Acute toxicity, 28-day repeated-dose toxicity and toxicokinetic study of timosaponin BII in beagle dogs.

The safety evaluation of timosaponin BII (TBII) in beagle dogs with toxicokinetic study was performed. For the acute oral toxicity study, the minimum lethal dose (MLD) of TBII was more than 2000 mg/kg and suggested the characteristics of absorption saturation. For the 28-day repeated dose oral toxicity and toxicokinetic studies, there was no significant effect on all test parameters except for prolonged APTT in the 60 and 180 mg/kg groups, which recovered after withdrawal. The increase of drug exposure of 180 mg/kg group was not proportional to the increase of administration dose, showing the characteristics of absorption saturation.

Comparative toxicity and toxicokinetic studies of oxiracetam and (S)-oxiracetam in dogs.

Oxiracetam (ORT) is known as a derivative of piracetam in the family of nootropics for treating memory impairment and cognition disorders. Given the chiral toxicological concerns surrounding ORT and the absence studies of (S)-ORT, the toxicity and toxicokinetics of (S)-ORT, and comparative toxicology of oxiracetam were systematically investigated in dogs following acute and 13-week repeated oral dosing. The animal toxicity mainly manifested as loose stools in both the acute and the 13-week studies. The no-observed-adverse-effect level is proposed to be 100 mg/kg. The 13-week toxicokinetics study indicated that, in the (S)-ORT group, the time to peak concentration was delayed, elimination half-life extended, and apparent volume of distribution increased compared with the ORT group. The clearance rate increased at low- and mid-doses, but decreased in the high-dose group and was accompanied by drug accumulation. Compared with the same dose of ORT, (S)-ORT had a lower clearance rate and longer elimination half-life.

Upregulated microRNA-485 suppresses apoptosis of renal tubular epithelial cells in mice with lupus nephritis via regulating the TGF-ß-MAPK signaling pathway by inhibiting RhoA expression.

Lupus nephritis (LN) is a common and severe complication of systemic lupus erythematosus. Without intervention, LN may cause acute kidney injury and end-stage renal disease. This study aims to determine whether microRNA-485 (miR-485) affects renal tubular epithelial cells (RTECs) in LN mice via the TGF-ß-MAPK signaling pathway by targeting RhoA. Renal tissue samples were initially extracted from 15 LN and 15 normal mice. RTECs were cultivated in vitro and grouped after transfection of different mimics, inhibitors, or siRNA- RhoA. The target gene of miR-485 was confirmed by dual-luciferase reporter assay. Flow cytometry and MTT assay were applied to detect cell viability and apoptosis. It was determined that RhoA was a target gene of miR-485. We found that urine protein, creatinine, RhoA, interleukin 6 (IL-6), transforming growth factor-ß1 (TGF-ß1), and p38 mitogen-activated protein kinases (p38MAPK) were highly expressed in renal tissues of LN mice, while poor levels of miR-485 were recorded. The overexpression of miR-485 or siRNA- RhoA or the combination of miR-485 and siRNA- RhoA was demonstrated to lead to a reduction of levels of RhoA, IL-6, TGF-ß, and p38MAPK, as well as a promotion of RTECs proliferation and inhibition of RTECs apoptosis. Taken together, these findings indicated that overexpressed miR-485 downregulates RhoA which could promote cell viability and inhibit apoptosis of RTECs by regulating the RhoA-mediated TGF-ß-MAPK signaling pathway in LN mice.

Time-response characteristic and potential biomarker identification of heavy metal induced toxicity in zebrafish.

