Aristolactam I (AL-I), as a metabolite of AA-I, exhibits toxicity that remains a subject of significant debate. AL-I exhibits detectable distribution in both traditional Chinese medicines and environmental samples. Therefore, understanding the safety of AL-I, particularly its long-term toxicity, is crucial. The detection rate of AL-I were detected in wheat samples obtained from Serbia was 100%, with a maximum concentration of 0.409 ng/g, cumulative exposures could matter. In long-term toxicity testing, no animals in AL-I groups died. At week 24 administration, no pathological changes similar to AA-I group were found in AL-I-H group. However, mild renal tubular injury was observed in discontinuation period. Localized mild renal interstitial collagen deposition was noted in 80% kidneys at weeks 50 discontinuation, but no pathological changes were observed in other tissues and AL-I-L group. Additionally, results of bone marrow micronucleus assay for AL-I-H group were negative, and no SNP mutation changes were observed in kidney/liver/stomach tissues compared with the control. A variety of metabolites were detected in AA-I group, including intermediates with carcinogenic risks. However, the metabolites in AL-I group were only AL-Ia and its glucuronide/sulfated derivatives, and AL-I was not detected in kidneys. We infer the nephrotoxicity of AL-I is much lower than that of AA-I, and no obvious tumorigenic or mutagenic effects were observed, which could be associated with metabolism in the body. Meanwhile, AL-I accumulation in environmental samples from Balkan region does not reach levels associated with nephrotoxicity, suggesting minimal toxicological risk. However, it is essential to control dosage and duration of use when applying medicines containing AL-I in clinical practice.

BACKGROUND:Chronic atrophic gastritis (CAG) is a common chronic digestive disorder associated with the occurrence and development of gastric cancer (GC). Euodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (Fructus evodiae) is a traditional Chinese medicinal material often used to treat gastrointestinal diseases. However, the therapeutical effects of Fructus evodiae in CAG and underlying mechanisms remain unelucidated. PURPOSE:This study aimed to determine the efficacy of the water decoction of fructus evodiae in the treatment of CAG, reveal its mechanism of action, clarify its pharmacological material basis and regulatory pathways in the treatment of CAG, and provide a scientific basis for the clinical treatment of CAG with fructus evodiae. METHOD:First, we established CAG rat model and administered fructus evodiae decoction for treatment, monitoring serum inflammatory factors, hormones, and histopathology. Then, components of fructus evodiae decoction were determined using UHPLC-Q Exactive Orbitrap HRMS. Subsequently, the mechanism of fructus evodiae in the treatment of CAG were explored by proteomics analysis, network pharmacology and molecular docking. Finally, in vivo and in vitro validation was taken by western blot (WB) assays, ELISA, CCK-8, immunofluorescence staining, hematoxylin and eosin (HE) measurement, bile acid (BAs) analysed. RESULTS:Data showed that 25 compounds were identified in fructus evodiae decoction. The proteomics analysis, network pharmacology, and molecular docking results showed evodiamine (EVO) is a major active ingredient and PI3K/AKT/NF-κB pathway and gastrointestinal motility are key regulating pathways in fructus evodiae decoction used to treat CAG. In vivo and in vitro experiments confirmed that Fructus evodiae decoction and EVO significantly reduced the expression levels of IL-1β, IL-6, TNF-α, Gastrin-17 (G-17), somatostatin (SS), caudal type homeobox 2 (CDX2), and mucin 2 (MUC2) protein by inhibited PI3K/AKT/NF-κB signaling. Moreover, fructus evodiae decoction and EVO increased the expression of the gastrointestinal excitatory neurotransmitters ACh and 5-HT, promoted gastric emptying rate and small intestinal propulsion rate, and reduced the level of BAs in the stomach, thereby reducing further damage to the gastric mucosa. CONCLUSION:Fructus evodiae decoction exerts anti-CAG effects primarily by suppressing chronic inflammation and intestinal metaplasia through inhibition of the PI3K/AKT/NF-κB signaling pathway, thereby reducing bile acid accumulation and restoring gastrointestinal function. Importantly, evodiamine was identified as a key bioactive alkaloid of Fructus evodiae and was further validated as a critical mediator of these effects via targeted in vivo and in vitro experiments. By integrating proteomics, network pharmacology, molecular docking, and single-compound mechanistic validation, this study establishes evodiamine-centered regulation of the PI3K/AKT/NF-κB axis as a unifying mechanism underlying the therapeutic action of Fructus evodiae in MNNG-induced chronic atrophic gastritis.

