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Background: Triptolide (TP), a principal active substance from Tripterygium wilfordii, exhibits various pharmacological effects. However, its potential hepatotoxicity has always been a significant concern in clinical applications., Purpose: This research aimed to explore the involvement of ferroptosis in TP-mediated hepatic injury and the underlying mechanisms., Methods: In this study, in vitro and in vivo experiments were involved. Hepatocyte damage caused by TP was evaluated using MTT assays, liver enzyme measurement and H&E staining technique. Ferroptosis was assessed by measuring iron level, lipid peroxide, glutathione (GSH), mitochondrial morphology and the key protein/mRNA expression implicated in ferroptosis. To verify the contribution of ferroptosis to TP-induced liver damage, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) and a plasmid for overexpressing glutathione peroxidase 4 (GPX4) were employed. Subsequently, nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice and Nrf2 overexpression plasmid were utilized to investigate the underlying mechanisms. Nontargeted lipidomics was used to analyze lipid metabolism in mouse liver. Moreover, the cellular thermal shift assay (CETSA), cycloheximide (CHX) and MG132 treatments, and immunoprecipitation (IP) assays were applied to validate the binding of TP to Nrf2 and their interactions., Results: TP triggered ferroptosis in hepatocytes, as indicated by iron accumulation and lipid peroxidation. Ferroptosis was responsible for TP-induced hepatic injury. During the process of TP-induced liver damage, the Nrf2 signaling pathway was significantly suppressed. Notably, the deletion of Nrf2 in mice aggravated the extent of liver injury and ferroptosis associated with TP, whereas enhancing Nrf2 expression in cells significantly reduced TP-induced ferroptosis. Additionally, dysregulation of lipid metabolism was associated with TP-induced liver injury. TP may directly bind to Nrf2 and enhance its degradation through the ubiquitin-proteasome pathway, thereby inhibiting or reducing Nrf2 expression., Conclusion: In summary, the suppression of Nrf2 by TP facilitated the occurrence of ferroptosis, resulting in liver damage., (© 2024. The Author(s).)

Triptolide (TP), an active component isolated from the traditional Chinese herb Tripterygium wilfordii Hook F (TWHF), shows great promise for treating inflammation-related diseases. However, its potential nephrotoxic effects remain concerning. The mechanism underlying TP-induced nephrotoxicity is inadequately elucidated, particularly at single-cell resolution. Hence, single-cell RNA sequencing (scRNA-seq) of kidney tissues from control and TP-treated mice is performed to generate a thorough description of the renal cell atlas upon TP treatment. Heterogeneous responses of nephron epithelial cells are observed after TP exposure, attributing differential susceptibility of cell subtypes to excessive reactive oxygen species and increased inflammatory responses. Moreover, TP disrupts vascular function by activating endothelial cell immunity and damaging fibroblasts. Severe immune cell damage and the activation of pro-inflammatory Macro_C1 cells are also observed with TP treatment. Additionally, ligand-receptor crosstalk analysis reveals that the SPP1 (osteopontin) signaling pathway targeting Macro_C1 cells is triggered by TP treatment, which may promote the infiltration of Macro_C1 cells to exacerbate renal toxicity. Overall, this study provides comprehensive information on the transcriptomic profiles and cellular composition of TP-associated nephrotoxicity at single-cell resolution, which can strengthen the understanding of the pathogenesis of TP-induced nephrotoxicity and provide valuable clues for the discovery of new therapeutic targets to ameliorate TP-associated nephrotoxicity., (© 2024 Wiley‐VCH GmbH.)

Ethnopharmacological Relevance: Triptolide is a major bioactive and toxic ingredient isolated from the traditional Chinese herb Tripterygium wilfordii (T. wilfordii) Hook F. It exhibits potent antitumor, immunosuppressive, and anti-inflammatory biological activities; however, its clinical application is hindered by severe systemic toxicity. Two preparations of T. wilfordii, including T. wilfordii glycoside tablets and T. wilfordii tablets, containing triptolide, are commonly used in clinical practice. However, their adverse side effects, particularly hepatotoxicity, limit their safe use. Therefore, it is crucial to discover potent and specific detoxification medicines for triptolide., Aim of the Study: This study aimed to investigate the detoxification effects and potential mechanism of action of spironolactone on triptolide-induced hepatotoxicity to provide a potential detoxifying strategy for triptolide, thereby promoting the safe applications of T. wilfordii preparations in clinical settings., Materials and Methods: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Nuclear fragmentation was visualized using 4',6-diamidino-2-phenylindole (DAPI) staining, and protein expression was analyzed by Western blotting. The inhibitory effect of spironolactone on triptolide-induced hepatotoxicity was evaluated by examining the effects of spironolactone on serum alanine aminotransferase and aspartate aminotransferase levels, as well as liver pathology in a mouse model of triptolide-induced acute hepatotoxicity. Furthermore, a survival assay was performed to investigate the effects of spironolactone on the survival rate of mice exposed to a lethal dose of triptolide. The effect of spironolactone on triptolide-induced global transcriptional repression was assessed through 5-ethynyl uridine staining., Results: Triptolide treatment decreased the cell viability, increased the nuclear fragmentation and the cleaved caspase-3 levels in both hepatoma cells and hepatocytes. It also increased the alanine aminotransferase and aspartate aminotransferase levels, induced the hepatocyte swelling and necrosis, and led to seven deaths out of 11 mice. The above effects could be mitigated by pretreatment with spironolactone. Additionally, molecular mechanism exploration unveiled that spironolactone inhibited triptolide-induced DNA-directed RNA polymerase II subunit RPB1 degradation, consequently increased the fluorescence intensity of 5-ethynyl uridine staining for nascent RNA., Conclusions: This study shows that spironolactone exhibits a potent detoxification role against triptolide hepatotoxicity, through inhibition of RPB1 degradation induced by triptolide and, in turn, retardation of global transcriptional inhibition in affected cells. These findings suggest a potential detoxification strategy for triptolide that may contribute to the safe use of T. wilfordii preparations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Triptolide (TP) is a major active and toxic composition of the Chinese medicine Tripterygium wilfordii Hook. F. (TWHF), exhibiting various therapeutic bioactivities. Among the toxic effects, the hepatotoxicity of TP deserves serious attention. Previously, our research group proposed a new view of TP-related hepatotoxicity: hepatic hypersensitivity under lipopolysaccharide (LPS) stimulation. However, the mechanism of TP/LPS-induced hepatic hypersensitivity remains unclear. In this study, we investigated the mechanism underlying TP/LPS-induced hypersensitivity from the perspective of the inhibition of proteasome activity, activated endoplasmic reticulum stress (ERS)-related apoptosis, and the accumulation of reactive oxygen species (ROS). Our results showed that N-acetylcysteine (NAC), a common ROS inhibitor, decreased the expression of cleaved caspase-3 and cleaved PARP, which are associated with FLIP enhancement. Moreover, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was able to alleviate TP/LPS-induced hepatotoxicity by reducing ERS-related apoptosis protein expression (GRP78, p-eIF2α/eIF2α, ATF4, CHOP, cleaved caspase-3 and cleaved PARP) and ROS levels, with ATF4 being an indispensable mediator. In addition, the proteasome activity inhibitor MG-132 further aggravated ERS-related apoptosis, which indicated that the inhibition of proteasome activity also plays an important role in TP/LPS-related liver injuries. In summary, we propose that TP/LPS may upregulate the activation of ERS-associated apoptosis by inhibiting proteasome activity and enhancing ROS production through ATF4., (© 2024. The Author(s).)

