Drug pairs of Huangqi and Dnggui alleviates pyroptosis by promoting autophagy activity via AMPK/mTOR signaling pathway in middle-cerebral artery occlusion/reperfusion in rats

Article

Author: Wang, Ruikun ; Zhang, Yi ; Hou, Xianming ; Liu, Luyao ; Zhao, Yanmeng ; Gao, Weijuan ; Zhou, Xiaohong ; Jin, Xiaofei

ETHNOPHARMACOLOGICAL RELEVANCECerebral ischemia-reperfusion (I/R) injury is a common complication of ischemic stroke, with autophagy and pyroptosis playing key roles. Huangqi and Danggui (HQDG) are a commonly used drug pair of Chinese traditional medicine for clinical treatment of ischemic stroke.AIM OF THE STUDYThe study aims to investigate the interaction between autophagy and pyroptosis regulated by HQDG through the AMPK/mTOR signaling pathway during cerebral I/R injury.MATERIALS AND METHODSModel of middle-cerebral artery occlusion/reperfusion (MCAO/R) in SD rats was established using the Longa suture method. The components of traditional Chinese medicine were detected by liquid chromatography coupled to quadrupole orbitrap high resolution mass spectrometry (LC/MS). Neurological deficits were evaluated by neurological function score. Changes of cerebral blood flow were detected by a laser speckle blood flow imaging instrument. The volume of cerebral infarction was observed by 2,3,5-Chlorotriphenyltetrazolium (TTC) staining. The permeability of the blood-brain barrier was measured by Evans blue test. Neurovascular unit and autophagosomes in brain tissue were assessed by transmission electron microscopy. Neuronal pyroptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)/Caspase-1 staining. The expression of autophagy related proteins, pyroptosis related proteins, and AMPK/mTOR pathway related proteins were detected by Western blot.RESULTSAfter cerebral I/R injury, autophagy and pyroptosis, were characterized by increased number of autophagosomes and pyroptosis cells, upregulated expression of Beclin 1, LC3-II/LC3-I, NLRP3, cleaved Caspase-1, IL-1beta, IL-18 proteins, and downregulated expression of P62 proteins. HQDG significantly improved neurological function, reduced the volume of cerebral infarction, increased cerebral blood flow, improved blood-brain barrier permeability and the function of neurovascular units. Autophagy was further activated and pyroptosis was significantly inhibited by HQDG, which promoted increased number of autophagosomes, enhanced expression of Beclin 1, LC3-II/LC3-I proteins, reduced expression of P62, NLRP3, cleaved Caspase-1, IL-1beta, and IL-18 proteins, and downregulated the number of pyroptosis cells. On the other hand, after administering 3-Methyladenine (3-MA) to inhibit autophagy, the above effects of HQDG were significantly inhibited. Besides, HQDG promoted AMPK phosphorylation, and weakened mTOR phosphorylation. However, after the administration of AMPK inhibitor Compound C, HQDG caused increase in Beclin 1 and LC3-II/LC3-I, reduced P62 and NLRP3, and cleaved Caspase-1 protein expression, whereas cerebral blood flow decreased.CONCLUSIONHQDG alleviated pyroptosis by promoting autophagy via AMPK/mTOR signaling pathway after middle-cerebral artery occlusion/reperfusion in rats, showing its potential for treatment of cerebral I/R injury in humans.

31 Dec 2024·Pharmaceutical Biology

Study of the multitarget mechanism of Astragalus (HUANGQI) in the treatment of Alzheimer’s disease based on network pharmacology and molecular docking technology

Article

Author: Qin, Chunmeng ; Zheng, Xiangru ; Sun, Mei ; Lv, Feng ; Du, Dan ; Li, Wenjun