ObjectiveTo establish a mouse model of hypoxic pulmonary hypertension (HPH) through intermittent hypoxia induction, and to develop a comprehensive and reliable evaluation system for the HPH model, thereby providing experimental evidence for mechanistic studies and translational applications related to this disease.MethodsTwenty-four male specific pathogen-free (SPF) BALB/c mice were randomly divided into a control group and a model group, with 12 mice per group. Mice in the model group were placed in a hypobaric oxygen chamber control system to establish the HPH model, whereas mice in the control group received no intervention. After 28 days of modeling, a comprehensive evaluation of the pathophysiological characteristics of both groups was performed using a general condition scoring scale, echocardiography, hemodynamic measurements, blood gas analysis, hematological tests, organ coefficient determination, histopathological examination, and enzyme-linked immunosorbent assay (ELISA). In addition, correlation analyses were conducted among echocardiographic parameters, the contents of endothelin‑1 (ET‑1) and N‑terminal pro‑B‑type natriuretic peptide (NT‑proBNP) in lung tissue, and other measured indices.ResultsWith prolonged modeling duration time, body weight, water intake, and activity level of mice in the model group were significantly decreased compared with the control group (P<0.05). Echocardiography showed that, compared with the control group, the model group exhibited increased right ventricular dimensions (P<0.01), right ventricular anterior wall thickening (P<0.01), and widened main pulmonary artery diameter (P<0.01), whereas the peak systolic velocity across the pulmonary valve, tricuspid annular plane systolic excursion, and peak systolic velocity of the tricuspid annulus were significantly decreased (P<0.001); tricuspid regurgitation was observed in some model animals. Hemodynamic results revealed that right ventricular systolic pressure was elevated in the model group compared with the control group (P<0.001). Blood gas analysis showed that pH, partial pressure of oxygen, oxygen saturation, actual bicarbonate, and total carbon dioxide were all decreased in the model group compared with the control group (P<0.01). Hematological indices demonstrated that lymphocyte counts (P<0.05) and reticulocyte counts (P<0.001) were decreased in the model group compared with the control group. Compared with the control group, the organ coefficients of heart and lung in the model group were significantly increased (P<0.05 and P<0.001). Pathological examination revealed that the right ventricular hypertrophy index was significantly increased in the model group (P<0.001), with varying degrees of damage to the right ventricle, pulmonary artery, and pulmonary vessels; both the pulmonary artery wall thickness percentage and the pulmonary wall area percentage were significantly elevated (P<0.001). ELISA results showed that the levels of ET‑1 and NT‑proBNP in lung tissues were significantly increased in the model group compared with the control group (P<0.05 and P<0.001, respectively). Correlation analysis indicated that some echocardiographic parameters were highly correlated with multiple indices in the development of HPH (P<0.05).ConclusionEchocardiography can accurately assess a series of hemodynamic changes in HPH, including right ventricular structural, functional impairment, and pulmonary hypertension. Laboratory tests not only help verify whether the model has been successfully established, but also provide deeper insights into the pathogenesis of HPH, evaluate the effects of interventions, and offer scientific evidence for clinical outcomes. Pathological examination can further confirm the alterations in pulmonary vascular remodeling and increased right heart load. This multimodal analysis provides a reliable animal model and evaluation paradigm for both basic and translational research on HPH, and is of great significance for exploring disease mechanisms and developing precision therapeutic strategies.

BackgroundFunctional dyspepsia (FD) is a prevalent gastrointestinal disorder with limited long-term efficacy of conventional treatments. Ganhai Weikang Capsules (GHWKCs), a classic traditional Chinese medicine formula, has shown remarkable clinical efficacy in alleviating FD symptoms, but its underlying mechanisms, particularly on the gut microbiota-host metabolism axis, remain unclear.MethodsWe performed chemical profiling of GHWKCs using UPLC-LTQ-Orbitrap-MS and established an FD rat model mimicking the TCM pathogenesis of “spleen deficiency and qi stagnation”. We evaluated gastrointestinal motility, histopathology, serum gastrointestinal hormones, and tissue inflammatory factors. Integrated duodenal metabolomics, cecal 16S rRNA sequencing, and network pharmacology were applied to explore the regulatory mechanisms, with Spearman correlation analysis conducted between differential metabolites and gut microbiota.ResultsA total of 63 chemical metabolites were identified in GHWKCs. GHWKCs significantly improved gastric emptying and intestinal propulsion, normalized gastrointestinal hormone levels, and inhibited pro-inflammatory cytokine secretion in FD rats. Metabolomics revealed 18 differential metabolites mainly involved in fatty acid β-oxidation and primary bile acid biosynthesis. Gut microbiota analysis showed GHWKCs counteracted dysbiosis, particularly increasing Coriobacteriales and Actinobacteriota abundance. Correlation analysis confirmed a positive correlation between Coriobacteriales and glycocholic acid. Network pharmacology identified 31 active metabolites, 213 intersection targets, and key pathways including PI3K-Akt, MAPK, and Toll-like receptor signaling.ConclusionGHWKCs ameliorate FD through multi-target mechanisms involving improved motility, reduced inflammation, and restored microbiota-metabolic homeostasis. The Coriobacteriales-glycocholic acid axis represents a putative core regulatory pathway bridging TCM theory and modern molecular mechanisms, providing a preclinical mechanistic framework for TCM-based therapy of FD.