The present work aims to explore the time-response (from 24 h to 96 h) characteristic and identify early potential sensitive biomarkers of copper (Cu) (as copper chloride dihydrate), cadmium (Cd) (as cadmium acetate), lead (Pb) (as lead nitrate) and chromium (Cr) (as potassium dichromate) exposure in adult zebrafish, focusing on reactive oxygen species (ROS), SOD activity, lipid peroxidation and gene expression related to oxidative stress and inflammatory response. Furthermore, the survival rate decreased apparently by a concentration-dependent manner after Cu, Cr, Cd and Pb exposure, and we selected non-lethal concentrations 0.05 mg/L for Cu, 15 mg/L for Cr, 3 mg/L for Cd and 93.75µg/L for Pb to test the effect on the following biological indicators. Under non-lethal concentration, the four heavy metals have no apparent histological change in adult zebrafish gills. Similar trends in ROS production, MDA level and SOD activity were up-regulated by the four heavy metals, while MDA level responded more sensitive to Pb by time-dependent manner than the other three heavy metals. In addition, mRNA levels related to antioxidant system (SOD1, SOD2 and Nrf2) were up-regulated by non-lethal concentration Cu, Cr, Cd and Pb exposure. MDA level and SOD1 gene have a more delayed response to heavy metals. Genes related to immunotoxicity were increased significantly after heavy metals exposure at non-lethal concentrations. TNF-α and IL-1ß gene have similar sensibility to the four heavy metals, while IL-8 gene was more responsive to Cr, Cd and Pb exposure at 48 h groups and IFN-γ gene showed more sensitivity to Cu at 48 h groups than the other heavy metals. In conclusion, the present works have suggested that the IFN-γ gene may applied as early sensitive biomarker to identify Cu-induced toxicity, while MDA content and IL-8 gene may use as early sensitive biomarkers for evaluating the risk of Pb exposure. Moreover, IL-8 and IFN-γ gene were more responsive to heavy metals, which may become early sensitive and potential biomarkers for evaluating inflammatory response induced by heavy metals. This work reinforces the concept of the usefulness of gene expression assays in the evaluation of chemicals effects and helps to establish a background data as well as contributes to evaluate early environmental risk for chemicals, even predicting toxicity.

Anti-Psoriasis Effects and Mechanisms of Α-(8-Quinolinoxy) Zinc Phthalocyanine-Mediated Photodynamic Therapy.

BACKGROUND/AIMS: The aim of this study was to determine the anti-psoriasis effects of α-(8-quinolinoxy) zinc phthalocyanine (ZnPc-F7)-mediated photodynamic therapy (PDT) and to reveal its mechanisms. METHODS: HaCaT cells were used to observe the influence of ZnPc-F7-PDT on cell proliferation in vitro. The in vivo anti-psoriasis effects of ZnPc-F7-PDT were evaluated using a mouse vagina model, a propranolol-induced cavy psoriasis model and an imiquimod (IMQ)-induced nude mouse psoriasis model. Flow cytometry was carried out to determine T lymphocyte levels. Western blotting was performed to determine protein expression, and a reverse transcription-polymerase chain reaction test was performed to determine mRNA expression. RESULTS: The results showed that ZnPc-F7-PDT significantly inhibited the proliferation of HaCaT cells in vitro; when the light doses were fixed, changing the irradiation time or output power had little influence on the inhibition rate. ZnPc-F7-PDT significantly inhibited the hyperproliferation of mouse vaginal epithelium induced by diethylstilbestrol and improved propranolol- and IMQ-induced psoriasis-like symptoms. ZnPc-F7-PDT inhibited IMQ-induced splenomegaly and T lymphocyte abnormalities. ZnPc-F7-PDT did not appear to change T lymphocytes in the mouse vagina model. ZnPc-F7-PDT down-regulated the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), interleukin (IL)-17A mRNA and IL-17F mRNA, and up-regulated the expression of Bax. CONCLUSION: In conclusion, ZnPc-F7-PDT exhibited therapeutic effects in psoriasis both in vitro and in vivo and is a potential approach in the treatment of psoriasis. Potential mechanisms of these effects included the inhibition of hyperproliferation; regulation of PCNA, Bcl-2, Bax, IL-17A mRNA and IL-17F mRNA expression; and immune regulation.

Toxicokinetics and toxicity of potassium 2-(1-hydroxypentyl)-benzoate in beagle dogs.

Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a prodrug of 3-n-butylphthalide (dl-NBP) for treatment of cerebral ischemic stroke in China, which undergoes lactonization to form dl-NBP in plasma. And, the phase II-III clinical trial of dl-PHPB has been approved by China Food and Drug Administration (CFDA) in 2013. In this study, a toxicity and toxicokinetics evaluation of dl-PHPB was performed using beagle dogs at specially high-dose 108 mg/kg/day (65-fold higher than humans at MHRD) for 4 weeks by intravenous administration, with a 3-week recovery period. And the plasma concentrations of dl-PHPB along with its metabolite dl-NBP were determined by HPLC-UV method. The results showed that dl-PHPB was quickly metabolized into dl-NBP, and no significant accumulation was observed. A slight to moderate behavior-associated toxicity was revealed in the process of delivery; and recovered to normal at the end of administration. Changes in the blood hematological profiles included significantly increased NEUT levels and lower LYM% content. Meanwhile, a notable increase in TG content was also observed in the serum biochemical parameters at 4-week post-exposure. These findings were reversible during the recovery period. The information from these studies would be taken into consideration for the interpretation of toxicology findings and provide a reference for clinical safety assessment.

