XinJiaCongRongTuSiZiWan protects triptolide-induced rats from oxidative stress injury via mitophagy mediated PINK1/Parkin signaling pathway.

PURPOSE: XinJiaCongRongTuSiZiWan (XJCRTSZW) is a traditional Chinese medicine compound for invigorating the kidney, nourishing blood, and promoting blood circulation. This study aimed to explore the effect of XJCRTSZW on triptolide (TP)-induced oxidative stress injury. METHODS: Adult female Sprague-Dawley rats and human ovarian granulosa cell lines were treated with TP and XJCRTSZW. Hematoxylin and eosin staining, enzyme-linked immunosorbent assay, flow cytometry, CCK-8, JC-1 staining, transmission electron microscopy, reverse transcription-quantitative polymerase chain reaction, and Western blotting were performed in this study. RESULTS: XJCRTSZW treatment observably ameliorated the TP-induced pathological symptoms. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of estradiol, anti-Mullerian hormone, progesterone, superoxide dismutase, ATP content, mitochondrial membrane potential, p62, and Hsp60 mRNA, and protein levels in vivo and in vitro (p < 0.05). However, TP-induced elevation of follicle stimulating hormone and luteinizing hormone concentrations, malondialdehyde levels, reactive oxygen species levels, apoptosis rate, mitophagy, and the mRNA and protein expressions of LC3-II/LC3-I, PTEN-induced kinase 1 (PINK1), and Parkin were decreased (p < 0.05). In addition, XJCRTSZW treatment markedly increased cell viability in vitro (p < 0.05). CONCLUSIONS: XJCRTSZW protects TP-induced rats from oxidative stress injury via the mitophagy-mediated PINK1/Parkin pathway.

Antinociceptive and anti-inflammatory activity of extracts and α-pyrones isolated from Cantinoa stricta.

This study evaluated the composition and the antinociceptive and anti-inflammatory activity of the crude extracts and two isolated compounds, anamarine (ANA) and 10-epi-olguine (eOL), obtained from the leaves of Cantinoa stricta (Lamiaceae). Crude ethanolic extract (EEt) and dichloromethane extract (DCM), selected based on NMR data, were submitted to pharmacological tests in male Swiss mice. The oral administration of EEt and DCM significantly reduced the second phase of formalin-induced nociception (60%), lipopolysaccharide (LPS)-induced mechanical hyperalgesia (90%), and carrageenan (Cg)-induced edema (25%). ANA and eOL, the major compounds in EEt and DCM extracts, administered orally or locally (in the paw), also reduced the LPS-induced mechanical hyperalgesia (Oral ID50 1.9 and 3.9 mg/kg; Local ID50 93.4 and 677.3 ng, respectively) without changing the thermal acute nociception or the motor performance of the animals. Local administration of ANA and eOL also reduced Cg-induced edema (40 and 23%, respectively). These isolated compounds did not change the mechanical hyperalgesia induced by tumor necrosis factor-α, interleukin-1ß, prostaglandin E2, dibutyryl cyclic AMP, or forskolin but reversed the hyperalgesia induced by dopamine, epinephrine, and phorbol 12-myristate 13-acetate. The hyperalgesia induced by epinephrine was reversed in male but not in female mice, in which this response is not dependent on protein kinase C (PKC). These results suggest that C. stricta extracts possess antinociceptive and anti-inflammatory activity which is related to the presence of ANA and eOL. Differently from the known analgesics, these substances seem to exert their action mainly interfering with the sympathetic component of pain, possibly with PKC.

Group 1 innate lymphoid cell activation via recognition of NKG2D and liver resident macrophage MULT-1: Collaborated roles in triptolide induced hepatic immunotoxicity in mice.

