OBJECTIVES: To study the therapeutic mechanism of <i>Tuihuangi> Mixture against cholestasis. METHODS: Forty-eight Wistar rats were randomized equally into blank group, model group, ursodeoxycholic acid group and <i>Tuihuangi> Mixture group. Except for those in the blank group, all the rats were given α‑naphthylisothiocyanate (ANIT) to establish rat models of cholestasis, followed by treatments with indicated drugs or distilled water. Serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL of the rats were determined, and hepatic expressions IL-1β, IL-18, FXR, NLRP3, ASC, Caspase-1 and GSDMD were detected using q-PCR, ELISA or Western blotting. Histopathological changes of the liver tissues were observed using HE staining. RESULTS: The rat models of cholestasis had significantly increased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL with increased mRNA and protein expressions of IL-1β and IL-18, decreased protein and mRNA expressions of FXR, and increased protein expressions of NLRP3 and Caspase-1 and mRNA expressions of NLRP3, ASC, Caspase-1 and GSDMD in the liver tissue, showing also irregular arrangement of liver cells, proliferation of bile duct epithelial cells and inflammatory cells infiltration. Treatment of the rat models with <i>Tuihuangi> Mixture significantly decreased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL, lowered IL-1β and IL-18 and increased FXR protein and mRNA expressions, and reduced NLRP3, ASC, Caspase-1 and GSDMD proteins and NLRP3, ASC and Caspase-1 mRNA expressions in the liver tissue. <i>Tuihuangi> Mixture also significantly alleviated hepatocyte injury, bile duct epithelial cell proliferation and inflammatory cell infiltration in the liver of the rat models. CONCLUSIONS <i>Tuihuangi> Mixture can effectively improve cholestasis in rats possibly by inhibiting NLRP3 inflammatosome-mediated pyroptosis via regulating FXR.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats ; Receptors, Cytoplasmic and Nuclear/metabolism* ; Cholestasis/drug therapy* ; Rats, Wistar ; Inflammasomes/metabolism* ; 1-Naphthylisothiocyanate ; Drugs, Chinese Herbal/therapeutic use* ; Male ; Interleukin-18/metabolism* ; Caspase 1/metabolism* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

OBJECTIVES: To investigate the therapeutic mechanism of <i>Huoxue Shufengi> Granules (HXSFG) for alleviating central sensitization in a mouse model of chronic migraine (CM). METHODS: We analyzed the main chemical components of HXSFG through literature review and explored their pharmacological mechanisms by bioinformatics analyses. In a male C57BL/6J mouse model of CM established by intraperitoneal injections of nitroglycerin (10 mg/kg) every other day (5 injections), the effects of gavage with low, and high doses of HXSFG or intraperitoneal injections of topiramate for ameliorating central sensitization were evaluated using Von Frey test and a hot plate apparatus; the changes in expressions of inflammatory factors, the proteins in the TLR4/NF‑κB signaling pathway, and activation of c-Fos and CGRP were detected using RT-qPCR, Western blotting and immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that the main active components in HXSFG for alleviating CM included formononetin, paeoniflorin, quercetin, and tanshinone. Gene Ontology (GO) enrichment analysis identified 492 GO entries, comprising 366 biological processes, 46 cellular components, and 80 molecular functions. KEGG pathway enrichment analysis indicated that the Toll-like receptor and NF‑κB signaling pathways were crucial in mediating the therapeutic effects of HXSFG on CM. In the mouse models of CM, both topiramate and HXSFG treatments alleviated the symptoms of central sensitization, evidenced by improved mechanical and thermal pain thresholds in the mice. HXSFG significantly reduced the expression of c-Fos and CGRP, improved inflammatory markers, and downregulated the expressions of TLR4, p-NF‑κB, IL-1β, and TNF‑α proteins in the mouse models. CONCLUSIONS HXSFG effectively alleviates central sensitization in CM mice by modulating the inflammatory pathways and inhibiting the TLR4/ NF-κB signaling pathway, suggesting its potential as a therapeutic option for CM.