Comparison of Tripterygium wilfordii Hook F with methotrexate in the treatment of active rheumatoid arthritis (TRIFRA): a randomised, controlled clinical trial.

OBJECTIVES: To compare the efficacy and safety of Tripterygium wilfordii Hook F (TwHF) with methotrexate (MTX) in the treatment of active rheumatoid arthritis (RA). METHODS: Design: a multicentre, open-label, randomised controlled trial. All patients were assessed by trained investigators who were unaware of the therapeutic regimen. INTERVENTION: 207 patients with active RA were randomly allocated (1:1:1) to treatment with MTX 12.5 mg once a week, or TwHF 20 mg three times a day, or the two in combination. At week 12, if reduction of the 28-joint count Disease Activity Score (DAS28) was <30% in the monotherapy groups, the patient was switched to MTX+TwHF. The primary efficacy point was the proportion of patients achieving an American College of Rheumatology (ACR) 50 response at week 24. RESULTS: 174/207 (84.1%) patients completed 24 weeks of the trial. In an intention-to-treat analysis, the proportion of patients reaching the ACR50 response criteria was 46.4% (32/69), 55.1% (38/69) and 76.8% (53/69), respectively, in the MTX, TwHF and MTX+TwHF groups (TwHF vs MTX monotherapy, p=0.014; MTX+TwHF vs MTX monotherapy, p<0.001). Similar statistically significant patterns at week 24 were found for ACR20, ACR70, clinical Disease Activity Index good responses, EULAR good response, remission rate and low disease activity rate. Significant improvement in the Health Assessment Questionnaire and 36-item Short-Form Health Survey questionnaire scores from baseline to week 24 was seen in each treatment arm (p<0.05), though no significant difference was found among the treatment arms (p>0.05). The result of per-protocol analysis agreed with that seen in the intention-to-treat analysis. Seven, three and five women in the TwHF, MTX and combination groups, respectively, developed irregular menstruation (TwHF vs MTX monotherapy, p=0.216). CONCLUSIONS: TwHF monotherapy was not inferior to, and MTX+TwHF was better than, MTX monotherapy in controlling disease activity in patients with active RA. TRIAL REGISTRATION NUMBER: NCT01613079.

[Study on anti-oxidative effect of extracts from Cichorium endivia on HepG2 cells and its mechanism].

OBJECTIVE: To investigate the protective effect of extracts from Cichorium endivia (CEE) in H2O2-induced HepG2 cell oxidative stress injury, and explore the antioxidant mechanism of CEE in HepG2 cells. METHOD: The viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidant-response element (ARE)-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells. RESULT: The cell viability reduced, while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE, the intracellular ARE activity could increase in a concentration-dependent manner. In addition, the mRNA expressions of regulatory genesGCLC, GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE. CONCLUSION: CEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE's antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.

Early detection of pancreatic cancer in patients with recurrent pancreatitis: A case report.

BACKGROUND: Pancreatic cancer presents a challenge with its low early diagnosis and treatment rates, leading to high metastasis and mortality rates. The median survival time for advanced pancreatic cancer is a mere 3 months. However, there's hope: small pancreatic cancers diagnosed at an early stage (T1) or those less than or equal to 1 cm in diameter boast an impressive 5-year survival rate of nearly 100%. This underscores the critical importance of early pancreatic cancer detection for significantly improving prognosis. CASE SUMMARY: Pancreatic cancer, a malignant tumor of the digestive tract, poses challenges in both diagnosis and treatment due to its occult and atypical clinical symptoms. Clinically, patients with recurrent pancreatitis should be vigilant, as it may be indicative of pancreatic cancer, particularly in middle-aged and elderly patients. Here, we presented the case of a patient who experienced recurrent acute pancreatitis within a span of 2 months. During the initial episode of pancreatitis, routine imaging failed to identify the cause of pancreatic cancer. However, upon recurrence of acute pancreatitis, endoscopic ultrasonography (EUS) revealed a space-occupying lesion approximately 1 cm in size in the pancreatic body. Subsequent EUS coupled with fine-needle aspiration examination demonstrated atypical pancreatic gland epithelium. Ultimately, the patient underwent surgery and was diagnosed with an intraductal papillary mucinous tumor of the pancreas (severe epithelial dysplasia, focal cancer). CONCLUSION: We recommend EUS for patients with recurrent pancreatitis of unknown etiology to exclude early pancreatic cancer.