Triptolide (TP) is the major bioactive component of traditional Chinese medicine Tripterygium wilfordii Hook. F., a traditional Chinese medicinal plant categorized within the Tripterygium genus of the Celastraceae family. It is recognized for its therapeutic potential in addressing a multitude of diseases. Nonetheless, TP is known to exhibit multi-organ toxicity, notably hepatotoxicity, which poses a significant concern for the well-being of patients undergoing treatment. The precise mechanisms responsible for TP-induced hepatotoxicity remain unresolved. In our previous investigation, it was determined that TP induces heightened hepatic responsiveness to exogenous lipopolysaccharide (LPS). Additionally, natural killer (NK) cells were identified as a crucial effector responsible for mediating hepatocellular damage in this context. However, associated activating receptors and the underlying mechanisms governing NK cell represented innate lymphoid cell (ILC) activation remained subjects of inquiry and were not yet investigated. Herein, activating receptor Killer cell lectin like receptor K1 (NKG2D) of group 1 ILCs was specifically upregulated in TP- and LPS-induced acute liver failure (ALF), and in vivo blockade of NKG2D significantly reduced group 1 ILC mediated cytotoxicity and mitigated TP- and LPS-induced ALF. NKG2D ligand UL16-binding protein-like transcript 1 (MULT-1) was found upregulated in liver resident macrophages (LRMs) after TP administration, and LRMs did exhibit NK cell activating effect. Furthermore, M1 polarization of LRMs cells was observed, along with an elevation in intracellular tumor necrosis factor (TNF)-α levels. In vivo neutralization of TNF-α significantly alleviated TP- and LPS-induced ALF. In conclusion, the collaborative role of group 1 ILCs and LRMs in mediating hepatotoxicity was confirmed in TP- and LPS-induced ALF. TP-induced MULT-1 expression in LRMs was the crucial mechanism in the activation of group 1 ILCs via MULT-1-NKG2D signal upon LPS stimulation, emphasizing the importance of infection control after TP administration.

Triptolide induces apoptosis and cytoprotective autophagy by ROS accumulation via directly targeting peroxiredoxin 2 in gastric cancer cells.

Triptolide, a natural bioactive compound derived from herbal medicine Tripterygium wilfordii, has multiple biological activities including anti-cancer effect, which is being tested in clinical trials for treating cancers. However, the exact mechanism by which Triptolide exerts its cytotoxic effects, particularly its specific protein targets, remains unclear. Here, we show that Triptolide effectively induces cytotoxicity in gastric cancer cells by increasing reactive oxygen species (ROS) levels. Further investigations reveal that ROS accumulation contributes to the induction of Endoplasmic Reticulum (ER) stress, and subsequently autophagy induction in response to Triptolide. Meanwhile, this autophagy is cytoprotective. Interestingly, through activity-based protein profiling (ABPP) approach, we identify peroxiredoxins-2 (PRDX2), a component of the key enzyme systems that act in the defense against oxidative stress and protect cells against hydroperoxides, as direct binding target of Triptolide. By covalently binding to PRDX2 to inhibit its antioxidant activity, Triptolide increases ROS levels. Moreover, overexpression of PRDX2 inhibits and knockdown of the expression of PRDX2 increases Triptolide-induced apoptosis. Collectively, these results indicate PRDX2 as a direct target of Triptolides for inducing apoptosis. Our results not only provide novel insight into the underlying mechanisms of Triptolide-induced cytotoxic effects, but also indicate PRDX2 as a promising potential therapeutic target for developing anti-gastric cancer agents.

Cyperus rotundus L.: Invasive weed plant with insecticidal potential against Aphis craccivora Koch and Planococcus lilacinus (Cockerell).

