Suanzaoren Decoction inhibits neuroinflammation and alleviates hippocampal neuronal injury in APP/PS1 transgenic mice by regulating the Hippo-YAP pathway

doi:10.12122/j.issn.1673-4254.2026.10.14

Abstract

Abstract:

Objective To investigate the mechanism by which Suanzaoren Decoction (SZRD) ameliorates hippocampal neuronal injury in APP/PS1 mice. Methods Thirty 7-month-old APP/PS1 transgenic mice were randomized into 3 groups for daily gavage with saline or SZRD at 12.96 or 25.92 g/kg (n=10) for 28 consecutive days, with 10 saline-treated age-matched male C57BL/6J mice as the blank control group. Spatial learning and memory abilities of the mice were evaluated using Morris water maze test. Neuronal integrity and amyloid β (Aβ) deposition were examined with Nissl staining and Thioflavin S staining, respectively, and hippocampal IBA1 expression was detected by immunohistochemistry. IBA1/Aβ immunofluorescence double staining was used to evaluate the spatial relationship between hippocampal microglia and Aβ plaques. RNA sequencing was employed to identify the key targets. Western blotting was performed to detect hippocampal expressions of nuclear factor kappa-B (NF‑κB p65), p-NF‑κB p65-ser536, interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), and the Hippo-YAP pathway proteins including mammalian sterile 20-like kinase 1 (MST1), large tumor suppressor 1 (LATS1), Yes-associated protein (YAP1), and p-YAP1- s127. Results In APP/PS1 mice, SZRD at both the low and high doses significantly improved learning and memory performance and neuronal organization, increased hippocampal neuronal number, and reduced amyloid-β (Aβ) deposition and IBA1 expression. The treatments also significantly decreased IBA1-positive area, Aβ-positive area, and IBA1/Aβ co-localization in the transgenic mice. RNA sequencing revealed that SZRD downregulated the key genes in the Hippo signaling pathway and inflammation-related genes in APP/PS1 mice. Western blotting demonstrated that SZRS treatment significantly lowered hippocampal expressions of MST1, LATS1, p-YAP1-s127/YAP1, p-NF-κB p65-Ser536/NF-κB p65, IL-1β, IL-6, and TNF-α in APP/PS1 mice. Conclusion SZRD ameliorates hippocampal neuronal injury in APP/PS1 mice by regulating the Hippo-YAP signaling pathway and inhibiting neuroinflammation.

Key words: Suanzaoren Decoction, APP/PS1 mice, RNA sequencing, hippo-YAP signaling pathway, neuroinflammation

Qixuan NIU, Dan WU, Bixuan LAI, Junhua CHEN, Zhengyu WANG, Qinghua LONG. Suanzaoren Decoction inhibits neuroinflammation and alleviates hippocampal neuronal injury in APP/PS1 transgenic mice by regulating the Hippo-YAP pathway[J]. Journal of Southern Medical University, 2026, 46(10): 2447-2455.

Figures/Tables 7

Fig.1 Results of Morris water maze test. A: Representative paths of the mice in each group in positioning navigation test and spatial probe test. B: Analysis of first 5-day water maze test in each group. C: Analysis of the day 7 water maze test in each group. n=10. *P<0.05, **P<0.01.

Fig.2 Nissl staining of mouse hippocampal tissues. A: Nissl staining in the CA1 and CA3 region of the hippocampus of the mice in each group (Scale bar=20 μm). B: Quantitative analysis of the results of Nissl staining. n=3. *P<0.05, **P<0.01

Fig.3 Aβ staining of mouse hippocampal tissues. A: Aβ Thioflavin S staining in each group (Scale bar=100 μm). B: Quantification of Aβ positive areas in each group (n=3). *P<0.05, **P<0.01

Fig.4 Immunohistochemical staining of mouse hippocampal tissues. A: IBA1 immunohistochemistry in each group (Scale bar=50 μm). B: Quantification of IBA1 positive areas in each group (n=3). *P<0.05, **P<0.01.

Fig.5 Representative images of IBA1/Aβ immunofluorescence double staining of mouse hippocampal tissues. A: Double immunofluorescence staining for IBA1/Aβ in each group (Scale bar=20 μm). B: Quantification of Aβ-positive area (%), IBA1-positive area (%), and IBA1/Aβ co-localization (n=3). *P<0.05, **P<0.01.

Fig.6 Differential gene expression and functional analysis. A: Volcano plot of the differentially expressed genes (DEGs). B: Heatmap of DEGs. C: GO functional enrichment analysis of DEGs. D: KEGG pathway enrichment analysis of DEGs.

