Influence of Zhuanggu Guanjie Pill on Seven Cytochrome P450 Enzymes Based on Probe Cocktail and Pharmacokinetics Approaches.

BACKGROUND: The use of herbal medicines has tremendously increased over the past few decades. Case reports and controlled clinical investigations of herbal-drug interactions have been reported. Since Cytochrome P450 (CYP) enzymes play an important role in drug interactions. The evaluation of the influence of herbal medicines on the activities of CYPs is beneficial to promote scientific and rational clinical use of herbal medicines. OBJECTIVE: Herein, we aimed to develop and validate a method to simultaneously quantify seven CYP cocktail probe drugs consisting of phenacetin (PNC), bupropion (BPP), losartan potassium (LK), omeprazole (OMP), dextromethorphan (DM), chlorzoxazone (CZZ) and midazolam (MDZ) and their respective metabolites in a single acquisition run and use this method to evaluate the influence of Zhuanggu Guanjie Pill (ZGGJP) on seven CYPs. METHODS: A cost-effective and simple UHPLC-(±)ESI-MS/MS method for simultaneous determination of seven probe drugs and metabolites in rat plasma was developed and validated. Male and female rats were randomly divided into three groups and treated with 1.2 g/kg/d ZGGJP, 5 g/kg/d ZGGJP and 0.5% CMC-Na for 14 consecutive days. After 24 h of the last administration, all rats were administrated orally with probe drugs. The influence of ZGGJP on the CYPs was carried out by comparing the metabolic ratio (Cmax, AUC0-t) of metabolites/probe drugs in rats. RESULTS: The calibration curves were linear, with correlation coefficient > 0.99 for seven probe drugs and their corresponding metabolites. Intra- and inter-day precisions were not greater than 15% RSD and the accuracies were within ±15% of nominal concentrations. The ZGGJP showed significant inductive effect on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats. CONCLUSION: ZGGJP had inductive effects on CYP1A2, CYP2B6, CYP2C9 and CYP3A in male and female rats.

iTRAQ Quantitative Proteomic Analysis of Different Expressed Proteins and Signal Pathways in Bakuchiol-Induced Hepatotoxicity.

Bakuchiol (BAK) is an abundant natural compound. BAK has been reported to have several biological activities such as anticancer, antiaging, anti-inflammatory, and prevention of bone loss. However, it causes hepatotoxicity, the mechanism of which is not known. In this study, we explored the mechanism of BAK hepatotoxicity by treating rats with 52.5 mg/kg and 262.5 mg/kg of BAK, administered continuously for 6 weeks. We examined the liver pathology and biochemical composition of bile to determine toxicity. Mechanisms of BAK hepatotoxicity were analyzed based on relative and absolute quantification (iTRAQ) protein equivalent signatures and validated in vitro using LO2 cells. iTRAQ analysis revealed 281 differentially expressed proteins (DEPs) in liver tissue of the BAK-treated group, of which 215 were upregulated, and 66 were downregulated. GO and KEGG enrichment analysis revealed that bile secretion, lipid metabolism, and cytochrome P450 signaling pathways were enriched in DEPs. Among them, peroxisome proliferator-activated receptor α (PPARα), farnesoid X receptor (FXR), and cholesterol 7α-hydroxylase (CYP7a1) were closely associated with the development and progression of BAK-induced hepatic metabolic dysfunction and abnormal bile metabolism. This study shows that BAK can induce hepatotoxicity through multiple signaling pathways.

