Bacterial melanin in rat models of Parkinson's disease: a potential neuroprotective strategy

Melanins are widely used in medicine, pharmacology and cosmetics. Different technologies have been used to obtain melanin including： chemical synthesis based on oxidation of tyrosine and its derivatives; extraction from animal materials; alkaline extraction from plant material; and microbiological synthesis. A few number of works have been published that were focused on purification of water insoluble 3,4-dihy- droxy-phenylalanine-melanins （Kukulianskaia et al., 2002）. The majority of synthetic and natural melanins are insoluble in wa- ter that significantly complicates preparation of pharmacolog- ical and cosmetic preparations. Obtaining of low-cost soluble biotechnological melanin can speed up application of melanin in medicine and other fields. For the first time, melanin-syn-thesizing strain with high level of pigment synthesis - Bacillus thuringiensis was obtained. The ecologically safe technology of biosynthesis, isolation and purification of the bacterial melanin has been elaborated.

Effects of ginsenoside of stem and leaf combined with choline on learning and memory ability of rat models with Alzheimer diseases

BACKGROUND： Central adrenergic nerve and 5-serotonergic nerve can influence central cholinergic nerve on learning and memory and make easy for study; however, ginsenoside of stem and leaf （GSL） can improve functions of central adrenergic nerve; moreover, 5-serotonergic nerve and the combination with choline can produce synergistic effect and enhance learning and memory ability so as to improve learning and memory disorder of patients with Alzheimer disease （AD）. OBJECTIVE ： To observe the effects of GSL combining with choline on learning and memory of AD model rats DESIGN ： Randomized grouping design and controlled animal study SETIING ： Department of Pharmacology, Taishan Medical College MATERIALS ： The experiment was carried out in the Pharmacological Department of Medical College of Jilin University from October 1996 to January 1997. Forty healthy male Wistar rats of clean grade were randomly divided into 5 groups, including sham-injury group, model group, GSL group, choline group and combination group, with 8 rats in each group. Main medications： GSL with the volume more than 92.8% was provided by Department of Chemistry, Norman Bethune Medical College of Jilin University. Panaxatriol, the main component, was detected with thin layer scanning technique and regarded as the index of GSL quality [（55±1）%, CV= 2%, n = 5]. Choline was provided by the Third Shanghai Laboratory Factory. METHODS ： 150 nmol quinolinic acid was used to damage bilateral Meynert basal nuclei of adult rats so as to establish AD models. Rats in GSL, choline and combination groups were intragastric administrated with 400 mg/kg GSL, 200 mg/kg choline （20 mL/kg）, and both respectively last for 17 days starting from two days before operation. Rats in sham-injury group and model group were perfused with the same volume of distilled water once in each morning for the same days. （1） Passive avoidance step-down test： Five minutes later, rats jumped up safe platform when they were shocked with 36 V alternating current. If rats jumped down from the platform and the feet touched railings, the response was wrong. Numbers of wrong response were recorded within 3 minutes, and then the test was redone after 24 hours. （2） Morris water-maze spatial localization task： Swimming from jumping-off to platform directly was regarded as right response. Additionally, 4 successively right responses were regarded as the standard. Each rat was trained 10 times a day with 120 s per time for 3 successive days. The interval was 30 s. Three days later, numbers of right response were recorded. The training times were increased to 30 for unlearned rats. （3） Measurement of activity of choline acetylase in cerebral cortex： Rats were sacrificed at 17 days after operation to obtain cerebral cortex to measure activity of choline acetylase with radiochemistry technique. （4） Synergistic effect： It was expressed as Q value： Q value = factual incorporative effect/anticipant incorporative effect; Q ≥ 1 was regarded as synergistic effect. Anticipant incorporative effect = （EA＋EB-EA·EB）, EA and EB were single timing effect, respectively in GSL group and choline group. E（step-down test and Morris water maze test） = （x in model group - factual value in medicine groups）/x in model group; E （activity of choline acetylase） = （factual value in medicine groups -xin model group）/xin model group. MAIN OUTCOME MEASURES ： （1） Passive avoidance step-down test and Morris water-maze spatial localization task in the study of learning and memory; （2） activity of choline acetylase. RESULTS ： All 40 rats were involved in the final analysis. （1） Passive avoidance response： At learning phase on first day and retesting phase on the next day, numbers of wrong responses within 3 minutes were more in model group than sham operation group, and there was significant difference [（5.88±1.46）, （2.25±0.87） times; （2.63±1.06）, （0.50±0.53） times; P 〈 0.01]; numbers of wrong responses within 3 minutes were less in combination group than model group, and there was significant difference [learning phase： （1.12±0.83）, （5.88±1.46） times; retesting phase： （0.38±0.74）, （2.63±1.06）times, P 〈 0.01]; moreover, effect was stronger than that in GSL group and choline group. The Q value was 1.07 and 1.59, respectively and it showed synergistic effect. Spatial localization task： Training times were more in model group than sham operation group, and there was significant difference [（2.9±2.5）, （12.6±3.5） times; P 〈 0.01]. Training times were less in combination group than model group, and there was significant difference [（11.8±2.4）, （27.9±2.5） times, P 〈 0.01]; moreover, effect was stronger than that in GSL group and choline group. The Q value was 1.07 and it showed synergistic effect. （3） Activity of choline acetylase： Activity was lower in model group than sham operation group, and there was significant difference [（30.56±8.33）, （61.11 ±8.33） nkat/g; P 〈 0.01]. Activity was higher in combination group than model group and there was significant difference [（50.00±8.33）, （30.56±8.33） nkat/g, P 〈 0.01];moreover, effect was stronger than that in GSL group and choline group. The Q value was 1.5 and it showed synergistic effect. CONCLUSZON： GSL in combination with choline can synergically improve the disorder of learning and memory of AD model rats. Its mechanism may be involved in enhancing the function of central cholinergic system.

