[Clinical effects of free anterolateral thigh perforator flap pedicled with descending genicular artery in repairing wounds after lower leg limb-sparing surgery].

Objective: To explore the clinical effects of free anterolateral thigh perforator flap pedicled with descending genicular artery in repairing wounds after lower leg limb-sparing surgery. Methods: A retrospective observational study was conducted. From January 2019 to June 2021, 12 patients with wounds after lower leg limb-sparing surgery who met the inclusion criteria were admitted to Suzhou Ruihua Orthopedic Hospital, including 6 males and 6 females, aged 17 to 74 years, with original wound area ranging from 17 cm×9 cm to 40 cm×15 cm. Five patients had infection in wounds. The wounds were all repaired by free anterolateral thigh perforator flap from contralateral thigh, with area of 18 cm×10 cm to 37 cm×9 cm. The artery of flap was anastomosed with the descending genicular artery, and the wounds in donor areas were sutured directly. Seven patients were transplanted with split-thickness skin grafts from the contralateral thigh to cover the remaining wounds that can not be covered by flap and the wounds in donor areas were covered with gauze. During the operation, the types of perforating branch carried by flap and the types of arteries and veins in recipient areas were recorded. The survival and occurrence of vascular crisis of flap, the survival of skin graft, the wound healing in donor and recipient areas, and the length of hospital stay after flap transplant surgery were recorded. During follow-up, the color and texture of flap, reinfection in lower leg, and fracture healing were recorded. At the last follow-up, the limb salvage function of patients was evaluated according to the functional evaluation criteria of Chen Zhongwei's amputated limb replantation. Results: The types of perforating branches carried by flaps were as follows: 6 cases of only carrying the descending branch of the lateral circumflex femoral artery, 3 cases of only carrying the oblique branch of the lateral circumflex femoral artery, and 3 cases of carrying the descending branch of the lateral circumflex femoral artery and oblique branch of the lateral circumflex femoral artery after internal pressurization anastomosis. The types of arteries in the recipient area of flap were as follows: one case of main trunk of the descending genicular artery, 8 cases of the saphenous branch of the descending genicular artery, and 3 cases of the articular branch of the descending genicular artery. The types of veins in the recipient area of flap were as follows: 8 cases of one accompanying vein of the descending genicular artery and one branch of the great saphenous vein, and 4 cases of two branches of the great saphenous vein. All the flaps survived without vascular crisis, and all the skin grafts also survived. The wounds in the donor and recipient areas were all healed. The length of hospital stay of patient after flap transplant surgery ranged from 13 to 79 days. During the follow-up of 6 to 23 months, the color and texture of flap were both good, with no infection in lower leg wound. Internal or external fixation were removed after fracture healing in 5 patients, and bone graft internal fixation was performed in 7 patients whose fractures were not healed after surgery and all the incisions healed without infection. At the last follow-up, the limb salvage effect of patients was evaluated as followings: excellent in 7 patients, good in 4 patients, and fair in one patient. Conclusions: Free anterolateral thigh flap pedicled with descending genicular artery can effectively repair the wounds after lower leg limb-sparing surgery and control infection with short length of hospital stay, while not increasing the risk of secondary injury of distal limb vessels. Thus, it can obtain satisfactory limb salvage effect which is worthy of clinical promotion.

[Repeated stellate ganglion blockade for the treatment of ventricular tachycardia storm in patients with nonischemic cardiomyopathy: a new therapeutic option for patients with malignant arrhythmias].

