Progress in experimental models to investigate the in vivo and in vitro antidiabetic activity of drugs

Diabetes mellitus is one of the world's most prevalent and complex metabolic disorders,and it is a rapidly growing global public health issue.It is characterized by hyperglycemia,a condition involving a high blood glucose level brought on by deficiencies in insulin secretion,decreased activity of insulin,or both.Prolonged effects of diabetes include cardiovascular problems,retinopathy,neuropathy,nephropathy,and vascular alterations in both macro-and micro-blood vessels.In vivo and in vitro models have always been important for investigating and characterizing disease pathogenesis,identifying targets,and reviewing novel treatment options and medications.Fully understanding these models is crucial for the researchers so this review summarizes the different experimental in vivo and in vitro model options used to study diabetes and its consequences.The most popular in vivo studies involves the small animal models,such as rodent models,chemically induced diabetogens like streptozotocin and alloxan,and the possibility of deleting or overexpressing a specific gene by knockout and transgenic technologies on these animals.Other models include virally induced models,diet/nutrition induced diabetic animals,surgically induced models or pancreatectomy models,and non-obese models.Large animals or non-rodent models like porcine(pig),canine(dog),nonhuman primate,and Zebrafish models are also outlined.The in vitro models discussed are murine and human beta-cell lines and pancreatic islets,human stem cells,and organoid cultures.The other enzymatic in vitro tests to assess diabetes include assay of amylase inhibition and inhibition ofα-glucosidase activity.

The Identification of Phenylalanine Ammonia-Lyase(PAL)Genes from Pinus yunnanensis and an Analysis of Enzyme Activity in vitro

Phenylalanine ammonia lyase(PAL)is the rate-limiting and pivotal enzyme of the general phenylpropanoid path-way,but few reports have been found on PAL genes in Pinus yunnanensis.In the present study,three PAL genes were cloned and identified from P.yunnanensis seedlings for thefirst time,namely,PyPAL-1,PyPAL-2,and PyPAL-3.Our results indicated that the open-reading frames of PyPAL genes were 2184,2157,and 2385 bp.Phylogenetic tree analysis revealed that PyPALs have high homology with other known PAL genes in other plants.In vitro enzymatic analysis showed that all three PyPAL recombinant proteins could catalyze the deamination of L-phenylalanine to form trans-cinnamic acid,but only PAL1 and PAL2 can catalyze the conversion of L-tyrosine toρ-coumaric acid.Three PyPAL genes were expressed in different tissues in 1-year-old P.yunnanensis,and such genes had different expression patterns.This study lays a foundation for further understanding of the biosynthesis of secondary metabolites in P.yunnanensis.

Changes in Polyphenols and Antioxidant Activities of Yingshan Yunwu Tea during Digestion in Vitro

[Objectives]To explore the change rule of polyphenol content and antioxidant activity of coarse old leaves of Yingshan Yunwu Tea in the process of human digestion.[Methods]The coarse and old leaves of Yunwu tea in Yingshan,Huanggang,Hubei Province were selected as the research object,and their digestion in vitro was simulated.The total polyphenol content was determined by Folin-phenol reagent colorimetric method,and the DPPH radical scavenging activity and total antioxidant activity were determined.[Results]After simulated gastrointestinal digestion in vitro,the polyphenol content and antioxidant activity of coarse old leaf tea soup showed a downward trend.After gastrointestinal digestion,the polyphenol content in tea infusion separately decreased by 31.8%and 8.5%;the scavenging rate of DPPH free radical was 97%before digestion,decreased to 92%after gastric digestion and 65%after intestinal digestion,which decreased by 5%and 27%,respectively;after gastrointestinal digestion,the total antioxidant capacity of tea soup decreased by 4.7%and 3.1%,respectively.[Conclusions]This study provided a reference for the development and application of coarse old leaves of Yingshan Yunwu tea,and provided a reference for the nutritional value evaluation and comprehensive utilization of coarse old leaves,so as to make the best use of coarse tea leaves and reduce the waste of resources.

Research progress of in vitro activation mechanism and clinical application of female ovarian tissue

The activation and development of primordial follicles is the key to the maturation of female gametes.Premature ovarian insufficiency(POI)patients are unable to complete the primordial follicle activation and development due to follicular dormancy and unbalanced developmental regulation in the body,leading to female infertility.Ovarian tissue in vitro activation(IVA)technology has become a new way to solve the problem of patients who cannot auto-activate primordial follicles to obtain their own mature oocytes.In IVA research,signaling pathways such as PI3K/PTEN/Akt and Hippo have become the focus of current research.This review will describe the relevant research progress and clinical application of the IVA mechanism,and provide a reference for clinical research on ovarian tissue culture and activation in vitro.