Fig.7 Effects of Suanzaoren Decoction on the Hippo-YAP pathway and neuroinflammation-related markers in mouse hippocampus. A: Protein bands of MST1, LATS1, p-YAP1-s127, YAP1, p-NF-κB p65-ser536, NF-κB p65, IL-6, IL-1β, TNF-α in mouse hippocampus. B: Expression levels of MST1, LATS1, p-YAP1-s127, YAP1, p-NF-κB p65-ser536, NF-κB p65, IL-6, IL-1β, TNF-α proteins in mouse hippocampus (n=3). *P<0.05, **P<0.01.

References 31

[1]	Zhu YL, Guo XM, Li SM, et al. Naringenin ameliorates amyloid-β pathology and neuroinflammation in Alzheimer's disease[J]. Commun Biol, 2024, 7: 912. doi：10.1038/s42003-024-06615-6
[2]	2024 Alzheimer's disease facts and figures[J]. Alzheimer's Dement, 2024, 20(5): 3708-821. doi：10.1002/alz.13809
[3]	Wang CC, Zong S, Cui XL, et al. The effects of microglia-associated neuroinflammation on Alzheimer's disease[J]. Front Immunol, 2023, 14: 1117172. doi：10.3389/fimmu.2023.1117172
[4]	Terry BK, Kim S. The role of Hippo-YAP/TAZ signaling in brain development[J]. Dev Dyn, 2022, 251(10): 1644-65. doi：10.1002/dvdy.504
[5]	Mia MM, Cibi DM, Abdul Ghani SAB, et al. YAP/TAZ deficiency reprograms macrophage phenotype and improves infarct healing and cardiac function after myocardial infarction[J]. PLoS Biol, 2020, 18(12): e3000941. doi：10.1371/journal.pbio.3000941
[6]	Zheng MJ, Li RG, Song J, et al. Hippo-Yap signaling maintains sinoatrial node homeostasis[J]. Circulation, 2022, 146(22): 1694-711. doi：10.1161/circulationaha.121.058777
[7]	Tanaka H, Homma H, Fujita K, et al. YAP-dependent necrosis occurs in early stages of Alzheimer's disease and regulates mouse model pathology[J]. Nat Commun, 2020, 11: 507. doi：10.1038/s41467-020-14353-6
[8]	Sahu MR, Ahmad MH, Mondal AC. MST1 selective inhibitor Xmu-mp-1 ameliorates neuropathological changes in a rat model of sporadic Alzheimer's Disease by modulating Hippo-Wnt signaling crosstalk[J]. Apoptosis, 2024, 29(9/10): 1824-51. doi：10.1007/s10495-024-01975-0
[9]	王新, 冯诗瑶, 吴昊, 等. 张仲景治虚烦不眠方之证治概要与方药分析[J]. 中国民族民间医药, 2019, 28(3): 59-62.
[10]	Long QH, Li T, Zhu QH, et al. SuanZaoRen decoction alleviates neuronal loss, synaptic damage and ferroptosis of AD via activating DJ-1/Nrf2 signaling pathway[J]. J Ethnopharmacol, 2024, 323: 117679. doi：10.1016/j.jep.2023.117679
[11]	龙清华, 朱麒行, 麦合丽娅·艾斯卡尔, 等. 酸枣仁汤通过激活AMPK/SIRT1/PGC-1α信号通路改善阿尔茨海默病模型小鼠线粒体功能障碍[J]. 中国药理学通报, 2023, 39(7): 1256-62.
[12]	Long QH, Wu YG, He LL, et al. Suan-Zao-Ren Decoction ameliorates synaptic plasticity through inhibition of the Aβ deposition and JAK2/STAT3 signaling pathway in AD model of APP/PS1 transgenic mice[J]. Chin Med, 2021, 16: 14. doi：10.1186/s13020-021-00425-2
[13]	龙清华, 丁莉, 赵宾宾, 等. 酸枣仁汤通过抑制APP/PS1双转基因小鼠海马神经炎症发挥神经保护作用[J]. 中国实验方剂学杂志, 2019, 25(20): 1-6. doi：10.13422/j.cnki.syfjx.20191936
[14]	龙清华, 赵宾宾, 丁莉, 等. 酸枣仁汤通过抑制神经炎症和改善神经营养水平促进APP/PS1痴呆小鼠海马神经发生[J]. 世界科学技术-中医药现代化, 2021, 23(9): 3014-22.
[15]	吴丹, 赖碧璇, 王政喻, 等. 酸枣仁汤对APP/PS1模型小鼠海马神经元保护作用及PI3K/Akt信号通路的影响[J]. 中国中药杂志, 2026, 51(1): 173-80.
[16]	黄继汉, 黄晓晖, 陈志扬, 等. 药理试验中动物间和动物与人体间的等效剂量换算[J]. 中国临床药理学与治疗学, 2004, 9(9): 1069-72. doi：10.3969/j.issn.1009-2501.2004.09.026
