Effect of an educational video about ERAS on reducing preoperative anxiety and promoting recovery.

Video propaganda is reported effectively improving patients' understanding of operation. However, whether a video introducing patients' most concerns can reduce preoperative anxiety and promote recovery stays unsealed. In this study, we investigated the effects of complementary therapy of educational video during preoperative visit. The results showed that thirty-five (23.2%) parents in Group Control were diagnosed as anxiety according to SAS, and nineteen (12.3%) patients were diagnosed after video intervention. The APAIs anxiety score and APAIs information score in Group Video were lower than those in Group Control. Compared with Group Control, video visit helped to increase the first-attempt pass rate of the knowledge retention exam and solve the patient's most worried concerns, and decrease incidence of emergence agitation, total cost of hospitalization and length of hospital stay. Moreover, video visit improved satisfaction degrees of patients and their main family members. Briefly, our study demonstrated video visit can improve patients' knowledge of anesthesia and decrease their preoperative anxiety, which may represent an important complementary therapy to routine preoperative visits.

[Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway].

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 µmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 µmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Response of soil microbial community to plant composition changes in broad-leaved forests of the karst area in Mid-Subtropical China.

The rapid growth and expansion ofCryptomeria japonica (Thunb. ex L. f.) D. Don in karst area strongly affects plant composition of native deciduous broad-leaved forest, which seriously threat ecosystem function and service. Given the importance of soil microorganisms in regulating nutrients cycling and plant species coexistence, understanding soil microbial attributes and their relationships with soil and vegetation features in forests harboring different C. japonica abundance will help understanding the drivers of ecosystem function changes. Here we examined the diversity and composition of soil bacterial and fungal communities and their correlations with plant diversity as well as soil physicochemical properties in karst broad-leaved forests with different relative abundances of C. japonica (i.e., a high, moderate, low and no proportion level with a stem density of 1,487, 538, 156 and 0 plant/hm2, respectively) in Mid-Subtropical China. We found that soil pH decreased while soil water content (SWC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) tended to increase with the increase in C. japonica abundance. In contrast, soil available nitrogen (AN), available phosphorus (AP) and available potassium (AK) content declined by 26.1%∼49.3% under the high level of C. japonica abundance. A gradual decrease in relative abundance of Acidobacteria and Chloroflexi while a pronounced increase in relative abundance of Ascomycota and Basidiomycota were observed with increase of C. japonica abundance. Alternations in bacterial composition were closely related to changes in AP and AK, while the change of fungal structure was mainly related to SWC, soil organic carbon (SOC) and pH, indicating that bacterial community was sensitive to declines in soil available nutrients and fungal structure was sensitive to changes in soil physicochemical properties (i.e., pH and SWC) and organic carbon resource. Understory plants had the highest α-diversity in forest containing moderate abundance of C. japonica, which might be related to the high bacterial diversity. Our findings suggest conservation of soil bacterial and fungal taxa that are responsible for nutrients availability and carbon sequestration is of great significance for improving the resistance of natural deciduous broad-leaved forests to the rapid spread of C. japonica in karst areas. Moreover, Acidobacteria, Chloroflexi, Ascomycota and Basidiomycota are potential indicators for soil properties changes, which should be taken into consideration in karst forest managements.

Downregulation of p38 MAPK Activation and Radiation-Sensitive 52 Expression Enhances 5-Fluorouracil and Erlotinib-Induced Cytotoxicity in Human Lung Squamous Cells.