Background: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure., Methods: Male mice were subjected to TP at doses of 15, 30, and 60 μg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining., Results: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells., Conclusion: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: The proper application of toxic medicines is one of the characteristics of traditional Chinese medicines, and the use of traditional Chinese medicines follows the principle of dialectical treatment. It is necessary to combine different "syndrome" or "disease" states with the toxicity of traditional Chinese medicines to form a reliable toxicity evaluation system. Fuzi, the lateral root of Aconitum carmichaelii Debx, is recognized as a panacea for kidney yang deficiency syndrome, however, its toxic effects significantly limit its clinical application., Aim of the Study: Herein, our research aimed to explore the toxic effects of Fuzi on syndrome models, and tried to reveal the underlying mechanisms., Materials and Methods: Firstly, the mouse model of kidney yang deficiency syndrome was established through intramuscular injection of 25 mg/kg hydrocortisone per day for 10 consecutive days. Then, the acute toxicity of Fuzi in normal mice and kidney yang deficiency model mice was explored. Finally, the plasma metabolite concentrations and liver CYP3A4 enzyme activity were analyzed to reveal the possible mechanisms of the different pharmacological and toxicological effects of Fuzi in individuals with different physical constitutions., Results: It was found that the treatment with Fuzi (138 g/kg) had serious toxic effects on kidney yang deficiency mice, leading to the death of 80% of the mice, whereas it showed no lethal toxicity in normal mice. This indicates that Fuzi induced greater toxicity in kidney yang deficiency mice than in normal ones. The liver CYP3A4 enzyme activity in kidney yang deficiency mice was decreased by 20% compared to the controls, resulting in slower metabolism of the toxic diester diterpenoid alkaloids in Fuzi., Conclusion: In conclusion, our study showed that changes of the metabolic enzyme activity in individuals with different syndromes led to different toxic effects of Chinese medicines, emphasizing the crucial importance of considering individual physical syndromes in the clinical application of traditional Chinese medicine, and the significance of conducting safety evaluations and dose predictions on animal models with specific syndromes for traditional Chinese medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Triptolide has a significant antitumor activity, but its toxicity limits its clinical application. As the mitochondrion-targeting strategy showed an advantage in selective antitumor effect based on the higher mitochondrial membrane potential (MMP) in tumor cells than normal cells, the lipophilic cations triphenylphosphonium and E -4-(1 H -indol-3-yl vinyl)- N -methylpyridinium iodide (F16) were selected as targeting carriers for structural modification of triptolide. The derivatives bearing F16 generally retained most antitumor activities, overcame its inhibition plateau phenomena, and enhanced its selective antitumor effect in lung cancer. The representative derivative F9 could accumulate in the mitochondria of NCI-H1975 cells, inducing apoptosis and a dose-dependent increase in intracellular reactive oxygen species and reducing MMP. Moreover, no effects were observed in normal cells BEAS-2B. In vivo studies showed that the developmental, renal, and liver toxicities of F9 to zebrafish were significantly lower than those of triptolide. This study provides a promising idea to relieve the toxicity problem of triptolide.

Triptolide is a predominant active component of Triptergium wilfordii Hook. F, which has been used for the treatment of cancers and autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and diabetic nephropathy. Therefore, triptolide and its derivates are considered to have promising prospects for development into drugs. However, the clinical application of triptolide is limited due to various organ toxicities, especially liver toxicity. The potential mechanism of triptolide-induced hepatotoxicity has attracted increasing attention. Over the past five years, studies have revealed that triptolide-induced liver toxicity is involved in metabolic imbalance, oxidative stress, inflammations, autophagy, apoptosis, and the regulation of cytochrome P450 (CYP450) enzymes, gut microbiota and immune cells. In this review, we summarize the pharmacological applications and hepatotoxicity mechanism of triptolide, which will provide solid theoretical evidence for further research of triptolide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this study, two tigliane diterpenoids, 12-deoxyphorbol-13-hexadecanoate and 12-deoxyphorbol-13-acetate (prostratin), were identified from the methanol extract of the roots of Euphorbia fischeriana and were found to have the ability to significantly reduce the survival of Caenorhabditis elegans . It was determined that exposure to these two compounds had toxic effects on the growth, reproduction, locomotion behavior, and accumulation of lipids and lipofuscin of the nematodes. Moreover, the transcription levels of the genes associated with lipid accumulation, apoptosis, insulin, and nuclear hormone synthesis in C. elegans were significantly influenced. Interestingly, 12-deoxyphorbol-13-hexadecanoate produced exposure toxicity at lower concentrations than that of prostratin. Pearson correlation analysis indicates that the elevated exposure toxicity of 12-deoxyphorbol-13-hexadecanoate may be the result of differing transcription levels, which result from the differential expression of fat-6 , egl-38 , and cep-1 . These results reveal that esterification with a long-chain fatty acid elevates the exposure toxicity of this tigliane diterpenoid, thus providing a basis for the application of tigliane diterpenoids in plant-derived nematicides.

Aim: Triptolide (TRI) is an active diterpenoid lactone compound isolated from Tripterygium wilfordii,We focused on investigating the effect and mechanism of Triptolide (TRI) on liver injury., Methods: The toxic dose (LD50 = 100 μM) of TRI on liver Kupffer cells was explored, and network pharmacological analysis was performed to identify Caspase-3 as the target of TRI-induced liver injury. Regarding the pyroptosis research, we examined the level of TRI-induced pyroptosis in Kupffer cells, including inflammatory cytokine detection, protein assay, microscopic cell observation and LDH toxicity test. The effect of TRI on pyroptosis was assessed after knocking out GSDMD, GSDME and Caspase-3 in cells, respectively. We also investigated the liver injury-inducing action of TRI at the animal level., Results: Our experimental results were consistent with those predicted by network pharmacology, indicating that TRI could bind to Caspase-3-VAL27 site to promote the cleavage of Caspase-3, and Cleaved-Caspase-3 induced pyroptosis of Kupffer cells through GSDME cleavage. GSDMD was not involved in TRI's action. TRI could promote Kupffer cell pyroptosis, elevate the inflammatory cytokine levels, and facilitate the expressions of N-GSDME and Cleaved-Capase 3. After the mutation of VAL27, TRI could not bind to Caspase-3. Animal-level results showed that TRI could induce liver injury in mice, while Caspase-3 knockout or Caspase-3 inhibitors could antagonize the action of TRI., Conclusion: We find that the TRI-induced liver injury occurs primarily through the Caspase-3-GSDME pyroptosis signal. TRI can promote Caspase - 3 maturation and regulate kupffer cell pyroptosis. The present findings offer a new idea for the safe use of TRI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Drug-induced organ injury is one of the key factors causing organ failure and death in the global public. Triptolide (TP) is the main immunosuppressive component of Tripterygium wilfordii Hook. f. (Leigongteng, LGT) for the first-line management of autoimmune conditions, but it can cause serious multi-organ injury. Lysimachia christinae (Jinqiancao, JQC) is a detoxifying Chinese medicine and could suppress LGT's toxicity. It contains many immune enhancement and organ protection components including chlorogenic acid (CA), rutin (Rut), and quercetin (Que). This study aimed to explore the protection of combined treatments of these organ-protective ingredients of JQC on TP-induced liver, kidney, and heart injury and initially explore the mechanisms. Molecular docking showed that CA, Rut, and Que bounded protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway-related molecules intimately and might competitively antagonize TP. Corresponding in vivo results showed that the combination activated TP-inhibited protein of AKT/mTOR pathway, and reversed TP-induced excessive ferroptosis (excessive Fe 2+ and lipid peroxidation malondialdehyde accumulation, decreased levels of antioxidant enzymes catalase, glutathione peroxidase, glutathione-s transferase, reduced glutathione, and superoxide dismutase, and down-regulated P62/nuclear factor erythroid-2-related factor 2/heme oxygenase-1 pathway), and apoptosis (activated apoptotic factor Fas and Bax and inhibited Bcl-2) in the organ of mice to varying degrees. In conclusion, the combined treatments of CA, Rut, and Que from JQC inhibited TP-induced multi-organ injury in vivo, and the mechanism may largely involve immunomodulation and activation of the AKT/mTOR pathway-mediated cell death reduction including ferroptosis and apoptosis inhibition., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: The transformations that occur in diterpenoid alkaloids during the process of sand frying for Chinese herbal medicine preparation have yet to be clarified. This study investigated the structural changes that take place in 3-acetylaconitine during a simulation of heat-processing and evaluated the toxicity and biological activity of the pyrolysis products., Methods: The diterpenoid alkaloid 3-acetylaconitine was heated at 180 °C for 15 min to simulate the process of sand frying. The pyrolysis products were separated using column chromatography, and their structures were investigated using high-resolution electrospray ionization mass spectroscopy and nuclear magnetic resonance spectroscopy. Further, in vivo cardiotoxicity and acute toxicity of 3-acetylaconitine and its pyrolysis products were compared, and the aconitine-induced arrhythmia model was employed to evaluate the antiarrhythmic effect of the pyrolysis products., Results: Two new diterpenoid alkaloids, pyroacetylaconitine and 16-epi-pyroacetylaconitine, a pair of epimers at C-16, were isolated. After comparing the structures of these compounds, possible transformation pathways were proposed. Compared with the prototype compound, 3-acetylaconitine, the cardiotoxicity and acute toxicity of the heat-transformed products were significantly decreased. In the biological activity assay, the two pyrolysis products exhibited an effective increase in ventricular premature beat latency, a reduction in the occurrence of ventricular tachycardia, as well as an increase in the rate of arrhythmia inhibition, implying strong antiarrhythmic activity., Conclusion: Compared with 3-acetylaconitine, its pyrolysis products displayed lower toxicity and good antiarrhythmic effects; thus, they have potential for being developed into antiarrhythmic medicines. Please cite this article as: Wang YJ, Wang Y, Tao P. Structural characterization, in vivo toxicity and biological activity of two new pyro-type diterpenoid alkaloids derived from 3-acetylaconitine. J Integr Med. 2023; 21(3): 302-314., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.)