Objectives: This study sought to describe our institutional experience of repeated percutaneous stellate ganglion blockade (R-SGB) as a treatment option for drug-refractory electrical storm in patients with nonischemic cardiomyopathy (NICM). Methods: This prospective observational study included 8 consecutive NICM patients who had drug-refractory electrical storm and underwent R-SGB between June 1, 2021 and January 31, 2022. Lidocaine (5 ml, 1%) was injected in the vicinity of the left stellate ganglion under the guidance of ultrasound, once per day for 7 days. Data including clinical characteristics, immediate and long-term outcomes, and procedure related complications were collected. Results: The mean age was (51.5±13.6) years. All patients were male. 5 patients were diagnosed as dilated cardiomyopathy, 2 patients as arrhythmogenic right ventricular cardiomyopathy and 1 patient as hypertrophic cardiomyopathy. The left ventricular ejection fraction was 37.8%±6.6%. After the treatment of R-SGB, 6 (75%) patients were free of electrical storm. 24 hours Holter monitoring showed significant reduction in ventricular tachycardia (VT) episodes from 43.0 (13.3, 276.3) to 1.0 (0.3, 34.0) on the first day following R-SGB (P<0.05) and 0.5 (0.0, 19.3) after whole R-SGB process (P<0.05). There were no procedure-related major complications. The mean follow-up was (4.8±1.1) months, and the median time of recurrent VT was 2 months. Conclusion: Minimally invasive R-SGB is a safe and effective method to treat electrical storm in patients with NICM.

[Clinical characteristics and long-term follow-up results of radiofrequency ablation for the treatment of ventricular tachycardia in patients with arrhythmogenic left ventricular cardiomyopathy].

Objective: To investigate the acute and long-term outcome of catheter ablation for the treatment of ventricular tachycardia (VT) in patients with arrhythmogenic left ventricular cardiomyopathy (ALVC). Methods: This retrospective, cross-sectional study enrolled ALVC patients undergoing radiofrequency ablation for the treatment of VT at the First Affiliated Hospital of Nanjing Medical University from January 2011 to December 2018 and collected their clinical characteristics and intraoperative electrophysiological examination. Patients were followed up every 6 months after radiofrequency ablation until August 2021. Echocardiographic results and VT recurrence post radiofrequency ablation were analysed. Results: Totally 12 patients were enrolled (mean age: (42±15) years, 11 males(11/12)). The mean of left ventricular end diastolic diameter (LVDd) and left ventricular ejection fraction (LVEF) were (51±5)mm and (65±5)%, respectively. Twelve VTs were induced in 10 patients during the electrophysiological study, and the mean tachycardia cycle length was (293±65) ms. Three-dimensional substrate mapping revealed the diseased area at endocardial site in one patient, at epicardial sites in the other 11 patients (involved endocardial sites in 2 cases) with the basal part near the mitral annulus being the predilection for the substrate (10/11). After the catheter ablation at the endocardial and epicardial sites respectively, the complete procedure endpoint was achieved in all patients (VT cannot be induced post ablation). The median follow-up time was 65 (25, 123) months. One patient was lost to follow-up, and the other 11 patients survived without VT. No significant cardiac function deterioration was detected by the echocardiographic examination ((51±5)mm vs. (52±5)mm, P>0.05 for LVDd, (65±5)% vs. (60±6)%, P>0.05 for LVEF) at the end of follow-up. Conclusion: After radiofrequency ablation, the complete procedure endpoint is achieved in ALVC patients, and the catheter ablation provides long-term ventricular tachycardia control during the long-term follow-up.

MiR-21 relieves rheumatoid arthritis in rats via targeting Wnt signaling pathway.