Jujuboside A ameliorates tubulointerstitial fibrosis in diabetic mice through down-regulating the YY1/TGF-ß1 signaling pathway.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus, which is characterized in renal tubulointerstitial fibrosis (TIF). The current study was designed to investigate the protective effect of Jujuboside A (Ju A) on TIF in type 2 diabetes (T2DM) mice, and explore its underlying anti-fibrosis mechanism. A mouse T2DM model was established using high fat diet (HFD) feeding combined with intraperitoneal injection of streptozotocin (STZ). Then, diabetic mice were treated with Ju A (10, 20 and 40 mg·kg-1·d-1, i.g.) for 12 weeks. Results showed that administration of Ju A not only down-regulated fasting blood glucose (FBG) levels, but also improved hyperlipidemia and renal function in diabetic mice. Moreover, the reduced ECM accumulation was observed in the renal cortex of Ju A treated diabetic mice, while the TIF progression was also attenuated by Ju A through blocking the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs). Further mechanism studies showed that Ju A treatment effectively down-regulated the protein expression and subsequent nuclear translocation of Yin Yang 1 (YY1) in the renal cortex of diabetic mice, and reduced the levels of transforming growth factor-ß1 (TGF-ß1) in the serum and renal cortex of Ju A treated mice. According to invitro studies, the up-regulated YY1/TGF-ß1 signaling pathway was restored by Ju A in high glucose (HG) cultured HK-2 cells. Taken together, these findings demonstrated that Ju A can ameliorate the TIF of DN through down-regulating the YY1/TGF-ß1 signaling pathway.

Association between serum vitamin D status and the anti-seizure treatment in Chinese children with epilepsy.

Objective: To compare the serum 25-OH-VitD levels, the major marker of vitamin D (VitD) status, between healthy children and children with epilepsy before initiation of and during anti-seizure medications (ASMs) treatment and to evaluate the potential influence factors on 25-OH-VitD levels. Another major aim was to assess the potential role of VitD supplementation. Methods: For comparison, we finally enrolled and collected data from 6,338 healthy children presenting to Health Care Department and 648 children visiting primary care pediatricians with symptoms of epilepsy in Children's Hospital of Nanjing Medical University from January 2019 to June 2021. The demographic and biochemical characteristics of each child were extracted from the hospital information system. Results: Serum 25-OH-VitD levels in 648 children with epilepsy were significantly lower than those of 6,338 healthy children (P < 0.0001), and the percentage of VitD insufficiency and deficiency status in pediatric patients was 49.19%. Of note, the serum 25-OH-VitD levels in children with newly diagnosed epilepsy before receiving any ASMs treatment were also significantly lower than those in healthy controls. Interestingly, ASMs therapy, alone or in combination, did not consistently reduce baseline serum VitD levels in children with epilepsy. The lower serum VitD levels in pediatric patients than those in healthy children might be related to the disease itself, rather than the ASMs treatment. As expected, VitD supplementation substantially increased the serum 25-OH-VitD levels (P < 0.0001). More critically, children with epilepsy receiving VitD supplementation achieved good seizure control in our study. Significance: In this retrospective study, the childhood epilepsy before initiation of and during ASMs treatment decreased the serum 25-OH-VitD concentrations, suggesting a clear association between epileptic disease and the risk of VitD deficiency. ASMs coadministration and long-term valproic acid treatment did not worse VitD-deficiency status, but in the small group receiving VitD supplementation, there was a significant improvement in reduction of seizure frequency. Therefore, pediatric clinicians are urged to raise public awareness of epilepsy-associated VitD deficiency.

[Determination of concentrations and toxicokinetics of triptolide in plasma and liver of mice by UHPLC-MS/MS].