Cyperus rotundus L. is a widely distributed invasive weed plant with vast traditional medicinal uses. Herein, the methanolic root extract of C. rotundus and its fractions (n-hexane, chloroform, n-butanol, and aqueous) were evaluated for insecticidal activity against nymphs of Aphis craccivora Koch and crawlers of Planococcus lilacinus (Cockerell) to find promising lead (s). In contact topical assay, among extract/fractions, n-hexane fraction exhibited more toxicity against A. craccivora (LD50 = 1.12 µg/insect) and P. lilacinus (LD50 = 0.94 µg/insect). The chemical analysis of n-hexane fraction revealed a volatile composition similar to that of the essential oil (EO) of C. rotundus roots. Hence, EO was extracted using water and deep eutectic solvents (DESs) as cosolvent, which revealed enhancement in EO yield (from 0.28 to 0.46% w/w) on implementing DESs. A total of 35 diverse volatile metabolites were identified in all EO samples, accounting for 85.0 to 91.8% of chemical composition, having cyperotundone, cyperene mustakone, isolongifolen-5-one, boronia butenal as major constituents. The EO obtained with DES-7 [choline chloride: ethylene glycol (1:4)] and DES-6 [choline chloride: lactic acid (1:3)] were found effective against A. craccivora (LD50 = 0.62-0.87 µg/insect) and P. lilacinus (LD50= 0.59-0.67 µg/insect) after 96 h. NMR analysis of EO revealed cyperotundone as a major compound, which was isolated along with cyperene and cyperene epoxide. All the molecules were found effective against P. lilacinus, whereas against A. craccivora cyperotundone, cyperene and cyperene epoxide showed promising toxicity (LD50 = 0.74-0.86 µg/insect). Extract/fractions, EO, and isolated molecules showed a significant reproductive inhibition rate of A. craccivora at higher concentrations. All the tested concentrations of cyperotundone showed significant inhibition of acetylcholinesterase (AChE) and glutathione-S-transferase (GST) in A. craccivora and P. lilacinus. Based upon the present study, C. rotundus can be recommended to control targeted insects in the greenhouse/field conditions after performing bio-efficacy and phytotoxicity studies.

Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer.

Paclitaxel resistance is associated with a poor prognosis in non-small cell lung cancer (NSCLC) patients, and currently, there is no promising drug for paclitaxel resistance. In this study, we investigated the molecular mechanisms underlying the chemoresistance in human NSCLC-derived cell lines. We constructed paclitaxel-resistant NSCLC cell lines (A549/PR and H460/PR) by long-term exposure to paclitaxel. We found that triptolide, a diterpenoid epoxide isolated from the Chinese medicinal herb Tripterygium wilfordii Hook F, effectively enhanced the sensitivity of paclitaxel-resistant cells to paclitaxel by reducing ABCB1 expression in vivo and in vitro. Through high-throughput sequencing, we identified the SHH-initiated Hedgehog signaling pathway playing an important role in this process. We demonstrated that triptolide directly bound to HNF1A, one of the transcription factors of SHH, and inhibited HNF1A/SHH expression, ensuing in attenuation of Hedgehog signaling. In NSCLC tumor tissue microarrays and cancer network databases, we found a positive correlation between HNF1A and SHH expression. Our results illuminate a novel molecular mechanism through which triptolide targets and inhibits HNF1A, thereby impeding the activation of the Hedgehog signaling pathway and reducing the expression of ABCB1. This study suggests the potential clinical application of triptolide and provides promising prospects in targeting the HNF1A/SHH pathway as a therapeutic strategy for NSCLC patients with paclitaxel resistance. Schematic diagram showing that triptolide overcomes paclitaxel resistance by mediating inhibition of the HNF1A/SHH/ABCB1 axis.

Three-membered ring formation catalyzed by α-ketoglutarate-dependent nonheme iron enzymes.

Epoxides, aziridines, and cyclopropanes are found in various medicinal natural products, including polyketides, terpenes, peptides, and alkaloids. Many classes of biosynthetic enzymes are involved in constructing these ring structures during their biosynthesis. This review summarizes our current knowledge regarding how α-ketoglutarate-dependent nonheme iron enzymes catalyze the formation of epoxides, aziridines, and cyclopropanes in nature, with a focus on enzyme mechanisms.

Triptolide inhibits the proinflammatory potential of myeloid-derived suppressor cells via reducing Arginase-1 in rheumatoid arthritis.

