Tripterygium glycosides extract (TGE), the primary active component of tripterygium glycosides tablets, is widely used for immune-related disorders but raises significant clinical concerns regarding cholestatic drug-induced liver injury. As conventional models fail to fully recapitulate the complex pathogenesis of traditional Chinese medicine toxicity, this study aimed to elucidate the mechanisms of TGE-induced cholestatic injury using a biomimetic microfluidic liver-on-a-chip platform. The chip integrated rat precision-cut liver slices (PCLSs) and human endothelial cells (EA.hy926) to simulate the hepatic sinusoidal microenvironment. Following TGE exposure (15-135 μg/mL for 12 and 24 h), vascular barrier integrity was maintained, while liver injury markers (ALT, AST, TBA, DBIL) significantly increased in a dose- and time-dependent manner, accompanied by progressive histopathological deterioration in PCLSs. Mechanistically, TGE triggered severe oxidative stress (decreased SOD/GSH/GSH-Px and increased MDA) and upregulated pro-inflammatory cytokines (IL-4 and IL-1β). Consequently, the expression of the bile acid receptor FXR and transporters (BSEP and MRP2) was significantly downregulated. In conclusion, TGE induces cholestatic liver injury via a sequential pathway: oxidative stress initiates an immune-inflammatory response, which subsequently suppresses the FXR/BSEP/MRP2 axis. Future studies should focus on developing fully humanized liver-on-a-chip systems to further validate these mechanisms and improve clinical translational significance.

ETHNOPHARMACOLOGICAL RELEVANCE:The Kai-Xin-San (KXS), as an ancient classic prescription, has been used for the treatment of amnesia for thousands of years. Modern clinical and non-clinical pharmacological studies have found that it has significant therapeutic effects on dementia and depression, but there are relatively few studies on its safety. AIM OF THE STUDY:Subacute and chronic toxicity studies were conducted to investigate the symptoms, severity, target organs, development and recovery of toxic reactions, as well as the toxic dose. These studies provide technical data for ensuring the safety of KXS. MATERIALS AND METHODS:In the sub-acute toxicity study, rats were orally administered KXS at doses of 0.80, 1.61, 3.22, and 6.43 g/kg body weight for a duration of 4 weeks. In the chronic toxicity study, rats were orally administered KXS at doses of 0.27, 0.81, and 2.43 g/kg body weight for a duration of 26 weeks, and a withdrawal study was conducted for a period of 4 weeks after the treatment.The rats were observed daily for clinical signs and mortality. Changes in body weight, food consumption, and water consumption were periodically monitored. Additionally, urinalysis results, hematological and biochemical parameters, relative organ weights, and pathology were monitored at specific observation time points. RESULTS:In the sub-acute toxicity study, necropsy of dead and moribund rats revealed evident distension and swelling of the gastrointestinal tract, as well as thinning of the intestinal wall. The main adverse reactions observed included flatulence, piloerection, abnormal breathing sounds, and emaciation. Doses of 1.61 g/kg and below did not cause animal death. The gastrointestinal system is the main target organ of toxicity. In the chronic toxicity study, the no-observed-adverse-effect-level (NOAEL) of KXS was 0.27 g/kg, and its toxic effects were primarily concentrated in the gastrointestinal system. This led to secondary pathological changes in the immune system, hematopoietic system, and heart, suggesting that relevant indicators should be monitored when large doses are used clinically for an extended period of time. CONCLUSIONS:During the rodent toxicity evaluation, severe gastrointestinal damage was observed when KXS, powdered with crude drugs, was administered. The NOAEL for rats was found to be 0.27 g/kg/day.

Ethnopharmacological relevance: Tripterygium glycosides (TG), which are extracted from the traditional Chinese medicine, Tripterygium wilfordii Hook F. (TwHF), has promising applications in the treatment of renal diseases; however, since its active components exerts bidirectional kidney toxicity, its clinical application is severely restricted.Aim of the study:: Recent investigations have demonstrated definite toxicity-reducing effects from glycyrrhizic acid glycosides (GA) when combined with TG; however, the mechanism remains unclear. To our knowledge, this is the first study to investigate the specific molecular mechanism by which GA alleviates TG-induced renal toxicity from the perspective of tight junctions.Materials and methods: Dynamic analyses, which investigated the changes in kidney toxicity biomarkers for different combinations and concentrations of TG and GA, were conducted for three weeks on SD rats and renal tissue structural changes were examined after three weeks of administration. Additionally, the transcription and translation levels of the relevant tight junctions and RhoA/ROCK1/MLC signalling proteins were analysed in HK -2 cells. Results: Our study showed that TG can cause transient tubulotoxicity at certain doses, and that the combined application of GA and TG can repair tight junction structures by regulating the key factors in the RhoA/ROCK1/ MLC signalling pathway, thus reducing TG-induced nephrotoxicity.Conclusions: Overall, this study provides a new strategy to reduce TG-induced toxicity by protecting renal tight junctions.