Processing-induced reduction in dianthrones content and toxicity of Polygonum multiflorum: Insights from ultra-high performance liquid chromatography triple quadrupole mass spectrometry analysis and toxicological assessment.

BACKGROUND: Polygonum multiflorum-induced liver injury (PM-DILI) has significantly hindered its clinical application and development. METHODS: This study investigates the variation in content and toxicity of dianthrones, the toxic components of P. multiflorum, during different processing cycles. We employed the ultra-high-performance liquid chromatography triple quadrupole mass spectrometry method to quantify six dianthrones in raw P. multiflorum and formulations processed with a method called nine cycles of steaming and sunning. Additionally, toxicity assessments were conducted using human normal liver cell line L02 and zebrafish embryos. RESULTS: Results indicate a gradual reduction in dianthrones content with increasing processing cycles. Processed formulations exhibited significantly reduced cytotoxicity in L02 cells and hepatotoxicity in zebrafish embryos. CONCLUSIONS: Our findings elucidate the relationship between processing cycles and P. multiflorum toxicity, providing theoretical support for its safe use.

Vasorelaxant effects of the extracts and some flavonoids from the buds of Coreopsis tinctoria.

CONTEXT: The buds of Coreopsis tinctoria Nutt (Compositae) are used in the treatment of hypertension in the Uyghur folk medicine in China. OBJECTIVE: To investigate vasorelaxant properties of extracts and some flavonoids from C. tinctoria (CT) and their underlying mechanisms in isolated rat thoracic aortic rings. MATERIALS AND METHODS: Vasorelaxant effects of ethanol extracts of CT (CTA) and its flavonoids as well as water-ethanol eluates from CTA by AB-8 resins (CTAA∼CTAF) were evaluated on rat aortic rings pre-contracted with phenylephrine (PE, 1 µM) or high KCl (60 µM). We evaluated the effect of CTA, CTAD and CTAE on PE-induced contraction in a Ca²âº-free medium and a dose-effect curve of Ca²âº in pre-contracted ring with high KCl. RESULTS: Endothelial removal did not modify the effect of CTAD and CTAE (3.00 g/L) neither on PE-pre-contracted rings (164.78 ± 21.44 and 191.47 ± 16.75%) nor on KCl-pre-contracted rings (75.68 ± 10.76 and 125.14 ± 17.41%) compared with intact-endothelium rings pre-contracted with high KCl (100.49 ± 17.30 and 110.81 ± 16.33%). CTAD and CTAE (3.00 g/L) down-regulated the dose-effect curve of Ca²âº in pre-contraction with high KCl, and inhibited the pre-contraction with PE in a Ca²âº-free medium (p < 0.05). Seven flavonoids were obtained from CTAD, of which luteolin (5) and quercetin (6) were found to be the most effective relaxation in rings precontracted with PE (EC50: 0.006 and 0.039 g/L, p < 0.05) or high KCl (EC50: 0.023 and 0.045 g/L, p < 0.05). DISCUSSION AND CONCLUSION: These data demonstrated the vasorelaxant effect of CT, and its mechanism is likely due to an inhibitory effect on calcium movements through cell membranes.

[A multicenter study of coronary artery disease and its risk factors in rheumatoid arthritis in China].