INTRODUCTION: 5-Fluorouracil (5-FU) is used to treat various cancers, including non-small-cell lung cancer (NSCLC). It inhibits nucleotide synthesis and induces single- and double-strand DNA breaks. In the homologous recombination pathway, radiation-sensitive 52 (Rad52) plays a crucial role in DNA repair by promoting the annealing of complementary single-stranded DNA and stimulating Rad51 recombinase activity. Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor with clinical activity against NSCLC cells. However, whether the combination of 5-FU and erlotinib has synergistic activity against NSCLC cells is unknown. METHODS: After the 5-FU and/or erlotinib treatment, the expressions of Rad52 mRNA were determined by quantitative real-time polymerase chain reaction analysis. Protein levels of Rad52 and phospho-p38 MAPK were determined by Western blot analysis. We used specific Rad52 or p38 MAPK small interfering RNA and p38 MAPK inhibitor (SB2023580) to examine the role of p38 MAPK-Rad52 signal in regulating the chemosensitivity of 5-FU and/or erlotinib. Cell viability was assessed by MTS assay and trypan blue exclusion assay. RESULTS: In 2 squamous cell carcinoma cell lines, namely, H520 and H1703, 5-FU reduced Rad52 expression in a p38 MAPK inactivation-dependent manner. Enhancement of p38 MAPK activity by transfection with MKK6E (a constitutively active form of MKK6) vector increased the Rad52 protein level and cell survival by 5-FU. However, in human lung bronchioloalveolar cell adenocarcinoma A549 cells, 5-FU reduced Rad52 expression and induced cytotoxicity independent of p38 MAPK. Moreover, 5-FU synergistically enhanced the cytotoxicity and cell growth inhibition of erlotinib in NSCLC cells; these effects were associated with Rad52 downregulation and p38 MAPK inactivation in H520 and H1703 cells. CONCLUSION: The results provide a rationale for combining 5-FU and erlotinib in lung cancer treatment.

A Systematic Approach for Liposome and Lipodisk Preclinical Formulation Development by Microfluidic Technology.

Lipid nanoparticles have transformed the drug delivery field enhancing the therapeutic drug performance of small molecules and biologics with several approved drug products. However, in industry, these more complex drug delivery systems such as liposomes require more material and time to develop. Here, we report a liposome and lipodisk decision tree with model compounds of diverse physicochemical properties to understand how to resourcefully optimize encapsulation efficiency (EE) for these lipid-based drug delivery systems. We have identified trends with physicochemical properties such as Log P, where higher Log P compounds such as curcumin were able to efficiently load into the lipid bilayer resulting in high EE with altering the drug/lipid (D/L) ratio. Moderate Log P compounds such as cyclosporine A and dexamethasone had significantly higher encapsulation in lipodisks, which contain higher amounts of PEG lipid compared to liposomes. The EE of negative Log P compounds, like acyclovir, remained low regardless of altering the D/L ratio and PEG concentrations. In this study, microfluidic techniques were employed to fabricate liposomes and lipodisks formulations allowing for a reproducible strategy for formulation development. Both liposome and lipodisk of curcumin demonstrated enhanced in vivo performance compared with a conventional formulation in the rat pharmacokinetic study. This combination of approaches with multiple model compounds and lipid-based drug delivery systems provides a systematic guidance to effective strategies to generate higher EE with minimal drug waste and expedite the process for preclinical development when applied to industry compounds.

Investigating the Utility of Humanized Pregnane X Receptor-Constitutive Androstane Receptor-CYP3A4/7 Mouse Model to Assess CYP3A-Mediated Induction.