Background: Compound Qinlan oral liquid (CQOL) is a traditional Chinese medicinal formulation with potent anti-inflammatory effects for treating acute bronchiolitis. However, its specific active components and mechanisms of action remain unclear. Objectives: This study aimed to identify potential active components and targets of CQOL. Methods: UPLC-Q-TOF-MS was used to detect and characterize its major chemical constituents, alongside an investigation of active compounds and metabolic characteristics in rats. Network pharmacology and molecular docking analyses were conducted to elucidate the complex mechanisms underlying CQOL’s effects. Finally, the anti-inflammatory activities of selected monomers and compounds were validated. Results: A total of 118 chemical components were identified in the CQOL freeze-dried powder, while 24 components and 37 metabolites were detected in rat serum. Network pharmacology analysis of the serum components revealed key active components targeting EGFR, STAT3, SRC, HSP90AA1, AKT1, tumor necrosis factor, ESR1, HSP90AB1, MAPK1, and PIK3CA. Molecular docking results indicated that HSPA8, HSP90AA1, and HSP90AB1 are critical targets mediating CQOL’s anti-inflammatory effects. In addition, CQOL demonstrated the most significant anti-inflammatory response compared to any single component tested. Conclusions: These findings highlight CQOL’s potential as a complementary medicine for respiratory infections and provide a theoretical foundation for further development and clinical application.

Background/Objectives: Acute lung injury (ALI) is a severe condition driven largely by inflammation and has limited therapeutic options. Although saikosaponin B1 (SSB1), a primary bioactive saponin from Bupleurum Radix, has demonstrated anti-inflammatory properties, its efficacy against ALI and its corresponding molecular mechanisms remain largely unexplored. This study employed an integrated approach combining network pharmacology, transcriptomics, and metabolomics to decipher the protective mechanisms of SSB1 against ALI. Methods: Potential targets were identified via network pharmacology, and core targets were validated through molecular docking, dynamics simulations, and independent GEO transcriptomic datasets. Experimental validation was performed in an LPS-induced murine ALI model, combining histopathology, ELISA, and integrated transcriptomic and metabolomic analyses. Results: Integrated analyses identified IL1B, TNF, and IL6 as core targets through which SSB1 exerts its anti-ALI effects. These targets were validated by high-affinity binding in simulations, confirmed in independent GEO transcriptomic datasets, and shown to be normalized by SSB1 treatment in vivo. Mechanistically, SSB1 appears to modulate the NOD-like receptor and cGAS-STING signaling pathways and rectify the key metabolic pathways orchestrated by these targets, including glycerophospholipid, arachidonic acid, and linoleic acid metabolism. Conclusions: This study systematically investigates the therapeutic effects of SSB1 against ALI by identifying its potential targets and underlying pathways. These results provide crucial mechanistic insights and robust experimental support, thereby paving the way for the clinical translation of SSB1.

BACKGROUND:Geniposide (GE) has potential efficacy in treating ulcerative colitis (UC). However, its reactivity can be affected by rapid degradation after oral administration. Furthermore, increasing oral doses may lead to hepatotoxicity. Thus, We used enema administration, characterized by smaller dose and higher localized concentration in the lesion, to improve the above situation. PURPOSE:We aimed to confirm that enema administration is a better modality than oral administration for GE against UC and to explore its mechanism. STUDY DESIGN/METHOD:We established UC mouse model, monitoring Disease Activity Index (DAI), inflammatory cytokines levels, and histopathology. Macrogenomics and bile acid (BAs) metabolomics analysed the major intestinal flora and BAs. Simultaneouslly, we conducted quantitative proteomics analysis and screened core proteins and pathway. In vitro validation was taken by qPCR, immunofluorescence and immunoblotting experiments. RESULTS:GE via enema alleviate UC by inhibiting inflammatory factor production through downregulating S100A8/S100A9/NF-κB pathway. Analysis of the intestinal flora and BAs revealed that the enhanced abundance of Lachnospiraceae, which improves the ratio of primary to secondary BAs, and the reduced abundance of Provocaceae, which increases intestinal permeability and promotes inflammation, favored the restoration of the intestinal barrier. In addition, in vitro experiments confirmed that the key BA metabolites (mainly UDCA, DCA, and LCA) stimulated TGR5 signal to inhibit the assembly of the NLRP3 inflammasome and alleviated inflammation. CONCLUSION:We firstly confirmed that GE alleviates UC via the enema route in a better manner than the oral route, through enhancing the intestinal barrier, restoring intestinal flora and BAs homeostasis, and inhibiting inflammatory injury. This study initially revealed that GE can alleviate UC through elevating UDCA, DCA, and LCA levels at the colonic site to activate TGR5 receptor for inhibiting the NLRP3 inflammasome, in addition to downregulating the S100A8/S100A9/-TLR4-NF-κB pathway related inflammatory response directly. The evidences offer a promising strategy and profround meaning for UC treatment.