Triptolide (TPT) is widely used in the treatment of rheumatoid arthritis (RA). However, its regulatory mechanisms are not fully understood. This study demonstrated that Myeloid-derived suppressor cells (MDSCs) were expanded in both RA patients and arthritic mice. The frequency of MDSCs was correlated with RA disease severity and T helper 17 (Th17) responses. MDSCs from RA patients promoted the polarization of Th17 cells in vitro, which could be substantially attenuated by blocking arginase-1 (Arg-1). TPT inhibited the differentiation of MDSCs, particularly the monocytic MDSCs (M-MDSCs) subsets, as well as the expression of Arg-1 in a dose dependent manner. Alongside, TPT treatment reduced the potential of MDSCs to promote the polarization of IL-17+ T cell in vitro. Consistently, TPT immunotherapy alleviated adjuvant-induced arthritis (AIA) in a mice model, and reduced the frequency of MDSCs, M-MDSCs and IL-17+ T cells simultaneously. The presented data suggest a pathogenic role of MDSCs in RA and may function as a novel and effective therapeutic target for TPT in RA.

Icariside II prevents kidney fibrosis development in chronic kidney disease by promoting fatty acid oxidation.

Renal interstitial fibrosis (RIF) is the main pathological basis for the progression of chronic kidney disease (CKD), however, effective interventions are limited. Here, we investigated the effect of Icariside II (ICA-II) on RIF and explored the underlying mechanisms. Rats receiving 5/6 ablation and infarction (A/I) surgery were gavaged with ICA-II (5 or 10 mg/kg) for 8 weeks. In vitro, TGF-ß1-stimulated NRK-52E cells were treated with ICA-II and (or) oleic acid, etomoxir, ranolazine, fenofibrate, and GW6471. The effects of ICA-II on RIF, fatty acid oxidation, lipid deposition, and mitochondrial function were determined by immunoblotting, Oil red O staining, colorimetric, and fluorometric assays. Using adeno-associated virus injection and co-culture methods, we further determined mechanisms of ICA-II anti-RIF. ICA-II ameliorated the fibrotic responses in vivo and in vitro. RNA-seq analysis indicated that ICA-II regulated fatty acid degradation and PPAR pathway in 5/6 (A/I) kidneys. ICA-II attenuated lipid accumulation and up-regulated expression of PPARα, CPT-1α, Acaa2, and Acadsb proteins in vivo and in vitro. Compared to ICA-II treatment, ICA-II combined with Etomoxir exacerbated mitochondrial dysfunction and fibrotic responses in TGF-ß-treated NRK-52E cells. Importantly, we determined that ICA-II improved lipid metabolism, fatty acid oxidation, mitochondrial function, and RIF by restoring PPARα. Co-culture revealed that ICA-II decreased the expression of Fibronectin, Collagen-I, α-SMA, and PCNA proteins in NRK-49F cells by restoring PPARα of renal tubular cells. ICA-II may serve as a promising therapeutic agent for RIF in 5/6 (A/I) rats, which may be important for the prevention and treatment of CKD.

Pharmacological activity and clinical progress of Triptolide and its derivatives LLDT-8, PG490-88Na, and Minnelide: a narrative review.

Triptolide, a compound isolated from a Chinese medicinal herb, has potent antitumor, immunosuppressive, and anti-inflammatory properties. Due to its interesting structural features and diverse pharmacological activities, it has attracted great interest by the Society of Organic Chemistry and Pharmaceutical Chemistry. However, its clinical potential is greatly hampered by limited aqueous solubility and oral bioavailability, and multi-organ toxicity. In recent years, various derivatives of Triptolide have made varying degrees of progress in the treatment of inflammatory diseases, autoimmune diseases, and cancer. The most researched and potentially clinically valuable of them were (5R)-5-hydroxytriptolide (LLDT-8), PG490-88Na (F6008), and Minnelide. In this review, we provide an overview of the advancements made in triptolide and several of its derivatives' biological activity, mechanisms of action, and clinical development. We also summarized some prospects for the future development of triptolide and its derivatives. It is hoped to contribute to a better understanding of the progress in this field, make constructive suggestions for further studies of Triptolide, and provide a theoretical reference for the rational development of new drugs.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide.

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

Cardioprotective mechanisms of cytochrome P450 derived oxylipins from ω-3 and ω-6 PUFAs.