OBJECTIVE: To investigate the influence of micro ribonucleic acid (miR)-21 on rats with rheumatoid arthritis (RA) through the Wnt signaling pathway. MATERIALS AND METHODS: A total of 30 rats were divided into three groups: control group (healthy rats, n=10), model group (rat model of RA, n=10), and MiR group (rat model of RA injected with miR-21 lentivirus, n=10). The paw volume, arthritis indexes, and protein expression level in each group were analyzed by means of paw volume and arthritis index measurement, reverse transcription-polymerase chain reaction (RT-PCR) assay, and fluorescent Western blotting. RESULTS: The expression levels of inflammatory factors declined in MiR group compared with those in model group, while they were higher in model group than those in control group and MiR group (p<0.05). At 15 d after transfection with lentivirus, the paw volume in MiR group was smaller than that in model group, which was decreased markedly with the extended time of transfection (p<0.05). On the 30th d, MiR group had a remarkably smaller paw volume than model group. In comparison with that in control group, the paw volume in model group was increased notably from the 7th d and displayed a significant difference in the 30th d (p<0.05). The arthritis indexes in MiR group were lower than those in model group; however, there were no apparent inflammations at the joints at 15 d after drug administration. Moreover, the longer the time of drug administration was, the less apparent the inflammations at the joints will be. The inflammations at the joints were ameliorated evidently on the 30th d in MiR group (p<0.05). Compared with those in control group, the inflammations in model group were increased significantly from the 7th d, with significant differences in the 30th d (p<0.05). The messenger RNA (mRNA) expression levels of interleukin-6 (IL-6), IL-8, and Wnt in MiR group were higher than those in control group, but lower than those in model group (p<0.05), while they were higher in model group than those in control group (p<0.05). The expression level of Wnt protein was decreased in MiR group compared with that in model group (p<0.05), and model group had a prominently elevated expression level of Wnt protein in comparison with control group (p<0.05). CONCLUSIONS: MiR-21 overexpression can repress the expressions of IL-6 and IL-8 and relieve the symptoms of RA by down-regulating the Wnt signal.

Neuroprotection of ulinastatin on transient cerebral ischemia via antioxidative mechanisms.

Ulinastatin [also called urinary trypsin inhibitor (UTI)] has beneficial effects on cerebral ischemic injury evoked by cardiac arrest (CA). However, the underlying mechanisms are unknown. The purpose of this report was to determine the involvement of antioxidative signal pathway of the hippocampus in effects of UTI in the process of neurological functions after transient cerebral ischemia. CA was induced by asphyxia followed by cardiopulmonary resuscitation in rats. Western blot analysis and ELISA were used to examine expression of Nrf2-antioxidant response element (ARE) and superoxide dismutases (SOD), and the levels of products of oxidative stress. In addition, the modified neurological severity score (mNSS) and spatial working memory performance were employed to assess neurological deficiencies in CA rats. Our results show that CA impaired Nrf2-ARE and SOD in the hippocampus CA1 region and amplified products of oxidative stress, namely 8-isoprostaglandin F2α (8-iso PGF2α) and 8-hydroxy-2’-deoxyguanosine (8-OHdG). Systemic administration of UTI largely restored Nrf2-ARE and SOD, and this also attenuated amplification of 8-iso PGF2α and 8-OHdG induced by cerebral ischemia and thereby alleviated neurological deficits with increasing survival of CA rats. Our data suggest that UTI improves Nrf2-ARE signals and inhibits products of oxidative stress in the hippocampus, which is linked to improvement of neurological deficiencies in transient cerebral ischemia. UTI plays a beneficial role in modulating cerebral ischemic injury via antioxidative mechanisms.

The effect of selenium supplementation on coronary heart disease: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Selenium is a crucial mineral with antioxidant and immune functions, and selenium deficiency may increase the risk of coronary heart disease (CHD). However, the effect of selenium supplementation on CHD is still controversial according to numerous randomized controlled trials (RCTs). The aim of our meta-analysis study was to investigate the impact of selenium on CHD. METHODS: PUBMED, EMBASE, MEDLINE, and the Cochrane Central Register of Controlled Trials databases were systematically searched to identify RCTs evaluating the effect of selenium supplementation on CHD mortality, blood lipid profile (high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total cholesterol), serum C-reactive protein (CRP), and the level of glutathione peroxidase (GSH-PX) from inception until September 20, 2016. Odds ratio of CHD mortality and the associated 95% confidence intervals (CIs) were calculated using the fixed effect model. Weighted mean difference or standardized mean difference (SMD) and 95% confidence intervals (CIs) were calculated to determine the lipid profile, serum CRP, and GSH-PX using fixed effect or random effect models depending on the observed heterogeneity. RESULTS: A total of 16 eligible RCTs with 43998 participants were included. Significant effects were observed for serum CRP (SMD=-0.48; 95% CI, -0.96 to 0; p=0.049) and GSH-PX (SMD=0.5; 95% CI, 0.36-0.64; p<0.001) after selenium supplementation. However, selenium supplementation was not statistically associated with CHD mortality and an aberrant lipid profile. CONCLUSION: Selenium supplementation decreased serum CRP and increased the GSH-PX level, suggesting a positive effect on reducing oxidative stress and inflammation in CHD. However, selenium supplementation is not sufficient to reduce mortality and to improve the lipid status.

