Ethanol extracts of Cinnamomum migao H.W. Li attenuates neuroinflammation in cerebral ischemia-reperfusion injury via regulating TLR4-PI3K-Akt-NF-κB pathways.

Cinnamomum migao H.W. Li, commonly known as migao (MG), is used in the Miao region of China for treating cardiovascular and cerebrovascular diseases, attributed to its detoxifying (Jiedu in Chinese), activating blood circulation (Huoxue in Chinese), and promoting Qi circulation (Tongqi in Chinese) properties. However, its therapeutic potential for ischemic stroke (IS) remains unexplored. Therefore, this study was to explore the protective effect of MG against cerebral ischemia-reperfusion injury caused by IS. The aim of this study was to investigate whether ethanol extract of MG (EEMG) attenuates cerebral ischemia-reperfusion injury, and explored the underlying mechanisms. Middle cerebral artery occlusion and reperfusion (MCAO/R) was established, and the efficacy of EEMG was evaluated using triphenyltetrazolium chloride (TTC), immunofluorescence, hematoxylin-eosin (HE) staining, and real-time quantitative PCR (qRT-PCR). Qualitative analysis of EEMG was analyzed for chemical composition by liquid chromatography-mass spectrometry (LC-MS). The molecular mechanism of EEMG was explored by metabolomics, network pharmacology, immunoblotting, immunofluorescence staining, gene knockdown, and agonist treatment. The results showed that EEMG alleviates ischemic injury in MCAO/R-operated rats and reduces neuronal damage of OGD/R-treated SH-SY5Y cells. Specifically, EEMG inhibited the release of inflammatory factors and reversed serum metabolic profile disorders of MCAO/R rats. Network pharmacology analysis showed that the PI3K-Akt and NF-κB signaling pathways play a role in the neuroprotective effects of EEMG against ischemic injury and in mitigating the inflammatory response. Consistent with our expectations, EEMG activated PI3K-AKT and suppressed NF-kB signaling pathways both in MCAO/R-operated rats and OGD/R-treated BV2 cells. The results showed that knockdown of TLR4 abolished the EEMG-mediated inhibition on neuroinflammation in OGD/R-treated BV2 cells. After treating BV2 cells with the TLR4 agonist neoseptin 3, EEMG showed a trend toward inhibiting neuroinflammation, though the effect was not statistically significant. Additionally, EEMG was found to improve liver injury caused by cerebral ischemia-reperfusion, which is associated with NF-κB signaling pathway in this study. Collectively, this study demonstrated that EEMG attenuates neuroinflammation in cerebral ischemia-reperfusion injury via regulating TLR4-PI3K-Akt-NF-κB pathways.

Wu W ，Xu L ，Mu D ，Wang D ，Tan S ，Liu L ，Li Y ，Chai H ，Hou Y ... -  《-》

Ammopiptanthus nanus (M. Pop.) Cheng f. stem ethanolic extract ameliorates rheumatoid arthritis by inhibiting PI3K/AKT/NF-κB pathway-mediated macrophage infiltration.

Ammopiptanthus nanus (M. Pop.) Cheng f. (A. nanus), a traditional Kirgiz medicinal plant, its stem has shown potential in treating rheumatoid arthritis (RA) in China, either through oral medication or by topical application directly to the affected joints, but its underlying mechanism of action remains unexplored. The purpose of this study is to elucidate pharmacological mechanism of A. nanus in ameliorating RA using a comprehensive approach that combines network pharmacology, molecular docking and experimental evaluations. Firstly, the major constituents of A. nanus stem ethanolic extract were identified and quantified by High-Performance Liquid Chromatography (HPLC). Disease target data from Gene Cards database was then used to define RA-associated targets. A protein-protein interaction (PPI) network was created via STRING database. The DAVID database powered gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to gain functional insights. In vitro, RAW264.7 cells were treated with A. nanus to investigate the roles of target proteins and pathways during lipopolysaccharide (LPS) - induced inflammation. Immunofluorescence assays were performed to assess the effects of A. nanus on macrophage infiltration. The key targets and signalling pathways were validated using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), molecular docking, immunohistochemical analysis, western blotting and immunofluorescence. Finally, the therapeutic potential of A. nanus in RA was evaluated in a carrageenan-induced rat model. Network analysis identified 31 potential targets of A. nanus associated with RA, including 10 hub targets. KEGG analysis highlighted the involvement of PI3K/AKT signaling pathway. In vivo experiments demonstrated that A. nanus treatment significantly protected against carrageenan-induced inflammatory paw tissue and attenuated macrophage infiltration. Both in vivo and in vitro experiments confirmed that A. nanus significantly downregulated the protein expression of COX-2, iNOS and IL-1β, and inhibited PI3K/AKT/NFκB pathway, which are closely linked to RA. Furthermore, molecular docking and cellular thermal shift assay revealed that licoflavanone showed a strong binding affinity with key targets. In summary, this study provides the first evidence of the potent anti-inflammatory activity of A. nanus in experimental RA. The mechanism of action appears to involve inactivation of the PI3K/AKT/NF-κB pathway-mediated macrophage infiltration. These findings indicate that A. nanus has significant potential as a therapeutic potential agent for RA treatment and offer novel insights for future research and drug development in this field.