This study was to investigate the potential mechanism of triptolide-induced hepatotoxicity. We found a novel and variable role of p53/Nrf2 crosstalk in triptolide-induced hepatotoxic process. Low doses of triptolide led to adaptive stress response without obvious toxicity, while high levels of triptolide caused severe adversity. Correspondingly, at the lower levels of triptolide treatment, nuclear translocation of Nrf2 as well as its downstream efflux transporters multidrug resistance proteins and bile salt export pump expressions were significantly enhanced, so did p53 pathways that also increased; at a toxic concentration, total and nuclear accumulations of Nrf2 decreased, while p53 showed an obvious nuclear translocation. Further studies showed the cross-regulation between p53 and Nrf2 after different concentrations of triptolide treatment. Under mild stress conditions, Nrf2 induced p53 highly expression to maintain the pro-survival outcome, while p53 showed no obvious effect on Nrf2 expression and transcriptional activity. Under high stress conditions, the remaining Nrf2 as well as the largely induced p53 mutually inhibited each other, leading to a hepatotoxic result. Nrf2 and p53 could physically and dynamically interact. Low levels of triptolide enhanced the interaction between Nrf2 and p53. Reversely, p53/Nrf2 complex dissociated at high levels of triptolide treatment. Altogether, variable p53/Nrf2 crosstalk contributes to triptolide-induced self-protection and hepatotoxicity, modulation of which may be a potential strategy for triptolide-induced hepatotoxicity intervention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Triptolide (TP) is the major active ingredient of Tripterygium wilfordii Hook, a traditional Chinese herb that possesses various pharmacological activities and has been used to treat autoimmune and inflammatory diseases for thousands of years. However, the clinical application of TP is limited due to its multiorgan toxicity, and in particular, its negative impact on female fertility. To date, the specific toxic mechanisms on reproduction induced by TP remain unclear. In the current study, an LC-MS/MS-based metabolomic approach was adopted to study TP-induced reproductive toxicity and its mechanism. Histopathological examination of the ovaries showed that TP significantly induced follicular atresia and decreased the numbers of corpus luteum in rats, as well as reducing the gonadal index and destroying the microstructure of the ovary. Immunohistochemical staining revealed that TP significantly induced apoptosis of rat follicle cells. Metabolomics analysis revealed that 67 and 74 small molecule metabolites in the ovaries and serum, respectively (fold-changes > 1.5, p < 0.05), were significantly different in TP-treated rats compared to CON group rats. Target profiling identified the metabolites arachidonic acid, prostaglandin D2, prostaglandin H2 and prostaglandin E2 as potential serum biomarkers for TP-induced ovary damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Tripterygium glycosides tablets (TGT) and Tripterygium wilfordii tablets (TWT) have been used to treat autoimmune diseases clinically, however, the side effects of TWT are higher than TGT, especially for hepatotoxicity., The Aim of the Study: This study aims to determine the mechanism of TWT-induced liver injury., Materials and Methods: We performed metabolomic analysis of samples from mice with liver injury induced by TGT and TWT. Ppara-null mice were used to determine the role of PPARα in TWT-induced liver injury., Results: The results indicated that TWT induced the accumulation of medium- and long-chain carnitines metabolism, which was associated with the disruption of PPARα-IL6-STAT3 axis. PPARα agonists fenofibrate could reverse the liver injury from TWT and TP/Cel, and its protective role could be attenuated in Ppara-null mice. The toxicity difference of TWT and TGT was due to the different ratio of triptolide (TP) and celastrol (Cel) in the tablet in which TP/Cel was lower in TWT than TGT. The hepatotoxicity induced by TP and Cel also inhibited PPARα and upregulated IL6-STAT3 axis, which could be alleviated following by PPARα activation., Conclusions: These results indicated that PPARα plays an important role in the hepatotoxicity of Tripterygium wilfordii, and PPARα activation may offer a promising approach to prevent hepatotoxicity induced by the preparations of Tripterygium wilfordii., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Six diterpenoids including three ent-kauranes (1-2, 4) and three cleistanthanes (3, 5-6) were isolated from the roots and stems of Phyllanthus acidus (L.) Skeels. Of them, (16S)-ent-16,17,18-tri-hydroxy-19-nor-kaur-4-en-3-one (1), phyllanthone A (2), and 6-hydroxycleistanthol (3) are new compounds, while the ent-kaurane diterpenoids were reported from the titled plant for the first time. Their structures were elucidated on the basis of the extensive spectroscopic analyses. Compounds 2 and 4-6 displayed cytotoxic potential with IC 50 values ranging from 1.96 to 29.15 μM. They also showed moderate anti-inflammatory activities (IC 50  = 6.30-12.05 μM). Particularly, the new ent-kaurane 2 displayed cytotoxic potential against HL-60 (IC 50  = 2.00 μM) and MCF-7 (IC 50  = 3.55 μM) cells, and anti-inflammatory activity (IC 50  = 6.47 μM)., (Copyright © 2022. Published by Elsevier B.V.)