[The function of NLRP1 in noninfectious pulmonary injury following allogeneic hematopoietic stem cell transplantation].

Objective: To explore the function of NLRP1 in noninfectious pulmonary injury (nonIPI) after allogeneic stem cell transplantation (allo-HSCT) . Methods: In this study, we established the model of allo-HSCT with C57BL/6 and NLRP(-/-) mouse as recipients. Chimera rate was measured by flow cytometry. The HE staining was used to observe the pathology changes in the lungs. NLRP1 and relevant inflammatory proteins were measured by Western Blot. Results: On the day 14 after allo-HSCT, the chimera rate was more than 96%, HSCs of donors had been successfully transplanted into recipients. HE staining showed that nonIPI occurred after allo-HSCT. The degrees of injuries reached the peak on day 21. In addition, the expressions of MPO, NLRP1, p20, Mature-IL-1ß and Mature-IL-18 had same tends with the degrees of nonIPI. When we knocked out NLRP1 gene of recipients, the degrees of nonIPI reduced and the expressions of MPO, p20, Mature-IL-1ß and Mature-IL-18 were less than in non-knockout group. Conclusion: allo-HSCT could cause nonIPI and high expressions of MPO, p20, IL-1ß, IL-18, NLRP1. Knocking out NLRP1 gene could alleviate the degrees of nonIPI and reduce the expressions of relevant inflammatory proteins, indicating that NLRP1 might be one of factors contributed to nonIPI after allo-HSCT.

Development and Validation of a Nomogram for Predicting Incidence of Early Allograft Dysfunction Following Liver Transplantation.

OBJECTIVE: Early allograft dysfunction (EAD) is frequent complication post-liver transplantation and is closely related to recipient's mortality and morbidity. We sought to develop a nomogram for predicting incidence of EAD. METHODS: Based on multivariate analysis of donor, recipient, and operation data of 199 liver transplants from deceased donors between 2013 and 2015, we identified 5 significant risk factors for EAD to build a nomogram. The model was subjected to prospective validation with a cohort of 42 patients who was recruited between January and June 2016. The predictive accuracy and discriminative ability were measured by area under the receiver operating characteristic curve (AUC). The agreement between nomogram prediction and actual observation was showed by the calibration curve. RESULTS: Incidence rate of EAD in the training set and validation cohort were 55.91% (104/199) and 54.76% (23/42), respectively. In the training set, according to the results of univariable and multivariable analysis, 5 independent risk factors including donor gender, donor serum gamma-glutamyl transpeptidase level, donor serum urea level, donor comorbidities (respiratory, cardiac, and renal dysfunction), and recipient Model for End-stage Liver Disease score were identified and assembled into the nomogram. The AUC of internal validation using bootstrap resampling and prospective validation using the external cohort of 42 patients was 0.74 and 0.60, respectively. The calibration curves for probability of EAD showed acceptable agreement between nomogram prediction and actual observation. According to the score table, the probability of EAD was under 30% when the total point tally was under 72. But when the total was up to 139, the risk of EAD increased to 60%. CONCLUSION: We've established and validated a nomogram that can provide individual prediction of EAD for liver transplant recipients. The practical prognostic model may help clinicians to qualify the liver graft accurately, making a more reasonable allocation of organs.

Sensitive and selective LC-MS/MS assay for quantitation of flutrimazole in human plasma.