Clinical induction liability is assessed with human hepatocytes. However, underpredictions in the magnitude of clinical induction have been reported. Unfortunately, in vivo studies in animals do not provide additional insight because of species differences in drug metabolizing enzymes and their regulatory pathways. To circumvent this limitation, transgenic animals expressing human orthologs were developed. The aim of this work was to investigate the utility of mouse models expressing human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 (Tg-Composite) in evaluating clinical induction. Rifampin, efavirenz, and pioglitazone, which were employed to represent strong, moderate, and weak inducers, were administered at multiple doses to Tg-Composite animals. In vivo CYP3A activity was monitored by measuring changes in the exposure of the CYP3A probe substrate triazolam. After the in vivo studies, microsomes were prepared from their livers to measure changes of in vitro CYP3A4 activity. In both in vivo and in vitro, distinction of clinic induction was recapitulated as rifampin yielded the greatest inductive effect followed by efavirenz and pioglitazone. Interestingly, with rifampin, in vivo CYP3A activity was approximately 4-fold higher than in vitro activity. Conversely, there was no difference between in vivo and in vitro CYP3A activity with efavirenz. These findings are consistent with the report that, although rifampin exhibits differential inductive effects between the intestines and liver, efavirenz does not. These data highlight the promise of transgenic models, such as Tg-Composite, to complement human hepatocytes to enhance the translatability of clinical induction as well as become a powerful tool to further study mechanisms of drug disposition. SIGNIFICANCE STATEMENT: Underprediction of the magnitude of clinical induction when using human hepatocytes has been reported, and transgenic models may improve clinical translatability. The work presented here showcases the human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 model, which was able to recapitulate the magnitude of clinical induction and to differentiate tissue-dependent induction observed with rifampin but not with efavirenz. These results not only foreshadow the potential application of such transgenic models in assessing clinical induction but also in further investigation of the mechanism of drug disposition.

[Effect of Tripterygium Glycosides Tablets on reproductive toxicity in male rats with â ¡ type collagen induced arthritis].

The aim of this paper was to observe the toxic effect of Tripterygium Glycosides Tablets on the reproductive system of Ⅱ type collagen induced arthritis(CIA) male rats, and to explore the toxic mechanism preliminarily. Fifty SD rats were randomly divided into normal control group(Con), model group(CIA), Tripterygium Glycosides Tablets clinical equivalent dose groups of 1, 2, 4 times(9, 18, 36 mg·kg~(-1)), 10 rats in each group, and were given by gavage once a day for 42 days after the first immunization. The organ index of testis and epididymis were calculated on days 21 and 42. Histopathological and morphological changes of testis and epididymis were observed under optical microscope. Sperm count, sperm malformation rate and sperm kinetic parameters in epididymal tissues were observed by computer assisted sperm analysis(CASA). The concentration of testosterone(T), nitric oxide synthase(NOS) and aromatase(CYP19 A1) in serum were detected by ELISA. Immunohistochemistry was used to observe the expression of Bax and Bcl-2 related proteins in the apoptosis pathway of testis and epididymis. The results showed that, compared with Con group, CIA group significantly increased the rate of testicular spermatogenic tubule lesion and sperm malformation, decreased the average path speed, and no significant changes were observed in other groups. Tripterygium Glycosides Tablets at 4 times clinical equivalent dose can significantly reduce the testis index(P<0.01), each dose group can reduce the epididymis index(P<0.05). Each dose group of Tripterygium Glycosides Tablets could cause different degrees of damage to the testis and epididymis, the proportion of testicular histopathology lesions increased, the number of spermatogenic cells in the seminiferous tubules decreased, and so on. It could reduce the number of sperm, increase the rate of sperm deformity, make the parameters of sperm dynamics abnormal, and so on. Tripterygium Glycosides Tablets at 4 times dose could significantly reduce the content of serum sex hormone T and key enzyme of androgen synthesis(P<0.05 or P<0.01), but had no effect on CYP19 A1. The expression of Bax and Bcl-2 in testis and epididymis were increased by 2 and 4 times doses of Tripterygium Glycosides Tablets(P<0.05, P<0.01 or P<0.01). The results showed that 21 d administration of Tripterygium Glycosides Tablets at equal or higher doses could induce obvious toxic effect to the reproductive organs of CIA male rats, and lower the level of serum sex hormone T and the key enzyme of androgen synthesis, NOS. The mechanism of abnormal changes of Bax and Bcl-2 in Testis and epididymis is still to be elucidated.

[Overview of hepatotoxicity studies on Tripterygium wilfordii in recent 20 years].