Animals ; Toll-Like Receptor 4/metabolism* ; NF-kappa B/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Male ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Migraine Disorders/metabolism* ; Disease Models, Animal ; Inflammation

OBJECTIVES: To explore the therapeutic mechanism of compound <i>Centella asiaticai> formula (CCA) for alleviating <i>Schistosoma japonicumi> (Sj)-induced liver fibrosis in mice. METHODS: The active components and targets of CCA were identified using the TCMSP database with cross-analysis of Sj-related liver fibrosis targets. A "drug-component-target-pathway-disease" network was constructed using Cytoscape 3.9.1. Functional enrichment analysis (GO/KEGG) was performed using DAVID. Molecular docking study was carried out to validate interactions between the core targets and the key compounds. For experimental validation of the results, 36 mice were divided into control group, Sj-infected model group, and CCA-treated groups. In the latter two groups, liver fibrosis was induced via abdominal infection with Sj cercariae for 8 weeks, followed by 8 weeks of daily treatment with CCA decoction or saline. Hepatic pathology of the mice was assessedwith HE and Masson staining, and hepatic expressions of collagen-I and collagen-III were detected using immunohistochemistry; serum IL-6 and TNF-α levels were determined with ELISA. Hepatic expressions of TLR4 and MyD88 proteins were analyzed with Western blotting. RESULTS: We identified a total of 107 bioactive CCA components and 791 targets, including 37 intersection targets linked to Sj-induced fibrosis. The core targets included TNF, TP53, JUN, MMP9, and CXCL8, involving the IL-17 signaling, lipid metabolism, TLR4/MyD88 axis, and cancer pathways. Molecular docking study confirmed strong binding affinity between quercetin (a primary CCA component) and TNF/TP53/JUN/MMP9. In Sj-infected mouse models, CCA treatment significantly attenuated hepatic inflammatory cell infiltration, reduced collagen-I and collagen-III deposition, improved tissue architecture, reduced serum IL-6 and TNF-α levels, and downregulated TLR4 and MyD88 expressions in the liver. CONCLUSIONS CCA mitigates Sj-induced liver fibrosis by targeting TNF, TP53, JUN, and MMP9 to modulate the TLR4/MyD88 pathway, thereby suppressing pro-inflammatory cytokine release, inhibiting hepatic stellate cell activation, reducing collagen deposition, and preventing granuloma formation in the liver.
Animals ; Toll-Like Receptor 4/metabolism* ; Mice ; Myeloid Differentiation Factor 88/metabolism* ; Schistosoma japonicum ; Liver Cirrhosis/parasitology* ; Schistosomiasis japonica ; Signal Transduction ; Molecular Docking Simulation ; Inflammation ; Centella/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVES: To evaluate the protective effect of <i>Bufei Yisheni> Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. METHODS: BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. RESULTS: CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. CONCLUSIONS BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.
Humans ; Epithelial Cells/cytology* ; Drugs, Chinese Herbal/pharmacology* ; NF-kappa B/metabolism* ; Bronchi/cytology* ; Smoke/adverse effects* ; Apoptosis/drug effects* ; Mucin 5AC/metabolism* ; Cell Line ; Toll-Like Receptor 4/metabolism* ; Mucin-5B/metabolism* ; Signal Transduction/drug effects* ; Nicotiana ; Rats ; Thiocarbamates/pharmacology* ; Animals

OBJECTIVES: To investigate the therapeutic effect of electroacupuncture (EA) at Zusanli (ST36) acupoint on hyperlipidemia in mice and explore the underlying mechanisms. METHODS: Thirty C57BL/6J mice were equally randomized into normal diet group, high-fat diet (HFD) group, and EA group. The changes in blood lipids and serum malondialdehyde (MDA) content of the mice were evaluated, and histopathological changes and lipid accumulation in the liver were observed using Oil red O staining (ORO). The expressions of NLRP3, TLR4, and IL-1β proteins in the colon tissues were detected with Western blotting, and gut microbiota changes were analyzed using 16S rDNA sequencing. RESULTS: In mice with HFD feeding, 16 weeks of EA treatment significantly lowered body weight and serum TC, TG, LDL-C and MDA levels, obviously reduced lipid accumulation in the liver, and ameliorated HFD-induced elevations of protein expressions of NLRP3, TLR4, and IL-1β. 16S rRNA sequencing revealed that EA significantly altered gut microbiota composition, and increased the diversity and relative abundance of beneficial bacterial groups such as <i>Muribaculaceaei> and <i>Lachnospiraceaei> NK4A136_group. CONCLUSIONS Electroacupuncture at ST36 alleviates blood lipid disorders in hyperlipidemic mice possibly by improving intestinal microbiota structure, promoting degradation of high-caloric carbohydrates, cholesterol lipid metabolism and intestinal mucosa repair, and reducing toxin leakage, lipid peroxides, and liver fat deposition.