The seminal discovery that cytochrome P450 enzymes (CYPs) can oxidize polyunsaturated fatty acids (PUFAs) sparked a new area of research aimed at discovering the role of these metabolites in cardiac physiology and pathophysiology. CYPs metabolize arachidonic acid, an ω-6 PUFA, to alcohols and epoxides with the latter providing cardioprotection following myocardial infarction, hypertrophy, and diabetes-induced cardiomyopathy through their anti-inflammatory, vasodilatory and antioxidant properties. Despite their protective properties, the use of EETs as therapeutic agents is hampered mainly by their rapid hydrolysis to less active vicinal diols by soluble epoxide hydrolase (sEH). Several approaches have been investigated to prolong EET signaling effects using small molecule sEH inhibitors, chemically and biologically stable analogs of EETs and more recently, through the development of an sEH vaccine. Alternatively, research investigating the cardioprotective outcomes of ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), mainly focused on dietary intake or supplementation studies. EPA and DHA have overlapping but distinct effects on myocardial function and merit separate studies to fully understand their mechanism of cardiac protection. In contrast to EETs, relatively fewer studies examined the protective mechanisms of EPA and DHA derived epoxides to determine if some protective effects are in part due to the CYP mediated downstream metabolites. The actions of CYPs on PUFAs generate potent oxylipins utilizing diverse cardioprotective mechanisms and the extent of their full potential will be important for the future development of therapeutics to prevent or treat cardiovascular disease.

A comprehensive review on celastrol, triptolide and triptonide: Insights on their pharmacological activity, toxicity, combination therapy, new dosage form and novel drug delivery routes.

Celastrol, triptolide and triptonide are the most significant active ingredients of Tripterygium wilfordii Hook F (TWHF). In 2007, the 'Cell' journal ranked celastrol, triptolide, artemisinin, capsaicin and curcumin as the five natural drugs that can be developed into modern medicinal compounds. In this review, we collected relevant data from the Web of Science, PubMed and China Knowledge Resource Integrated databases. Some information was also acquired from government reports and conference papers. Celastrol, triptolide and triptonide have potent pharmacological activity and evident anti-cancer, anti-tumor, anti-obesity and anti-diabetes effects. Because these compounds have demonstrated unique therapeutic potential for acute and chronic inflammation, brain injury, vascular diseases, immune diseases, renal system diseases, bone diseases and cardiac diseases, they can be used as effective drugs in clinical practice in the future. However, celastrol, triptolide and triptonide have certain toxic effects on the liver, kidney, cholangiocyte heart, ear and reproductive system. These shortcomings limit their clinical application. Suitable combination therapy, new dosage forms and new routes of administration can effectively reduce toxicity and increase the effect. In recent years, the development of different targeted drug delivery formulations and administration routes of celastrol and triptolide to overcome their toxic effects and maximise their efficacy has become a major focus of research. However, in-depth investigation is required to elucidate the mechanisms of action of celastrol, triptolide and triptonide, and more clinical trials are required to assess the safety and clinical value of these compounds.

Epoxidation of Methyl Esters as Valuable Biomolecules: Monitoring of Reaction.

The paper is focused on the epoxidation of methyl esters prepared from oil crops with various profiles of higher fatty acids, especially unsaturated, which are mainly contained in the non-edible linseed and Camelina sativa oil (second generation). The novelty consists in the separation and identification of all products with oxirane ring formed through a reaction and in the determination of time course. Through the epoxidation, many intermediates and final products were formed, i.e., epoxides with different number and/or different position of oxirane rings in carbon chain. For the determination, three main methods (infrared spectroscopy, high-pressure liquid chromatography and gas chromatography with mass spectrometry) were applied. Only gas chromatography enables the separation of individual epoxides, which were identified on the base of the mass spectra, molecule ion and time course of products. The determination of intermediates enables: (i) control of the epoxidation process, (ii) determination of the mixture of epoxides in detail and so the calculation of selectivity of each product. Therefore, the epoxidation will be more environmentally friendly especially for advanced applications of non-edible oil crops containing high amounts of unsaturated fatty acids.

Occurrence of glycidyl esters in infant formula products on the Canadian market between 2015 and 2019.