Tripterygium wilfordii is widely used in the treatment of rheumatism with curative effect. However,its toxicity and adverse reactions,especially the hepatotoxicity,rank the first in the herbs induced liver injury,is the key factors hindering its clinical application. This paper reviewed the literatures related to the hepatotoxicity of T. wilfordii in recent 20 years,and summarized the characteristic of hepatotoxicity induced by T. wilfordii,the factors causing liver injury,the mechanism of toxicity,and the measures to reduce toxicity. In animal experiments,the T. wilfordii induced-hepatotoxicity in physiological state was more serious than pathological state. The T. wilfordii induced-hepatotoxicity is related to various toxic components contained in it,but alkaloids are the most toxic one.Overdose and cumulative overdose are the lead causing of hepatotoxicity induced by T. wilfordii. The theory of oxidative stress is still an important mechanism of T. wilfordii induced-hepatotoxicity,and Nrf2,as a key regulatory enzyme of oxidative stress,has become an important target for drugs to against T. wilfordii induced-hepatotoxicity. Mitochondrial autophagy and liver hypersensitivity are new mechanisms of liver injury induced by T. wilfordii. The measures such as dosage control,drug compatibility and dosage form variations can help to reduce the hepatotoxicity induced by T. wilfordii. This paper clarified the current situation and shortcomings of safety research on T. wilfordii,so as to propose new research strategies and provide ideas for rational evaluation of safety and clinical safe drug use of T. wilfordii.

[Effect of Tripterygium Glycosides Tablets on synovial angiogenesis in rats with type â ¡ collagen induced arthritis].

To observe the effect of Tripterygium Glycosides Tablets on angiogenesis of rats with type Ⅱ collagen-induced arthritis( CIA) and on the tube formation of human umbilical vein endothelial cells( HUVEC) in vitro. The HUVEC were induced by 20 µg·L-1 vascular endothelial growth factor( VEGF) in vitro,and were treated with 0. 1,1,10 mg·L-1 Tripterygium Glycosides Tablets continuously for 7 hours. The numbers of branches of tube formation were measured. SD rats were immunized to establish CIA. CIA rats were treated with 9,18,36 mg·kg-1·d-1 Tripterygium Glycosides Tablets for 42 days. Histopathological examination( HE) was performed to observe the vascular morphology and vascular density in the synovial membrane of the inflamed joints. Immunohistochemistry and immunofluorescence were performed to observe the expression of platelets-endothelial cell adhesion molecule( CD31) and αsmooth muscle actin( αSMA) in synovial membrane. Immunohistochemistry and Western blot were performed to observe the expression of hypoxia-inducible factors 1α( HIF1α) and angiotensin 1( Ang1) in the synovial tissue. The results showed that the numbers of branches of tube formation of HUVEC induced by VEGF were improved,and declined significantly after treated by Tripterygium Glycosides Tablets. Compared with the normal group,the vascular density,CD31 positive expression,CD31 +/αSMA-immature and total vascular positive expression in the synovial membrane of the model group were significantly increased,and so as HIF1α and Ang1 in the synovium. Tripterygium Glycosides Tablets reduced the synovial vascular density and inhibited the positive expression of CD31,CD31+/αSMA-immature blood vessels and total vascular,but has no effect on CD31+/αSMA+mature blood vessels. Tripterygium Glycosides Tablets also inhibited the expression of HIF1α and Ang1 in synovial membrane of inflammatory joints. Our results demonstrated that Tripterygium Glycosides Tablets could inhibit the angiogenesis of synovial tissue in CIA rats and the tube formation of HUVEC,which is related to the down-regulation of HIF1α/Ang1 signal axis.

[Effect of Tripterygium Glycosides Tablets on reproductive toxicity in female rats with â ¡ type collagen induced arthritis].