Animals ; Electroacupuncture ; Gastrointestinal Microbiome ; Hyperlipidemias/blood* ; Mice, Inbred C57BL ; Mice ; Diet, High-Fat ; Toll-Like Receptor 4/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Acupuncture Points ; Male ; Lipids/blood* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

OBJECTIVES: To explore the mechanism of cinnamic acid (CA) for improving doxorubicin-induced myocardial injury (DIC) in mice. METHODS: Network pharmacology analysis was used to obtain the key targets of CA and DIC. Male C57BL/6J mice were randomized into Sham, DOX, CA (25, 50 and 100 mg/kg)+DOX, and CA+Ferrostatin-1+DOX groups, and their myocardial function and pathology were examined by echocardiography and HE staining. Serum levels of CK-MB, LDH, MDA, IL-6, TNF‑α and myocardial ROS level were detected, and the expression levels of TLR4 and ferroptosis pathway proteins in myocardial tissue were detected by Western blotting. Cultured murine cardiomyocytes (HL-1 cells) with or without transfection with a small interfering RNA targeting TLR4 (si-TLR4) were treated with DOX or Erastin, and the cellular ROS content was measured by DCFH-DA staining; the expression level of GPX4 was detected using immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that CA may improve DIC through TLR4 signaling. DOX treatment caused obvious myocardial injury in mice, which showed significantly increased serum levels of CK-MB, LDH, MDA, IL-6, TNF-α and myocardial ROS level with decreased myocardial levels of SLC7A11 and GPX4 proteins and increased levels of TLR4 and PTGS2 proteins. All these changes in the mouse models were significantly alleviated by treatment with CA, and the mice receiving CA or ferrostatin-1 treatment exhibited increased myocardial expressions of SLC7A11 and GPX4 proteins and lowered expressions of TLR4 and PTGS2 proteins. In cultured HL-1 cells, treatment with DOX and Erastin both obviously increased intracellular ROS level and decreased cellular GPX4 expression level, and these changes were strongly attenuated by TLR4 interference. CONCLUSIONS CA, as a potent herbal monomer, can effectively alleviate DIC in mice by inhibiting TLR4-mediated ferroptosis.
Animals ; Ferroptosis/drug effects* ; Toll-Like Receptor 4/metabolism* ; Myocytes, Cardiac/metabolism* ; Mice, Inbred C57BL ; Mice ; Male ; Doxorubicin/adverse effects* ; Cinnamates/pharmacology* ; Signal Transduction ; Reactive Oxygen Species/metabolism*

OBJECTIVES: To explore the therapeutic mechanism of puerarin for alleviating synovitis in rats with collagen-induced arthritis (CIA). METHODS: In a SD rat model of CIA, we tested the effects of daily gavage of puerarin at low, moderate and high doses (10, 30, and 100 mg/kg, respectively) for 3 weeks, with tripterygium glycosides (GTW, 10 mg/kg) as the positive control, on swelling in the hind limb joints regions evaluated by arthritis index scoring. Mass fraction of the liver of the rats was calculated, and pathologies in joint synovial membrane were observed with HE staining. The expressions of transforming growth factor β‑activated kinase-1 (TAK1), Toll-like receptor 4 (TLR4), and nuclear factor kappa-Bp65 (NF‑κB p65) at the mRNA and protein levels in the synovial tissues were detected using Real-time PCR and Western blotting. RESULTS: Compared with those in the model group, the rats in GTW group and high-dose puerarin group showed significantly reduced mass fraction of the liver. Treatment with GTW and puerarin at the 3 doses all significantly alleviated plantar swelling, lowered arthritis index scores, and improved synovitis in CIA rats (<i>Pi><0.05), and the effects of puerarin showed an obvious dose dependence. Both GTW and puerarin treatments significantly lowered TAK1, TLR4, and NF‑κB p65 mRNA and protein expressions in the synovium of CIA rats. CONCLUSIONS Puerarin alleviates synovium damages in CIA rats possibly by suppressing the TLR4/NF‑κB signaling pathway via downregulating TAK1 expression.