Novel translational rat models of dopamine transporter deficiency

Dopamine （DA） is one of the brain＇s fundamental neurotransmitters. Despite the fact that the dopaminergic synapses constitute less than 1% of all brain synapses, DA is implicated in a number of critical physiological functions and in the pathogenesis of important psychiatric diseases such as schizophrenia, attention-deficit/hyperactivity disorder （ADHD）, Parkinson＇s disease （PD） and others.

A review of rat models of periodontitis treated with natural extracts

Objective:To review treatment methods using natural extracts applied in rat models of periodontitis to establish a direction for the design of future experiments.Methods:An electronic search of PubMed was carried out using the keywords“periodontitis,”“natural”,“extracts”,“herb*”,“plants”and“rats.”Articles were screened against inclusion and exclusion criteria by two independent researchers.Data describing the characteristics of rats,method of periodontitis inducement,extract administration,and outcome measures were extracted and analyzed by more than two authors manually.Results:Of the 864 articles identified,33 studies were included.The use of SpragueeDawley rats(51.2%)and male rats(90.9%)was preferred.The most common experimental methods were ligature placement(72.7%)and oral administration(66.7%).Alveolar bone loss was evaluated mainly by photography(51.5%)and micro-computed tomography(39.4%).Factors related to bone remodeling and inflammatory processes,such as interleukin-1b,tumor necrosis factor-a,receptor activator of nuclear factor-kB,and osteoprotegerin,were also measured.Conclusion:Many diverse experimental periodontitis models have been used.However,few articles observed bone formation,immune responses,antibacterial effects,and toxicity.Future studies to assess natural extracts for the treatment of periodontitis should be robust and well-designed.

Genetic Rat Models of Schizophrenia-Relevant Symptoms

It is recognized that developing valid animal models is essential for the research on the neurobiological mechanisms of (and treatments for) psychiatric disorders, even when these are as complex as schizophrenia. To be considered a valid analogue of the disorder, a given model should present good face validity (i.e. similarity of symptoms), good predictive validity (i.e. similarity of treatment effects and potential for discovering novel treatments) and enough construct validity (i.e. the model should help discover neurobiological mechanisms underlying the disorder or some relevant symptoms). The complexity of symptoms (positive, negative and cognitive) of schizophrenia makes it a very difficult task for a model to mimic all the main features of the disorder, but some rodent (mouse and rat) models have behavioural and even neurobiological phenotype characteristics resembling positive-like symptoms, cognitive symptoms and some neurochemical features of schizophrenia. As several recent works have already reviewed the main behavioural and developmental models, as well as the most used drug-induced, lesion-induced and genetic mouse models, the present review focuses on describing the most relevant genetically-based rat models of schizophrenia-relevant symptoms. Thus, we discuss several selective breeding programs leading to rat lines/strains which present impaired prepulse inhibition (PPI) of the acoustic startle response and (in some cases) latent inhibition deficits (both of which may be considered as endophenotypes of schizophrenia related with pre-attentive processes and attention, respectively), as well as other schizophrenia-relevant symptoms (e.g. learning deficits). Evidence is presented for the effects of genetic background on PPI (and other symptoms/phenotypes), as well as for environmental influences on genetic predisposition to enhanced apomorphine (mixed dopamine receptor agonist) effects. Some of the described rat models appear to present face validity and, to a certain extent, construct validity. While efforts should be made to evaluate the predictive validity of these genetic rat models, we propose that they have the advantage (over mouse knockouts, for example) of better representing “normal” genetic, neurobiological and phenotype variation, thus allowing the study of associations among them by means of genetic mapping or gene expression studies.

