Introduction: Hemorrhoids significantly impair the quality of life of patients, primarily manifesting as pain, bleeding, and prolapse. Detoxifying fumigation and washing granules (DFWG), a traditional Chinese medicine, have demonstrated potential for alleviating hemorrhoids; however, its underlying mechanisms of action have yet to be fully elucidated. This study aimed to investigate the putative active constituents and possible mechanisms of DFWG in the treatment of hemorrhoids. Methods: Active compounds in DFWG were identified through network pharmacology, and targets were screened using protein-protein interaction (PPI) network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify core signaling pathways, and in vivo validation was conducted in a rat model of perianal edema. Results: Network pharmacology identified 328 active compounds and 1020 targets from DFWG, along with 314 hemorrhoid-related targets, yielding 46 overlapping targets. PPI analysis revealed 10 core targets, while GO and KEGG analyses indicated roles in regulating apoptosis and inflammation. Molecular docking results indicated that the binding energies between core target proteins and compounds were consistently below −5.0 kcal/mol. In vivo, DFWG reduced perianal swelling and rectoanal coefficient in model rats, lowered serum IL-1β, TNF-α, and IL-6, and suppressed p-NF-κB p65/NF-κB p65 expression in perianal tissues. Discussion: The findings of network pharmacology indicate that DFWG primarily alleviates hemorrhoidal symptoms by modulating key inflammatory mediators. This mechanism is further supported by the observed reduction in inflammatory responses in model rats, along with concurrent improvements in pathological manifestations. Conclusion: DFWG exerts protective effects against hemorrhoids in rats, likely by suppressing inflammation.
Introduction: To investigate the differences in efficacy between Hormone Replacement Therapy (HRT) combined with Acupoint Catgut Embedding (ACE) and HRT combined with Sham Acupoint Embedding (SAM) in patients with Diminished Ovarian Reserve (DOR). Methods: A prospective, multicenter, randomized, single blind, controlled trial design was employed. Participants were randomly allocated to either the HRT + ACE group or the HRT + SAM group. Active treatment lasted 3 menstrual cycles, followed by 3 cycles of follow-up. Serum AMH, AFC, Ovarian volume, and serum Testosterone (T), FSH, LH, Estradiol (E2), Prolactin (PRL), and Cytokines (IL-2, IL-6, IL-17) levels were measured pre-treatment and post 3- treatment cycles and at follow-up for 3 treatment cycles. Results: In the treatment group, after 3 treatment cycles, AMH levels increased from baseline (P < 0.05). AFC, ovarian volume, and E2 all increased significantly in both groups compared with baseline (P < 0.05). Basal FSH (bFSH), basal LH (bLH), and IL-2 levels significantly decreased in the two groups (P < 0.05). All indicators in the treatment group at both post-treatment and 3- month follow-up were significantly better than baseline (P < 0.05). The treatment group was better than the control group in terms of AMH, AFC, bFSH, bLH, and IL-2 after 3 months and 6 months follow-up (P < 0.05). No serious adverse events occurred in either group. Discussion: This study demonstrates that HRT combined with ACE can regulate neuroendocrine function, stimulate the reproductive axis, and modulate HPO axis function to improve ovarian function. Although ACE cannot normalize ovarian function, it optimizes follicle development and menstruation. It is a promising low-risk alternative for HRT-intolerant DOR patients. Conclusion: HRT combined with ACE can improve certain ovarian reserve indicators, including increasing AMH levels, AFC, and ovarian volume in DOR patients, with effects superior to those of HRT combined with SAM, suggesting that HRT combined with ACE may enhance ovarian function in DOR patients.