Animals ; Toll-Like Receptor 4/metabolism* ; Rats, Sprague-Dawley ; Rats ; MAP Kinase Kinase Kinases/metabolism* ; Signal Transduction/drug effects* ; Arthritis, Rheumatoid/drug therapy* ; NF-kappa B/metabolism* ; Isoflavones/therapeutic use* ; Male ; Arthritis, Experimental/drug therapy* ; Transcription Factor RelA/metabolism* ; Synovial Membrane/metabolism*

OBJECTIVES: To explore the therapeutic effect of <i>LuoFuShani> Rheumatism Plaster (LFS) on neuropathic pain (NP) and its molecular mechanism. METHODS: Mouse models of sciatic nerve chronic constriction injury (CCI) were treated with low, medium, and high doses (2.2, 4.4, and 8.8 cm², respectively) of LFS by topical application for 14 consecutive days. The therapeutic effects were assessed by evaluating the mechanical withdrawal threshold (MWT), paw withdrawal latency (PWL), plasma IL-6 and TNF-α levels, and histopathology of the sciatic nerve. Network pharmacology and molecular docking were used to identify the key targets and signaling pathways. The key targets were verified by RT-qPCR and immunohistochemistry. The biosafety of LFS was evaluated by measuring the organ indices and damage indicators of the heart, liver, and kidneys. RESULTS: Compared with the CCI group, LFS dose-dependently increased MWT and PWL, reduced plasma IL-6 and TNF-α levels, and alleviated sciatic nerve inflammation in the mouse models. Network pharmacology identified 378 bioactive compounds targeting 279 NP-associated genes enriched in TLR and TNF signaling. Molecular docking showed that quercetin and ursolic acid in LFS could stably bind to TLR4 and TNF‑α. In the mouse models of sciatic nerve CCI, LFS significantly downregulated the mRNA expression levels of <i>Tlr4i> and <i>Tnf-αi> in the spinal cord in a dose-dependent manner and lowered the protein expressions of TLR4 and TNF-α in the sciatic nerve. LFS treatment did not cause significant changes in the organ indices or damage indicators of the heart, liver and kidneys as compared with those in the CCI model group and sham-operated group. CONCLUSIONS LFS alleviates NP in mice by suppression of TLR4/TNF-α-mediated neuroinflammation with a good safety profile.