This study aims to establish an ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) method for determining the concentrations of triptolide(TP) in plasma and liver, and to explore the toxicokinetics of TP and the relationship between TP exposure and liver injury in C57 BL/6 mice, so as to provide reference for dissecting the toxicity mechanism of TP. The liquid chromatography was conducted with ZORBAX SB-C_(18) column(3.0 mm×100 mm, 3.5 µm) and the mobile phase of methanol-0.05 mmol·L~(-1) ammonium acetate. Electrospray ionization(ESI) and multiple reaction monitoring(MRM) mode were employed for mass spectrometry. After oral administration of TP(toxic dose 600 µg·kg~(-1)), the blood and liver tissues of the C57 BL/6 mice were collected at different time points to measure the TP concentrations in plasma and liver tissues. Furthermore, the blood biochemical indexes, including alkaline phosphatase(ALP), alanine aminotransferase(ALT), aspartate aminotransferase(AST), and total bile acid(TBA), were determined. After being processed by DAS 2.0, the experiment data showed that the TP in mice had the toxicokinetic parameters of T_(max)=5 min, C_(max)=14.38 ng·mL~(-1), t_(1/2)=0.76 h, AUC_(0-t)=5.63 h·ng·mL~(-1), MRT_(0-t)=0.56 h, and CL_(Z/F)=103.19 L·h~(-1)·kg~(-1). The trend of TP concentration in mouse liver tissue was consistent with that in plasma. The concentration of TP peaked at the time point of 5 min and then decreased until TP was completely metabolized. The plasma biochemical indexes(ALT, AST, ALP, and TBA) showed no significant changes within 3 h after TP administration. TP had high clearance rate and short residence time and did not significantly increase the blood biochemical indexes in mice. The results suggested that the exposure amount of free TP in vivo cannot directly cause liver injury, which might be caused by the binding of TP to some substances or the stimulation of inflammation and immune response.

Triptolide Induces Liver Injury by Regulating Macrophage Recruitment and Polarization via the Nrf2 Signaling Pathway.

Triptolide (TP) has limited usage in clinical practice due to its side effects and toxicity, especially liver injury. Hepatic macrophages, key player of liver innate immunity, were found to be recruited and activated by TP in our previous study. The nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway exerts a protective role in TP-induced liver damage, but its effect on the functions of hepatic macrophage has not been elucidated. Here, we determined whether TP can regulate the recruitment and polarization of hepatic macrophages by inhibiting Nrf2 signaling cascade. Our results demonstrated that TP inhibited the Nrf2 signaling pathway in hepatic macrophages. The changes in hepatic macrophages were responsible for the increased susceptibility toward inflammatory stimuli, and hence, TP pretreatment could induce severe liver damage upon the stimulation of a nontoxic dose of lipopolysaccharides. In addition, the Nrf2 agonist protected macrophages from TP-induced toxicity and Nrf2 deficiency significantly aggravated liver injury by enhancing the recruitment and M1 polarization of hepatic macrophages. This study suggests that Nrf2 pathway-mediated hepatic macrophage polarization plays an essential role in TP-induced liver damage, which can serve as a potential therapeutic target for preventing hepatotoxicity induced by TP.

Tripterygium wilfordii multiglycoside-induced hepatotoxicity via inflammation and apoptosis in zebrafish.

Tripterygium wilfordii multiglycoside (GTW) is a commonly used compound for the treatment of rheumatoid arthritis (RA) and immune diseases in clinical practice. However, it can induce liver injury and the mechanism of hepatotoxicity is still not clear. This study was designed to investigate GTW-induced hepatotoxicity in zebrafish larvae and explore the mechanism involved. The 72 hpf (hours post fertilization) zebrafish larvae were administered with different concentrations of GTW for three days and their mortality, malformation rate, morphological changes in the liver, transaminase levels, and histopathological changes in the liver of zebrafish larvae were detected. The reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the levels of microRNA-122 (miR-122) and genes related to inflammation, apoptosis, cell proliferation and liver function. The results showed that GTW increased the mortality of zebrafish larvae, while significant malformations and liver damage occurred. The main manifestations were elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), significant liver atrophy, vacuoles in liver tissue, sparse cytoplasm, and unclear hepatocyte contours. RT-PCR results showed that the expression of miR-122 significantly decreased by GTW; the mRNA levels of inflammation-related genes il1ß, il6, tnfα, il10, cox2 and ptges significantly increased; the mRNA level of tgfß significantly decreased; the mRNA levels of apoptosis-related genes, caspase-8 and caspase-9, significantly increased; the mRNA level of bcl2 significantly decreased; the mRNA levels of cell proliferation-related genes, top2α and uhrf1, significantly reduced; the mRNA levels of liver function-related genes, alr and cyp3c1, significantly increased; and the mRNA level of cyp3a65 significantly decreased. In zebrafish, GTW can cause increased inflammation, enhanced apoptosis, decreased cell proliferation, and abnormal expression of liver function-related genes, leading to abnormal liver structure and function and resulting in hepatotoxicity.