The aim of this paper was to observe the toxic effect of Tripterygium Glycosides Tablets( TG) on the reproductive system of Ⅱ type collagen induced arthritis( CIA) male rats,and to explore the toxic mechanism preliminarily. Fifty SD rats were randomly divided into normal control group( Con),model group( CIA),Tripterygium Glycosides Tablets clinical equivalent dose groups of 1,2,4 times( 9,18,36 mg·kg-1),10 rats in each group,and were given by gavage once a day for 42 days after the first immunization.The organ indexes of uterine and ovarian were calculated on days 21 and 42. Histopathological and morphological changes of uterine and ovarian were observed under optical microscope. The concentration of estradiol( E2),follicle-stimulating hormone( FSH),luteinizing hormone( LH),17α-hydroxylase( CYP17 A1) and cytochrome P450 19 A1( CYP19 A1) in serum were detected by ELISA. Immunohistochemistry was used to observe the expression of Bax and Bcl-2 related proteins in the apoptosis pathway of uterus and ovary. The results showed that compared with the Con group,CIA group could reduce the number of uterine glands( P<0.05),but no significant changes were observed in other groups. Compared with the CIA group,there were no significant changes in the coefficients of uterus and ovary in the Tripterygium Glycosides Tablets groups. The number of uterine glands,total follicles in the ovary,mature follicles and corpus luteum,the distribution of blood vessels and mitochondria had a certain inhibitory trend,and also slightly increased the number of atresia follicles,but the histopathological quantitative indicators were not statistically different. Except that 2 times clinical dose of Tripterygium Glycosides Tablets could significantly reduce the content of CYP19 A1( P<0. 05) after 42 d administration,there were no significant changes in serum estrogen E2,FSH,LH and estrogen synthesis key enzymes CYP17 A1 in each administration group. Medium and high doses of Tripterygium Glycosides Tablets could increase the expression of apoptotic protein Bax in uterine and ovarian tissues( P<0. 05,P<0. 01),and all the administration groups could inhibit the expression of apoptotic inhibiting protein Bcl-2( P <0. 05,P<0. 01,P<0.001),42 d was more obvious than 21 d. In conclusion,4 times and less than 4 times Tripterygium Glycosides Tablets did not cause obvious toxicity and histopathological changes in the reproductive organs of CIA rats,but it could reduce the level of serum estrogen synthesis key enzyme CYP19 A1 and affect the content of apoptosis-related proteins Bax and Bcl-2 in uterus and ovary tissues. The relevant mechanism needs further study.

[Comparative study on dose-toxicity-effect of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets on CIA model rats].

The aim of this paper was to compare the properties of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets from dose-effect-toxicity on type Ⅱ collagen-induced arthritis( CIA) in rats. SD rats were randomly divided into eight groups,including normal group,model group,Tripterygium Glycosides Tablets groups( 1 times equivalent dose 0.009 g·kg-1,4 times equivalent dose 0.036 g·kg-1,16 times equivalent dose 0.144 g·kg-1),Tripterygium wilfordii Tablets groups( 1 times equivalent dose 0.007 5 mg·kg-1,4 times equivalent dose 0.030 mg·kg-1,16 times equivalent dose 0.120 mg·kg-1). Beginning on the first immunization,Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets administered intraperitoneally once a day. After the second immunization,the symptoms such as redness and swelling of joints were observed,and the clinical score and incidence of arthritis were evaluated. HE and Masson staining were used to examine the histopathological changes of joints. The expression level of anti-type Ⅱ collagen antibody Ig G in serum was detected by ELISA,routine testing of blood components,the concentration of ALP( alkaline phosphatase),ALT( alanine aminotransferase),AST( aspartate aminotransferase),GGT( gamma-glutamyltransferase),TBi L( total bilirubin),CRE( creatinine) and UREA( urea) in serum were detected by enzymatic assay. The rate of sperm deformity in the epididymis was evaluated under light microscope. The extent of damage to the testis and ovarian tissue was assessed by HE staining. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets attenuated the inflammation,redness,swelling and deformity of joints and reduced the clinical score and incidence of arthritis in CIA rats. Meanwhile,it also exhibited obvious reduction in all pathological features such as joint synovitis,pannus,cartilage erosion and bone destruction. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets reduced Ig G in a dose-dependent manner,and Tripterygium Glycosides Tablets is better than Tripterygium wilfordii Tablets( P<0.05 or P<0.01). The high doses of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could significantly increase the organ coefficient of liver and spleen and reduced RBC and HGB in CIA rats( P<0.01),and severity leading to death. Gastric mucosal injury and morphological changes of liver and kidney were not observed in CIA rats of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets treatment group. The 4 and 16 times doses of Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could significantly increase serum ALT,GGT and decrease CRE( P<0.05 or P<0.01). Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets could increase the sperm deformity rate and damage the testicular seminiferous tubules of CIA male rats. Severity increased with dose and time increasing. The effect of Tripterygium Glycosides Tablets( 16 times) is more significant than Tripterygium wilfordii Tablets( 16 times). Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets significantly delayed onset of arthritis and inhibited the paw edema and arthritic score. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets also caused male reproductive damage,high dose affected hematopoiesis,and maximum dose leading to death. Tripterygium Glycosides Tablets and Tripterygium wilfordii Tablets all depended on dose-effect-toxicity manner. Anti-arthritis effect of Tripterygium Glycosides Tablets is better than Tripterygium wilfordii Tablets,but the toxicity of Tripterygium Glycosides Tablets maximum dose is more obvious. The relevant conclusions of our study will provide experimental references for clinical rational use of drugs,and further clinical studies are needed to confirm our conclusions.

Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses.

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies (MAbs) targeted to the hemagglutinin (HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.

Evaluating the Utility of Canine Mdr1 Knockout Madin-Darby Canine Kidney I Cells in Permeability Screening and Efflux Substrate Determination.

Permeability assays are commonly conducted with Madin-Darby canine kidney (MDCK) cells to predict the intestinal absorption of small-molecule drug candidates. In addition, MDCK cells transfected to overexpress efflux transporters are often used to identify substrates. However, MDCK cells exhibit endogenous efflux activity for a significant proportion of experimental compounds, potentially leading to the underestimation of permeability and confounded findings in transport studies. The goal of this study was to evaluate canine Mdr1 knockout MDCK (gMDCKI) cells in permeability screening and human MDR1 substrate determination in a drug discovery setting. The gMDCKI cells were established by CRISPR-Cas9-mediated knockout of the canine Mdr1 gene in MDCKI wildtype (wt) cells. A comparison of efflux ratios (ER) between MDCKI wt and gMDCKI showed that out of 135 compounds tested, 38% showed efflux activity in MDCKI wt, while no significant efflux was observed in gMDCKI cells. Apparent permeability (Papp) from apical-to-basolateral (A-to-B) and basolateral-to-apical were near unity in gMDCKI cells, which approximated passive permeability, and 17% of compounds demonstrated increases in their Papp A-to-B values. Overexpression of human MDR1 in gMDCKI (gMDCKI-MDR1) cells enabled substrate determination without the contribution of endogenous efflux, and the assay was able to deconvolute ambiguous results from MDCKI-MDR1 and identify species differences in substrate specificity. An analysis of 395 and 474 compounds in gMDCKI and gMDCKI-MDR1, respectively, suggested physicochemical properties that were associated with low permeability correlated with MDR1 recognition. Poorly permeable compounds and MDR1 substrates were more likely to be large, flexible, and more capable of forming external hydrogen bonds. On the basis of our evaluation, we concluded that gMDCKI is a better cell line for permeability screening and efflux substrate determination than the MDCK wt cell line.

Molluscicidal activity of Solidago canadensis L. extracts on the snail Pomacea canaliculata Lam.