Aristolochic acid II (AAII), a major nephrotoxic and carcinogenic component of aristolochic acids (AAs), has been less studied compared with its well-characterized analog, aristolochic acid I (AAI). Although AAs are known to induce carcinogenesis via DNA adduct formation, the toxicity mechanisms, environmental prevalence, and long-term health impacts of AAII remain poorly understood. This study aimed to systematically evaluate AAII’s acute and chronic toxicity, carcinogenic mechanisms, and environmental exposure patterns using integrated murine models and phytochemical analyses to clarify its toxicological profile and associated health risks. C57BL/6J mice were used in the following experiments: (1) determination of AAII content in 3 commonly used Aristolochia medicinal materials via liquid chromatography-mass spectrometry/mass spectrometry; (2) acute toxicity testing with single doses of 10, 20, or 40 mg/kg; and (3) chronic exposure with 1 or 10 mg/kg administered every other day for 24 weeks, followed by 21 to 40 weeks of postexposure monitoring. Histopathological examination, whole-exome sequencing, biochemical assays, and micronucleus tests were performed to assess multi-organ damage, tumorigenesis, genomic mutation signatures, and direct clastogenicity. Phytochemical analyses were used to evaluate environmental distribution. (1) A single 40 mg/kg dose of AAII induced dose-dependent renal tubular degeneration without hepatotoxicity; (2) the 10 mg/kg group showed significant mortality (20%), tumor incidence (33.3%, primarily forestomach and bladder transitional cell carcinomas), persistent renal interstitial fibrosis, and subclinical hepatic injury. Chronic exposure to 1 mg/kg still induced 13.3% mortality and 15.5% tumor incidence over a 64-week period; (3) whole-exome sequencing revealed a predominance of C>T mutations and pathway enrichment in chemical carcinogenesis and cytochrome P450-mediated metabolism, indicating reactive metabolite-driven mechanisms distinct from classical AA-DNA adducts; and (4) no histopathological changes were observed in nontarget organs (brain, heart, and testes), and micronucleus assays confirmed the absence of direct clastogenicity. This study highlights the delayed carcinogenic risks of low-dose chronic AAII exposure and emphasizes the need to update regulatory frameworks to ensure the safe use of aristolochiaceae-containing herbal products.

Emodin is a hydroxyanthraquinone compound that is widely distributed and has multiple pharmacological activities, including anti-diarrheal, anti-inflammatory, and liver-protective effects. Research indicates that emodin may be one of the main components responsible for inducing hepatotoxicity. However, studies on the mechanisms of liver injury are relatively limited, particularly those related to bile acids(BAs) metabolism. This study aims to systematically investigate the effects of different dosages of emodin on BAs metabolism, providing a basis for the safe clinical use of traditional Chinese medicine(TCM)containing emodin. First, this study evaluated the safety of repeated administration of different dosages of emodin over a 5-week period, with a particular focus on its impact on the liver. Next, the composition and content of BAs in serum and liver were analyzed. Subsequently, qRT-PCR was used to detect the mRNA expression of nuclear receptors and transporters related to BAs metabolism. The results showed that 1 g·kg~(-1) emodin induced hepatic damage, with bile duct hyperplasia as the primary pathological manifestation. It significantly increased the levels of various BAs in the serum and primary BAs(including taurine-conjugated and free BAs) in the liver. Additionally, it downregulated the mRNA expression of farnesoid X receptor(FXR), retinoid X receptor(RXR), and sodium taurocholate cotransporting polypeptide(NTCP), and upregulated the mRNA expression of cholesterol 7α-hydroxylase(CYP7A1) in the liver. Although 0.01 g·kg~(-1) and 0.03 g·kg~(-1) emodin did not induce obvious liver injury, they significantly increased the level of taurine-conjugated BAs in the liver, suggesting a potential interference with BAs homeostasis. In conclusion, 1 g·kg~(-1) emodin may promote the production of primary BAs in the liver by affecting the FXR-RXR-CYP7A1 pathway, inhibit NTCP expression, and reduce BA reabsorption in the liver, resulting in BA accumulation in the peripheral blood. This disruption of BA homeostasis leads to liver injury. Even doses of emodin close to the clinical dose can also have a certain effect on the homeostasis of BAs. Therefore, when using traditional Chinese medicine or formulas containing emodin in clinical practice, it is necessary to regularly monitor liver function indicators and closely monitor the risk of drug-induced liver injury.