Natural products have played a pivotal role for the discovery of anticancer drugs. Tonantzitlolones are flexibilan-type diterpenes rare in nature; therefore, few reports have shown antiviral and cytotoxic activities. This study aimed to investigate the in vivo antitumor action of Tonantzitlolone B (TNZ-B) and its toxicity. Toxicity was evaluated in mice (acute and micronucleus assays). Antitumor activity of TNZ-B (1.5 or 3 mg/kg intraperitoneally - i.p.) was assessed in Ehrlich ascites carcinoma model. Angiogenesis and reactive oxygen species (ROS) and nitric oxide (NO) production were also investigated, in addition to toxicological effects after 7-day treatment. The LD 50 (lethal dose 50%) was estimated at around 25 mg/kg (i.p.), and no genotoxicity was recorded. TNZ-B reduced the Ehrlich tumor's volume and total viable cancer cell count (p < 0.001 for both). Additionally, TNZ-B reduced peritumoral microvessel density (p < 0.01), suggesting antiangiogenic action. Moreover, a decrease was observed on ROS (p < 0.05) and nitric oxide (p < 0.001) levels. No significant clinical findings were observed in the analysis of biochemical, hematological, and histological (liver and kidney) parameters. In conclusion, TNZ-B exerts antitumor and antiangiogenic effects by reducing ROS and NO levels and has weak in vivo dose-repeated toxicity. These data contribute to elucidate the antitumor action of TNZ-B and point the way for further studies with this natural compound as an anticancer drug., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Grandiflorenic acid (GFA) is one of the main kaurane diterpenes found in different parts of Sphagneticola trilobata. It has several biological activities, especially antiprotozoal action. In turn, Chagas disease is a complex systemic disease caused by the protozoan Trypanosoma cruzi, and the drugs available to treat it involve significant side effects and impose an urgent need to search for therapeutic alternatives. In this context, our goal was to determine the effect of GFA on trypomastigote and intracellular amastigote forms. Our results showed that GFA treatment led to significantly less viability of trypomastigote forms, with morphological and ultrastructural changes in the parasites treated with IC 50 of GFA (24.60 nM), and larger levels of reactive oxygen species (ROS), mitochondrial depolarization, lipid droplets accumulation, presence of autophagic vacuoles, phosphatidylserine exposure, and plasma membrane damage. In addition, the GFA treatment was able to reduce the percentage of infected cells and the number of amastigotes per macrophage (J774A.1) without showing cytotoxicity in mammalian cell lines (J774A.1, LLCMK 2 , THP-1, AMJ2-C11), in addition to increasing TNF-α and reducing IL-6 levels in infected macrophages. In conclusion, the GFA treatment exerted influence on trypomastigote forms through an apoptosis-like mechanism and by eliminating intracellular parasites via TNF-α/ROS pathway, without generating cellular cytotoxicity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The thunder god vine, Tripterygium hypoglaucum, is a toxic nectar plant distributed across China. A terpenoid, called triptolide (TRP), found in nectar can impair honeybees' foraging responses, dance communication, and olfactory learning. In the present study, we tested the tolerances of the native honeybee Apis cerana and the introduced honeybee A. mellifera to short-term and long-term exposure to TRP. The results showed that introduced A. mellifera is more vulnerable in fatality to high concentrations of TRP sucrose solution (5 and 10 µg TRP mL -1 ) than A. cerana. We also compared the short-term and long-term exposure effects of TRP on olfactory learning and memory between the two honeybee species, and the olfactory learning and memory of both honey bee species showed impaired performance after both 2 h or 7 days of being fed with TRP sucrose solution. However, A. cerana showed a higher tolerance and resistance to TRP toxin than A. mellifera. Our results support a coevolution hypothesis in that the native species A. cerana has higher toxin tolerance than the introduced species A. mellifera., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Liver toxicity induced by Triptolide (TP) has limited its clinical application on rheumatoid arthritis (RA). Saponins have been proved as an efficacious remedy to mitigate hepatotoxicity. However, the mechanism of reducing hepatotoxicity by saponins intervention remains incompletely characterized. Tryptophan (Trp) metabolites activate transcriptional regulators to mediate host detoxification responses. Our study aimed to investigate whether Clematichinenoside AR (C-AR) could attenuate TP-induced liver damage by regulating Trp metabolism. We used targeted metabolomics to quantify Trp metabolites in the serum and liver samples of collagen-induced arthritis rats treated by TP. Multiple comparison analyses helped the evaluation of promising biomarkers. The pronounced changed levels of Trp, indole acetic acid, and indole-3-carboxaldehyde in the serum and indole acetic acid, indole, and tryptamine in the liver are relevant to TP-induced liver injury. Intervention with C-AR could relieve TP-induced hepatotoxicity evidenced by ameliorative serum parameters and hepatic histology. In addition, C-AR regulated the levels of these indoles biomarker candidates to normal. Therapeutic modulation with natural compounds might be a useful clinical strategy to ameliorate toxicity induced by TP. Deciphering Trp metabolism will facilitate a better understanding of the pathogenesis of diseases and drug responding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Triptolide (TP), the main bioactive and toxic ingredient of Tripterygium wilfordii Hook F, causes severe toxicity, particularly for hepatotoxicity. However, the underlying mechanisms for its hepatotoxicity are not entirely clear., Aim of the Study: The purpose of the study was to explore the role of miR-155, a microRNA closely related to various liver injuries and a regulator of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway, in TP-induced liver injury in vitro and in vivo., Materials and Methods: First, in vitro L02 cells were treated with different concentrations of TP. The protein levels of Nrf2 and its downstream genes Heme oxygenase1 (HO-1) were determined by Western blot. The mRNA expression of miR-155, Nrf2, NAD(P)H: quinone oxidoreductase 1 (NQO1) and HO-1 were measured using qRT-PCR. And we transfected miR-155 inhibitor and miminc before TP treatment to determine the mRNA and/or protein levels of miR-155, Nrf2 and HO-1. Then, we further confirmed the interaction between miR-155 and Nrf2 pathway in TP-induced hepatic injury in BALB/C mice. The degree of liver injury was determined by HE staining and serum biochemical. The mRNA expression of miR-155 was examined with qRT-PCR and Nrf2 and HO-1 gene expression in liver were evaluated by immunohistochemistry and/or Western blot., Results: The results showed that TP significantly induced the expression of miR-155 both in L02 cells and in rodents liver tissue, and the inhibition of miR-155 could mitigate the hepatic damages caused by TP. Further experiments demonstrated that the inhibition of miR-155 reversed the down-regulation of Nrf2 and HO-1 by TP, while the miR-155 mimic enhanced the effects of TP. Animal experiments also showed that the inhibition of miR-155 by miR-155 antagomir reversed the decrease of Nrf2 induced by TP administration., Conclusions: These results indicated that miR-155 played an important role in TP-induced hepatotoxicity by regulating the Nrf2 signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In recent decades, herbal medicines have played more and more important roles in the healthcare system in the world because of the good efficacy. However, with the increasing use of herbal medicines, the toxicity induced by herbal medicines has become a global issue. Therefore, it is needed to investigate the mechanism behind the efficacy and toxicity of herbal medicines. In this study, using Aconiti Lateralis Radix Praeparata (Fuzi) as an example, we adopted a systems pharmacology approach to investigate the mechanism of Fuzi in treating rheumatoid arthritis and in inducing cardiac toxicity and neurotoxicity. The results showed that Fuzi has 25 bioactive compounds that act holistically on 61 targets and 27 pathways to treat rheumatoid arthritis, and modulation of inflammation state is one of the main mechanisms of Fuzi. In addition, the toxicity of Fuzi is linked to 32 compounds that act on 187 targets and 4 pathways, and the targets and pathways can directly modulate the flow of Na + , Ca 2+ , and K + . We also found out that non-toxic compounds such as myristic acid can act on targets of toxic compounds and therefore may influence the toxicity. The results not only reveal the efficacy and toxicity mechanism of Fuzi, but also add new concept for understanding the toxicity of herbal medicines, i.e., the compounds that are not directly toxic may influence the toxicity as well., (© 2021. The Author(s).)

Spodoptera litura Fab. is a polyphagous pest causing damage to many agriculture crops leading to yield loss. Recurrent usage of synthetic pesticides to control this pest has resulted in resistance development. Plant-derived diterpenoid compound andrographolide was isolated from the leaves of Andrographis paniculata. It was analysed by gas chromatography-mass spectroscopy and quantified by HPLC. Nutritional indices and digestive enzymatic profile were evaluated. Third, fourth and fifth instar larvae were treated with different concentrations of andrographolide. At 3, 6 and 9 ppm-treated concentrations the larvae showed decreased RGR, RCR, ECI, ECD values with adverse increase in AD. The digestive enzymes were significantly inhibited when compared with control. Conspicuously, andrographolide showed pronounced mortality of S. litura by inhibition of enzyme secretion and intake of food. The binding ability of andrographolide with CYTP450 showed high affinity with low binding energy. Andrographolide has the potential to be exploited as a biocontrol agent against S. litura as an eco-friendly pesticide.

As a widely existing traditional Chinese medicine component, TP (triptolide) has serious reproductive toxicity which causes severe damage to the reproductive system and limits its application prospect. TP and MET (metformin) have shown great potential in combined with each other in anticancer and anti-inflammatory. Whether metformin can resist the reproductive toxicity caused by triptolide, the effects of MET on TP-induced reproductive capacity has not been reported. In this study, metformin was used to investigate the therapeutic effect on reproductive toxicity induced by TP in rat. The results showed that metformin had significant therapeutic effects on oxidative stress damage, destruction of the blood-testosterone barrier and apoptosis. And it proved that its therapeutic effect is mainly to restore the structural and functional stability of testis through antioxidant stress. It will provide guidance for the treatment of reproductive toxicity caused by TP and the adjuvant detoxification of TP application., (Copyright © 2021. Published by Elsevier Inc.)

The liver is an important organ for amino acid metabolism, and its damage can be reflected in the changes of amino acid level in the body. Triptolide (TP) has broad anti-inflammatory and anti-tumor activities, but its clinical application is limited due to hepatotoxicity. In this work, a simple, accurate and sensitive gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS/MS) method was developed and validated for evaluating the serum levels of amino acids from control and TP-induced liver injured rats, and chemometric analysis was employed for amino acid metabolic profiles analysis. It was found that 11 amino acids showed significant changes after TP administration, and they were mainly involved in 5 metabolic pathways that are phenylalanine, tyrosine and tryptophan biosynthesis, alanine, aspartate and glutamate metabolism, glutamine and glutamate metabolism, phenylalanine metabolism and arginine biosynthesis. Five amino acids including tyrosine, glutamine, glutamic acid, tryptophan and alanine were identified as biomarkers of TP hepatotoxicity by further analysis. These results indicated that the novel amino acid metabolic profiling study based on the GC-QqQ-MS/MS provided not only exact concentrations of serum amino acids, but also a prospective methodology for evaluation of chemically induced liver injury.