Aim: Tripterygium glycosides (TG) extracted from the plant Tripterygium wilfordii Hook F has been used to treat chronic kidney diseases for many years. However, hepatotoxicity limits its clinical application. Glycyrrhizic acid glycosides (GA) can reduce TG hepatotoxicity, however, further investigation into the underlying molecular mechanisms by which GA attenuates TG-induced hepatotoxicity is required. Methods: Sprague‒Dawley rats were randomly divided into the control group, the TG groups (TG189 mg/kg group, TG472.5 mg/kg group), and the TG + GA groups (TG189 mg/kg + GA20.25 mg/kg group, TG472.5 mg/kg + GA20.25 mg/kg group). After 21 consecutive days of intragastric administration, structural and molecular changes in hepatocytes were detected. Results: After 21 days of TG treatment, the serum level of the total bilirubin, triglyceride, total cholesterol, and low-density lipoprotein cholesterol increased in the TG189 mg/kg and TG472.5 mg/kg groups when compared to the control group. High-density lipoprotein cholesterol levels were reduced in both TG groups. The ultrastructure of hepatocytes and the structural integrity of the liver were compromised. In addition, the relevant molecular level of the peroxisome proliferators-activated receptor α (PPARα) and acyl-CoA synthetase long-chain family members (ACSLs) pathway was modulated. With the addition of 20.25 mg/kg GA, the serum biochemical indexes and liver tissue structure ultrastructure of hepatocytes were improved, and the PPARα-ACSLs pathway was corrected. Conclusion: The combined application of GA and TG improved abnormal lipid metabolism, repaired liver structure, reduced lipid deposition in hepatocytes, and reduced TG-induced hepatotoxicity.

目的 明确雷公藤多苷(tripterygium glycosides,TG)肝毒性的剂量-时间-效应关系,基于细胞色素P450酶的调控研究TG致大鼠肝损伤的毒性机制,探讨复方甘草酸苷(compound glycyrrhizin,CG)配伍TG的减毒机制,为TG临床安全应用提供理论依据.方法 雄性SD大鼠30只,按体重随机分为对照组、TG 189.0 mg·kg-1组、TG 472.5 mg·kg-1组、TG 189.0 mg·kg-1和TG 472.5 mg·kg-1剂量分别与CG 20.25 mg·kg-1剂量联合用药组,共5组,每组6只.每日给药1次,连续3周.分别于给药后1、2、3周,测定血清中总蛋白(TP)、白蛋白(ALB)、谷草转氨酶(AST)、谷丙转氨酶(ALT)含量;末次给药结束,计算肝系数,行组织病理学检查,对CYP2B1和CYP3A1基因的转录水平进行检测.结果 给药1周后,TG 189.0、472.5 mg·kg-1组血清中ALB水平均降低(P<0.05),TG 472.5 mg·kg-1组ALT升高(P<0.01);至给药3周,除上述指标的改变外,TG189.0、472.5 mg·kg-1组进一步引起大鼠血清TP含量的降低(P<0.05);与CG联合用药3周后,TG472.5 mg·kg-1剂量联合用药组与同等剂量的TG单独用药组比较,可降低ALT含量(P<0.01),升高ALB、TP含量(P<0.05).至给药3周,TG 189.0 mg·kg-1组的大鼠肝细胞轻微肿胀,汇管区少量淋巴细胞浸润;TG 472.5 mg·kg-1组的大鼠肝重量和肝系数较对照组增加(P<0.05,P<0.01),肝被膜下见片状坏死灶伴出血,肝细胞空泡变性、肿胀,汇管区淋巴细胞浸润.与CG联合用药后,大鼠肝脏的组织结构得到修复.189.0、472.5 mg·kg-1剂量的TG均可导致大鼠肝组织中CYP2B1和CYP3A1 mRNA水平的升高(P<0.05,P<0.01);TG与CG联合用药后,CYP2B1 mRNA水平下调.结论 连续3周给予大鼠189.0、472.5 mg·kg-1剂量的TG,可对大鼠造成不同程度的肝损伤.其中,TG 472.5 mg·kg-1组肝损伤更为严重,给药3周较给药1周时肝毒性更显著,具有剂量-时间-效应关系.大鼠肝细胞内细胞色素P450酶CYP2B1和CYP3A1 mRNA过表达为其毒性机制之一;CG可通过下调CYP2B1 mRNA水平,减轻肝毒性.

