Neuroprotective effects of Tiaogeng decoction against H2O2-induced oxidative injury and apoptosis in PC12 cells via Nrf2 and JNK signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiaogeng decoction (TGD), a mixture of 10 traditional Chinese herbs, has been used clinically for over 30 years in treating menopause-related symptoms such as cognitive changes, mood disorders, vasomotor symptoms, and sleep disorders. These central nervous system symptoms are closely associated with declined ovarian function, which dramatically increases the risk of neurodegenerative disease. Previous studies revealed that TGD may have anti-oxidative and anti-apoptotic properties, potentially preventing neurodegenerative conditions; however, the underlying pharmacological mechanism remains unclear. AIM OF THE STUDY: This study aimed to examine whether TGD could activate the Nrf2 and C-Jun N-terminal kinase (JNK) signaling pathways to effectively reduce oxidative injury and apoptosis in PC12 cells and elucidate the mechanism by which this medicine may prevent neurodegenerative disease. MATERIALS AND METHODS: PC12 cells were exposed to different concentrations of TGD (125, 250, 500 µg/mL) and H2O2 (150 µM). 17ß-estradiol (0.05 µg/mL) was used as the positive control. A cell counting kit-8 (CCK-8) and a lactate dehydrogenase (LDH) assay were used to detect cell viability and cytotoxicity, while Hoechst and flow cytometry were performed to evaluate apoptosis levels. Mitochondrial function was assessed by measuring mitochondrial membrane potential (MMP), and superoxide dismutase (SOD), and reactive oxygen species (ROS) levels were used to measure oxidative stress (OS). Western blot analysis was used to identify the levels of Nrf2, phospho-JNK (p-JNK), phospho-mitogen-activated protein kinase kinase 7 (p-MKK7), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), Caspase3 (Casp3), Caspase9 (Casp9), Bax, and Bcl-2 proteins. Moreover, JNK agonist anisomycin and Nrf2 inhibitor ML385 were used to validate pathways. RESULTS: TGD pretreatment significantly alleviated H2O2-induced cytotoxicity, apoptosis, MMP, and OS levels. H2O2 stimulated the activation of Nrf2 and JNK signaling pathways, but TGD increased the extent of Nrf2 antioxidant activation, decreased the activation of JNK, and eventually reversed the H2O2-induced protein expression of p-MKK7, Keap1, HO-1, Cleaved Caspase3 (CL-Casp3), Cleaved Caspase9 (CL-Casp9), Bax, and Bcl-2. CONCLUSIONS: This study's findings suggest that TGD may attenuate oxidative injury and apoptosis via the Nrf2 and JNK signaling pathways, making it a potential therapeutic candidate for neurodegenerative diseases.

A Network Pharmacology Approach to Estimate Potential Targets of the Active Ingredients of Epimedium for Alleviating Mild Cognitive Impairment and Treating Alzheimer's Disease.

BACKGROUND: The present study made use of a network pharmacological approach to evaluate the mechanisms and potential targets of the active ingredients of Epimedium for alleviating mild cognitive impairment (MCI) and treating Alzheimer's disease (AD). METHODS: The active ingredients of Epimedium were acquired from the Traditional Chinese Medicine System Pharmacology database, and potential targets were predicted using the TCMSP target module, SwissTargetPrediction, and PharmMapper database. Target proteins correlating with MCI and AD were downloaded from the GeneCards, DisGeNet, and OMIM databases. The common targets of Epimedium, MCI, and AD were identified using the Jvenn online tool, and a protein-protein interaction (PPI) network was constructed using the String database and Cytoscape. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the common targets was performed using DAVID, and molecular docking between active ingredients and target genes was modeled using AutoDock Vina. RESULTS: A total of 20 active ingredients were analyzed, and 337 compound-related targets were identified for Epimedium. Out of 236 proteins associated with MCI and AD, 54 overlapped with the targets of Epimedium. The top 30 interacting proteins in this set were ranked by topological analysis. GO and KEGG enrichment analysis suggested that the common targets participated in diverse biological processes and pathways, including cell proliferation and apoptosis, inflammatory response, signal transduction, and protein phosphorylation through cancer pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, sphingolipid signaling pathway, FoxO signaling pathway, and TNF signaling pathway. Molecular docking analysis suggested that the 20 active ingredients could bind to the top 5 protein targets. CONCLUSIONS: The present study provides theoretical evidence for in-depth analysis of the mechanisms and molecular targets by which Epimedium protects against MCI, AD, and other neurodegenerative diseases and lays the foundation for pragmatic clinical applications and potential new drug development.