Ethnopharmacological Relevance: Aconitum carmichaelii Debeaux, a famous traditional medicinal herb for collapse, rheumatic fever, and painful joints, always raises global concerns about its fatal toxicity from toxic alkaloids when improperly processed. Therefore, it is urgent to clarify the internal molecular mechanism of processing detoxification on Aconitum and develop simple and reliable approaches for clinical application, which is also of great significance to the rational medicinal use of Aconitum., Aim of the Study: The study aimed at developing a complete molecular mechanism exploration strategy in complex medicinal herb decocting system, clarifying the internal molecular mechanism of processing detoxification on Aconitum, and exploring valid approaches for detoxification., Materials and Methods: Aconiti Lateralis Radix Praeparata (Fuzi) was selected as the model for exploring the complex Aconitum detoxification mechanism using an advanced online real-time platform based on extractive electrospray ionization mass spectrometry. The methods realized the sensitive capture of dynamic trace intermediates, accurate qualitative and quantitative analysis, and real-time and long-term monitoring of multi-components with satisfactory accuracy and resistance to complex matrices., Results: Components in the complex Aconitum decocting system were real-timely characterized and fat meat was discovered and verified to directionally detoxify Aconitum while reserving the therapy effect. More importantly, the dynamic detoxification mechanism in the chemically complex Aconitum decoction was molecularly profiled. A novel reaction pathway based on nucleophilic substitution reaction mechanism was proposed. As confirmed by the theoretic calculations at DFT B3LYP/6-31G (d) levels, fatty acids (e.g., palmitic acid) acted as a green, cheap, and high-performance catalyst and promote the decomposition of toxic diester alkaloids to non-toxic and active benzoyl-monoester alkaloids through the discovered mechanism., Conclusion: The study exposed a novel detoxification molecular mechanism of Aconitum and provided an effective method for the safe use of Aconitum, which could effectively guide the development of traditional processing technology and compatibility regulation of the toxic herb and had great value to the modernization and standardization development of traditional medicine., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Treatment of neuropathic pain (NP) resulting from nerve injury is one of the most complicated and challenging in modern practice. Pharmacological treatments for NP are not fully effectively and novel approaches are requisite. Recently, transplantation of bone mesenchymal stem cells (BMSCs) has represented a promising approach for pain relief and neural repair, but how it produces beneficial effects on resiniferatoxin (RTX) induced nerve injury is still unclear. Here, we identified the BMSCs' analgesic effects and their potential mechanisms of microglial cells activation on RTX induced neuralgia. Immunostaining, biochemical studies demonstrated that microglia rather than astrocyte cells activation involved in RTX induced mechanical hyperalgesia, whereas the GFP-labeled BMSCs alleviated this mechanical hyperalgesia. Moreover, pain-related TRPA1, PKCδ, CaMKIIɑ (Calcium/calmodulin dependent protein kinase II), P38/MAPK (mitogen-activated protein kinase), p-P65 activation and expression in the spinal cord were significantly inhibited after BMSC administration. In addition, BMSCs treated RTX mice displayed a lower density of mushroom dendritic spines. Our research suggested that activation of PKCδ-CaMKIIɑ-P38/MAPK-p-P65 pathway and mushroom dendritic spines abnormal increase in the spinal cord is the main mechanism of RTX induced neuropathic pain, and transplant of BMSCs to the damaged nerve may offer promising approach for neuropathic pain., (Copyright © 2021. Published by Elsevier Inc.)

Geranylgeraniol (GGOH) is an isoprenoid compound found in annatto seeds and an intermediate of the mevalonate pathway found within organisms serving various functions. Toxicological studies on its safety profile are not readily available. To assess the safety of GGOH, a molecularly distilled, food grade annatto oil, consisting of approximately 80% trans-GGOH, was subjected to a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test in order to investigate its genotoxic potential and a 90-day repeated-dose oral toxicity study in rats in order to investigate its potential subchronic toxicity and identify any target organs. No evidence of mutagenicity or genotoxic activity was observed under the applied test systems. In the 90-day study, male and female Hsd. Han Wistar rats were administered daily doses of 0, 725, 1450, and 2900 mg/kg bw/day by gavage. Treatment-related adverse effects were observed in the forestomach at all dose levels and in the liver at the intermediate- and high-dose levels. Based on these results, the lowest observed adverse effect level (LOAEL) for local effects and the no observed adverse effect level (NOAEL) for systemic effects were determined as 725 mg/kg bw/day., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Even today, weeds continue to be a considerable problem for agriculture. The application of synthetic herbicides produces serious environmental consequences, and crops suffer loss of their activity due to the appearance of new resistant weed biotypes. Our aim is to develop new effective natural herbicides that improve the problem of resistance and do not harm the environment. This work is focused on a bioassay-guided isolation and the characterization of natural products present in Moquiniastrum pulchrum leaves with phytotoxic activity and its preliminary application in weeds. Moquiniastrum pulchrum was selected for two reasons: it is an abundant species in the Cerrado region (the second most important ecosystem in Brazil, after the Amazon)-the explanation behind its being a dominant species is a major focus of interest-and it has traditional employment in folk medicine. Six major compounds were isolated in this plant: one flavone and five diterpenes, two of which are described for the first time in the literature. Four of the six compounds exhibited phytotoxic activity in the bioassays performed. The results confirmed the phytotoxic potential of this plant, which had not been investigated until now.

Ethnopharmacological Relevance: Aconiti Lateralis Radix Praeparata (Chinese name: Fuzi), the root of Aconitum carmichaelii Debx., is a representative medicine for restoring yang and rescuing patient from collapse. However, less studies had been reported on the reproductive toxicity and genotoxicity of Fuzi. According to the principle of reducing toxicity and preserving efficiency, only processed products of Fuzi are commonly applied in clinic, including Baifupian, Heishunpian and Danfupian. However, whether processing could alleviate the reproductive toxicity and genotoxicity of Fuzi had not been revealed., Aim of the Study: To assess the effect and possible mechanism of Fuzi and its processed products on reproductive toxicity and genotoxicity in male mice., Materials and Methods: Aqueous extracts of Fuzi and its processed products (Baifupian, Heishunpian and Danfupian, 5.85 g/kg) were administrated by gavage once daily for fourteen consecutive days. The reproductive toxicity was evaluated by testis weight, testis ratio, testis histopathology, sperm count, sperm viability rate and sperm deformity rate. The genotoxicity was evaluated by comet assay and micronucleus test in sperm, peripheral blood cell and bone marrow cell. Possible mechanisms of attenuating toxicity by processing were analyzed by detecting the level of testosterone, superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and catalase (CAT)., Results: Fuzi significantly caused different degrees of reproductive toxicity and genotoxicity, specifically reducing the weight and testicular coefficient of testis, causing obvious pathological changes in testicular tissue, reducing sperm count and sperm viability rate, increasing sperm deformity rate and DNA damage in sperm/peripheral blood cells/bone marrow cells. Moreover, Fuzi decreased the level of testosterone, SOD, GSH and CAT, while increased the level of MDA in serum. Notably, the reproductive toxicity and genotoxicity induced by the processed products, especially Heishunpian and Danfupian, were significantly lowered compared to Fuzi. Processing could increase the level of testosterone, SOD, GSH, CAT and decrease the level of MDA compared to Fuzi., Conclusion: Fuzi and its processed products had reproductive toxicity and genotoxicity, but the toxicity of processed products was significantly weakened compared to Fuzi. The protective mechanism of processing to reduce the toxicity of Fuzi might be related to increasing the level of testosterone and decreasing oxidative stress., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Systemic treatment with resiniferatoxin (RTX) induces small-fiber sensory neuropathy by damaging TRPV1-expressing primary sensory neurons and causes distinct thermal sensory impairment and tactile allodynia, which resemble the unique clinical features of postherpetic neuralgia. However, the synaptic plasticity associated with RTX-induced tactile allodynia remains unknown. In this study, we found that RTX-induced neuropathy is associated with α2δ-1 upregulation in the dorsal root ganglion (DRG) and increased physical interaction between α2δ-1 and GluN1 in the spinal cord synaptosomes. RNAscope in situ hybridization showed that RTX treatment significantly increased α2δ-1 expression in DRG neurons labeled with calcitonin gene-related peptide, isolectin B4, NF200, and tyrosine hydroxylase. Electrophysiological recordings revealed that RTX treatment augmented the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of evoked EPSCs in spinal dorsal horn neurons, and these effects were reversed by blocking NMDA receptors with AP-5. Inhibiting α2δ-1 with gabapentin, genetically ablating α2δ-1, or targeting α2δ-1-bound NMDA receptors with α2δ-1Tat peptide largely normalized the baseline frequency of mEPSCs and the amplitude of evoked EPSCs potentiated by RTX treatment. Furthermore, systemic treatment with memantine or gabapentin and intrathecal injection of AP-5 or Tat-fused α2δ-1 C terminus peptide reversed allodynia in RTX-treated rats and mice. In addition, RTX-induced tactile allodynia was attenuated in α2δ-1 knock-out mice and in mice in which GluN1 was conditionally knocked out in DRG neurons. Collectively, our findings indicate that α2δ-1-bound NMDA receptors at presynaptic terminals of sprouting myelinated afferent nerves contribute to RTX-induced potentiation of nociceptive input to the spinal cord and tactile allodynia. SIGNIFICANCE STATEMENT Postherpetic neuralgia (PHN), associated with shingles, is a distinct form of neuropathic pain commonly seen in elderly and immunocompromised patients. The synaptic plasticity underlying touch-induced pain hypersensitivity in PHN remains unclear. Using a nonviral animal model of PHN, we found that glutamatergic input from primary sensory nerves to the spinal cord is increased via tonic activation of glutamate NMDA receptors. Also, we showed that α2δ-1 (encoded by Cacna2d1 ), originally considered a calcium channel subunit, serves as an auxiliary protein that promotes activation of presynaptic NMDA receptors and pain hypersensitivity. This new information advances our understanding of the molecular mechanism underlying PHN and suggests new strategies for treating this painful condition., (Copyright © 2021 the authors.)