Yao Y ，Wang J ，Zhang H ，Peng T ，Sun Y ，Zhang R ，Meng X ，Lu X ，Gao Y ，Jin Y ，Zhang Y ，Chen L ... -  《-》

Gnetupendin A protects against ischemic stroke through activating the PI3K/AKT/mTOR-dependent autophagy pathway.

Autophagy has been recently emerged as a prominent factor in the pathogenesis of ischemic stroke (IS) and is increasingly being considered as a potential therapeutic target for IS. Gnetum parvifolium has been identified as a potential therapeutic agent for inflammatory diseases such as rheumatism and traumatic injuries. However, the pharmacological effects of Gnetupindin A (GA), a stilbene compound isolated from Gnetum parvifolium, have not been fully elucidated until now. Here we identified the therapeutic potential of GA for IS, deeply exploring the possible mechanisms related to its regulation of autophagy. The mouse model of middle cerebral artery occlusion-reperfusion (MCAO/R) and the oxygen-glucose deprivation reperfusion (OGD/R)-exposed cells served as models to study the protection of GA against IS. The adeno-associated virus (AAV) encoding shAtg5, in conjunction with autophagy inhibitor 3-Methyladenine (3-MA) were utilized to explore the role of GA in regulating autophagy following IS. Molecular docking, CETSA, and DARTS were used to identify the specific therapeutic target of GA. PI3K inhibitor LY294002 was employed to test the participation of PI3K in GA-mediated autophagy and neuroprotective effects following IS. Our findings revealed that treatment with GA significantly alleviated the brain infract volume, edema, improved neurological deficits and attenuated apoptosis. Mechanistically, we found that GA promoted autophagic flow both in vivo and in vitro after IS. Notably, neural-targeted knockdown of Atg5 abolished the neuroprotective effects mediated by GA. Inhibition of autophagy using 3-MA blocked the attenuation on apoptosis induced by GA. Moreover, molecular docking, CETSA, and DARTS analysis demonstrated that GA specifically targeted PI3K and further inhibited the activation of PI3K/AKT/mTOR signaling pathway. LY294002, which inhibits PI3K, reversed GA-induced autophagy and neuroprotective effects on OGD/R-treated cells. We demonstrated, for the first time, that GA protects against IS through promoting the PI3K/AKT/mTOR-dependent autophagy pathway. Our findings provide a novel mechanistic insight into the anti-IS effect of GA in regulating autophagy.

Mu D ，Liu J ，Mi Y ，Wang D ，Xu L ，Yang Y ，Liu Y ，Liang D ，Hou Y ... -  《-》

Investigating the possible mechanism of Cornus officinalis in the therapy of ischemic stroke by UHPLC-Q-TOF-MS, network pharmacology, molecular docking, and experimental verification.