Acute promyelocytic leukemia (APL) is highly malignant and progresses rapidly. In recent years, several studies have shown that oral arsenic, the primary component is realgar, could effectively alleviate APL, with therapeutic effects not inferior to those of intravenous arsenic and a higher safety profile, but currently the active substances and mechanism of realgar's anti-APL effect are unclear, making oral arsenic agents containing realgar lack sufficient scientific basis clinically. The clinical trials on oral arsenic agents containing realgar for the treatment of APL over the past 20 years were reviewed, suggesting it had good therapeutic effect and relatively high safety. Quantitative analysis was conducted on the accumulation of arsenic metabolites in different tissues during the longest 90 day period after realgar administration in rats, and it was found that dimethylarsenic (DMA) was the most predominant form of arsenic metabolites in the body. The DMA concentration slightly increased and remained stable with prolonged administration time, even if discontinuation, the DMA concentration still remained at a certain level. Distribution and accumulation of DMA were significantly higher in blood than in organs. Futhermore, DMA significantly prolonged the survival time of APL mice, induced cell apoptosis, inhibited NB4 cell proliferation, promoted the differentiation of leukemia cells, and downregulated the expression of PML-RARα fusion protein and wild-type RARα protein. The study concluded that DMA could be the main active substance of realgar and is responsible for its significant anti-APL effects, suggesting realgar has good efficacy for blood-related diseases and have low hepatorenal toxicity.

Background:Psoralea corylifolia L.(Buguzhi,BGZ),known for its efficacy in supporting pregnancy and preventing miscarriage,has been used in China for over 1000 years.Recently,BGZ has been identified as a potential cause of drug-induced liver injury.However,its safety during pregnancy remains unclear,which significantly hinders its routine clinical application. Objective:To investigate the effects of BGZ administration during pregnancy on the liver of mouse mothers and their weaned 21-day-old offspring. Methods:Mice were orally administered BGZ at doses of 2.5 and 10 g/kg during pregnancy,with BGZ withdrawal during the lactation period.Liver histopathology(hematoxylin-eosin staining),biochemical analysis,and evaluation of liver bile acid metabolism were per-formed after the lactation period. Results:BGZ administration at doses of 2.5 and 10 g/kg during pregnancy,followed by withdrawal during the lactation period,caused mild liver damage in both mothers and their 21-day-old offspring.Serum total bile acid(TBA)levels were elevated compared with those in the control group.Additionally,changes were observed in the levels and proportions of various bile acids(BAs)in the liver,suggesting mild effects on BA metabolism. Conclusion:BGZ administration during pregnancy caused mild liver damage and increased serum TBA levels in both mouse mothers and their 21-day-old offspring.This phenomenon may be associated with imbalanced BA metabolism in the liver.Based on the present study and the limited toxicological research on BGZ,pregnant women should avoid prolonged use of BGZ.If BGZ is administered dur-ing pregnancy,serum TBA levels should be monitored,and if elevated,BGZ should be discontinued.

Background: Acute lung injury (ALI) often leads to serious respiratory diseases with high incidence rates and mortality. For centuries, Xiebai San (XBS) has been a classical traditional Chinese medicine (TCM) about respiratory illness such as pneumonia in children. However, the related mechanism of XBS against ALI remains indistinct. Purpose: To reveal specific targets of XBS in lipopolysaccharide (LPS)-induced ALI mice using integrated pharmacology. Study Design: The integrated method was to expound mechanism and targets of XBS inhibited ALI. Methods: The primary components in XBS were identified by ultra high performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-QTOF-MS). The potential drug targets were established using network pharmacology. The anti-ALI effect of XBS was evaluated in mice. Additionally, therapeutic targets were screened by integrating metabolome and transcriptome and verified in lung tissue. Results: In total, 163 chemical components were identified in XBS, and a network of "3 drugs-18 components-86 targets" for XBS against ALI was constructed. In ALI mice, XBS alleviated lung inflammation by decreasing permeation and expression of neutrophils, tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta ) in bronchoalveolar lavage fluid (BALF), serum, and lung tissue. Next, the transcriptome of lung tissue was analyzed and enriched, indicating the importance of mitogen-activated protein kinase (MAPK), Janus kinase-signal transducer and activator of transcription (JAK-STAT), and others, which was consistent with network pharmacology prediction. Also, western blotting and immunohistochemistry results showed that XBS was against ALI mainly by inhibiting extracellular signal regulated kinase (ERK) and signal transducer and activator of transcription 3 (Stat3) phosphorylation. In addition, the metabolome of lung tissue revealed that XBS mainly regulated pathways involved in arachidonic acid, glycerophospholipid, and tryptophan metabolisms. The expression levels of leukotriene, phosphatidylcholine, kynurenine, and others were also verified.