Introduction: This study aimed to investigate the mechanisms by which Danggui Shaoyao powder (DSP) treats metabolic dysfunction-associated fatty liver disease (MAFLD). Methods: The MAFLD rats were established by feeding a high-fat diet (HFD). After 12 weeks of modeling, DSP treatment was administered simultaneously with the modeling for 4 weeks. Parameters assessed included lipid metabolism, liver function, oxidative stress, inflammatory factors, and reactive oxygen species (ROS) levels. Hepatic histopathological changes were also evaluated. Proteins associated with the AMP-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/nuclear factor-κB (NF-κB) signaling pathway were analyzed using western blot. Additionally, the involvement of the AMPK/Nrf2/NF-κB signaling pathway in palmitic acid (PA)-induced HepG2 cells was confirmed using an AMPK activator and AMPK and Nrf2 inhibitors. Results: Findings demonstrated that DSP effectively regulated lipid metabolism, improved liver function, and reduced oxidative injury and inflammation in MAFLD rats. In PA-treated HepG2 cells, DSP significantly decreased lipid accumulation, oxidative stress, and inflammation. Furthermore, DSP increased AMPK phosphorylation, leading to Nrf2 activation and inhibition of NF-κB phosphorylation in both MAFLD rats and HepG2 cells. Conclusion: The results indicate that DSP mitigates oxidative damage and inflammation associated with MAFLD, likely by modulating the AMPK/Nrf2/NF-κB signaling pathway.
Introduction: Alcohol-induced gastric injury is a common clinical problem with few good preventive options. In this study, we examined whether Xiangsha Yangwei Pills (XS), a traditional herbal formula used for digestive issues, could protect against ethanol-induced gastric damage and sought to elucidate how it might work. Methods: We combined network toxicology and network pharmacology to identify the main active compounds in XS and their potential biological targets. Two key components that emerged from this analysis, quercetin and borneol, were then examined using computational methods to predict their antioxidant activity—specifically their electron-acquisition ability and frontier orbital energy gaps. We also conducted in vitro experiments to directly test how well XS scavenges free radicals and chelates metal ions. For in vivo testing, we used a mouse model of alcohol-induced gastric injury and measured parameters such as gastric mucosal lesion severity, ulcer index scores, and total ulcer area to assess whether XS had a protective effect. Results: The network analysis identified nineteen central targets, many of which were antioxidantrelated enzymes such as superoxide dismutase, catalase, and myeloperoxidase. Our computational results suggested that the main active compounds have strong antioxidant potential, which was supported by the in vitro findings, showing that XS effectively scavenges free radicals and chelates metal ions. In the animal model, administering XS beforehand significantly reduced gastric mucosal damage, reducing both ulcer scores and the affected area by more than 80%. At the molecular level, XS treatment increased the activity of the body's own antioxidant enzymes (superoxide dismutase, catalase) and reduced tissue levels of oxidative stress markers such as malondialdehyde and myeloperoxidase. Discussion: The integration of network toxicology and pharmacology revealed 19 overlapping targets, predominantly antioxidant enzymes, suggesting that the gastroprotective effect of XS is mechanistically mediated by mitigating oxidative stress. This computational prediction was empirically validated by the observed increase in superoxide dismutase and catalase activities and the reduction in malondialdehyde and myeloperoxidase levels in vivo. Notably, the high binding affinities of core components such as quercetin and borneol to key targets (e.g., AKT1, TNF, TP53) suggest that XS may exert synergistic, multi-target modulation beyond direct radical scavenging, potentially influencing cell survival and inflammatory pathways. These findings position XS as a promising multi-component strategy for managing alcohol-induced gastric injury, bridging traditional use with modern mechanistic evidence. Conclusion: Overall, our findings show that XS offers strong protection against alcohol-induced gastric injury. The main mechanism appears to be strengthening the antioxidant defense system in the gastric mucosa. These results provide scientific support for using XS as a potential approach to help reduce alcohol- related gastric damage.