Ethnopharmacological Relevance: Andrographis paniculata (Burm.f.) Nees is widely used all over the world, especially in subtropical regions such as India, Thailand, Vietnam, and China. As a traditional folk Chinese medicine, A. paniculata has been extensively utilized for the treatment of cold, fever, sore throat, cough, carbuncle, and sores, and it is commonly employed for 'clearing heat and resolving toxicity'. Typical symptoms of 'heat and toxicity' include swollen, painful gums, associated with virus-related diseases to a great extent. In vivo and in vitro experiments have demonstrated the potential antiviral properties of A. paniculata and identified its major active constituents against various viruses., Aim of the Study: This review focuses on connecting the traditional 'clearing heat and resolving toxicity' effect to compelling recent research advances on the antiviral effects of A. paniculata, explaining its major antiviral mechanisms, and assessing the shortcomings of existing work. Besides, ethnobotany, ethnopharmacological uses, phytochemicals, and toxicology of A. paniculata have been researched., Materials and Methods: The information about A. paniculata was collected from various sources including classic books about Chinese herbal medicine, and scientific databases including WEB OF SCIENCE, PubMed, ScienceDirect, Springer, ACS, SCOPUS, CNKI, CSTJ, and WANFANG., Results: In this review, the underlying mechanisms of antiviral effect mainly involve the regulation of virus entry, gene replication, and synthesis of functionally mature proteins. Also, A. paniculata is a safe agent without obvious toxicity. Ethnobotany, ethnopharmacological uses, and chemical constituents have been summarized., Conclusion: Andrographis paniculata (Burm.f.) Nees could be used as an imperative complementary medicine for the treatment of diverse virus infection, efforts should be made to gain insights into its antiviral properties., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Non-alcoholic Fatty Liver Disease (NAFLD) is one of the growing epidemics of the globe. This study was aimed to evaluate the anti-NAFLD effect of selected IAN derivatives using in silico, in vitro and in vivo models. In silico tools viz., DataWarrior, SwissADME and Gaussian 09 were used to predict the pharmacokinetic properties and electronic distribution patterns of the derivatives; docking analysis was done with Autodock against PPARα. Toxicities of the derivatives were assessed in HepG2 cells using MTT assay. Anti-NAFLD efficacies of the derivatives were assessed in free fatty acid induced steatotic HepG2 cells. In vivo anti-NAFLD effect of active isoandrographolide (IAN) derivative, 19-propionyl isoandrographolide (IAN-19P) was assessed in High Fat Diet fed rats. In silico and in vitro studies indicated that IAN-19P showed improved drug-likeness and drug score. The toxicity of IAN-19P to HepG2 cells was comparatively less than IAN and other derivatives. In free fatty acid induced steatotic HepG2 cells, treatment with IAN-19P significantly lowered intracellular triglyceride content and leakage of LDH and transaminases. Treating High Fat Diet fed animals with IAN-19P significantly lowered plasma lipids, transaminases, LDH and GGT levels. The treatment with IAN-19P upregulated the expressions of PPARα and CPT-1. IAN-19P did not produce any noticeable adverse effect till 2 g/kg concentration in acute and 250 mg/kg concentration in subacute toxicity studies. This study indicated the beneficial effect of IAN-19P for the treatment of NAFLD; however robust investigations are needed to establish the potential of IAN-19P to treat NAFLD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Triptolide (TP), an active component of Tripterygium wilfordii Hook. F, has been widely used in China for treating autoimmune and inflammatory diseases, and has also been validated by modern science and developed as a candidate anti-cancer treatment. However, liver toxicity of TP has seriously hindered its use and development, the clinical features and primary toxicological mechanism have been unclear. Considering the major target regulation mechanism of TP is the suppression of global transcription regulated by RNAPII, which is closed related with the detoxification of drugs. This paper tries to verify the synergistic liver injury and its mechanism of TP when co-administered with CYP3A4 substrate drug. The experiments showed that TP dose-dependently blocked transcriptional activation of CYP3A4 in both hPXR and hPXR-CYP3A4 reporter cell lines, lowered the mRNA and protein expression of PXR target genes such as CYP3A1, CYP2B1, and MDR1, and inhibited the functional activity of CYP3A in a time- and concentration-dependent manner in sandwich-cultured rat hepatocytes (SCRH) and female Sprague-Dawley (f-SD) rats. Furthermore, TP combined with atorvastatin (ATR), the substrate of CYP3A4, synergistically enhanced hepatotoxicity in cultured HepG2 and SCRH cells (CI is 0.38 and 0.29, respectively), as well as in f-SD rats, with higher exposure levels of both drugs. These results clearly indicate that TP inhibits PXR-mediated transcriptional activation of CYP3A4, leading to a blockade on the detoxification of itself and ATR, thereby greatly promoting liver injury. This study may implies the key cause of TP related liver injury and provides experimental data for the rational use of TP in a clinical scenario., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Colon cancer (CRC) was a malignant tumor and there were about 25% of patients with tumor metastasis at diagnosis stage. Chemotherapeutic agents for metastatic CRC patients were with great side effects and the clinical treatment results of advanced CRC were still not satisfactory. Human epidermal growth factor receptor 2 (HER2) is overexpressed in some CRC patients and is an effective target for CRC patient treatment. Anti-HER2 therapy had a beneficial role in the treatment of HER2-positive metastatic CRC with fewer side effects. CRC patients with BRAF mutations were resistant to HER2 antibodies treatment. Therefore, there was an urgent need to develop new therapeutic agents., Methods: HER2 targeted nanoparticles (TPLNP) drug delivery system loading triptolide (TPL) were prepared and identified. The effects of TPLNP and free TPL on cell viability, targeting and cell cycle progression on HT29 (BRAF mutation) with HER2 overexpression, were evaluated by Cell Counting Kit-8 (CCK8), Fluorescence Activating Cell Sorter (FACS) and immunofluorescence methods, respectively. The anti-tumor efficacies of TPLNP were evaluated in subcutaneous xenograft model of colon cancer and the survival rate, tumor volume, liver and kidney indexes of tumor-bearing mice were measured., Results: TPLNP was small in nanosize (73.4±5.2nm) with narrow size distribution (PDI=0.15±0.02) and favorable zeta potential (pH=9.6, zeta potential: -57.3±6.69mV; pH=7.0, zeta potential: -28.7±5.1mV; pH=5.6, zeta potential: -21.1±4.73mV). Comparing with free TPL treatment group, TPLNP developed stranger colon cancer-killing efficiency in a dose- and time-dependent manner detected with CCK8 method; achieved good in vitro colon cancer targeting detected with flow cytometry and immunofluorescence experiments; enhanced more HT29-HER2 apoptosis and induced more cell cycle arrested in G1-S phase detected with FACS in vitro. As for in vivo antitumor response, TPLNP remarkably inhibited the growth of colon cancer in the colon cancer xenograft model, significantly improved the survival rate and did not exhibit significant liver and kidney toxicity in contrast with free TPL in vivo., Conclusion: TPLNP was effectively against colon cancer with HER2 overexpression and BRAF mutation in pre-clinical models. In summary, the TPLNP appeared to be a promising treatment option for CRC in clinical application based on improved efficacy and the favorable safety profile., Competing Interests: Xu-Quan Li is an employee of Shanghai Henlius Biopharmaceuticals Co., Ltd. The authors report no other potential conflicts of interest for this work., (© 2021 Yalikong et al.)