OBJECTIVE: To learn about the prevalence and risk factors of coronary artery disease (CAD) in rheumatoid arthritis (RA). METHODS: Data were obtained from a 12-month retrospective investigation of the patients with RA, randomly selected from Departments of Rheumatology and Immunology in 21 big hospitals in China. The data were collected about their social conditions, clinical conditions, medications associated with RA, such as disease modifying anti-rheumatic drugs (DMARDs), non steroidal anti-inflammatory drugs (NSAIDs), glucocorticoid, biologic agents. A nonparameter test and multivariate logistic regression analysis were performed. RESULTS: In the study, 960 patients were enrolled. The prevalence of CAD was 3.5% in China, which was obviously higher than that of normal people. The prevalence of overweight and obesity, smoking, hypertension, diabetes mellitus, hypercholesterolemia and cerebrovascular disease were 35.1%, 12.3%, 17.0%, 7.7%, 0.4% and 3.0%, respectively. Compared with the control group, the CAD group had higher age [(64.7±9.3) years vs. (52.3±14.0) years,P<0.001], more rheumatoid nodules (14.7% vs. 3.1%,P=0.005), lower rate of hydroxychloroquine (HCQ) use (5.9% vs. 22.6%,P=0.021), higher prevalence rates of lung interstitial disease (17.5% vs. 7.0%,P<0.001), diabetes mellitus and hypertension (29.4% vs. 7.0%,P<0.001; 38.2% vs. 16.2%,P=0.001). There was no obvious correlation of CAD in RA with joint deformity, rheumatoid factor (RF) titer, glucocorticoid use, hypercholesterolemia and body mass index (BMI). Multivariate analysis showed higher age, diabetes mellitus and hypertension were independent predictors of CAD, and the use of HCQ was a protective factor of CAD. CONCLUSION: The prevalence of CAD is 3.5%. Higher age, diabetes mellitus and hypertension are independent predictors of CAD, and the use of HCQ is a protective factor of CAD.

[Survey of tumor necrosis factor inhibitors application in patients with rheumatoid arthritis in China].

OBJECTIVE: To investigate the current status of tumor necrosis factor (TNF) inhibitors application in rheumatoid arthritis (RA) patients in China and to analyze the related factors. METHODS: A retrospective survey was conducted in 21 hospitals from different parts of China. The patients with RA were randomly enrolled. Data of their social backgrounds, clinical conditions, usage and adverse effects of TNF inhibitors were collected. The costs of TNF inhibitors and the indirect costs of the disease were calculated. A multivariate Logistic regression analysis was performed to analyze the factors related to TNF inhibitors application. RESULTS: In the study, 1 095 RA patients from July 2009 to November 2010 were enrolled, of whom 112 had received TNF inhibitors, representing 10.2% of the total patients. The patients who received etanercept and infliximab were 7.4% (86/1 095) of the patients and 2.4% (26/1 095), respectively. There were 0.5% of the patients (5/1 095) who had received both of the TNF inhibitors. The patients who had accepted etanercept and treatment duration for less than 3 months and 3-6 months accounted for 38.5% and 25.0% respectively, while those treated with Infliximab were 38.1%. Their health assessment questionnaire (HAQ) scores were 1.1, 0.5 and 0.1, corresponding to treatment duration of infliximab for less than 3, 3-6 and 6-9 months and those were 1.3, 1.0, 0.3 corresponding to treatment duration of etanercept, respectively. Infliximab costs were RMB 24 525.0, 69 300.0 and 96 800.0 Yuan and etanercept costs were RMB 7 394.8, 9 158.6, 54 910.9 Yuan, respectively. Indirect costs for RA patients who accepted infliximab for less than 3, 3-6 and 6-9 months were RMB 365.6, 0 and 158.9 Yuan and those who accepted etanercept were RMB 2 158.4, 288.5 and 180.1 Yuan, respectively. Allergy and infection were the main side-effects of etanercept and both happened in 3.5% of all the patients. Liver damage happened in 2.3% of all the patients, while allergy and infection happened in 6.5% of all the patients who accepted infliximab. Logistic regression analysis showed that patients with higher education experience increased the odds of entering the TNF inhibitors group (OR: 1.292, 95%CI: 1.132-1.473, P=0.000). CONCLUSION: About one-tenth of RA patients in China have accepted TNF inhibitors. Higher education experience is the key factor for using TNF inhibitors.

[Sulphasalazine in patients with rheumatoid arthritis in China: a cross-sectional study].