OBJECTIVE: A highly sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of flutrimazole in human plasma. This study was to investigate the application of sensitive and selective LC-MS/MS method for quantitation of flutrimazole in human plasma. MATERIALS AND METHODS: The analysis and internal standard were extracted with ether and hexane (v:v, 1:1) followed by a rapid isocratic elution with a 0.1% formic acid/methanol (v:v, 20:80) on a C18 column (50 mm × 2.1 mm I.D.) and subsequent analysis by mass spectrometry in the multi-reaction-monitoring mode. The precursor to production transitions of m/z 279.0 → 183.1 and m/z 441.0 → 295.1 were used to measure the analyte and the internal standard. RESULTS: The assay was linear over the concentration range of 0.996-99.6 ng•mL-1 for flutrimazole in human plasma. The lower limit of quantification was 0.996 ng•mL-1 and the extraction recovery was larger than 78.83% for flutrimazole. The inter- and intra-day precision of the method at three concentrations was less than 9.26%. CONCLUSIONS: The LC-MS/MS method was firstly applied to quantitation of flutrimazole in human plasma.

Multidisciplinary Cooperation in a Simultaneous Combined Liver and Kidney Transplantation Patient of Primary Hyperoxaluria 1.

Primary hyperoxaluria type 1 is an autosomal recessive hereditary glyoxylate metabolism disorder characterized by excessive production of oxalate, caused by the deficiency of liver specific peroxisomal enzyme: alanineglyoxylate aminotransferase. For patients with end-stage renal disease, combined liver and kidney transplantation was needed. This report describes one patient, with a diagnosis of end-stage renal disease and primary hyperoxaluria 1 confirmed by PCR and direct sequencing with genomic DNA, received the simultaneous combined liver and kidney transplantation after seven months' waiting. However, there were several complications observed post surgery, such as protracted bleeding, common bile duct anastomotic stenosis, biliary calculi and recurrence of urolithiasis. All these were well solved by relevant department, and finally a satisfactory outcome was achieved. Multidisciplinary cooperation plays an important role on the PH1 patient management, especially when multiple complications are encountered.

An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

Doubts and difficulties in studying blood pressure variability.

The blood pressure (BP) fluctuation was first noticed in the 18th century. However, its clinical significance did not get attention until recent years. The increase in BP variability (BPV) is possibly more valuable than the increase in BP level for predicting damages in target organs. Moreover, attenuating BPV is more important than decreasing the BP level. However, the concept of BPV was not used in any related guideline for diagnosing, defining, and grading the risk of hypertension, which is due to the understanding of BPV is not profound, and blind areas and misunderstanding still exist in the definition, features, and classification of BPV. In this paper, the doubts and difficulties in studying BPV are analyzed, which may conduce to understand BPV and thus help for the clinical diagnosis and treatment of hypertension.

Calpain 2-mediated autophagy defect increases susceptibility of fatty livers to ischemia-reperfusion injury.

Hepatic steatosis is associated with significant morbidity and mortality after liver resection and transplantation. This study focuses on the role of autophagy in regulating sensitivity of fatty livers to ischemia and reperfusion (I/R) injury. Quantitative immunohistochemistry conducted on human liver allograft biopsies showed that, the reduction of autophagy markers LC3 and Beclin-1 at 1 h after reperfusion, was correlated with hepatic steatosis and poor survival of liver transplant recipients. In animal studies, western blotting and confocal imaging analysis associated the increase in sensitivity to I/R injury with low autophagy activity in fatty livers. Screening of autophagy-related proteins showed that Atg3 and Atg7 expression levels were marked decreased, whereas calpain 2 expression was upregulated during I/R in fatty livers. Calpain 2 inhibition or knockdown enhanced autophagy and suppressed cell death. Further point mutation experiments revealed that calpain 2 cleaved Atg3 and Atg7 at Atg3Δ92-97 and Atg7Δ344-349, respectively. In vivo and in vitro overexpression of Atg3 or Atg7 enhanced autophagy and suppressed cell death after I/R in fatty livers. Collectively, calpain 2-mediated degradation of Atg3 and Atg7 in fatty livers increases their sensitivity to I/R injury. Increasing autophagy may ameliorate fatty liver damage and represent a valuable method to expand the liver donor pool.

Genome-wide differential expression of synaptic long noncoding RNAs in autism spectrum disorder.