Animals ; Toll-Like Receptor 4/metabolism* ; Neuralgia/metabolism* ; Mice ; Signal Transduction/drug effects* ; Tumor Necrosis Factor-alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Sciatic Nerve/injuries* ; Male ; Molecular Docking Simulation ; Disease Models, Animal ; Interleukin-6

OBJECTIVES: To investigate the effect of hypaphorine (HYP) on Crohn's disease (CD)‑like colitis in mice and its molecular mechanism. METHODS: Thirty male C57BL/6J mice were equally randomized into WT, TNBS, and HYP groups, and in the latter two groups, mouse models of CD-like colitis were established using TNBS with daily gavage of 15 mg/kg HYP or an equivalent volume of saline. The treatment efficacy was evaluated by assessing the disease activity index (DAI), body weight changes, colon length and histopathology. The effect of HYP was also tested in a LPS-stimulated Caco-2 cell model mimicking intestinal inflammation by evaluating inflammatory responses and barrier function of the cells using qRT-PCR and immunofluorescence staining. GO and KEGG analyses were conducted to explore the therapeutic mechanism of HYP, which was validated in both the cell and mouse models using Western blotting. RESULTS: In the mouse models of CD-like colitis, HYP intervention obviously alleviated colitis as shown by significantly reduced body weight loss, colon shortening, DAI and inflammation scores, and expressions of pro-inflammatory factors in the colon tissues. HYP treatment also significantly increased the TEER values, reduced bacterial translocation to the mesenteric lymph nodes, liver, and spleen, lowered serum levels of I-FABP and FITC-dextran, increased the number of colonic tissue cup cells, and upregulated colonic expressions of MUC2 and tight junction proteins (claudin-1 and ZO-1) in the mouse models. In LPS-stimulated Caco-2 cells, HYP treatment significantly inhibited the expressions of pro-inflammatory factors and increased the expressions of tight junction proteins. Western blotting showed that HYP downregulated the expressions of the key proteins in the TLR4/MyD88 signaling pathway in both the <i>in vitroi> and <i>in vivoi> models. CONCLUSIONS HYP alleviates CD-like colitis in mice possibly by suppressing intestinal epithelial inflammation and improving gut barrier function.
Animals ; Male ; Mice, Inbred C57BL ; Crohn Disease/drug therapy* ; Mice ; Humans ; Caco-2 Cells ; Intestinal Mucosa/metabolism* ; Colitis/drug therapy* ; Disease Models, Animal ; Inflammation ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; Intestinal Barrier Function

Triggering receptor expressed on myeloid cells 2 (TREM2)-mediated microglial phagocytosis is an energy-intensive process that plays a crucial role in amyloid beta (Aβ) clearance in Alzheimer's disease (AD). Energy metabolic reprogramming (EMR) in microglia induced by TREM2 presents therapeutic targets for cognitive impairment in AD. Jiawei Xionggui Decoction (JWXG) has demonstrated effectiveness in enhancing energy supply, protecting microglia, and mitigating cognitive impairment in APP/PS1 mice. However, the mechanism by which JWXG enhances Aβ phagocytosis through TREM2-mediated EMR in microglia remains unclear. This study investigates how JWXG facilitates microglial phagocytosis and alleviates cognitive deficits in AD through TREM2-mediated EMR. Microglial phagocytosis was evaluated through immunofluorescence staining in vitro and in vivo. The EMR level of microglia was assessed using high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) kits. The TREM2/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) signaling pathway was analyzed using Western blotting in BV₂ cells. TREM2^-/- BV₂ cells were utilized for reverse validation experiments. The Aβ burden, neuropathological features, and cognitive ability in APP/PS1 mice were evaluated using ELISA kits, immunohistochemistry (IHC), and the Morris water maze (MWM) test. JWXG enhanced both the phagocytosis of EMR disorder-BV₂ cells (EMRD-BV₂) and increased EMR levels. Notably, these effects were significantly reversed in TREM2^-/- BV₂ cells. JWXG elevated TREM2 expression, adenosine triphosphate (ATP) levels, and microglial phagocytosis in APP/PS1 mice. Additionally, JWXG reduced Aβ-burden, neuropathological lesions, and cognitive deficits in APP/PS1 mice. In conclusion, JWXG promoted TREM2-induced EMR and enhanced microglial phagocytosis, thereby reducing Aβ deposition, improving neuropathological lesions, and alleviating cognitive deficits.
Drugs, Chinese Herbal/pharmacology* ; Microglia/drug effects* ; Phagocytosis ; Cognitive Dysfunction/drug therapy* ; Metabolic Reprogramming ; Animals ; Mice ; Cell Line ; Receptors, Immunologic/metabolism* ; Membrane Glycoproteins/metabolism* ; Signal Transduction ; Amyloid beta-Peptides/metabolism* ; Energy Metabolism