In Vitro Anti-Bacterial and Anti-Fungal Activities of Extracts from Different Parts of 7 Zingiberaceae Plants

This study aimed to explore the anti-bacterial and anti-fungal activities of extracts from different parts of plants in the Zingiberaceae family.The inhibitory rate,minimum inhibitory concentration(MIC),and minimum bactericidal concentration(MBC)of leaf and stem,and root and rhizome extracts from Alpinia katsumadai Hayata,Alpinia oxyphylla Miq×Alpinia henryi K.Schumann,Alpinia oblongifolia Hayata,Alpinia nigra(Gaertn.)Burtt,Amomum villosum Lour,Alpinia zerumbet(Pers.)Burtt.et Smith and Alpinia oxyphylla Miq were determined using the fungus cake method and double dilution method.The seven Zingiberaceae plants exhibited characteristic antibacterial activities against pathogenic bacteria and fungi.At a 1.5 mg mL^(−1),A.zerumbet root and rhizome extracts exhibited strong inhibitory activity against S.aureus and E.coli,with 83.23%and 79.62%,respectively.In addition,A.zerumbet leaf and stem extracts had an inhibitory rate of 90.85%against P.aeruginosa.At the same concentration,the leaf and stem,root and rhizome extracts of A.katsumadai had the best anti-bacterial effect against F.oxysporum,with inhibition rates of 84.46%and 84.73%,respectively.Moreover,A.katsumadai and A.zerumbet leaf and stem extracts had the most significant antibacterial effect against S.aureus,with a MIC of 0.063 mg mL^(−1).Thus,both A.katsumadai and A.zerumbet extracts had significant antibacterial activity.In addition,by comparing the inhibitory effect of extracts from different parts,it was found that the inhibitory rate and average inhibitory rate of extracts from leaf and stem were higher than those from root and rhizome.The chemical constituents of A.katsumadai and A.zerumbet,determined by the high-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS),revealed that citric acid(CA),alpinetin,and pinocembrin(PNCB)were the functional constituents yielding the antibacterial activity.Overall,A.katsumadai and A.zerumbet have the potential to be developed as new plant fungicides and bactericides.

Design,delivery and efficacy testing of therapeutic nucleic acids used to inhibit hepatitis C virus gene expression in vitro and in vivo

Despite major achievements in the treatment ofchronic hepatitis C with the combination ofinterferons and the nucleoside analog ribavirin themajority of patients with chronic hepatitis C virus(HCV) infection cannot be treated effectively.Toimprove this response rate we used antisensetechnologies to inhibit HCV translation as possibleadditional option for experimental treatment.Antisense oligodeoxynucleotides(ODN) are

Double-layered osmotic pump controlled release tablets of actarit: In vitro and in vivo evaluation

The aim of the study was to develop actarit double-layered osmotic pump tablets to overcome the weak points of actarit common tablets, such as short half-life and large plasma concentration fluctuations. Single factor experiment and orthogonal test were applied to optimize the formulation;the pharmacokinetic study was performed in beagle dogs adopting actarit common tablets as reference tablets. The optimal formulation was as follows: drug layer: 150 mg actarit, 240 mg PEO-N80, 50 mg NaCl;push layer: 140 mg PEO-WSR303, 20 mg NaCl;coating solution: 30 g cellulose acetate and 6 g PEG 4000 in 1000 ml 94% acetone solution, 60 mg coating weight gain. The pharmacokinetic study showed that T max was prolonged by the contrast of commercial common tablets with constant drug release rate, but the bioavailability was equivalent. And a good in vivo –in vitro correlation of the actarit osmotic pump tablets was also established. The designed actarit osmotic pump tablets can be applied for rheumatoid arthritis, proposing a promising replacement for the marked common products.