[2,3,5,4'-Tetrahydroxystibane-2-O-ß-D-glucoside induces liver injury by disrupting bile acid homeostasis and phospholipids efflux].

Polygonum multiflorum is a traditional Chinese herbal medicine and has many biological activities such as hair-blacking, anti-atherosclerosis, anti-inflammatory and anti-aging. However, the liver injury induced by P. multiflorum has aroused wide attention in recent years. 2,3,5,4'-tetrahydroxystibane-2-O-ß-D-glucoside(TSG) is a main component of P. multiflorum, but the role of TSG in inducing liver injury is unclear. The aim of present study was to evaluate TSG's potential liver injury and effects on bile acid homeostasis and phospholipids efflux. C57 BL/6 J mice received intraperitoneal administration of 400 mg·kg~(-1) of TSG daily for 15 days, and then biochemical indexes of liver injury and changes of phospholipid content were detected. The changes of bile acid compositions were detected by LC-MS/MS. The results showed TSG 400 mg·kg~(-1) significantly increased the content of serum total bile acid(TBA) and alkaline phosphatase(ALP). Elevated free bile acid levels were observed in TSG-treated groups, including ß-muricholic acid(ß-MCA), ursodeoxycholic acid(UDCA), hyodeoxycholic acid(HDCA), chenodeoxycholic acid(CDCA), deoxcholic acid(DCA) in serum and ß-MCA, CDCA in liver. TSG inhibited the protein expression of farnesoid X receptor(FXR) and down stream bile salt export pump(BSEP), which may result in the accumulation of bile acid. TSG also inhibited the expression of 25-hydroxycholesterol-7 alpha-hydroxylase(CYP7 B1), which may disturb the alternative pathway for bile acid synthesis. In addition, intraperitoneal injection of TSG 400 mg·kg~(-1) significantly decreased the content of phospholipids in bile. The research showed that TSG significantly inhibited the expression of multidrug resistance protein 2(MDR2) and destroyed the regular distribution of MDR2 on the bile duct membrane of liver. In vitro results showed that the IC_(50) of TSG on HepG2 cells was about 1 500 µmol·L~(-1) and TSG at 500 µmol·L~(-1)(for 24 h) could destroy the distribution of MDR2 on the bile duct membrane of liver. In conclusion, TSG induced liver injury by disrupting bile acid homeostasis and phospholipids efflux.

Altered integrity of hepatocyte tight junctions in rats with triptolide-induced cholestasis.

Triptolide (TP), an active component of Tripterygium wilfordiiHook. f. (TWHF), has been widely used for centuries as a traditional Chinese medicine. However, the clinical application of TP has been restricted due to multitarget toxicity, such as hepatotoxicity. In this study, 28 days of oral TP administration (100, 200, or 400 µg·kg-1·d-1) induced the occurrence of cholestasis in female Wistar rats, as evidenced by increased serum levels of γ-glutamyl transpeptidase (γ-GGT), alkaline phosphatase (ALP) and hepatic total bile acids (TBAs). In addition, the heptocyte polarity associated with the strcture of tight junctions (TJs) was disrupted in both rats and sandwich-cultured primary hepatocytes. Immunoblotting revealed decreased expression of the TJ-associated proteins occludin, claudin-1, and zonula occludens protein (ZO-1), and downregulated mRNA levels of these TJs was also detected by real-time PCR. An immunofluorescence analysis showed abnormal subcellular localization of occludin, claudin-1 and ZO-1, which was also confirmed by transmission electron microscopy. Moreover, the concentration of FITC-dextran, a marker of paracellular penetration, was found to increase rapidly in bile increased rapidly (within 6 minutes) after treatment with TP, which indicated the functional impairment of TJs. Taken together, these results suggest that the administration of TP for 28 consecutive days to rats could induce cholestatic injury in the liver, and the increased paracellular permeability might play an important role in these pathological changes.