Introduction: The therapeutic efficacy of Xianglian Anchang Decoction (XLAC) in Ulcerative Colitis (UC) has been established, but the underlying pharmacological mechanisms have remained poorly elucidated. In this study, we employed an integrated strategy combining network pharmacology, transcriptomic profiling, and in vivo experimentation to reveal these mechanisms, with a particular focus on how XLAC modulates the IL-17/AP-1 signaling axis to attenuate intestinal inflammation and promote mucosal repair. materials and methods: 2.1 Experimental Animals Fifty 8-week-old male C57BL/6J mice were purchased from Beijing Huafukang Bio-Technology Co., Ltd. (Beijing, China; certificate No. SCXK [Jing] 2024-0003). Animals were housed in a specific pathogen-free (SPF) facility under controlled temperature (25 ± 0.5 °C) and humidity (50 ± 5%), with a 12-h light/dark cycle and free access to standard chow and water. All experimental procedures were approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Hebei University of Chinese Medicine (Approval No. IACUCHPHCM-2024078). 2.2 Preparation of XLAC XLAC consists of 15 herbal ingredients. All crude herbs were obtained from the First Affiliated Hospital of Hebei University of Chinese Medicine with batch authentication. Based on human-to-mouse dose conversion (9.1× the clinical equivalent dose), two concentrations were prepared: 1.24 g/mL (low dose, XLAC-L) and 2.48 g/mL (high dose, XLAC-H). Standardized extraction was performed by soaking the herbs in water at a ratio of 1:10 (w/v), followed by two cycles of atmospheric-pressure decoction (boiling for 1 h each), vacuum concentration at 70 °C, and storage at 4 °C. In subsequent experiments, the extracts were administered by oral gavage for 7 consecutive days. Quality control included Latin botanical authentication, monitoring of extraction yield (68 ± 3%), and HPLC fingerprint analysis to ensure batch-to-batch consistency. 2.3 Experimental Design The 50 male C57BL/6J mice were randomly divided into a control group (n = 10) and a model group (n = 40). After one week of acclimatization, the model group received 2% (w/v) dextran sulfate sodium (DSS; molecular weight 36–50 kDa; MP Biomedicals, Santa Ana, CA, USA) in drinking water to induce acute UC. Drug interventions were initiated simultaneously with DSS administration. The model mice were further randomly assigned to four groups: DSS model group, XLAC low-dose group (XLAC-L, 12.4 g/kg), XLAC high-dose group (XLAC-H, 24.8 g/kg), and sulfasalazine group (SASP, 5 g/kg). Control and model groups received equal volumes of distilled water by gavage. Treatments were administered once daily for 7 days. General condition and body weight were monitored daily. After the final administration, mice were fasted for 24 h with free access to water. Animals were then anesthetized by intraperitoneal injection of 3% pentobarbital sodium (45 mg/kg). Colon tissues were collected, snap-frozen in liquid nitrogen and stored at −80 °C, or fixed in 10% (w/v) formalin. Colon length was measured. The overall experimental workflow is shown in Fig. 1A. 2.4 Disease Activity Index (DAI) Body weight loss, stool consistency, and fecal bleeding were recorded daily. Disease activity was evaluated using the disease activity index (DAI), calculated as: DAI = (weight loss score + stool consistency score + fecal blood score)/3。 2.5 Hematoxylin and Eosin (H&E) Staining H&E staining was performed to assess histological changes in colon tissues. Colon samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sectioned at 4 μm thickness, deparaffinized in xylene, rehydrated through graded ethanol, and stained with hematoxylin and eosin. Histopathological scoring of inflammatory infiltration and mucosal damage was performed under a light microscope (Leica Microsystems, Wetzlar, Germany). 2.6 Periodic Acid–Schiff (PAS) Staining Intestinal mucosal barrier integrity was evaluated using a PAS staining kit (Beyotime, China) according to the manufacturer’s instructions. Paraffin sections were deparaffinized, rehydrated, oxidized with periodic acid, stained with Schiff reagent, and counterstained with hematoxylin. Goblet cells producing mucins were observed under a light microscope (Leica Microsystems, Germany). 