Insight into the preformed albumin corona on in vitro and in vivo performances of albumin-selective nanoparticles

Preformed albumin corona of albumin-nonselective nanoparticles(NPs)is widely exploited to inhibit the unavoidable protein adsorption upon intravenous administration.However,very few studies have concerned the preformed albumin corona of albumin-selective NPs.Herein,we report a novel type of albumin-selective NPs by decorating 6-maleimidocaproyl polyethylene glycol stearate(SA)onto PLGA NPs(SP NPs)surface,taking albuminnonselective PLGA NPs as control.PLGA NPs and SP NPs were prepared by emulsion-solvent evaporation method and the resultant NPs were in spherical shape with an average diameter around 180 nm.The corresponding albumin-coating PLGA NPs(PLGA@BSA NPs)and albumin-coating SP NPs(SP@BSA NPs)were formulated by incubating SP NPs or PLGA NPs with bovine serum albumin solution,respectively.The impact of albumin corona on particle characteristics,stability,photothermal effect,cytotoxicity,cell uptake,spheroid penetration and pharmacokinetics was investigated.In line with previous findings of preformed albumin coating,PLGA@BSA NPs exhibited higher stability,cytotoxicity,cell internalization and spheroid penetration performances in vitro,and longer blood circulation time in vivo than those of albumin-nonselective PLGA NPs,but albumin-selective SP NPs is capable of achieving a comparable in vitro and in vivo performances with both SP@BSA NPs and PLGA@BSA NPs.Our results demonstrate that SA decorated albumin-selective NPs pave a versatile avenue for optimizing nanoparticulate delivery without preformed albumin corona.

Development of cryptotanshinone-loaded pellets for angina chronotherapy:In vitro/in vivo prediction and evaluation

The clinical manifestations of variant angina is unevenly distributed during the 24 h, thusthe in vivo performance of drugs should be tailored according to the angina circadianrhythm. Cryptotanshinone(CTN) is one of the representative bioactive lipid-soluble com-ponents of Danshen which has been commonly used for cardiovascular diseases such asangina pectoris. The aim of this study was to develop a novel CTN sustained-released pel-lets(CTN-SRPs) to precisely synchronize the CTN plasma concentrations with predictedoccurrence of angina pectoris for angina chronotherapy. A deconvolution-based methodwas applied to develop and optimize the CTN-SRPs. The plasma concentration-time curveof CTN immediate-released formulation after oral administration in rats was used as theweight function. The predicted plasma concentration-time curve of CTN-SRPs simulatedaccording to the incidence of variant angina during 24 h was used as the response func-tion. Then the desired drug release profile of CTN-SRPs was calculated based on deconvo-lution using weight function and response function, and subsequently used for guiding theformulation optimization. CTN-SRPs were prepared with the combinations of PVP, polox-amer 127 and EC as matrix using fluidized bed technology. An orthogonal design was em-ployed to obtain the optimal formulation with its release profile similar with the desiredone. Pharmacokinetic studies validated that the actual plasma concentration-time curve ofthese optimized CTN-SRPs was similar with the predicted one. In addition, the percent er-rors(%PE) of CTN plasma concentrations in 8–12 h were less than 10%. In conclusion, thisdeconvolution-based method could be applied to adjust the in vivo performance of drugs forangina chronotherapy.

The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-（2,3-dibromo-4,5-dihydroxybenzyl）-2,3-dibromo-4,5-dihydroxy benzyl methyl ether （1）, （＋）-3-（2,3-dibromo-4,5-dihydroxyphenyl）-4-bromo-5,6-dihydroxy-1,3- dihydroisobenzofuran （2）, 3-bromo-4-（2,3-dibromo-4,5-dihydroxybenzyl）-5-methoxymethyl-pyrocatechol （3）, 2,2＇,3,3＇-tetrabromo-4,4＇,5,5＇-tetrahydroxy-diphenylmethane （4）, bis（2,3-dibromo-4,5-dihydroxybenzyl） ether （5）, 2,2＇,3-tribromo-3＇,4,4＇,5-tetrahydroxy-6＇-ethyloxymethyldiphenylmethane （6） were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTF assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Be17402 and HCT-8. Their inhibitory activity against protein tyrosine kinase （PTK） with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana （EELN） was evaluated on S180-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC50 below 10 pg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4% respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer.