Glycidyl fatty acid esters (GEs) are processing contaminants formed during refining steps of vegetable oils. 'In vivo' hydrolysis of GEs releases potentially carcinogenic and genotoxic glycidol (2,3-epoxy-1-propanol). Occurrence of GEs in vegetable oils used for infant formula manufacturing may pose a potential health concern for formula-fed infants. Refined oils are commonly used as the main fat ingredient in formula manufacturing. For this study, different infant formula products (powders, concentrates and ready-to-feed formula products) were purchased and analysed in 2015 (35 samples) and 2019 (33 samples). Seven individual GEs were analysed by LC-MS/MS via direct approach by stable isotope dilution analysis, and total bound glycidol concentrations were calculated. Concentrations of bound glycidol in reconstituted formula reached maxima of 40.3 ng/g in the 2015 samples and 31.5 ng/g in the samples collected in 2019, with respective means of 8.7 ng/g and 6.7 ng/g. The analysed bound glycidol concentrations are comparable with concentration ranges from other studies, but are higher than observed in studies from the European market. Temporal trend data show a reduction of bound glycidol concentrations in 2019. GE concentrations were compared across different manufacturers.

Stereoselective effects of chiral epoxiconazole on the metabolomic and lipidomic profiling of leek.

Epoxiconazole is widely used in agriculture to control plant diseases; however, its effect on the nutritional quality of crops is poorly understood. In the present study, the stereoselective effects of epoxiconazole and its enantiomers on leek metabolites and lipids were clarified using metabolomics and lipidomics based on ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Multivariate analysis revealed significant differences in 123 compounds of leek exposed to chiral epoxiconazole compared to the control group. The order of the degree of influence was (+)-epoxiconazole > racemic-epoxiconazole>(-)-epoxiconazole, indicating significant stereoselectivity. The differential expression of metabolites indicated the activation of stress defense systems, including the antioxidant defense system and signaling pathways and phenylpropanoid metabolism. Alterations in the levels of compounds associated with nutritional quality, such as amino acids, vitamins, phenylpropanoids, flavonoids and lipids indicated changes in the nutritional quality of leek. In general, the nutritional quality of leek decreased after exposure to epoxiconazole.

[Protective effect and mechanism of Astragalus membranaceus and Angelica sinensis compatibility against triptolide-induced hepatotoxicity by regulating Keap1/Nrf2/PGC-1α].

This paper aims to investigate the protective effect and mechanism of Astragalus membranaceus and Angelica sinensis before and after compatibility against triptolide(TP)-induced hepatotoxicity. The experiment was divided into a blank group, model group, Astragalus membranaceus group, Angelica sinensis group, and compatibility groups with Astragalus membranaceus/Angelica sinensis ratio of 1∶1, 2∶1, and 5∶1. TP-induced hepatotoxicity model was established, and corresponding drug intervention was carried out. The levels of alanine transaminase(ALT), aspartate transaminase(AST), and alkaline phosphatase(ALP) in serum were detected. Pathological injuries of livers were detected by hematoxylin-eosin(HE) staining. The levels of malondialdehyde(MDA), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and reduced glutathione(GSH) in the liver were measured. Wes-tern blot method was used to detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(Keap1), peroxisome proliferator-activated receptor gamma, coactivator-1 alpha(PGC-1α), heme oxygenase-1(HO-1), and NAD(P)H quinone dehydrogenase 1(NQO1) in livers. Immunofluorescence was used to detect the expression of Nrf2 and PGC-1α in livers. The results indicated that Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 could significantly reduce the levels of serum AST, ALT, and ALP, improve the pathological damage of liver tissue, increase the levels of GSH and GSH-Px, and reduce the content of MDA in liver tissue. Astragalus membranaceus/Angelica sinensis ratio of 1∶1 and 2∶1 could significantly improve the level of SOD. Astragalus membranaceus and Angelica sinensis before and after compatibility significantly increased the protein expression of HO-1 and NQO1, improved the protein expression of Nrf2 and PGC-1α, and decreased the protein expression of Keap1 in liver tissue. The above results confirmed that the compatibility of Astragalus membranaceus and Angelica sinensis had antioxidant effects by re-gulating Keap1/Nrf2/PGC-1α, and the Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 had stronger antioxidant effect and significantly reduced TP-induced hepatoto-xicity.