2.7 Immunofluorescence (IF) Staining Paraffin-embedded colon sections (4 μm) were deparaffinized in xylene (3 × 10 min), rehydrated through graded ethanol (100%, 95%, 85%, 75%; 5 min each), and rinsed with distilled water. Antigen retrieval was performed in 0.01 M citrate–EDTA buffer (P0086, Beyotime, China; 1:50) using microwave heating. Endogenous peroxidase was blocked with 3% H₂O₂ at 37 °C for 10 min. Sections were blocked with 5% BSA (ST023, Beyotime, China) at room temperature for 30 min and incubated overnight at 4 °C with anti–ZO-1 antibody (GB111402, Servicebio, China; 1:500). After washing, sections were incubated with Alexa Fluor 488–conjugated secondary antibody (GB25303, Servicebio, China; 1:500) and Coralite594-conjugated secondary antibody (SA00013-3-100, Proteintech, China; 1:300) for 1 h at room temperature in the dark. Fluorescence images were captured using a Leica inverted fluorescence microscope. 2.8 Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) Total RNA was isolated from colon tissues using the TransZol Up Plus RNA Kit (TransGen Biotech, ER501-01, China). cDNA was synthesized from 1 μg total RNA using the GoScript™ Reverse Transcription System (Promega, A5001, USA). RT-qPCR was performed using MonAmp™ ChemoHS qPCR Mix (Monad, MQ00401S, China). All procedures followed the manufacturers’ instructions. 2.9 Immunohistochemistry (IHC) Paraffin-embedded colon sections (4 μm) were deparaffinized, rehydrated, and subjected to heat-mediated antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min. Sections were blocked with 10% goat serum for 30 min and incubated overnight at 4 °C with primary antibodies against FOS (1:200, ab190289, Abcam, UK) or JUN (1:100, ab31419, Abcam, UK). After incubation with HRP-conjugated secondary antibody (1:800, #7074, Cell Signaling Technology, USA), staining was visualized using DAB substrate, counterstained with hematoxylin, dehydrated, and mounted. Images were acquired under a light microscope. 2.10 Flow Cytometry Colon tissues were processed into single-cell suspensions using RPMI 1640 medium supplemented with 10 Methods: UC was induced in mice by 2% Dextran Sulfate Sodium (DSS) administration in drinking water. The animals were randomly divided into five groups: control (untreated), model (DSS only), XLAC-L (12.4 g/kg/day XLAC extract), XLAC-H (24.8 g/kg/day XLAC extract), and sulfasalazine reference (SASP) (5 g/kg/day sulfasalazine reference drug). Following a 7-day treatment period, various parameters were evaluated, including body mass fluctuations, colon dimensions, microscopic tissue alterations, and intestinal barrier integrity biomarker levels. A computational target prediction analysis was performed to elucidate the pharmacological mechanisms of XLAC, with subsequent transcriptome sequencing verification. Analytical techniques including fluorescence-activated cell sorting, protein immunoblotting, and tissue staining were employed to investigate the modulatory impact of XLAC on UC via the IL-17/AP-1 molecular cascade. Molecular docking and molecular dynamics simulations were performed to evaluate constituent–target binding and complex stability. Results: XLAC significantly prevented DSS-induced body weight loss, preserved colon length, and reduced the severity of colonic histopathological injury. The XLAC intervention suppressed the secretion of inflammatory mediators such as IL-6 and TNF-α, while concurrently promoting the expression of the tight junctional protein ZO-1. Integrated network pharmacology and transcriptomic profiling identified the IL-17 signaling pathway as a potential mechanism underlying the therapeutic effect of XLAC against UC. Further experiments validated that XLAC decreased IL-17A levels and inhibited both the expression and phosphorylation of the AP-1 transcription factors c-Jun and c-Fos. Molecular docking revealed stable binding between the principal bioactive constituents of XLAC (β-sitosterol and quercetin) and the c-Jun/c-Fos protein complex. Molecular dynamics simulations demonstrate the structural stability of the β-sitosterol-c-FOS complex. Discussion: Our findings reveal a critical role of the IL‑17/AP‑1 signaling axis in the pathogenesis of Ulcerative Colitis (UC) and demonstrate that XLAC may exert its therapeutic effects through this pathway. The multi‑target regulatory mechanism of XLAC provides a molecular basis for its superior efficacy and fewer side effects compared with conventional single‑target therapies. These data support the potential of XLAC as a candidate drug for UC treatment. Conclusion: XLAC exerted protective effects against UC by regulating the IL-17/AP-1 signaling pathway, suppressing the inflammatory responses, and promoting intestinal mucosal barrier repair. These findings provide a mechanistic basis that supports the clinical application of XLAC in UC treatment.