In vitro antioxidant and antimicrobial activity cycloart-23-ene-3β,25-diol(B2) isolated from Pongamia pinnata(L.Pierre)

Objective:To evaluate the in-vitro antioxidant and antimicrobial activity of cvcloart-23- ene-3β,25-diol(called as B2) isolated from stem bark of Pongamia pinnata.Methods:In vitro antioxidant activity of B2 was determined by methods for determination of Dl’PH radical scavenging,reducing power,superoxide anion radical scavenging,hydroxyl radical scavenging, hydrogen peroxide scavenging,metal chelating and nitric oxide radical scavenging at the doses of 20.40.60.80 and 100μg/mL,respectively.3 -tocopherol with same concentration was used as a standard antioxidant.In vitro antimicrobial activity of B2 was determined by cup plate method in different concentration range of 10-100μg/mL.Results:The results indicated that dose dependent%reduction against DPPH radical,reducing power,superoxide anion radical scavenging,hydroxyl radical scavenging,metal chelating,hydrogen peroxide scavenging and nitric oxide radical scavenging by B2 andβ-tocopherol.Conclusions:It is concluded that cycloart 23-ene-3β,25 diol(B2) showed dose dependent antioxidant activity.B2 showed more DPPH radical scavenging,reducing power,superoxide scavenging,hydroxyl radical scavenging, metal chelating scavenging,hydrogen peroxide radical scavenging and nitric oxide radical scavenging activity thanβ-tocopherol and in case of antimicrobial activity B2 exhibited broad-spectrum activ ity against bacteria and strong activity against yeast type of fungi.

In vitro Inhibitory Activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and Its Biofilms

[Objective] This study aimed to analyze the in vitro inhibitory activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and its biofilms. [Result] By agar diffusion test, in vitro inhibitory activity of 5. chinensis and P. sibiricum against V. harveyi was investigated. The minimal inhibitory concentration （ MIC） and minimal bactericidal concentration （MBC） of 5. chinensis and P. sibiricum against V. harveyi were determined by doubling dilution meth-od. The inhibitory activity of 5. chinensis and P. sibiricum on the formation of V. harveyi biofilms was evaluated by modified MTT assay. [ Result ] Both 5. chinen-sis and P. sibiricum had inhibitory activity against V. harveyi. The inhibition zone diameter of 5. chinensis against V. harveyi was 17. 95 mm; MIC and MBC of 5. chinensis were both 3.125 mg/ml. The inhibition zone diameter of P. sibiricum against V. harveyi was 12. 22 mm; MIC and MBC of P. sibiricum were 3.125 and 6.250 mg/ml, respectively. When the concentration was higher than 6. 25 mg/ml, 5. chinensis decoction had extremely significant inhibitory activity against V. harveyi （P 〈 0. 01） ; when the concentration was higher than 3. 125 mg/ml, P. sibiricum had extremely significant inhibitory activity against V. harveyi （P 〈0. 01）. [ Conclusion] 5. chinensis and P. sibiricum could significantly inhibit V. harveyi and its biofilms.

Sustained release donepezil loaded PLGA microspheres for injection:Preparation,in vitro and in vivo study

The purpose of this study was to develop a PLGA microspheres-based donepezil(DP)formulation which was expected to sustain release of DP for one week with high encapsulation efficiency(EE).DP derived from donepezil hydrochloride was encapsulated in PLGA microspheres by the O/W emulsion-solvent evaporation method.The optimized formulation which avoided the crushing of microspheres during the preparation process was characterized in terms of particle size,morphology,drug loading and EE,physical state of DP in the matrix and in vitro and in vivo release behavior.DP microspheres were prepared successfully with average diameter of 30m,drug loading of 15.92±0.31%and EE up to 78.79±2.56%.Scanning electron microscope image showed it has integrated spherical shape with no drug crystal and porous on its surface.Differential scanning calorimetry and X-ray diffraction results suggested DP was in amorphous state or molecularly dispersed in microspheres.The Tg of PLGA was increased with the addition of DP.The release profile in vitro was characterized with slow but continuous release that lasted for about one week and fitted well with first-order model,which suggested the diffusion governing release mechanism.After single-dose administration of DP microspheres via subcutaneous injection in rats,the plasma concentration of DP reached peak concentration at 0.50 d,and then declined gradually,but was still detectable at 15 d.A good correlation between in vitro and in vivo data was obtained.The results suggest the potential use of DP microspheres for treatment of Alzheimer’s disease over long periods.