Background The phyllosphere mycobiome plays a crucial role in plant fitness and ecosystem functions. The complex microbial ecological networks (MEN) formed by these fungi remain poorly understood, particularly with regard to their organization strategy and their contributions to plant secondary metabolites such as saponin. Results In this study, we constructed six MENs from leaf epiphytic and endophytic mycobiomes of three Panax species distributed in the northeast and southwest ends of mainland China. Hub nodes were absent in these MENs, which were significantly more complex, robust, and less efficient compared to random graphs ( P < 0.05), indicating a hub-independent high-robustness strategy to maintain structural homeostasis. The important roles of specific MEN modules in shaping leaf saponin profiles of each Panax species were proved by multiple machine learning algorithms. Positive regulation modules (PRMs) of total saponin content were further identified, which exhibited more deterministic ecological assembly and comprised of highly connected nodes as well as higher proportion of plant-associated fungal guilds compared to other network members, indicating their tight links with host plant. The significant and direct effects ( P < 0.05) of PRMs on total saponin accumulation were validated by well-fitted structural equation models (χ 2 < 0.3, P > 0.5). Taxonomic analysis revealed that Pleosporales and Chaetothyriales were significantly overrepresented by positive regulation taxa (PRT) of total saponin content (FDR < 0.05). Across PRT identified in three Panax species, Epicoccum and Coniothyrium were conservatively present, while species-specific taxa such as Agaricales were also found, indicating the conservatism and specificity of plant-fungi interactions associated with leaf saponin accumulation in Panax genus. Conclusions These findings provide a foundation for understanding mechanisms maintaining the steady state of phyllosphere mycobiome in healthy plant, and offer clues for engineering phyllosphere mycobiome to improve the accumulation of bioactive secondary metabolites on the basis of network modules.

Background: Skeletal muscles are organs with high energy requirements, especially during vigorous exercise. Adequate mitochondrial function is essential to meet the high energy needs of skeletal muscle cells. Recent studies have reported that red ginseng can significantly improve chronic fatigue; however, the specific mechanism of action is still not clear. Methods: A chronic fatigue syndrome mouse model was developed using C57BL/6J mice through long-term compound stimulation of stress factors. Following this, the animals were orally administered 200, 400, or 600 mg/kg red ginseng extracts for 28 days. Skeletal muscle lactate acid, serum lactate dehydrogenase, urea concentrations, ATP level, mitochondrial membrane potential, activities of Na+-K+-ATPase and cytochrome c oxidase were determined using assay kits or an automatic biochemical analyser detection system. Skeletal muscle mitochondria morphology was observed using electron microscopy and the expression of p-AMPK, PGC-1α, ACO2 and complex I in skeletal muscle protein was determined by western blotting. Results: Oral administration of 400 or 600 mg/kg red ginseng extract in mice with chronic fatigue reduced lactic acid, lactate dehydrogenase and urea, rescued the density and morphology of skeletal muscle mitochondria, increased the activities of Na+-K+-ATPase and cytochrome c oxidase, and activated the AMPK/PGC-1α cascade pathway, resulting in improved skeletal muscle mitochondrial function by restoring ATP level, mitochondrial membrane potential, complex I and mitochondrial biogenesis. Conclusion: The anti-fatigue effects of red ginseng are partly related to its potent mitochondrial improving activity, including decreasing mitochondrial swelling and mitochondrial membrane permeability, increasing mitochondrial biogenesis, thus ameliorating mitochondrial dysfunction.

Danlu tongdu tablets (DLTD) is a listed Chinese patent medicine collected in the Pharmacopoeia of the People’s Republic of China (version 2020). This prescription has been applied in clinics in China for lumbar spinal stenosis and lumbosacral disc herniations. The wide application of Danlu tongdu in therapy has raised some clinical adverse reactions, such as significant elevation of alanine transaminase (ALT) and aspartate transaminase (AST) in individual patients after use. The present study aimed to investigate the safety of Danlu tongdu and analyze its adverse effects on the liver. The maximum feasible dose (MFD) was used to carry out the acute toxicity tests. Mortality, adverse effects, body weight and food consumption were recorded for up to 14 days post treatment. In the 6-month chronic toxicity test, sprague-dawley rats were randomly divided into four groups according body weight, the experimental groups were administrated to rats at the concentrations of 1.67, 3.34 and 6.67 g/kg/day, whereas the control group was received the ultrapure water (vehicle) only, 10 ml/kg, once a day. The animal’s body weight, food consumption was monitored weekly. In addition, their hematological and biochemical parameters, body and organ weights and histopathology, were all measured at specific observation time points. Additionally, we further explored the adverse effects mechanism of Danlu tongdu on the liver through transcriptome analysis. No deaths or substance-relative toxicity were observed in the acute toxicity study or the 6-month chronic toxicity study with doses of 1.67 g/kg and 3.34 g/kg, respectively. We found that mild hypertrophy and hyperplasia of hepatic interlobular bile ducts were detected in some rats with doses of 6.67 g/kg after repeated oral administration of Danlu tongdu for 13 and 26 weeks, but the above changes in liver were reversible. The results of transcriptome sequencing showed that Danlu tongdu had a significant effect on cytochrome P450 enzymes in rat liver, especially cytochrome P450 1 (CYP1) subtype. Therefore, the toxic target organ of Danlu tongdu is the liver and the mechanism of mild liver injury is closely related to the up-regulation of cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1A2 (CYP1A2) expression.