[17]	郭帆, 吴东南, 刘玲, 等. 酸枣仁汤防治睡眠剥夺性大鼠学习记忆功能变化及其对NLRP3通路的影响[J]. 中国实验方剂学杂志, 2021, 27(2): 22-7.
[18]	Sun XY, Li LJ, Dong QX, et al. Rutin prevents tau pathology and neuroinflammation in a mouse model of Alzheimer's disease[J]. J Neuroinflammation, 2021, 18: 131. doi：10.1186/s12974-021-02182-3
[19]	Wang CY, Chen SS, Guo HY, et al. Forsythoside a mitigates Alzheimer's-like pathology by inhibiting ferroptosis-mediated neuroinflammation via Nrf2/GPX4 axis activation[J]. Int J Biol Sci, 2022, 18(5): 2075-90. doi：10.7150/ijbs.69714
[20]	Singh D. Astrocytic and microglial cells as the modulators of neuroinflammation in Alzheimer's disease[J]. J Neuroinfla-mmation, 2022, 19: 206. doi：10.1186/s12974-022-02565-0
[21]	Freitag K, Sterczyk N, Wendlinger S, et al. Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model[J]. J Neuroinflammation, 2022, 19: 172. doi：10.1186/s12974-022-02534-7
[22]	Chen D, Wigglesworth-Littlewood S, Gunn-Moore FJ. The Hippo signaling pathway as a therapeutic target in Alzheimer's disease[J]. Mol Neurodegener, 2025, 20(1): 100. doi：10.1186/s13024-025-00891-4
[23]	Cui DQ, Liu HX, Cao LL, et al. MST1, a novel therapeutic target for Alzheimer's disease, regulates mitochondrial homeostasis by mediating mitochondrial DNA transcription and the PI3K-Akt-ROS pathway[J]. J Transl Med, 2024, 22: 1056. doi：10.1186/s12967-024-05852-x
[24]	Wang H, Shang YC, Wang EL, et al. MST1 mediates neuronal loss and cognitive deficits: a novel therapeutic target for Alzheimer's disease[J]. Prog Neurobiol, 2022, 214: 102280. doi：10.1016/j.pneurobio.2022.102280
[25]	Lei L, Cheng YL, Yin AQ, et al. Aging-dependent YAP1 reduction contributes to AD pathology by upregulating the Nr4a1-AKT/GSK-3β axis[J]. Transl Neurodegener, 2025, 14(1): 29. doi：10.1186/s40035-025-00487-4
[26]	Xu XX, Shen XY, Wang JJ, et al. YAP prevents premature senescence of astrocytes and cognitive decline of Alzheimer's disease through regulating CDK6 signaling[J]. Aging Cell, 2021, 20(9): e13465. doi：10.1111/acel.13465
[27]	Chen SX, Wang LM, Yuan Y, et al. Electroacupuncture regulates microglia polarization via lncRNA-mediated hippo pathway after ischemic stroke[J]. Biotechnol Genet Eng Rev, 2023, 39(2): 1379-95. doi：10.1080/02648725.2023.2177046
[28]	Qing J, Liu XH, Wu Q, et al. Hippo/YAP pathway plays a critical role in effect of GDNF against a β‑induced inflammation in microglial cells[J]. DNA Cell Biol, 2020, 39(6): 1064-71. doi：10.1089/dna.2019.5308
[29]	Evans RC, Dar NJ, Chen LJ, et al. Inhibition of hippo signaling through ablation of Lats1 and Lats2 protects against cognitive decline in 5xFAD mice via increasing neuronal resilience against ferroptosis[J]. Aging Cell, 2025, 24(11): e70218. doi：10.1111/acel.70218
[30]	She G, Du JC, Wu W, et al. Hippo pathway activation mediates chemotherapy-induced anti-cancer effect and cardiomyopathy through causing mitochondrial damage and dysfunction[J]. Theranostics, 2023, 13(2): 560-77. doi：10.7150/thno.79227
[31]	Yu HL, Cao X, Li W, et al. Targeting connexin 43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling[J]. J Neuroinflammation, 2020, 17(1): 322. doi：10.1186/s12974-020-01978-z