagy after cerebral ischemia/reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Tong-Qiao-Huo-Xue Decoction (TQHXD) is a traditional classic Chinese Medicinal Formula (CMF) used for clinical treatment of ischemic stroke. TQHXD leads to improvement in the symptoms of the acute period of cerebral infarction and recovery period after stroke. Our previous studies also showed that TQHXD produced a significant protective effect on the brain after cerebral ischemia-reperfusion (I/R) injury. It is reported that autophagy is closely related to ischemic brain injury; however, the functional contribution of TQHXD to brain microvascular endothelial cell (BMEC) autophagy and its underlying mechanism remains unclear. AIM OF THE STUDY: The purpose of this study was to investigate the effects and mechanism of TQHXD in inhibiting cerebral ischemia-induced endothelial autophagy. MATERIALS AND METHODS: The high-performance liquid chromatography (HPLC) fingerprint of the chemical constituents from TQHXD was established for the quality control, and the Longa method was used to evaluate the efficacy of TQHXD in rats with middle cerebral artery occlusion (MCAO). The expression of LC3 was determined by immunofluorescence double staining. To evaluate the protective effects of TQHXD-containing cerebrospinal fluid (CSF) on BMECs injured by oxygen-glucose deprivation and reperfusion, cell survival rate was determined using the CCK-8 assay and cell apoptosis was determined by fluorescein isothiocyanate (FITC)-Annexin V/PI. Autophagy was detected using transmission electron microscopy. RESULTS: The results showed that TQHXD-CSF significantly ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury in BMECs. Confocal microscopy and Western blot results showed that TQHXD-CSF reduced autophagy-related protein expression and autophagosome number. The results of the western blotting indicated that TQHXD-CSF caused a marked increase in the phosphorylation of protein kinase B and phosphoinsotide-3 kinase (Akt/p-Akt and PI3K/p-PI3K, respectively) and their expression levels were down-regulated after treatment with pathway inhibitor, ZSTK474. Furthermore, in a MCAO model in rats, TQHXD markedly increased p-PI3K, p-Akt and p-mTOR, whereas the autophagy related proteins decreased. CONCLUSIONS: Taken together, these findings demonstrate that TQHXD protects against ischemic insult by inhibiting autophagy through the regulation of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway and that TQHXD may have therapeutic value for protecting BMECs from cerebral ischemia.

A sesquiterpene lactone from a medicinal herb inhibits proinflammatory activity of TNF-α by inhibiting ubiquitin-conjugating enzyme UbcH5.

UbcH5 is the key ubiquitin-conjugating enzyme catalyzing ubiquitination during TNF-α-triggered NF-κB activation. Here, we identified an herb-derived sesquiterpene lactone compound IJ-5 as a preferential inhibitor of UbcH5 and explored its therapeutic value in inflammatory and autoimmune disease models. IJ-5 suppresses TNF-α-induced NF-κB activation and inflammatory gene transcription by inhibiting the ubiquitination of receptor-interacting protein 1 and NF-κB essential modifier, which is essential to IκB kinase activation. Mechanistic investigations revealed that IJ-5 preferentially binds to and inactivates UbcH5 by forming a covalent adduct with its active site cysteine and thereby preventing ubiquitin conjugation to UbcH5. In preclinical models, pretreatment of IJ-5 exhibited potent anti-inflammatory activity against TNF-α- and D-galactosamine-induced hepatitis and collagen-induced arthritis. These findings highlight the potential of UbcH5 as a therapeutic target for anti-TNF-α interventions and provide an interesting lead compound for the development of new anti-inflammation agents.

Sesquiterpene lactones from Inula helianthus-aquatica.

OBJECTIVE: To investigate the sesquiterpene lactones of the aerial parts of Inula helianthus-aquatica. METHOD: Compounds were isolated and purified by silica gel, Sephadex LH-20 and preparative HPLC. On the basis of physicochemical properties and spectroscopic data, their structures were identified. RESULT: Seven sesquiterpene lactones and four other compounds were obtained and identified as 2-desoxy-4-epi-pulchellin (1), 6-acetoxy-4-hydroxy-1, 10H-pseudoguaia-11 (13)-en-12,8-olide (2), 4-acetoxy-6-hydroxy-1, 10H-pseudoguaia-11(13)-en-12,8-olide (3), 8-epi-inuviscolide (4), 2,3,11,13-tetrahydroaromaticin (5), 11,13-dihydro-ergolide (6), 4-epipulchellin-2-O-acetate (7), 7-epiloliolide (8), loliolide (9), beta-sitosterol (10) and daucosterol (11). CONCLUSION: All the compounds were isolated from this plant for the first time.