• Curdione, neocurdione, and curcumol are reversible and competitive inhibitors of CYP2B6 . • Curdione, neocurdione, and curcumol contribute to the inhibition of zedoary turmeric oil on CYP2B6. • Curdione and neocurdione can be hydroxylated into hydroxycurdione and hydroxyneocurdione by CPY2B6, respectively .

Ethnopharmacological relevance: Xiebai San (XBS), a classic Chinese prescription, has been used for the clinical treatment of pneumonia-related diseases for thousands of years. However, the anti-pneumonia pharmacodynamic material basis of XBS and its underlying mechanisms remain unclear. Aim of the study: This study aimed to comprehensively investigate and verify the anti-pneumonia pharmacodynamic material basis and mechanisms of XBS. Materials and methods: This study explored the anti-pneumonia activity and key pneumonia targets of XBS in lipopolysaccharide (LPS)-induced zebrafish and RAW264.7 cells in vivo and in vitro through transcriptomics, western blotting, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chemical fingerprint of XBS was established using high-performance liquid chromatography, and the similarities and areas of characteristic peaks of 15 batches of XBS were analyzed. Based on the spectrum-efficacy relationship, the potential anti-inflammatory components were screened according to their peak areas and efficacy using principal component analysis (PCA), bivariate correlation, and partial least squares regression analysis. Active components that bind to core targets were further screened based on surface plasmon resonance (SPR). The binding mode of proteins and components was simulated via molecular docking, which enabled the identification of the primary active components of XBS, thereby elucidating its anti-pneumonia properties. Finally, the anti-inflammatory activities of these components were verified in vitro. Results: XBS decreased neutrophil aggregation in zebrafish and nitric oxide (NO) secretion in RAW264.7 cells as well as suppressed the release of downstream inflammatory cytokines such as iNOS, TNF-alpha, IL-1 beta, IL-18, and CXCL10 related to TNF and JAK-STAT signaling pathways. The phosphorylation of I kappa B alpha, Akt, and Stat3 was alleviated after XBS in cells. The fingerprint similarities of 15 batches of XBS ranged from 0.381 to 0.994, with a large difference. A total of 15 characteristic peaks were identified, and the relative standard deviation of their peak areas ranged from 24.1% to 70.7%. The results of in vitro anti-inflammatory activities of 15 batches of XBS showed that all samples inhibited the expression levels of NO and nine inflammatory markers. The antiinflammatory index of 15 batches of XBS was determined to be 0.69-0.96 based on transformation of the anti-inflammatory rate and composite index method via PCA. The spectrum-efficacy relationship model of 15 characteristic peak areas and the anti-inflammatory index showed that 7 main potential active components were related to the anti-inflammatory activity of XBS. Moreover, four components (mulberroside A, isoquercitrin, liquiritigenin, and glycyrrhizic acid) screened based on SPR had different affinities toward TNFR1, Akt1, and Stat3 proteins, and the binding modes were elucidated via molecular docking. Finally, in LPS-induced RAW264.7 cells, all four active components (at a concentration of 60 mu M) significantly inhibited the expression levels of NO and inflammatory markers. Conclusions: Based on the comprehensive strategy of spectrum-efficacy relationship and SPR, mulberroside A, isoquercitrin, liquiritigenin, and glycyrrhizic acid were identified as the primary pharmacodynamic active components involved in the anti-pneumonia activity of XBS and were found to intervene in TNF and JAK-STAT signaling pathways.