Cornus officinalis is a conventional Chinese medicine for tonifying liver and kidney in ancient China. The active ingredients from Cornus officinalis can delay the progression of cerebral aneurysms, alleviate experimental autoimmune encephalomyelitis, and show a good intervention effect on brain diseases. Loganin, the active ingredient of Cornus officinalis, has a neuroprotective effect on cerebral ischemia-reperfusion injury in mice. It is yet unknown, nevertheless, how Cornus officinalis works to treat ischemic stroke. Based on ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), network pharmacology and molecular docking, Cornus officinalis's mechanism of intervention in ischemic stroke is explored and verified by experiments. To examine the chemical components of Cornus officinalis, UHPLC-Q-TOF-MS was used. The network pharmacology was used to construct the "active ingredient-core target-main pathway" network of Cornus officinalis. Then, the link between the main active components and the key protein targets, as determined by network pharmacology, was verified through the application of molecular docking. The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model used in this study was created using the suture technique. The pharmacological effects of Cornus officinalis were explored by neurological function score, behavior, TTC staining, ultrasound and flow cytometry. Western blot and qPCR were used to confirm the core target. The outcomes of the investigation demonstrated that Cornus officinalis had a potent anti-ischemic stroke effect. UHPLC-Q-TOF-MS method was used to determine 24 chemical constituents in Cornus officinalis, of which 22 components had a close relationship with protein targets relevant to ischemic stroke. The 27 protein targets screened by "active ingredient-core target-main pathway" may be the possible targets of Cornus officinalis in the therapy of ischemic stroke. Most of the 27 protein targets had to do with the inflammatory response, apoptosis and energy metabolism. KEGG enrichment analysis showed that AGE/RAGE ranked high and was closely related to inflammatory response. Molecular docking predicted that the top 10 components in the network diagram had good binding with inflammatory factors IL6, IL-1β and TNF-α protein targets. Western blot research outcomes stated that Cornus officinalis could firmly impede the production of AGE, RAGE, and P-NFκB P65. Cornus officinalis had the potential to prevent ischemic stroke by drastically inhibiting the production of TNF-α, IL-1β, and IL-6, according to the results of qPCR study. This study found that Cornus officinalis can improve the brain injury, motor ability and blood flow velocity of MCAO/R rats and suppress the inflammatory reaction through the AGE/RAGE/NFκB pathway to exert the therapeutic effect on ischemic stroke.

Zhang Y ，Yuan PP ，Li PY ，Zheng YJ ，Li SF ，Zhao LR ，Ma QY ，Cheng JL ，Ma JS ，Feng WS ，Zheng XK ... -  《-》

20(R)-ginsenoside Rg3 protects against focal cerebral ischemia‒reperfusion injury by suppressing autophagy via PI3K/Akt/mTOR signaling pathway.

This study aimed to investigate the effect of 20(R)-ginsenoside Rg3 on autophagy induced by cerebral ischemia‒reperfusion injury (CIRI) in rats and explore its regulation of the PI3K/Akt signaling pathway. Middle cerebral artery occlusion/reperfusion (MCAO/R) in male rats was injected intraperitoneally with 20(R)-ginsenoside Rg3 (5, 10, 20 mg/kg) 12 h before modeling, 2 h after ischemia and 12 h after reperfusion. Neurobehavioral and neuronal morphological changes were detected 24 h after brain I/R. In vitro, the OGD/R-induced injury model is replicated in PC12 cells and different concentrations of 20(R)-ginsenoside Rg3 are administered to observe its effects on cell viability and autophagy and PI3K/Akt/mTOR-related protein expression. Our findings suggest that treatment with 20 mg/kg 20(R)-ginsenoside Rg3 significantly attenuated the neuronal injury, as evidenced by a decreased number of damaged neurons, reduced dissolution of Nissl corpuscles, a fewer autophagosomes, and downregulated expression of Beclin1 and LC3-II/I compared with the MCAO/R group. Furthermore, 20(R)-ginsenoside Rg3 treatment significantly upregulated the expression of p62, p-PI3K, p-AKT, and p-mTOR. In vitro, 20(R)-ginsenoside Rg3 significantly improved the survival rate of cells following OGD/R and markedly attenuated the LY294002 and OGD/R-induced upregulation of Beclin1 and LC3 gene expression. Moreover, 20(R)-ginsenoside Rg3 could rescued the LY294002 and OGD/R-induced downregulation of p62, p-PI3K, p-AKT, and p-mTOR expression. 20(R)-ginsenoside Rg3 attenuates neuronal injury and motor dysfunction following ischemia-reperfusion by inhibiting the activation of autophagy, and its mechanism is related to the upregulation of the PI3K/Akt/mTOR signaling pathway.

Tao D ，Li F ，Zhang X ，Guo H ，Yang R ，Yang Y ，Zhang L ，Shen Z ，Teng J ，Chen P ，He B ... -  《-》