CONTEXTIn China, HUANGQI is widely used for the treatment of Alzheimer's disease (AD). However, a comprehensive understanding of its mechanism of anti-AD effects is lacking.OBJECTIVETo explore the active ingredients of HUANGQI and its potential targets and mechanisms of action in AD.MATERIALS AND METHODSThe active ingredients and targets of HUANGQI were screened from databases (TCSMP, ETCM, and BATMan), and AD-related genes were obtained from DrugBank and GeneCards. The same target genes were screened, and a drug-target disease network was constructed. The PPI network was constructed and GO and KEGG pathway enrichment analyses of the targets. The Cell Counting Kit-8 (CCK-8) assay was used to determine suitable HUANGQI treatment concentrations for HT-22 cells between 0-480 μg/mL. CCK-8, FITC-phalloidin and propidium iodide (PI) assays were used to examine the protective effect of (0, 60, 120, 240 μg/mL) of HUANGQI on 20 μM Aβ1-42-induced HT-22 cell cytotoxicity.RESULTSTwelve active ingredients of HUANGQI were selected, with 679 common targets associated with AD. GO and KEGG analysis revealed that the therapeutic mechanisms of HUANGQI involve TNF, AGE, the NF-κB pathway, and nuclear receptor activity-related processes. The CCK-8 assay indicated that HUANGQI was not cytotoxic to HT-22 cells at concentrations less than 240 μg/mL and was able to attenuate Aβ1-42-induced cellular damage (EC₅₀ = 83.46 μg/mL). FITC-phalloidin and PI assays suggested that HUANGQI could alleviate 20 μM Aβ1-42-induced neuronal cell cytotoxicity in a dose-dependent manner.CONCLUSIONHUANGQI has a protective effect on Aβ1-42-induced nerve cell injury; further mechanism research was needed.

01 Oct 2024·Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology

[Huangqi Glycoprotein improves myocardial injury in rats with adjuvant arthritis by interfering with lncRNA GAS5/miR-21/TLR4 signaling axis to inhibit pyroptosis].

Article

Author: Cao, Yunxiang ; Fu, Wanlan ; Shu, Kaiyan ; Zhu, Nanfei

Objective To investigate the effect and mechanism of Huangqi glycoprotein (HQGP) on myocardial injury in rats with adjuvant arthritis (AA) based on the long non-coding RNA growth arrest-specific transcript 5/microRNA-21/Toll-like receptor 4 (lncRNA GAS5/miR-21/TLR4) signal axis. Methods SD rats were randomized into normal control, model, methotrexate (MTX) and HQGP groups, with 6 rats in each group. The AA rat model was established, and the drug was administered on the 19th day after the model was established, for 4 consecutive weeks. The degree of toe swelling and the arthritis index (AI) of AA rats in each group were measured. The pathological changes of rat myocardial tissue were observed by HE staining, and the changes in the ultrastructure of the myocardial tissue and the situation of pyroptotic vesicles were observed by transmission electron microscope. The levels of serum interleukin 6 (IL-6), IL-18, IL-1β and tumor necrosis factor α (TNF-α) were detected by ELISA. The release of lactate dehydrogenase (LDH) in myocardial tissue was detected by kit. Real-time quantitative PCR was used to detect the mRNA expression of nuclear factor-kappa B p65 (NF-κB p65), cysteine aspartate specific proteinase 1 (caspase-1), nucleotide binding oligomerization domain like receptor family pyrin domain containing 3 (NLRP3), gasdermin D (GSDMD), and molecules in the lncRNA GAS5/miR-21/TLR4 signaling axis in the myocardial tissue. The protein levels of TLR4, NF-κB p65, caspase-1, NLRP3 and GSDMD in myocardial tissue were detected by Western blot. Results Compared with the normal control group, the toe swelling and AI of the model group rats were significantly increased. The myocardial fibers of rats dissolved and broken, the structure of sarcomere was blurred, the arrangement of myocardial fibers was disordered, there was inflammatory cell infiltration between tissues, the mitochondrial cristae were significantly broken and sparse, and the number of pyroptotic vesicles increased. The expression of serum pro-inflammatory cytokines IL-6, IL-18, IL-1β and TNF-α increased significantly. In myocardial tissue, the expression of GAS5 reduced significantly, the expression of miR-21 increased significantly, the release of LDH, and the mRNA and protein levels of TLR4, NF-κB p65, caspase-1, NLRP3 and GSDMD increased significantly. Compared with the model group, the toe swelling, AI, and the pathological status of myocardial tissue in the HQGP group rats were improved significantly. The expressions of serum IL-6, IL-18, IL-1β and TNF-α reduced significantly. In myocardial tissue, the expression of GAS5 increased significantly, the expression of miR-21 decreased significantly, the release of LDH, the mRNA and protein levels of TLR4, NF-κB p65, caspase-1 and NLRP3 decreased significantly, and the mRNA expression of GSDMD reduced while the protein level significantly reduced. Conclusion HQGP improves myocardial injury in AA rats by inhibiting miR-21/TLR4 signalling through up-regulation of lncRNA GAS5, inhibiting pyroptosis, and reducing pro-inflammatory cytokine expression.

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