OBJECTIVE: To investigate the medication status of rheumatoid arthritis (RA) patients and to analyze the clinical use of sulphasalazine (SSZ) and the adverse effect. METHODS: A total of 1 096 outpatients and inpatients diagnosed with RA were investigated in 21 hospitals all over China from July 2009 to December 2010, including gender, age of onset, clinical manifestations, as well as the clinical characteristics and medication status of 160 RA patients who received SSZ therapy. RESULTS: In the group of 160 patients who received SSZ, the male-to-female ratio was 1:7, The average age at onset was (46.1±15.0) years, while the average course was (9.9±7.8) years. The average dose of sulphasalazine was (1.87±0.52) g/d for a mean duration of (26.3± 14.6) months. Only 17% (27/160) of the patients received SSZ monotherapy. Methotrexate (63.1%), leflunomide (36.2%) and hydroxychloroquine (18.1%) were most commonly used combination drugs. And 36.2% (58/160) of the patients used the two-drug combination of methotrexate plus sulphasalazine .In this group, 41.9% (67/160) once used SSZ but withdrew for adverse events and other reasons, while 17.5% (28/160) withdrew for adverse events, of which the most common were gastrointestinal (8.8%), skin (3.8%) and liver toxicity (3.1%). CONCLUSION: Sulphaszlazine is not a common choice in the RA therapeutics in China, and the average dose of SSZ is lower than the standard dose of 2 to 3 g/d . The adverse events of SSZ are common; however, there are few severe adverse events or threat to life,SSZ is relatively safe in clinical practice.

[Study on effects of microRNA-21 antisense oligonucleotide in vivo and in vitro on bionomics of human cervical squamous carcinoma cell lines SiHa].

OBJECTIVE: To explore the effect of microRNA-21 (miR-21) antisense oligonucleotide on the biological characteristics of human cervical squamous carcinoma cell lines SiHa in vivo and in vitro. METHODS: Specific phosphorothioate antisense oligodeoxynucleotides targeting miR-21 were synthesized and transfected into cervical cancer cells in vitro. Expression of miR-21 in SiHa after transfection was detected by real-time RT-PCR. The cell proliferation was evaluated by MTT assay and colony formation experiment. The cell apoptosis was analyzed by annexin V-FITC/PI analysis. The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by tumor growth curves and immunohistochemistry (MaxVision method). H-E staining was used to document morphological changes and fluorometric TUNEL assay was to detect the apoptotic activity. RESULTS: After the transfection of antisense miR-21, the expression of miR-21 decreased along with an obvious growth inhibition, compared with that of the control groups (P < 0.05). Colony formation of both cell lines was markedly inhibited with antisense miR-21 (55.6% ± 1.4%), as compared with that in the negative group (98.3% ± 2.0%, P < 0.05). Flow cytometry assay showed that antisense miR-21 expression significantly enhanced the cell apoptosis (6.7% ± 1.3% and 29.4% ± 1.7%, P < 0.05). The tumor-forming rates of miR-21 transfected group, and negative control groups were 3/8 and 6/8, respectively (P < 0.05). Ki-67 proliferative marker staining decreased significantly (42% vs 90%) in the transfected group compared with negative control groups. Extensive dead tumor cells were seen in the miR-21 transfected cells along with a marked increase of apoptosis (P < 0.05). CONCLUSION: Targeted antisense oligonucleotide miR-21 effectively suppresses the growth of cervical carcinoma SiHa cells both in vitro and in vivo through an induction of apoptosis.

[MicroRNA383 regulates expression of PRDX3 in human medulloblastomas].

OBJECTIVE: To investigate the effects of microRNA-383 (miR-383) on PRDX3 gene expression, cell proliferation and apoptosis of human medulloblastma. METHODS: PRDX3 and miR-383 RNA expression was detected by real-time quantitative RT-PCR in human medulloblastoma tumor tissue samples, Daoy cell line and normal brain tissue samples. Western blot was used to detect protein expression of PRDX3. Synthetic miR-383 mimics were transfected into Daoy cells by lipofectamine. Using Cell Counting Kit-8 (CCK-8) method, flow cytometry was used to investigate the cell proliferation and apoptosis, cells reactive oxgen species(ROS), mitochondrial membrane potential changes in each experimental groups. RESULTS: Of 15 cases of human medulloblastoma tumor, 13 cases had miR-383 expression levels significantly lower than that of normal brain tissue, and 14 had PRDX3 mRNA expression levels significantly higher than that of normal brain tissue. The expression levels of miR-383 and PRDX3 in Daoy cells were 0.353 and 1.315 times than those of normal brain tissue, respectively. The protein expression levels of PRDX3 were higher in human medulloblatoma tumors and Daoy cells than that of normal brain tissue. Transfected miR-383 mimics increased the expression level of miR-383 after 24 h and 48 h was significantly higher than that of the control. In contrast, PRDX3 gene mRNA and protein expression levels were significantly decreased at 48 h compared with the control group. Using CCK-8 assay, the cell proliferation rate in the experimental group was significantly lower than that of the control group (P < 0.05). Annexin V-FITC assay demonstrated that early apoptosis rate of the experimental group (11.60 ± 0.30)% was significantly higher than those of the control group (2.3 ± 0.20)% and negative control group (10.37 ± 0.25)% (P = 0.000) after 48 h of transfection. The intracellular ROS levels after transfection at 24 and 48 h significantly increased than those of the control group. Mitochondrial membrane potential level at 24 h after transfection significantly decreased, comparing with the blank control group and the negative control group. CONCLUSIONS: Compared with normal brain tissue, decreased expression of miR-383 but elevated expression of PRDX3 are medulloblastoma tumour and Daoy cell lines. Up-regulation of miR-383 knockdowns the expression of PRDX3, inhibits proliferation and promotes apoptosis of Daoy cells, leading to increased intracellular ROS and decreased levels of mitochondrial membrane potential.