Triptolide (TP) possesses a wide range of biological and pharmacological activities involved in the treatment of various diseases. However, widespread usages of TP raise the urgent issues of the severe toxicity, which hugely limits its further clinical application. The novel functional nanostructured delivery system, which is of great significance in enhancing the efficacy, reducing side effects and improving bioavailability, could improve the enrichment, penetration and controlled release of drugs in the lesion location. Over the past decades, considerable efforts have been dedicated to designing and developing a variety of TP delivery systems with the intention of alleviating the adverse toxicity effects and enhancing the bioavailability. In this review, we briefly summarized and discussed the recent functionalized nano-TP delivery systems for the momentous purpose of guiding further development of novel TP delivery systems and providing perspectives for future clinical applications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The dried roots of Euphorbia kansui (EK) are especially beneficial for the treatment of edema, but the severe toxicity limits their clinical applications. Euphorbia kansui stir-fried with vinegar (VEK) is traditionally employed to reduce the toxicity of EK. However, the material basis for the toxicity reduction with effectivity conservation is still unclear. Therefore, in this study, a rapid, sensitive, and reliable ultra-fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method was firstly established to simultaneously determine six ingenane-type diterpenoids, i.e. kansuiphorin C (1), 5-O-benzoyl-20-deoxyingenol (2), 20-deoxyingenol (3), 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (4), 20-O-(2'E,4'Z-decadienoyl)ingenol (5), and ingenol (6), in EK and VEK based on the processing conversion. Then, the toxicity evaluation on zebrafish embryos and modulation of the expression of aquaporin-3 (AQP3) proteins in HT-29 cells were employed to investigate the toxicity-activity of six compounds. Chromatographic separation was obtained on Waters BEH RP18 column (2.1 mm × 100 mm, 2.5 μm) with the mobile phase composed of 0.1 % formic acid in acetonitrile and water, respectively. The column temperature was 35 ℃ at a flow rate of 0.4 mL min -1 . Multiple reaction monitoring was conducted in both positive and negative modes for quantitative analysis. The method was then successfully used for the determination of six compounds in EK and VEK. In addition, 1, 2, 4, and 5 had evident cardiotoxicity, intestinal irritation and nutrient absorption disorders on zebrafish larvae, while no in-vivo toxicity was seen for groups given 3 and 6 (LC 50 > 200 μM). Meanwhile, 1, 2, 4, 5, and 6 significantly increased the expression of AQP3 protein (p < 0.05) to promote the excretion of water in the colon. This study demonstrated that toxic ingenane-type diterpenoids converted into the less toxic compounds with the same core structure through the breakage of multiple ester bonds in the side chain. At the same time, the laxative effect was retained, providing useful information for the optimization of the process of EK and quality evaluation of other similar toxic Chinese herbal medicines., Competing Interests: Declaration of Competing Interest All the authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Drugs used for the treatment of diseases associated with chronic inflammation, such as cancer and rheumatoid arthritis have the potential to cause undesirable side-effects, which might result in patients ending treatment prematurely. However, plants are a viable option for the treatment of inflammatory diseases. In this study, we assessed the in vivo and in vitro anti-inflammatory activity, and the antitumor effects of the chloroform extract of Salvia ballotiflora (ECL). The pro-apoptotic effects of ECL in CT26 cells were also determined., Methods: The chloroform extract of Salvia ballotiflora (ECL) was standardized using 19-deoxyicetexone (DEOX) as a phytochemical marker. The anti-inflammatory activity of ECL was determined on acute and chronic inflammatory models using the TPA-induced mouse ear edema assay. The antitumor activity of ECL was evaluated by the subcutaneous inoculation of CT26 cells on the back of Balb/c mice. In vitro CT26 cell death induced by ECL was determined by Annexin V/propidium iodide staining assay using flow cytometry. ECL and the diterpenes isolated from the chloroform extract included 19-deoxyicetexone (DEOX), icetexone (ICT), and 7,20-dihydroanastomosine (DAM), which were tested in LPS-stimulated J774A.1 macrophages to quantify pro-inflammatory cytokine levels. The in vitro anti-arthritic activity of ECL was determined using the bovine serum protein (BSP) denaturation assay., Results: ECL exerted anti-inflammatory activities in acute (84% of inhibition, 2 mg/ear) and chronic models (62.71%, at 100 mg/kg). ECL showed antitumor activity at 200 mg/kg and 300 mg/kg, reducing tumor volume by 30 and 40%, respectively. ECL (9.5 μg/mL) induced in vitro apoptosis in CT26 cells by 29.1% (48 h of treatment) and 93.9% (72 h of treatment). ECL (10 μg/ml) decreased levels of NO (53.7%), pro-inflammatory cytokines IL-6 (44.9%), IL-1β (71.9%), and TNF-α (40.1%), but increased the production of the anti-inflammatory cytokine IL-10 (44%). The diterpenes DEOX, ICT, and DAM decreased levels of NO (38.34, 47.63, 67.15%), IL-6 (57.84, 60.45, 44.26%), and TNF-α (38.90, 31.30, 32.83%), respectively. ECL showed in vitro antiarthritic activity (IC 50  = 482.65 μg/mL)., Conclusions: ECL exhibited anti-inflammatory and anti-tumor activities. Furthermore, the diterpenes DEOX, DAM, and ICT showed anti-inflammatory activity by reducing levels of NO, TNF-α, and IL-6.

SRS27, an andrographolide analogue, had been proven to have therapeutic properties at a dose of 3 mg/kg in both in vitro and in vivo asthma models of our previous study. The present study focuses on the pharmacokinetic and toxicity profile of this compound to provide further evidence for the development of this compound as an anti-asthma agent. A simple pharmacokinetic study was performed in female BALB/c mice to measure blood plasma concentration of the compound at therapeutic dose. At a single dose of 3 mg/kg, SRS27 had a relatively short half-life but was able to achieve a concentration range of 13-19 μM that is related to its in vitro bioactivities. With regard to toxicity profile, SRS27 appears to be safe, as no histopathological changes were observed in the liver, kidneys and ovaries of SRS27-treated female BALB/c mice. In addition, there was no significant change in the mean body weight and organ weight of the animals in the SRS27-treated groups compared with the vehicle-treated control group at the end of the treatment. This fully supports the absence of any significant changes in peripheral blood leukocyte counts of SRS27-treated mice. Rewardingly, this acute toxicity study also revealed that SRS27 has a wide therapeutic window as no toxicity symptoms were detected with a dose up to 60 mg/kg daily when tested for 14 days. These results provide strong justification for further investigation of SRS27 as a potential new anti-asthma agent.

As our research focuses on anticancer drugs, a series of novel derivatives of flexicaulin A (FA), an ent-kaurene diterpene, condensed with an aromatic ring were synthesized, and their antiproliferative activities against four human cancer cell lines (TE-1, EC109, MCF-7, and MGC-803) were evaluated. The activities of most of the new compounds were better than those of FA. Compound 2y exhibited the best activity with an IC50 value reaching 0.13 μM against oesophageal cancer cells (EC109 cells). The IC50 values for 2y in normal cells (GES-1 cells and HUVECs) were 0.52 μM and 0.49 μM, respectively. Subsequent mechanistic investigations found that compound 2y can inhibit the proliferation of cancer cells and cell cloning. In addition, 2y could reduce the mitochondrial membrane potential, increase the apoptosis rate, and increase the ROS level in EC109 cells. Moreover, 2y can upregulate the expression of ROS/JNK pathway-related proteins (p-ASK1, p-MKK4, p-JNK, and p-Cjun (ser63)) and pro-apoptotic proteins (Bax, Bad, and Bim). In vivo experiments showed that 2y can inhibit tumour growth in nude mice. The mechanism involves an increase in protein expression in the ROS pathway, leading to changes in apoptosis-related proteins. In addition, compound 2y shows low toxicity. These results indicate that compound 2y holds promising potential as an antiproliferative agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The purpose of this study is to evaluate in vitro and in vivo antitumor efficacy and subacute toxicity of triptolide (TP) prodrug, a conjugate between TP and carboxymethyl chitosan (CC). The CCTP conjugate contained 4∼ wt % TP and displayed excellent aqueous solubility (5 mg/mL) as compared to the native TP (17 μg/mL). In vitro cytotoxicity of CCTP conjugate was evaluated by CCK8 assay against human pancreatic cancer (PC) cell lines, showing comparable the half maximal inhibitory concentration (IC 50 ) values to the parent TP. In a mouse model of PC (BxPC-3), the CCTP conjugate administered orally (at dose levels as low as 0.2 mg TP equivalent/kg) showed comparable efficacy in reducing or eliminating xenograft tumor to the same dose of TP, but exhibited much lower subacute toxicity as seen in body weight loss and hematological toxicity.

Objectives: We aimed to determine the diurnal rhythm of Tripterygium wilfordii (TW) hepatotoxicity and to investigate a potential role of metabolism and pharmacokinetics in generating chronotoxicity., Methods: Hepatotoxicity was determined based on assessment of liver injury after dosing mice with TW at different circadian time points. Circadian clock control of metabolism, pharmacokinetics and hepatotoxicity was investigated using Clock-deficient (Clock -/- ) mice., Key Findings: Hepatotoxicity of TW displayed a significant circadian rhythm (the highest level of toxicity was observed at ZT2 and the lowest level at ZT14). Pharmacokinetic experiments showed that oral gavage of TW at ZT2 generated higher plasma concentrations (and systemic exposure) of triptolide (a toxic constituent) compared with ZT14 dosing. This was accompanied by reduced formation of triptolide metabolites at ZT2. Loss of Clock gene sensitized mice to TW-induced hepatotoxicity and abolished the time-dependency of toxicity that was well correlated with altered metabolism and pharmacokinetics of triptolide. Loss of Clock gene also decreased Cyp3a11 expression in mouse liver and blunted its diurnal rhythm., Conclusions: Tripterygium wilfordii chronotoxicity was associated with diurnal variations in triptolide pharmacokinetics and circadian expression of hepatic Cyp3a11 regulated by circadian clock. Our findings may have implications for improving TW treatment outcome with a chronotherapeutic approach., (© 2020 Royal Pharmaceutical Society.)