"Click" preparation of a chiral macrocycle-based stationary phase for both normal-phase and reversed-phase high performance liquid chromatography enantioseparation.

Chiral polyimine macrocycles (CPMs) constitute a new family of organic macrocycles that have defined cavities, rigid shapes, inherent chirality and multiple cooperative binding sites, and have shown great potential in diverse areas. However, the application of CPMs for high performance liquid chromatography (HPLC) enantioseparation has rarely been reported. In this work, a novel chiral stationary phase (CSP) for HPLC was prepared by chemical bonding of a CPM (C54H72N6O3) onto thiolated silica via thiol-ene click reaction. The CSP exhibited good enantioselectivity in both normal- and reversed-phase HPLC. Chiral compounds included alcohols, diols, ketones, organic acids, esters, ethers, amines, and epoxides were enantioseparated on the column in normal-phase mode (17 compounds) and reversed-phase mode (20 compounds). Importantly, broader chiral resolution was observed with the column than that obtained using our previously studied chiral macrocycle H3L-based column, indicating the potential to significantly improve and broaden applicability of this novel macrocycle-type CSPs. Moreover, the CSP exhibited good complementary enantioseparation to Chiralpak AD-H and Chiralcel OD-H columns, enabling separation of some racemates that could not be separated by the two popular chiral HPLC columns. In addition, the fabricated column exhibited good stability and reproducibility. The relative standard deviations (RSDs) (n = 5) of retention time and resolution after multiple injections were < 0.20 % and < 0.39 %, respectively. The results demonstrated the great potential of this type of CPM for HPLC separation of enantiomers.

Black Raspberry Extract Enhances Glutathione Conjugation of the Fjord-Region Diol Epoxide Derived from the Tobacco Carcinogen Dibenzo[def,p]chrysene in Human Oral Cells.

In a series of previous studies we reported that black raspberry (BRB) powder inhibits dibenzo[a,l]pyrene (DBP)-induced DNA damage, mutagenesis, and oral squamous cell carcinoma (OSCC) development in mice. In the present study, using human oral leukoplakia (MSK-Leuk1) and squamous cell carcinoma (SCC1483) cells, we tested the hypothesis that BRB extract (BRBE) will enhance the synthesis of glutathione (GSH) and in turn increase GSH conjugation of the fjord-region DBP diol epoxide (DBPDE) derived from DBP leading to inhibition of DBP-induced DNA damage. The syntheses of DBPDE-GSH conjugate, DBPDE-dA adduct, and the corresponding isotope-labeled internal standards were performed; LC-MS/MS methods were used for their quantification. BRBE significantly (p < 0.05) increased cellular GSH by 31% and 13% at 6 and 24 h, respectively, in OSCC cells; in MSK-LeuK1 cells, the levels of GSH significantly (p < 0.05) increased by 55% and 22%, at 1 and 6 h. Since BRBE significantly enhanced the synthesis of GSH in both cell types, subsequent experiments were performed in MSK-Leuk1 cells. Western blot analysis was performed to determine the types of proteins involved in the synthesis of GSH. BRBE significantly (p < 0.05) increased the protein expression (2.5-fold) of the glutamate-cysteine ligase catalytic subunit (GCLC) but had no effect on the glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthetase (GSS). LC-MS/MS analysis showed that pretreatment of cells with BRBE followed by DBPDE significantly (p < 0.05) increased the levels of DBPDE-GSH conjugate (2.5-fold) and decreased DNA damage by 74% measured by assessing levels of DBPDE-dA adduct formation. Collectively, the results of this in vitro study clearly support our hypothesis, and the LC-MS/MS methods developed in the present study will be highly useful in testing the same hypothesis initially in our mouse model and ultimately in smokers.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes.

BACKGROUND: Rheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PURPOSE: This study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. METHODS: We identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. RESULTS: Our results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. CONCLUSION: Our research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.