Tiao Geng decoction inhibits tributyltin chloride-induced GT1-7 neuronal apoptosis through ASK1/MKK7/JNK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiao Geng (TG) decoction is a Chinese herbal medicine extract that has been utilized for the treatment of menopausal symptoms for a history of over 30 years. In our previous study, we suggest that TG decoction possibly exerts an anti-apoptotic effect on hypothalamic neurons of ovariectomized rats via the ASK1/MKK7/JNK pathway. Tributyltin chloride (TBTC) causes oxidative damage and induces apoptosis of primary hypothalamic neurons in rats. AIM OF THE STUDY: The present work aimed to explore the inhibition of TG decoction on TBTC-induced GT1-7 cell apoptosis and its possible molecular mechanism. MATERIALS AND METHODS: The GT1-7 cell line was exposed to TG decoction at diverse doses (31.25, 62.5, 125 µg/mL) for 24 h and later with TBTC (1 mg/L) for 1 h, with 17ß-E2 (100 nM) treatment being the positive control. Then, CCK8 assay was conducted to evaluate cell viability, while flow cytometric analysis was conducted to examine the apoptosis level. Related pathways and differentially expressed proteins were identified by tandem mass tag (TMT)-based quantitative phosphoproteomics. qRT-PCR was carried out to examine mRNA levels of Bax and B-cell lymphoma-2 (Bcl-2). Western blotting was performed to detect the levels of Bax, Bcl-2, c-Jun, c-Jun N-terminal kinase (JNK), Caspase-3 (Casp3), Mitogen-activated protein kinase kinase 7 (MKK7), and apoptosis signal-regulating kinase 1 (ASK1) . Finally, cells were pretreated with SP600125, an inhibitor of JNK, later the expression of JNK and Casp3 was measured. RESULTS: Application of TG decoction mitigated the GT1-7 cell apoptosis and injury caused by TBTC; besides, it inhibited the activation of the ASK1/MKK7/JNK pathway. Moreover, Bcl-2/Bax ratio became higher, and the MKK7, ASK1, Casp3 and c-Jun levels were inhibited. Besides, TG decoction combined with SP600125 (the JNK inhibitor) more significantly inhibited GT1-7 cell apoptosis caused by TBTC. CONCLUSION: As discovered from the experiment in this study, TG decoction has a neuroprotective effect, which is achieved through inhibiting the ASK1/MKK7/JNK signal transduction pathway to reduce GT1-7 cell apoptosis.

Tiao Geng decoction for treating menopausal syndrome exhibits anti-aging effects likely via suppressing ASK1/MKK7/JNK mediated apoptosis in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: TG-decoction (Tiao Geng decoction) is the extract of a Chinese herb mixture that has been used for treating menopausal symptoms for over 30 years. We have previously reported anti-aging and anti-oxidative effects of the TG-decoction on hypothalamic neurons in ovariectomized (OVX) rats. AIM OF THE STUDY: The present study further investigates the effects of TG-decoction on the prevention of aging-related ultrastructural changes in menopausal hypothalamic neurons and the likely molecular mechanism. MATERIALS AND METHODS: A total of 120 four-month-old female SPF Sprague Dawley rats were divided into six groups. Five groups were ovariectomized (OVX) and one group served as a sham control. Three OVX groups received TG-decoction at three different doses. The remaining two OVX groups served as positive and negative controls by receiving estradiol valerate and saline solution. The sham group received saline. After one month, aging-related ultrastructural alterations in hypothalamic neurons were evaluated using transmission electron microscopy. Nissl staining was used to assess the pathomorphological changes of the hypothalamic neurons. Cell apoptosis was evaluated by TUNEL. Expression of Bcl-2 family genes was studied using qRT-PCR. Expression of the apoptosis-related proteins ASK1, MKK7, JNK, c-Jun, Bax, Casp3 and Bcl-2 was studied using western blotting. RESULTS: Ovariectomy of female rats led to visible damage and aging-like alterations in the mitochondria and endoplasmic reticulum as well as large deposits of lipofuscin in hypothalamic tissue. TG-decoction treatment prevented this visible damage and lipofuscin deposition, increased the number of nerve cells and normally-shaped Nissl bodies, and reduced the number of TUNEL-positive cells. Expression of Bcl-2 gene was increased, while Bax gene reduced. Expression of the proteins ASK1, MKK7, JNK, c-Jun, Bax and Casp3 was reduced, while that of Bcl-2 was increased. CONCLUSION: TG-decoction reduces aging-related ultrastructural changes in hypothalamic neurons, likely by suppressing ASK1/MKK7/JNK-mediated apoptosis in neuronal mitochondria or nuclei.