Catalpol counteracts the pathology in a mouse model of Duchenne muscular dystrophy by inhibiting the TGF-ß1/TAK1 signaling pathway.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by a mutation in the gene encoding the dystrophin protein. Catalpol is an iridoid glycoside found in Chinese herbs with anti-inflammatory, anti-oxidant, anti-apoptotic, and hypoglycemic activities that can protect against muscle wasting. In the present study we investigated the effects of catalpol on DMD. Aged Dystrophin-deficient (mdx) mice (12 months old) were treated with catalpol (100, 200 mg·kg-1·d-1, ig) for 6 weeks. At the end of the experiment, the mice were sacrificed, and gastrocnemius (GAS), tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL) muscles were collected. We found that catalpol administration dose-dependently increased stride length and decreased stride width in Gait test. Wire grip test showed that the time of wire grip and grip strength were increased. We found that catalpol administration dose-dependently alleviated skeletal muscle damage, evidenced by reduced plasma CK and LDH activity as well as increased the weight of skeletal muscles. Catalpol administration had no effect on dystrophin expression, but exerted anti-inflammatory effects. Furthermore, catalpol administration dose-dependently decreased tibialis anterior (TA) muscle fibrosis, and inhibited the expression of TGF-ß1, TAK1 and α-SMA. In primary myoblasts from mdx mice, knockdown of TAK1 abolished the inhibitory effects of catalpol on the expression levels of TGF-ß1 and α-SMA. In conclusion, catalpol can restore skeletal muscle strength and alleviate skeletal muscle damage in aged mdx mice, thus may provide a novel therapy for DMD. Catalpol attenuates muscle fibrosis by inhibiting the TGF-ß1/TAK1 signaling pathway.

Synthesis and Biological Evaluation of Celastrol Derivatives as Potential Immunosuppressive Agents.

Celastrol, a friedelane-type triterpenoid isolated from the genus Triperygium, possesses antitumor, anti-inflammatory, and immunosuppressive activities. A total of 42 celastrol derivatives (1a-1t, 2a-2l, and 3a-3j) were synthesized and evaluated for their immunosuppressive activities. Compounds 2a-2e showed immunosuppressive effects, with IC50 values ranging from 25 to 83 nM, and weak cytotoxicity (CC50 > 1 µM). Compound 2a, with a selectivity index value 31 times higher than that of celastrol, was selected as a lead compound. Further research showed that 2a exerted its immunosuppressive effects by inducing apoptosis and inhibiting cytokine secretion via Lck- and ZAP-70-mediated signaling pathways.

[Mechanism of psoralen in aggravating hepatotoxicity induced by CCl_4 by delaying liver regeneration].