Effect of Gamma Irradiation on Total Phenolic Content and <i>in Vitro</i>Antioxidant Activity of Pomegranate (<i>Punica granatum</i>L.) Peels

Pomegranate peels were studied for the effect of gamma irradiation on microbial decontamination along with its effect on total phenolic content and in vitro antioxidant activity. Gamma irradiation was applied at various dose levels (5.0, 10.0, 15.0 and 25.0 kGy) on pomegranate peel powder. Both the values of total phenolic content and in vitro antioxi- dant activity were positively correlated and showed a significant increase (p < 0.05) for 10.0 kGy irradiated dose level immediately after irradiation and 60 days of post irradiation storage. At 5.0 kGy and above dose level, gamma irradia- tion has reduced microbial count of pomegranate peel powder to nil. Post irradiation storage studies also showed that, the irradiated peel powder was microbiologically safe even after 90 days of storage period.

Application of custom anatomy-based nerve conduits on rabbit sciatic nerve defects: in vitro and in vivo evaluations

The intermingling of regenerated nerve fibers inside nerve grafts is the main reason for mismatched nerve fibers. This is one of the key factors affecting limb function recovery after nerve injury. Previous research has shown that the accuracy of axon regeneration can be improved by a bionic structural implant. To this aim, iodine and freeze-drying high-resolution micro-computed tomography was performed to visualize the 3D topography of the New Zealand rabbit sciatic nerve (25 mm). A series of 1-, 2-, 3-, and 4-custom anatomy-based nerve conduits (CANCs) were fabricated based on the anatomical structure of the nerve fascicle. The match index, luminal surface, and mechanical properties of CANCs were evaluated before implanting in a 10-mm gap of the sciatic nerve. Recovery was evaluated by histomorphometric analyses, electrophysiological study, gastrocnemius muscle weight recovery ratio, and behavioral assessments at 12 and 24 weeks postoperatively. The accuracy of nerve regeneration was determined by changes in fluorescence-labeled profile number during simultaneous retrograde tracing. Our results showed that the optimal preprocessing condition for high-resolution micro-computed tomography visualization was treatment of the sciatic nerve with 40% Lugol’s solution for 3 days followed by lyophilization for 2 days. In vitro experiments demonstrated that the match index was highest in the 3-CANC group, followed by the 2-, 1-, and 4-CANC groups. The luminal surface was lowest in the 1-CANC group. Mechanical properties (transverse compressive and bending properties) were higher in the 3- and 4-CANC groups than in the 1-CANC group. In vivo experiments demonstrated that the recovery (morphology of regenerated fibers, compound muscle action potential, gastrocnemius muscle weight recovery ratio, pain-related autotomy behaviors, and range of motion) in the 3-CANC group was superior to the other CANC groups, and achieved the same therapeutic effect as the autograft. The simultaneous retrograde tracing results showed that the percentages of double-labeled profiles of the 2-, 3-, and 4-CANC groups were comparatively lower than that of the 1-CANC group, which indicates that regenerated nerve fascicles were less intermingled in the 2-, 3-, and 4-CANC groups. These findings demonstrate that the visualization of the rabbit sciatic nerve can be achieved by iodine and freeze-drying high-resolution micro-computed tomography, and that this method can be used to design CANCs with different channels that are based on the anatomical structure of the nerve. Compared with the 1-CANC, 3-CANC had a higher match index and luminal surface, and improved the accuracy of nerve regeneration by limiting the intermingling of the regenerated fascicles. All procedures were approved by the Animal Care and Use Committee, Xinjiang Medical University, China on April 4, 2017 (ethics approval No. IACUC20170315-02).

Effects of Size and Surface Charge of Polymeric Nanoparticles on in Vitro and in Vivo Applications

Biodegradable polymeric materials are the most common carriers for use in drug delivery systems. With this trend, newer drug delivery systems using targeted and controlled release polymeric nanoparticles (NPs) are being developed to manipulate their navigation in complex in vivo environment. However, a clear understanding of the interactions between biological systems and these nanoparticulates is still unexplored. Different studies have been performed to correlate the physicochemical properties of polymeric NPs with the biological responses. Size and surface charge are the two fundamental physicochemical properties that provide a key direction to design an effective NP formulation. In this critical review, our goal is to provide a brief overview on the influences of size and surface charge of different polymeric NPs in vitro and to highlight the challenges involved with in vivo trials.