Proinflammatory cytokines are potent inhibitors of bile acid nuclear receptors and transporters. Triptolide (TP), an active ingredient of Tripterygium wilfordii Hook. f., exhibits unique efficacy for autoimmune diseases and tumors. While its clinical application is greatly constrained by hepatotoxicity. Therefore, we explored the mechanism of iNKT cells and associated immunoregulators in TP-induced cholestatic liver injury. TP was administered to both female C57BL/6 mice and Jα18 -/- mice. INKT cells released significantly increased Th2 cytokine IL-4 in C57BL/6 mice after TP administration. Blood biochemistry, histopathology and immunohistochemistry demonstrated that TP-induced cholestasis liver injury. In Jα18 -/- mice, cholestatic liver damage was alleviated due to the upregulation of type 2 NKT cells, nuclear receptor FXR, transporter OATP1B2 and CYP450, but also the downregulation of Cxcl10, ICAM-1 and Egr-1. Above results suggested that Th2 cytokines produced by iNKT cells suppressed type 2 NKT cells and promoted the expression of immunoregulatory factors represented by CXCL10, ICAM-1 and Egr-1, which in turn affected cholestasis-related nuclear receptor, transporter and enzymes, thus aggravated cholestatic liver injury. Our research contributes to better understanding of the role of iNKT cells in TP-induced cholestatic liver injury, thereby providing potential therapeutic targets for clinical prevention and treatment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The rhizome of Dioscorea bulbifera L. (DBL) is a popular traditional herb in the treatment of goiters, breast lumps, and tumors. Unfortunately, DBL can give rise to severe hepatotoxicity. More than 100 cases of liver injury, due to the usage of DBL in China, have been reported in the past half-century. The main toxic components of DBL are furanoditerpenoids diosbulbin B (DSB) as well as 8-epidiosbulbin E (EEA). This toxic effect requires metabolic oxidation of the furan ring mediated by cytochrome P450 enzymes, and the P450 3A subfamily is the main enzyme responsible for the reported hepatotoxicity. cis -Enedial intermediates resulting from furan ring oxidation can react with nucleophilic sites of macromolecules, such as protein and DNA, which may trigger the toxicities. This review illustrates the liver injury induced by DBL including metabolic activation of DSB and EEA, the essential components responsible for DBL-induced hepatotoxicity, along with biochemical mechanisms of their toxic actions. It will facilitate the development of approaches to prevent and intervene in liver injury induced by DBL for its safe use in clinical practice.

Objective: To investigate the protective effect of arctiin with anti-inflammatory bioactivity against triptolide-induced nephrotoxicity in rats and explore the underlying mechanism., Methods: Forty SD rats were divided into 4 groups for gastric lavage of normal saline, arctiin (500 mg/kg), triptolide (500 μg/kg), or both arctiin (500 mg/kg) and triptolide (500 μg/kg). Blood samples were collected for analysis of biochemical renal parameters, and the renal tissues were harvested for determining the kidney index and for pathological evaluation with HE staining. In the in vitro experiment, HK-2 cells were treated with arctiin and triptolide either alone or in combination, and the cell viability was determined with MTT assay; the cell morphological changes was observed using laser confocal microscopy, cell apoptosis was detected using flow cytometry, and the expressions of inflammation-related protein expression were detected by Western blotting., Results: In SD rats, arctiin significantly antagonized triptolide-induced elevation of BUN, Scr and kidney index ( P < 0.05) and obviously improved renal tissue damages induced by triptolide including cell swelling, vacuolization and spotty necrosis. Arctiin significantly inhibited triptolide-induced cytotoxicity in HK-2 cells and increased the cell viability at 24 h ( P < 0.05). Arctiin also attenuated triptolide-induced cell morphological changes, decreased cell apoptosis rate ( P < 0.05) and reversed the expressions of IκBα and nuclear p65 ( P < 0.05)., Conclusions: Arctiin can protect the kidney from triptolide-induced damages in rats possibly through the anti-inflammatory pathway.

Triptolide, a major active component of Triptergium wilfordii Hook. f, is used in the treatment of autoimmune disease. However, triptolide is associated with severe adverse reactions, especially hepatotoxicity, which limits its clinical application. To examine the underlying mechanism of triptolide-induced liver injury, a combination of dose- and time-dependent toxic effects, RNA-seq and metabolomics were employed. Triptolide-induced toxicity occurred in a dose- and time-dependent manners and was characterized by apoptosis and not necroptosis. Transcriptomics profiles of the dose-dependent response to triptolide suggested that PI3K/AKT, MAPK, TNFα and p53 signaling pathways were the vital steps in triptolide-induced hepatocyte apoptosis. Metabolomics further revealed that glycerophospholipid, fatty acid, leukotriene, purine and pyrimidine metabolism were the major metabolic alterations after triptolide exposure. Finally, acylcarnitines were identified as potential biomarkers for the early detection of triptolide-induced liver injury., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Triptolide is a multi-functional natural small molecular compound extracted from a traditional Chinese medicinal herb. Triptolide and its derivatives exhibit cytotoxicity through inducing DNA damage, therefore increasing sensitivity to DNA-damage based chemotherapy or radiotherapy in different types of cells. However, the regulatory mechanism of genotoxicity by triptolide, and the loss of genome integrity induced by triptolide are not fully understood. Here, we measured the effects of triptolide on genome integrity in a human fibroblast line HCA2-hTERT using the neutral comet assay. We demonstrated that treating cells with triptolide induced genomic instability in HCA2-hTERT cells. Furthermore, we observed the accumulation of γH2AX foci in triptolide treated cells than control cells at 24 h post ionizing radiation. Further mechanistic studies indicated that triptolide inhibited the enzymatic activity of DNA-PKcs, the critical nonhomologous end joining factor. In vitro kinase activity assays showed that triptolide suppressed the kinase activity of DNA-PKcs and molecular docking also predicted a potential interaction between triptolide and DNA-PKcs. As a consequence, we found that triptolide treatment enhanced the interaction between DNA-PKcs and KU80 and hampered the following recruitment of 53BP1. Altogether, our finding provides a new perspective about the toxicity of triptolide in non-cancer cells and highlights the necessity of taking genome effects of triptolide and its derivatives into consideration in the future clinical and research applications., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Tetracycline (TET) has been widely used in the treatment of Streptococcus suis ( S. suis ) infection. However, it was found that the efficacy of many antibiotics in S. suis decreased significantly, especially tetracycline. In this study, GML-12 (a novel pleuromutilin derivative) was used in combination with TET against 12 S. suis isolates. In the checkerboard assay, the TET/GML-12 combination exhibited synergistic and additive effects against S. suis isolates ( n = 12). In vitro time-killing assays and in vivo therapeutic experiments were used to confirm the synergistic effect of the TET/GML-12 combination against S. suis strains screened based on an FICI ≤ 0.5. In time-killing assays, the TET/GML-12 combination showed a synergistic effect or an additive effect against three isolates with a bacterial reduction of over 2.4-log 10 CFU/mL compared with the most active monotherapy. Additionally, the TET/GML-12 combination displayed potent antimicrobial activity against four isolates in a mouse thigh infection model. These results suggest that the TET/GML-12 combination may be a potential therapeutic strategy for S. suis infection.

Triptolide (TP), the main active ingredient of Tripterygium wilfordii Hook F., has great potential in the treatment of autoimmune diseases. However, it has been found that the side effects of TP involve multiple organs and systems, of which the most serious side effects relate to the kidney. The mechanism of nephrotoxicity caused by TP requires further investigation. In the present study, we integrated proteomic and metabolomic methods to identify proteins and small molecule metabolites associated with TP-induced nephrotoxicity. There was a significant difference ( p value <0.05) in the expression changes of 357 proteins for quantitative proteomics. In addition, high resolution metabolomic data showed significant changes in the levels of 9 metabolites, including hypoxanthine, PC(22:0/18:4), sphingosine, phenylalanine, etc. Finally, based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database for network analysis, it was determined that the 7 differentially expressed proteins were highly correlated with these 9 metabolites. Enrichment analysis revealed that the metabolic pathways involved purine and pyrimidine metabolism, glycerol and phospholipid metabolism, sphingolipid metabolism, and amino acid metabolism. The key target proteins were verified by Western blot technology, and the mechanism of TP-induced nephrotoxicity was further elucidated to provide a basis for safe and rational application.