This study aimed to investigate whether psoralen can aggravate hepatotoxicity induced by carbon tetrachloride(CCl_4) by inducing hepatocyte cycle arrest and delaying liver regeneration. Female C57 BL/6 mice aged 6-8 weeks were randomly divided into control group, model group(CCl_4 group), combined group(CCl_4+PSO group) and psoralen group(PSO group). CCl_4 group and CCl_4+PSO group were given CCl_4 intraperitoneally at a dose of 100 µL·kg~(-1) once; olive oil of the same volume was given to control group and PSO group intraperitoneally; 12 h, 36 h and 60 h after CCl_4 injection, PSO group and CCl_4+PSO group were administrated with PSO intragastrically at a dose of 200 mg·kg~(-1); 0.5% CMC-Na of the same volume was administrated to control group and PSO group intragastrically. The weight of mice was recorded every day. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were measured at 36 h, 60 h and 84 h after CCl_4 injection. Mice were sacrificed after collection of the last serum samples. Liver samples were collected, and liver weight was recorded. Histopathological and morphological changes of liver were observed by haematoxylin and eosin staining. The mRNA levels of HGF, TGF-ß, TNF-α, p53 and p21 in liver were detected by RT-qPCR. Western blot was used to detect the levels of cell cycle-related proteins. According to the results, significant increase of serum ALT and AST and centrilobular necrosis with massive inflammatory cell infiltration were observed in CCl_4+PSO group. After PSO administration in CCl_4 model, the mRNA levels of HGF(hepatocyte growth factor) and TNF-α were reduced, while the mRNA expressions of TGF-ß, p53 and p21 was up-regulated. The expression of PCNA(proliferating cell nuclear antigen) was significantly increased in CCl_4 and CCl_4+PSO group, while the relative protein level in CCl_4+PSO group was slightly lower than that in CCl_4 group. Compared with control and CCl_4 group, the expression of p27(cyclic dependent kinase inhibitor protein p27) was prominently increased in CCl_4+PSO group. These results indicated that hepatotoxicity induced by CCl_4 could be aggravated by intraperitoneal administration with PSO, and the repair process of liver could be delayed. The preliminary mechanism may be related to the inhibition of PCNA and regulation of some cell cycle-associated protein by psoralen, in which the significant up-regulation of p27, p53 and p21 may play important roles.

Organic anion transporter 1 and 3 contribute to traditional Chinese medicine-induced nephrotoxicity.

With the internationally growing popularity of traditional Chinese medicine (TCM), TCM-induced nephropathy has attracted public attention. Minimizing this toxicity is an important issue for future research. Typical nephrotoxic TCM drugs such as Aristolochic acid, Tripterygium wilfordii Hook. f, Rheum officinale Baill, and cinnabar mainly damage renal proximal tubules or cause interstitial nephritis. Transporters in renal proximal tubule are believed to be critical in the disposition of xenobiotics. In this review, we provide information on the alteration of renal transporters by nephrotoxic TCMs, which may be helpful for understanding the nephrotoxic mechanism of TCMs and reducing adverse effects. Studies have proven that when administering nephrotoxic TCMs, the expression or function of renal transporters is altered, especially organic anion transporter 1 and 3. The alteration of these transporters may enhance the accumulation of toxic drugs or the dysfunction of endogenous toxins and subsequently sensitize the kidney to injury. Transporters-related drug combination and clinical biomarkers supervision to avoid the risk of future toxicity are proposed.

Quantitative proteomics analysis of Fructus Psoraleae-induced hepatotoxicity in rats.

Fructus Psoraleae, which is commonly consumed for the treatment of osteoporosis, bone fracture, and leucoderma, induces liver injury. This study investigated the pathogenesis of the ethanol extract of Fructus Psoraleae (EEFP)-induced liver injury in rats. EEFP (1.35, 1.80, and 2.25 g·kg-1) was administrated to Sprague Dawley (SD) rats for 30 d. We measured liver chemistries, histopathology, and quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-based protein profiling. EEFP demonstrated parameters suggestive of liver injury with changes in bile secretion, bile flow rate, and liver histopathology. iTRAQ analysis showed that a total of 4042 proteins were expressed in liver tissues of EEFP-treated and untreated rats. Among these proteins, 81 were upregulated and 32 were downregulated in the treatment group. KEGG pathway analysis showed that the drug metabolic pathways of cytochrome P450, glutathione metabolism, glycerolipid metabolism, and bile secretion were enriched with differentially expressed proteins. The expression of key proteins related to the farnesoid X receptor (FXR), i.e., the peroxisome proliferators-activated receptor alpha (PPAR-α), were downregulated, and multidrug resistance-associated protein 3 (MRP3) was upregulated in the EEFP-treated rats. Our results provide evidence that EEFP may induce hepatotoxicity through various pathways. Furthermore, our study demonstrates changes in protein regulation using iTRAQ quantitative proteomics analysis.