Protective effects of Ecklonia cava extract on the toxicity and oxidative stress induced by hair dye in in-vitro and in-vivo models

Oxidative hair dyes containingρ-phenylenediamine(PPD)are reported to induce an allergic reaction by promoting oxidative stress when absorbed through the skin.Despite the associated risk,these hair dyes remain popular owing to their convenience and sharpness of color.This makes it important to minimize the cytotoxicity and oxidative stress induced by PPD-containing hair dyes.Ecklonia cava extract has been evaluated in different studies for its protective effects against external stress in fibroblasts and keratinocytes.Our study was aimed at using in-vitro and in-vivo models to investigate the extract’s effects on cytotoxicity of and oxidative stress induced by PPD-containing hair dyes.Analysis of CIEL*a*b*Color space was first used to determine the range of E.cava extract that would not interfere with the coloring ability of the dye upon addition.Subsequently,the set ranges of E.cava extract(5% and 7%)were added to the hair dye and their toxicity assessed by evaluating the viability of fibroblasts and keratinocytes.The effects on developmental phenotypes and induction of oxidative stress by hair dye were evaluated and compared with those of hair dyes containing different contents of E.cava extract using an in-vivo zebrafish model.Our results showed that E.cava extract in hair dye could significantly decrease the cytotoxicity and levels of oxidative stress caused by hair dyes containing PPD in both in-vitro and in-vivo models.These results suggest that the addition of 7% E.cava extract to 250μg/mL hair dye does not interfere with the coloring ability of the dye while showing significant protective eff ects against the hair dye.The study proposes that the use of E.cava extract as an adduct to hair dyes containing PPD reduces the cytotoxicity and oxidative stress induced by these hair dyes.

Gan Shen Fu Fang ameliorates liver fibrosis in vitro and in vivo by inhibiting the inflammatory response and extracellular signalregulated kinase phosphorylation

BACKGROUND Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines.The Chinese herbal medicine,Gan Shen Fu Fang(GSFF)is composed of salvianolic acid B and diammonium glycyrrhizinate.In this study,we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment.AIM To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase(ERK)phosphorylation.METHODS Common bile duct-ligated rats were used for in vivo experiments.Hepatic stellate cells-T6(HSC-T6)cells were used for in vitro experiments.Hematoxylin and eosin staining and Masson staining,biochemical assays,hydroxyproline(Hyp)assays,enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis,liver function,the inflammatory response and ERK phosphorylation.The CCK8 assay,immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells.RESULTS GSFF improved liver function and inhibited liver fibrosis in common bile ductligated rats after 3 wk of treatment,as demonstrated by histological changes,hydroxyproline assays and collagen I concentrations.GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines[tumor necrosis factor-α(TNF-α)and interlukin-1β]and NF-κB.In addition,GSFF decreased ERK phosphorylation.In vitro,GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factorβ1(TGF-β1)stimulation and decreased the synthesis of collagen I.GSFF had the greatest effect at a concentration of 0.5μmol/L.GSFF inhibited the expression ofα-smooth muscle actin(α-SMA),a marker of HSC activation,in HSC-T6 cells.Consistent with the in vivo results,GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB.CONCLUSION GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro.These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation.

Rabbit Defensin(NP-1) Gene Transformation of Wheat and In Vitro Microbicidal Activity Assay of Transgenic Plants

Monocot high expression vector pBARUNP1, harboring rabbit defensin(NP 1) gene and selective bar gene for resistance to the herbicide Basta, were constructed and then transferred into immature embryos of wheat (“Bobwhite” and “Zhong 60634”)via particle bombardment. Southern and RNA dot blots showed the stable integration and transcription of foreign NP 1 gene in the wheat genome. Furthermore, in vitro microbicidal activity assay indicated the proper translation of defensin. Crude protein extraction of transgenic plants exhibited to some extent cytotoxic to several pathogens including G. saubinetii, B. subtilis, E.coli, and A. tumefaciens.

Formation of Superoxide Radical and Hydrogen Peroxide Enhanced by Trinitrotoluene in Rat Liver, Brain, Kidney, and Testicle in Vitro and Monkey Liver in Vivo

Trinitrotoluene (TNT) increased the formation of adrenochrome from adrenaline and the formation of formaldehyde from methanol in rat liver mitochondria and microsomes in vitro as well as in monkey liver mitrochondria and microsomes in vivo. The effects were more prominent at higher TNT concentrations. These findings indicate that TNT enhances the production of superoxide radicals (O_2^-) and hydrogen peroxide (H_2O_2). The production of O_2^- was more prominent in systems containing added TNT than in those containing added benzyl viologen. H_2O_2 production by mitochondria was more pronounced in the liver than in other organs, but its production by microsomes was more pronounced in the brain than in other organs. The results suggest that TNT undergoes cycling reduction which produces oxidative stress. 1989 Academic Press, Inc.