Integrated spatially resolved metabolomics and network toxicology to investigate the hepatotoxicity mechanisms of component D of Polygonum multiflorum Thunb.

ETHNOPHARMACOLOGICAL RELEVANCE: The liver toxicity of Reynoutria multiflora (Thunb.) Moldenke. (Polygonaceae) (Polygonum multiflorum Thunb, PM) has always attracted much attention, but the related toxicity materials and mechanisms have not been elucidated due to multi-component and multi-target characteristics. In previous hepatotoxicity screening, different components of PM were first evaluated and the hepatotoxicity of component D [95% ethanol (EtOH) elution] in a 70% EtOH extract of PM (PM-D) showed the highest hepatotoxicity. Furthermore, the main components of PM-D were identified and their hepatotoxicity was evaluated based on a zebrafish embryo model. However, the hepatotoxicity mechanism of PM-D is unknown. AIM OF THE STUDY: This work is to explore the hepatotoxicity mechanisms of PM-D by integrating network toxicology and spatially resolved metabolomics strategy. MATERIALS AND METHODS: A hepatotoxicity interaction network of PM-D was constructed based on toxicity target prediction for eight key toxic ingredients and a hepatotoxicity target collection. Then the key signaling pathways were enriched, and molecular docking verification was implemented to evaluate the ability of toxic ingredients to bind to the core targets. The pathological changes of liver tissues and serum biochemical assays of mice were used to evaluate the liver injury effect of mice with oral administration of PM-D. Furthermore, spatially resolved metabolomics was used to visualize significant differences in metabolic profiles in mice after drug administration, to screen hepatotoxicity-related biomarkers and analyze metabolic pathways. RESULTS: The contents of four key toxic compounds in PM-D were detected. Network toxicology identified 30 potential targets of liver toxicity of PM-D. GO and KEGG enrichment analyses indicated that the hepatotoxicity of PM-D involved multiple biological activities, including cellular response to endogenous stimulus, organonitrogen compound metabolic process, regulation of the apoptotic process, regulation of kinase, regulation of reactive oxygen species metabolic process and signaling pathways including PI3K-Akt, AMPK, MAPK, mTOR, Ras and HIF-1. The molecular docking confirmed the high binding activity of 8 key toxic ingredients with 10 core targets, including mTOR, PIK3CA, AKT1, and EGFR. The high distribution of metabolites of PM-D in the liver of administrated mice was recognized by mass spectrometry imaging. Spatially resolved metabolomics results revealed significant changes in metabolic profiles after PM-D administration, and metabolites such as taurine, taurocholic acid, adenosine, and acyl-carnitines were associated with PM-D-induced liver injury. Enrichment analyses of metabolic pathways revealed tht linolenic acid and linoleic acid metabolism, carnitine synthesis, oxidation of branched-chain fatty acids, and six other metabolic pathways were significantly changed. Comprehensive analysis revealed that the hepatotoxicity caused by PM-D was closely related to cholestasis, mitochondrial damage, oxidative stress and energy metabolism, and lipid metabolism disorders. CONCLUSIONS: In this study, the hepatotoxicity mechanisms of PM-D were comprehensively identified through an integrated spatially resolved metabolomics and network toxicology strategy, providing a theoretical foundation for the toxicity mechanisms of PM and its safe clinical application.