The hypoglycemic mechanism of catalpol involves increased AMPK-mediated mitochondrial biogenesis.

Mitochondria serve as sensors of energy regulation and glucose levels, which are impaired by diabetes progression. Catalpol is an iridoid glycoside that exerts a hypoglycemic effect by improving mitochondrial function, but the underlying mechanism has not been fully elucidated. In the current study we explored the effects of catalpol on mitochondrial function in db/db mice and C2C12 myotubes in vitro. After oral administration of catalpol (200 mg·kg-1·d-1) for 8 weeks, db/db mice exhibited a decreased fasting blood glucose level and restored mitochondrial function in skeletal muscle. Catalpol increased mitochondrial biogenesis, evidenced by significant elevations in the number of mitochondria, mitochondrial DNA levels, and the expression of three genes associated with mitochondrial biogenesis: peroxisome proliferator-activated receptor gammaco-activator 1 (PGC-1α), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1). In C2C12 myotubes, catalpol significantly increased glucose uptake and ATP production. These effects depended on activation of AMP-activated protein kinase (AMPK)-mediated mitochondrial biogenesis. Thus, catalpol improves skeletal muscle mitochondrial function by activating AMPK-mediated mitochondrial biogenesis. These findings may guide the development of a new therapeutic approach for type 2 diabetes.

[Effect of triptolide on Th17/Treg cells in spleen].

Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 µg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 µg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.

[Progress of effect and mechanisms of Tripterygium wilfordii on immune system].

Traditional Chinese medicine Tripterygium wilfordii Hook.f( TWHF) is a natural botanical drug in China. It has complex chemical compositions and has been used for a long history. TWHF was used as an insecticide to protect crops at early stage,and it was later found to have significant effects in the treatment of rheumatoid arthritis,attaining great concerns. With further researches,it was found that TWHF can treat various diseases in the medical field due to a variety of pharmacological activities such as anti-cancer,neuroprotection,anti-inflammatory and immune-suppressing,particularly. Multiple extracts of TWHF have unique immunosuppressive function,playing an immune role through multi-target and multi-channel,with significant effect in the treatment of autoimmune diseases. As an immune-suppressing drug,TWHF is worthy of in-depth research due to its broad application prospects. While achieving good clinical efficacy,reports about its toxic effects to multiple systems of the body are also increasing,greatly hindering its clinical application. In order to fully understand the immune-suppressing function of TWHF and reduce or avoid the occurrence of toxic and side effects,we summarized recent progress of TWHF on the immune organs,cells and factors in recent years,as well as the pharmacology and toxic effects,hoping to provide a scientific and reasonable reference for its wider use in clinical treatment.

[Study on difference of liver toxicity and its molecular mechanisms caused by Tripterygium wilfordii multiglycoside and equivalent amount of triptolid in rats].

Tripterygium wilfordii multiglycoside( GTW),an extract derived from T. wilfordii,has been used for rheumatoid arthritis and other immune diseases in China. However its potential hepatotoxicity has not been investigated completely. Firstly,the content of triptolid( TP) in GTW was 0. 008% confirmed by a LC method. Then after oral administration of GTW( 100,150 mg·kg-1) and TP( 12 µg·kg-1) in female Wistar rats for 24 h,it was found that 150 mg·kg-1 GTW showed more serious acute liver injury than 12 µg·kg-1 TP,with the significantly increased lever of serum ALT,AST,TBA,TBi L,TG and bile duct hyperplasia even hepatocyte apoptosis. The expression of mRNA and proteins of liver bile acid transporters such as BSEP,MRP2,NTCP and OATP were down-regulated significantly by GTW to inhibit bile acid excretion and absorption,resulting in cholestatic liver injury. Moreover,GTW was considered to be involved in hepatic oxidative stress injury,although it down-regulated SOD1 and GPX-1 mRNA expression without significant difference in MDA and GSH levels. In vitro,we found that TP was the main toxic component in GTW,which could inhibit cell viability up to 80% in Hep G2 and LO2 cells at the dose of 0. 1 µmol·L-1. Next a LC-MS/MS method was used to detect the concentration of triptolid in plasma from rats,interestingly,we found that the content of TP in GTW was always higher than in the same amount of TP,suggesting the other components in GTW may affect the TP metabolism. Finally,we screened the substrate of p-glycoprotein( p-gp) in Caco-2 cells treated with components except TP extrated from GTW,finding that wilforgine,wilforine and wilfordine was the substrate of p-gp. Thus,we speculated that wilforgine,wilforine and wilfordine may competitively inhibit the excretion of TP to bile through p-gp,leading to the enhanced hepatotoxity caused by GTW than the same amount of TP.

