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Outline
The purpose of this study was to investigate the potential regulatory mechanisms underlying the neuroprotective effects of Gualou Guizhi Decoction (GLGZD) in cerebral ischemia-reperfusion (CI/R)-injured rats. The main chemical components of GLGZD were identified by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOFMS). Molecular docking was performed using the CB-Dock2 platform to evaluate the binding interactions between GLGZD components and Fn14. After the middle cerebral artery occlusion (MCAO) model was established in male SD rats, the therapeutic efficacy of GLGZD was evaluated using a combination of assessments. The modified neurological severity score (mNSS) and Catwalk test were conducted to evaluate neurological and motor functions of rats. MRI was used to measure cerebral infarction volume. Nissl staining was performed to observe the morphological structures of the neurons. Western blot was used to detect protein expression of tumor necrosis factor-like weak inducer of apoptosis (TWEAK), fibroblast growth factor-inducible immediate-early response protein 14 (Fn14), TNF receptor associated factor 2 (TRAF2), cellular inhibitor of apoptosis 1 (cIAP1), NF-κB inducing kinase (NIK), inhibitor of kappa B kinase α (IKKα), phosphorylated IKKα, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) in ischemic cortex. Immunofluorescence was used to analyze the expression of Fn14 in neurons, and immunohistochemistry was employed to examine the nuclear translocation of nuclear factor kappa B (NF-κB). The results showed that the 8 identified ingredients of GLGZD could bind to Fn14. Compared with the sham surgery group, the model group exhibited significantly reduced motor function, Nissl-positive cells, and protein expression levels of cIAP1, TRAF2, and total Bcl-2 (P < 0.01). Conversely, mNSS scores, cerebral infarction volume, TWEAK, Fn14, NIK, Bax protein expression levels, IKKα phosphorylation levels, and neuronal Fn14-positive cell counts all significantly increased (P < 0.01). GLGZD treatment markedly improved motor function in MCAO rats, increased the number of Nissl-positive cells in the ischemic cortex, and enhanced cIAP1, TRAF2, and total Bcl-2 protein expression (P < 0.05, P < 0.01). It also significantly reduced mNSS and cerebral infarction volume, decreased TWEAK, Fn14, NIK, and Bax protein expression in the ischemic cortex, lowered IKKα phosphorylation, and reduced neuronal Fn14-positive cell counts (P < 0.05, P < 0.01). In conclusion, GLGZD can improve neurological and motor function after stroke in MCAO rats. This neuroprotective effect may be mediated through the TWEAK/Fn14/NF-κB pathway, which reduces neuronal damage. The animal study was approved by the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (approval number: FJTCM IACUC 2021087).
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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