A genome-wide differential expression of long noncoding RNAs (lncRNAs) was identified in blood specimens of autism spectrum disorder (ASD). A total of 3929 lncRNAs were found to be differentially expressed in ASD peripheral leukocytes, including 2407 that were upregulated and 1522 that were downregulated. Simultaneously, 2591 messenger RNAs (mRNAs), including 1789 upregulated and 821 downregulated, were also identified in ASD leukocytes. Functional pathway analysis of these lncRNAs revealed neurological pathways of the synaptic vesicle cycling, long-term depression and long-term potentiation to be primarily involved. Thirteen synaptic lncRNAs, including nine upregulated and four downregulated, and 19 synaptic mRNAs, including 12 upregulated and seven downregulated, were identified as being differentially expressed in ASD. Our identification of differential expression of synaptic lncRNAs and mRNAs suggested that synaptic vesicle transportation and cycling are important for the delivery of synaptosomal protein(s) between presynaptic and postsynaptic membranes in ASD. Finding of 19 lncRNAs, which are the antisense, bi-directional and intergenic, of HOX genes may lead us to investigate the role of HOX genes involved in the development of ASD. Discovery of the lncRNAs of SHANK2-AS and BDNF-AS, the natural antisense of genes SHANK2 and BDNF, respectively, indicates that in addition to gene mutations, deregulation of lncRNAs on ASD-causing gene loci presents a new approach for exploring possible epigenetic mechanisms underlying ASD. Our study also opened a new avenue for exploring the use of lncRNA(s) as biomarker(s) for the early detection of ASD.

DRP1 inhibition rescues retinal ganglion cells and their axons by preserving mitochondrial integrity in a mouse model of glaucoma.

Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic neuropathies.

Gender Differences in the Toxicokinetics of Triptolide after Single- and Multiple-dose Administration in Rats.

Triptolide is a natural compound extracted from the traditional Chinese medicine Tripterygium wilfordii Hook F with distinguishing pharmacological activities and evident toxicities. We reported previously that 28 continuous days of oral administration of triptolide in rats resulted in gender dimorphic profiles in toxicities. To better understand this issue, the toxicokinetics of triptolide was observed in this study. Rats of both sexes were administered 400 µg/kg triptolide either as a single dose or multiple doses for 28 days. Triptolide concentrations in rat plasma were determined using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The plasma concentration-time curve and toxicokinetic parameters revealed gender differences after single and repeated triptolide administration, including significantly higher area under the plasma concentration-time curve (AUC0-∞) and peak plasma concentration (Cmax), lower clearance rate (CL) and longer terminal elimination half-life (t1/2) of triptolide in females, and lower drug exposure levels and greater CL in males. The gender differential disposition of triptolide may be the cause of increased toxicity in females. Moreover, auto-inhibition of metabolism and the resulting increase in drug exposure were observed after repeated dosing. The AUC0-∞ of triptolide was increased 6-fold in females and 2-fold in males, while the CL of triptolide was significantly decreased by 84% in females and 55% in males. These results indicated that gender-related differences existed in the toxicokinetics of triptolide and long-term oral administration of triptolide resulted in drug accumulation, which might account for the gender differences in the toxicities of triptolide.

Augmented efficacy with the combination of blockade of the Notch-1 pathway, bortezomib and romidepsin in a murine MT-1 adult T-cell leukemia model.

Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell lymphotropic virus-1. There is no accepted curative therapy for ATL. We have reported that certain ATL patients have increased Notch-1 signaling along with constitutive activation of the nuclear factor-κB pathway. Physical and functional interaction between these two pathways provides the rationale to combine the γ-secretase inhibitor compound E with the proteasome inhibitor bortezomib. Moreover, romidepsin, a histone deacetylase inhibitor, has demonstrated major antitumor action in leukemia/lymphoma. In this study, we investigated the therapeutic efficacy of the single agents and the combination of these agents in a murine model of human ATL, the MT-1 model. Single and double agents inhibited tumor growth as monitored by tumor size (P<0.05), and prolonged survival of leukemia-bearing mice (P<0.05) compared with the control group. The combination of three agents significantly enhanced the antitumor efficacy as assessed by tumor size, tumor markers in the serum (human soluble interleukin-2 receptor-α and ß2-microglobulin) and survival of the MT-1 tumor-bearing mice, compared with all other treatment groups (P<0.05). Improved therapeutic efficacy obtained by combining compound E, bortezomib and romidepsin supports a clinical trial of this combination in the treatment of ATL.