Extracts from the aerial parts of Solidago canadensis L. were evaluated for molluscicidal activity against Pomacea canaliculata Lam. using an immersion bioassay method. The petroleum ether fraction of the ethanolic extract (PEEE) from S. canadensis exhibited strong molluscicidal activity. The PEEE mode of action in the hepatopancreas tissue of P. canaliculata was tested at several concentrations. Biochemical parameters, namely, soluble sugar content, protein, malondialdehyde (MDA), acetylcholinesterase (AChE) activity, alanine aminotransferase (ALT), and aspartate transaminase (AST) were significantly decreased or increased after exposure to PEEE for 48 h (p<0.05). Histological assessment results showed that hepatopancreas tissue structure was destroyed by exposure to PEEE. Gas chromatography-mass spectrometry analysis (GC-MS) was used to identify 15 compounds that could contribute to the molluscicidal efficacy of the PEEE. Molluscicidal assay, biochemical tests and histological assessments suggest that the PEEE from S. canadensis has potential utility as a molluscicide.

Herb pairs containing Angelicae Sinensis Radix (Danggui): A review of bio-active constituents and compatibility effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Herb compatibility is one of the most important characteristics of traditional Chinese medicine (TCM). Rather than being used singly, Chinese herbs are often used in formulae to obtain synergistic effects or to diminish possible adverse reactions. Herb pair, the most fundamental and simplest form of multi-herb formulae, is a centralized representative of herb compatibility. Danggui (Angelicae Sinensis Radix), a widely used Chinese medicine, is usually combined with another herb to treat women's diseases in the clinic. A series of herb pairs containing Danggui have gradually become a focus of modern research, and they exhibit encouraging prospects for development. MATERIALS AND METHODS: A systematic search for studies related to herb pairs containing Danggui was performed via a library search (books, theses, reports, newspapers, magazines, and conference proceedings) and an electronic search (Web of Science, PubMed, and Google Scholar). These sources were scrutinized for information on Danggui herb pairs. RESULTS: Based on a previous statistical analysis, a database containing 16,529 formulae of Danggui from the "Dictionary of Traditional Chinese Medicine Formulae" was reviewed. The results showed a high frequency of compatibility between Danggui and other 22 herbs. The most common ratio among these chosen herb pairs was 1:1, and a majority of the pairs were applied for the treatment of diseases in internal medicine. The present paper reviews ethnopharmacology and advances in variations of the bio-active components and compatibility effects of the herb pairs containing Danggui, especially Danggui-Huangqi, Danggui-Chuanxiong, and Danggui-Shaoyao, which are used at high frequency. It was also observed that there were fewer studies of Danggui-Fuzi, Danggui-Huanglian, Danggui-Gancao, Danggui-Fangfeng and Danggui-Ganjiang, although they have been recorded in classical books as commonly used herb pairs. Moreover, some herb pairs such as Danggui-Niuxi and Danggui-Chaihu have been used at high frequency according to the statistical analysis, however, they were not recognized as herb pairs in many relevant books. CONCLUSIONS: Recently, several TCM researchers have become interested in investigating the bio-active constituents and compatibility effects of herb pairs. Thus, some methods for in-depth study of herb pairs are essential to be established. The in vitro or in vivo bio-active constituents of herb pairs may differ from those of the single herbs. Additionally, comparative methods should be applied to study not only the bio-active constituents but also the effects of herb pairs. Study of component compatibility may be considered when the bio-active constituents and effects of an herb pair have been definitively demonstrated. Overall, the goal of our basic study of herb pairs should be their clinical application and the development of new drugs.

Regulation of Cell and Gene Therapy Medicinal Products in Taiwan.

Owing to the rapid and mature development of emerging biotechnology in the fields of cell culture, cell preservation, and recombinant DNA technology, more and more cell or gene medicinal therapy products have been approved for marketing, to treat serious diseases which have been challenging to treat with current medical practice or medicine. This chapter will briefly introduce the Taiwan Food and Drug Administration (TFDA) and elaborate regulation of cell and gene therapy medicinal products in Taiwan, including regulatory history evolution, current regulatory framework, application and review procedures, and relevant jurisdictional issues. Under the promise of quality, safety, and efficacy of medicinal products, it is expected the regulation and environment will be more flexible, streamlining the process of the marketing approval of new emerging cell or gene therapy medicinal products and providing diverse treatment options for physicians and patients.