Toxic effects of triptolide on adrenal steroidogenesis in H295R cells and female rats.

Triptolide (TP), a major active ingredient of Tripterygium wilfordii, exerts potent immunosuppressive effects in the treatment of rheumatoid arthritis but is not widely used in clinical practice due to its multiorgan toxicity, particularly hepatotoxicity, nephrotoxicity, and reproductive toxicity. An LC-MS/MS approach was employed to explore the endocrine-disrupting effects of TP. The endocrine-disrupting effects of various concentrations (0-100 nM) of TP for 48 hour were firstly investigated using an in vitro model (H295R cell line). It was found that TP did not decrease cell viability. The transcriptional levels of steroidogenic enzymes in H295R cells were assessed by quantificational real-time polymerase chain reaction. The possible adrenal and endocrine effects of oral administration of TP (0, 50, and 500 µg/kg) for 28 days on both normal and collagen-induced arthritis (CIA) rats were also explored. The serum and adrenal tissue hormone levels (corticosterone and progesterone) and adrenal histopathology were analyzed, with the results that TP significantly decreased the level of cortisol in H295R cells and the level of plasma corticosterone in both normal and CIA rats. Histological alterations in adrenal cortex were observed at the dose of 500 µg/kg. Exposure to TP for 48 hour had an obvious inhibitory effect on the messenger RNA transcript levels of HSD3B2, CYP21A2, CYP17A1, and CYP11B1, which is essential for the synthesis of corticosteroids. In a word, TP leads to the disorder of corticosteroid synthesis and secretion, and corticosteroid may be a potential biomarker for the treatment of multiorgan toxicity of TP.

Preventive Effects of Total Flavonoid C-Glycosides from Abrus mollis on Nonalcoholic Fatty Liver Disease through Activating the PPARα Signaling Pathway.

Abrus pulchellus subsp. mollis (Hance) Verdc. (Leguminosae) is a well-known edible plant usually added to soups and beverages. In this study, vicenin-2 (1: ), isoschaftoside (2: ), schaftoside (3: ), and their enrichment fraction, total flavonoid C-glycosides, derived from the extracts of A. mollis, were firstly found to prevent nonalcoholic fatty liver disease both in vitro and in vivo. In the in vitro study, total flavonoid C-glycosides decreased the lipid accumulation in oleic acid-treated HepG2 cells. The mechanisms of total flavonoid C-glycosides are involved in the regulation of peroxisome proliferator-activated receptor α and its downstream, and the reduction of proinflammatory cytokines. In high-fat diet-induced fatty liver rats, total flavonoid C-glycosides decreased the levels of glutamic-oxalacetic transaminease and glutamic-pyruvic transaminase, and decreased the lipid accumulation both in the liver and blood without affecting food intake. In addition, total flavonoid C-glycosides also increased the activities of the antioxidant enzyme system in vivo. In conclusion, total flavonoid C-glycosides are active components of A. mollis on nonalcoholic fatty liver disease, and can be used in functional food and supplements for nonalcoholic fatty liver disease prevention and treatment.