Background: The pathogenesis of metabolic syndrome was strongly associated with compromised metabolism homeostasis and gut microbiota imbalance. NAFLD is a progressive metabolic liver disease for which effective interventions are lacking. Bile acids exhibited appreciable metabolic regulatory effects and selective antimicrobial activity. Aim of the study: This study was designed to investigate the effect of BBBP, which mainly contained bile acids, on NAFLD from the perspectives of gut microbiota and metabolomics. Materials and methods: The present study was initiated on the anti-NAFLD effect of BBBP in HFD-fed mice. The efficacy of BBBP was evaluated by mice phenotypes, liver histopathological analysis and serum lipid and glucose levels. The activation of bile acid receptors such as Nr1h4, Nr1i2 and S1pr2 were detected by qRT-PCR analysis. Subsequently, untargeted metabolomics coupled with microbiomics was used to explore the mechanism of BBBP against NAFLD. Human L02 hepatocytes induced by OA and PA were used to investigate the effect of GABA on reducing lipid accumulation in vitro. Results: BBBP significantly and dose-dependently alleviated the obese phenotype, lipid accumulation and liver injury in mice subjected to 18 weeks HFD diet. Untargeted metabolomics and microbiomics analysis revealed that BBBP could alleviate the disturbance of lipid and amino acid metabolism and the imbalance of gut microbiota. Furthermore, BBBP oral gavage activated liver bile acid receptors, as indicated by elevated mRNA levels of Nr1h4, Nr1i2 and S1pr2. Surprisingly, we determined that BBBP, which mainly contained bile acids that possessed antimicrobial activity, could promote the growth of Lactobacillus. Correlation analysis showed a remarkable correlation between Lactobacillus and endogenous metabolites such as valine, serine, glutamine, et al. Among them, GABA which could be produced by Lactobacillus significantly reduced the lipid accumulation in L02 cells. Conclusions: The role of BBBP in regulating lipid metabolism might be achieved by activating bile acid receptors, or partially by promoting the levels of Lactobacillus and its metabolites such as GABA. Our study provided evidence that BBBP could be a novel therapeutic candidate for the treatment of NAFLD.

Ethnopharmacological relevance: Cholestasis is the main manifestation of cholestatic liver disease, which has a risk of progression to end-stage liver disease. Gardeniae Fructus is the dried fruit of Gardeniae jasminoides Ellis, a plant of the Rubiaceae family. Gardeniae Fructus has shown therapeutic potential in cholestasis-related liver diseases and it is generally believed that Gardeniae Fructus ameliorates cholestasis, which could be related to its influence on bile acids (BAs) metabolism. However, the specific targets of Gardeniae Fructus and its impact on enterohepatic circulation of BAs have not yet been fully elucidated.Aim of the study: To systematically elucidate the mechanism by which Gardenia extract (GE, total iridoids in Gardeniae Fructus, which contains the predominant and characteristic phytoconstituents of Gardeniae Fructus) ameliorates alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury. Materials and methods: Sprague-Dawley rats were orally administered water, obeticholic acid (OCA, 2 mg/kg), or GE (21 and 42 mg/kg) once daily for five days. On the third day, the model was established by administration of a single dose of ANIT (40 mg/kg) by oral gavage. Biochemical and pathological analyses, BA metabolomics, transcriptomics, and qRT-PCR were performed. Results: The profile of BAs in serum and liver confirmed that GE attenuated ANIT-induced acute cholestasis by affecting BA metabolism in a dose-dependent manner. Liver transcriptomic analysis indicated that GE mainly influenced the primary bile acid (PBA) biosynthesis and bile secretion pathways. GE mainly affected PBA biosynthesis in liver by upregulating Cyp8b1 gene expression, thereby significantly reducing the level of total bile acids (TBA). GE mainly promoted PBA excretion from liver into duodenum by upregulating Fxr and Oatp1 gene expression, thereby increasing the excretion of PBA in feces, and inhibiting PBA in liver entering the blood by alternative routes to reduce TBA levels in serum and urine and improve the enterohepatic circulation of BAs.Conclusion: GE attenuated ANIT-induced hepatotoxicity and cholestasis in rats by upregulating Cyp8b1 expression to inhibit BA synthesis in the liver, while also promoting BA excretion via the intestinal-fecal route, and improving enterohepatic circulation of BAs.