Anti-anaphylactic potential of benzoylpaeoniflorin through inhibiting HDC and MAPKs from Paeonia lactiflora.

Guided by cell-based anti-anaphylactic assay, eighteen cage-like monoterpenoid glycosides (1-18) were obtained from the bioactive fraction of P. lactiflora extract. Among these, compounds 1, 5, 6, 11, 12, 15, and 17 significantly reduced the release rate of ß-HEX and HIS without or with less cytotoxicity. Furthermore, the most potent inhibitor benzoylpaeoniflorin (5) was selected as the prioritized compound for the study of action of mechanism, and its anti-anaphylactic activity was medicated by dual-inhibiting HDC and MAPK signal pathway. Moreover, molecular docking simulation explained that benzoylpaeoniflorin (5) blocked the conversion of L-histidine to HIS by occupying the HDC active site. Finally, in vivo on PCA using BALB/c mice, benzoylpaeoniflorin (5) suppressed the IgE-mediated PCA reaction in antigen-challenged mice. These findings indicated that cage-like monoterpenoid glycosides, especially benzoylpaeoniflorin (5), mainly contribute to the anti-anaphylactic activity of P. lactiflora by dual-inhibiting HDC and MAPK signal pathway. Therefore, benzoylpaeoniflorin (5) may be considered as a novel drug candidate for the treatment of anaphylactic diseases.

Research progress of berberine mediated photodynamic therapy.

Berberine (BBR) is a plant secondary metabolite that has been used in photodynamic therapy (PDT) in the last few decades. The present review aimed to discuss the research progress of BBR-mediated photodynamic actions. The following key words were searched in several databases: 'Berberine' combined with 'photodynamic therapy', 'sonodynamic therapy (SDT)', 'ultraviolet', 'reactive oxygen' and 'singlet oxygen'. The results demonstrated that both type I and type II reactions participated in the photodynamic progression of BBR derivatives. In addition, the photochemical characteristics of BBR derivatives were affected by the polarity, pH and O2 content of solvents. DNA binding increases the lifespan of the photoexcited BBR state and generation of singlet oxygen (1O2). The chemical properties of substituents in different positions of the BBR skeleton are pivotal for its photochemical properties, particularly the methylenedioxy group at the C-2 and C-3 positions. BBR is a promising agent for mediating both PDT- and SDT-treated diseases, particularly in tumors. However, further studies are required to validate their biological effects. In addition, the molecular mechanisms underlying the antitumor effects of BBR-PDT remain unclear and warrant further investigation. The structural modification and targeted delivery of BBR have made it possible to broaden its applications; however, experimental verification is required. Overall, BBR acts as a sensitizer for PDT and has promising development prospects.

UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity.

BACKGROUND: The raw and processed roots of Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researchers to explore methods to improve quality standards to ensure their quality and treatment effects. METHODS: In the present study, an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was optimized and validated for the determination of dianthrones in PMR and PMRP using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase [A: acetonitrile and B: water containing 0.1% formic acid (v/v)] at a flow rate of 0.25 mL/min was achieved on an Agilent ZORBAX SB-C18 column (2.1 mm × 50 mm, 1.8 µm). The triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Moreover, compounds 5 and 6 were further evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay. RESULTS: The UHPLC-QQQ-MS/MS method was first developed to simultaneously determine six dianthrones in PMR and PMRP, namely, polygonumnolides C1-C4 (1-4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1-6 in 90 batches of PMR were in the ranges of 0.027-19.04, 0.022-13.86, 0.073-15.53, 0.034-23.35, 0.38-83.67 and 0.29-67.00 µg/g, respectively. The contents of 1-6 in 86 batches of commercial PMRP were in the ranges of 0.020-13.03, 0.051-8.94, 0.022-7.23, 0.030-12.75, 0.098-28.54 and 0.14-27.79 µg/g, respectively. Compounds 1-4 were almost completely eliminated after reasonable processing for 24 h and the contents of compounds 5 and 6 significantly decreased. Additionally, compounds 5 and 6 showed inhibitory activity in HepaRG cells with IC50 values of 10.98 and 15.45 µM, respectively. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of potentially toxic markers, the standardization of processing methods, the development of limit standards and a risk-benefit assessment. CONCLUSION: The results of the cytotoxicity evaluation of the dianthrones indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as toxic markers of PMRP. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in the future.