Inhibition of cyclophilin D by cyclosporin A promotes retinal ganglion cell survival by preventing mitochondrial alteration in ischemic injury.

Cyclosporin A (CsA) inhibits the opening of the mitochondrial permeability transition pore (MPTP) by interacting with cyclophilin D (CypD) and ameliorates neuronal cell death in the central nervous system against ischemic injury. However, the molecular mechanisms underlying CypD/MPTP opening-mediated cell death in ischemic retinal injury induced by acute intraocular pressure (IOP) elevation remain unknown. We observed the first direct evidence that acute IOP elevation significantly upregulated CypD protein expression in ischemic retina at 12 h. However, CsA prevented the upregulation of CypD protein expression and promoted retinal ganglion cell (RGC) survival against ischemic injury. Moreover, CsA blocked apoptotic cell death by decreasing cleaved caspase-3 protein expression in ischemic retina. Of interest, although the expression level of Bcl-xL protein did not show a significant change in ischemic retina treated with vehicle or CsA at 12 h, ischemic damage induced the reduction of Bcl-xL immunoreactivity in RGCs. More importantly, CsA preserved Bcl-xL immunoreactivity in RGCs of ischemic retina. In parallel, acute IOP elevation significantly increased phosphorylated Bad (pBad) at Ser112 protein expression in ischemic retina at 12 h. However, CsA significantly preserved pBad protein expression in ischemic retina. Finally, acute IOP elevation significantly increased mitochondrial transcription factor A (Tfam) protein expression in ischemic retina at 12 h. However, CsA significantly preserved Tfam protein expression in ischemic retina. Studies on mitochondrial DNA (mtDNA) content in ischemic retina showed that there were no statistically significant differences in mtDNA content among control and ischemic groups treated with vehicle or CsA. Therefore, these results provide evidence that the activation of CypD-mediated MPTP opening is associated with the apoptotic pathway and the mitochondrial alteration in RGC death of ischemic retinal injury. On the basis of these observations, our findings suggest that CsA-mediated CypD inhibition may provide a promising therapeutic potential for protecting RGCs against ischemic injury-mediated mitochondrial dysfunction.

A chiral HPLC-MS/MS method for simultaneous quantification of warfarin enantiomers and its major hydroxylation metabolites of CYP2C9 and CYP3A4 in human plasma.

Warfarin is an oral anticoagulant that requires frequent therapeutic drug monitoring due to a narrow therapeutic window, considerable interindividual variability in drug response, and susceptibility to drug-drug and drug-diet interactions. Enantiomeric separation and quantification of warfarin enantiomers and clinically important major hydroxylation metabolites are essential for drug interaction studies and phenotypic characterization of CYP2C9 and CYP3A4, the major cytochrome P450 (CYP) enzymes involved in warfarin metabolism. Here, we describe the development and validation of a chiral high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)-based quantification of R-warfarin, S-warfarin, S-7-hydroxywarfarin (the major CYP2C9 metabolite) and (9R;10S)-10-hydroxywarfarin (the CYP3A4 metabolite) in human plasma. Simple protein precipitation-based extraction showed good recovery of analytes (82.9 - 96.9%). The developed method exhibited satisfactory intra-day and inter-day accuracy and precision. The lower limits of detection were 0.25 nM (or ~0.08 ng/mL) for the warfarin enantiomers and 0.1 nM (or ~0.04 ng/mL) for S-7-hydroxywarfarin and (9R;10S)-10-hydroxywarfarin using only 50 µL plasma during extraction. The validated method was successfully applied to analyze plasma samples obtained from a healthy human subject who enrolled in a clinical drug interaction study involving warfarin.