Aristolochic acid (AA)-IIIa is an AA analog present in Aristolochiaceae plants. To evaluate the chronic toxicity of AA-IIIa, mice were intragastrically administered with media control, 1 mg/kg AA-IIIa, and 10 mg/kg AA-IIIa, and designated as the control (CTL), AA-IIIa low dose (AA-IIIa-L), and AA-IIIa high dose (AA-IIIa-H) groups, respectively. AA-IIIa was administered three times a week, every other day, for 24 weeks (24-week time point). Thereafter, some mice were sacrificed immediately, while others were sacrificed 29 or 50 weeks after AA-IIIa withdrawal (53- or 74-week time point). Serum and organs were collected for biochemical and pathological analyses, respectively. Whole-genome sequencing was performed on the kidney, liver, and stomach tissues of AAIIIa-treated mice for single-nucleotide polymorphism (SNP) detection. AA-IIIa-H mice died at 66 weeks, and the remaining mice showed moribund conditions at the 69 weeks. AA-IIIa induced minor kidney tubule injury, fibroblast hyperplasia, and forestomach carcinoma in mice. Bladder, intestine, liver, heart, spleen, lung, and testis tissues were not pathologically altered by AA-IIIa. In addition, AA-IIIa increased the C:G > A:T mutation in the kidney; however, no SNP mutation changes were observed in the liver and forestomach tissues of AA-IIIa-H mice at the 24-week time point compared with control mice. Therefore, we suspect that AA-IIIa is potentially mutagenic for mice after overdose and long-term administration. On the other hand, the forestomach is a unique organ in mice, but it does not exist in humans; thus, we hypothesize that the stomach toxicity induced by AA-IIIa is not a suitable reference for toxicological evaluation in humans. We recommend that Aristolochiaceae plants containing AA-IIIa should be properly supervised, and overdosing and long-term administration of drugs containing AA-IIIa should be avoided.

ETHNOPHARMACOLOGICAL RELEVANCE:Kelisha capsules (KLS) are often used to treat acute diarrhoea, bacillary dysentery, heat stroke, and other diseases. One of its components, Asarum, contains aristolochic acid I which is both nephrotoxic and carcinogenic. However, the aristolochic acid (AA) content in KLS and its toxicity remain unclear. AIM OF THE STUDY:The aims of this study were to quantitatively determine the contents of five aristolochic acid analogues (AAAs) in Asarum and KLS, and systematically evaluate the in vivo toxicity of KLS in rats. MATERIALS AND METHODS:Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the content of the five AAAs in Asarum and KLS. Sprague-Dawley rats were administered KLS at 0, 0.75, 1.5, and 3.0 g/kg respectively, and then sacrificed after 4 weeks of administration or after an additional 2 weeks of recovery. The endpoints assessed included body weight measurements, serum biochemistry and haematology indices, and clinical and histopathological observations. RESULTS:The AAAs content in Asarum sieboldii Miq. (HB-ESBJ) were much lower than those of the other Asarums. The contents of AA I, AA IVa, and aristolactam I in KLS were in the ranges of 0.03-0.06 μg/g, 1.89-2.16 μg/g, and 0.55-1.60 μg/g, respectively, whereas AA II and AA IIIa were not detected. None of the rats showed symptoms of toxic reactions and KLS was well tolerated throughout the study. Compared to the control group, the activated partial thromboplastin time values of rats in the 1.5 and 3.0 g/kg groups significantly reduced after administration (P < 0.05). In addition, the serum triglycerides of male rats in the 0.75 and 1.5 g/kg groups after administration, and the 0.75, 1.5, 3.0 g/kg groups after recovery were significantly decreased (P < 0.01 or P < 0.001). No significant drug-related toxicological changes were observed in other serum biochemical indices, haematology, or histopathology. CONCLUSIONS:The AA I content in KLS met the limit requirements (<0.001%) of the Chinese Pharmacopoeia. Therefore, it is safe to use KLS in the short-term. However, for safety considerations, attention should be paid to the effects of long-term KLS administration on coagulation function and triglyceride metabolism.

Due to soared obesity population worldwide, hepatosteatosis is becoming a major risk factor for hepatocellular carcinoma (HCC). Undertaken molecular events during the progression of steatosis to liver cancer are thus under intensive investigation. In this study, we demonstrated that high-fat diet potentiated mouse liver AKT2. Hepatic AKT2 hyperactivation through gain-of-function mutation of Akt2 (Akt2E17K) caused spontaneous hepatosteatosis, injury, inflammation, fibrosis, and eventually HCC in mice. AKT2 activation also exacerbated lipopolysaccharide and D-galactosamine hydrochloride-induced injury/inflammation and N-Nitrosodiethylamine (DEN)-induced HCC. A positive correlation between AKT2 activity and SCD1 expression was observed in human HCC samples. Activated AKT2 enhanced the production of monounsaturated fatty acid which was dependent on SREBP1 upregulation of SCD1. Blockage of active SREBP1 and ablation of SCD1 reduced steatosis, inflammation, and tumor burden in DEN-treated Akt2E17K mice. Therefore, AKT2 activation is crucial for the development of steatosis-associated HCC which can be treated with blockage of AKT2-SREBP1-SCD1 signaling cascade.