Metabolic activation of the indoloquinazoline alkaloids evodiamine and rutaecarpine by human liver microsomes: dehydrogenation and inactivation of cytochrome P450 3A4.

Evodiamine and rutaecarpine are the main active indoloquinazoline alkaloids of the herbal medicine Evodia rutaecarpa, which is widely used for the treatment of hypertension, abdominal pain, angina pectoris, gastrointestinal disorder, and headache. Immunosuppressive effects and acute toxicity were reported in mice treated with evodiamine and rutaecarpine. Although the mechanism remains unknown, it is proposed that metabolic activation of the indoloquinazoline alkaloids and subsequent covalent binding of reactive metabolites to cellular proteins play a causative role. Liquid chromatography-tandem mass spectrometry analysis of incubations containing evodiamine and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed formation of a major GSH conjugate which was subsequently indentified as a benzylic thioether adduct on the C-8 position of evodiamine by NMR analysis. Several other GSH conjugates were also detected, including conjugates of oxidized and demethylated metabolites of evodiamine. Similar GSH conjugates were formed in incubations with rutaecarpine. These findings are consistent with a bioactivation sequence involving initial cytochrome P450-catalyzed dehydrogenation of the 3-alkylindole moiety in evodiamine and rutaecarpine to an electrophile 3-methyleneindolenine. Formation of the evodiamine and rutaecarpine GSH conjugates was primarily catalyzed by heterologously expressed recombinant CYP3A4 and, to a lesser extent, CYP1A2 and CYP2D6, respectively. It was found that the 3-methyleneindolenine or another reactive intermediate was a mechanism-based inactivator of CYP3A4, with inactivation parameters KI = 29 µM and kinact = 0.029 minute(-1), respectively. In summary, these findings are of significance in understanding the bioactivation mechanisms of indoloquinazoline alkaloids, and dehydrogenation of evodiamine and rutaecarpine may cause toxicities through formation of electrophilic intermediates and lead to drug-drug interactions mainly via CYP3A4 inactivation.

Utility of intersystem extrapolation factors in early reaction phenotyping and the quantitative extrapolation of human liver microsomal intrinsic clearance using recombinant cytochromes P450.

Reaction phenotyping using recombinant human cytochromes P450 (P450) has great utility in early discovery. However, to fully realize the advantages of using recombinant expressed P450s, the extrapolation of data from recombinant systems to human liver microsomes (HLM) is required. In this study, intersystem extrapolation factors (ISEFs) were established for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 using 11 probe substrates, based on substrate depletion and/or metabolite formation kinetics. The ISEF values for CYP2C9, CYP2D6, and CYP3A4 determined using multiple substrates were similar across substrates. When enzyme kinetics of metabolite formation for CYP1A2, 2C9, 2D6, and 3A4 were used, the ISEFs determined were generally within 2-fold of that determined on the basis of substrate depletion. Validation of ISEFs was conducted using 10 marketed drugs by comparing the extrapolated data with published data. The major isoforms responsible for the metabolism were identified, and the contribution of the predominant P450s was similar to that of previously reported data. In addition, phenotyping data from internal compounds, extrapolated using the rhP450-ISEF method, were comparable to those obtained using an HLM-based inhibition assay approach. Moreover, the intrinsic clearance (CL(int)) calculated from extrapolated rhP450 data correlated well with measured HLM CL(int). The ISEF method established in our laboratory provides a convenient tool in early reaction phenotyping for situations in which the HLM-based inhibition approach is limited by low turnover and/or unavailable metabolite formation. Furthermore, this method allows for quantitative extrapolation of HLM intrinsic clearance from rhP450 phenotyping data simultaneously to obtaining the participating metabolizing enzymes.