The bio-active components of the Mongolian medicine Horcha-6 and therapeutic mechanism in the rat migraine model

Background:The active components of Horcha-6 were identified using liquid chromatography with tandem mass spectrometry.Also,we investigated the potential mechanisms that explain why Horcha-6 may be effective in treating migraines through the use of network pharmacology and a rat migraine model.Methods:After identifying the active components of Horcha-6,the corresponding genes of the active components’target were obtained from the Universal Protein database,and a“compound-target-disease”network was constructed using Cytoscape 3.9.0 software.For the in vivo experiments,nitroglycerin was injected intraperitoneally into rats to create a migraine model.Pre-treatment with Horcha-6 was administered orally for 14 days,and rats were subjected to migraine-related behavior tests.RNA sequencing was performed to identify the gene expression regulated by Horcha-6 in the trigeminal nerve.Results:A total of 903 chemical components of Horcha-6 have been collected in the liquid chromatography with tandem mass spectrometry.We discovered 55 of the Horcha-6 bio-active components that were evaluated based on their Percent Human Oral Absorption(≥30%)and DL values(≥0.185)on the traditional Chinese medicine systems pharmacology database.The“compound-target-disease”network contained 163 intersection targets with the migraine state.Gene Ontology analysis indicated that these components significantly regulated the immune response,vascular function,oxidative stress,etc.When Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed,we observed that most of the target genes were significantly enriched in the inflammation and neuro-related signaling pathway,toll-like receptor signaling pathway,neuroactive ligand-receptor interaction,etc.These predictions were further demonstrated via in vivo animal model experiments.The RNA sequencing results showed that 41 genes were down-regulated(P<0.05)and 86 genes were up-regulated(P<0.05)in the Horcha-6 treated group compared with the untreated group.Those genes were mainly involved in neuromodulation,vascular function,and hormone metabolism.Conclusion:The 55 bio-active components in Horcha-6 regulate inflammation,hormone metabolism,and neurotransmitters and have potential as a therapy to treat migraines.

Effect of oxidative stress-associated damage to the lung tissue caused by different body mass index in the rat models

Objective To investigate the influence of different diets on serum protein expression levels of 4-hydroxynonenal(4-HNE),thioredoxin(Trx),thioredoxin reductase(TrxR)and the sctivities of Trx and TrxR,and to explore the effect of damage to the lung tissue and the underlying mechanisms of different body mass index caused by different diets in the rat models.Methods

Correlation between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models

AIM： To evaluate serum TIMP-1 level and the correlation between TIMP-1 expression and liver fibrosis in immuneinduced and CCL4-induced liver fibrosis models in rats. METHODS： Immune-induced and CCL4-induced liver fibrosis models were established by dexamethasone （0.01 mg） and CCL4 respectively. Serum TIMP-1 level was detected with ELISA, while histopathological grade of liver biopsy was evaluated. Spearman rankcorrelation test was used to analyse the difference of the correlation between the TIMP-1 expression and hepatic fibrosis in the two fibrosis models. Furthermore,in situ hybridization was used to determine the expression difference of TIMP-1 mRNA in the two models. RESULTS： Positive correlation existed between serum TIMP-1 level of immune induced group and the histopathological stages of fibrosis liver of corresponding rats （Spearman rank-correlation test, rs = 0.812, P 0.05）, and the positive in situ hybridization signal of TIMP-1 mRNA was strong. In CCL4-induced liver fibrosis model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant（Spearman rank-correlation test, rs = 0.229, P 〉 0.05）. And compared with immune-induced model, the positivein situ hybridization signal of TIMP-1 mRNA was weaker, while the expression variation was higher in hepatic fibrosis of the same severity. CONCLUSION： The correlations between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models are different. In immune-induced model, serum TIMP-1 level could reflect the severity of liver fibrosis, while in CCL4-induced model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant.

A Comparative Study on Several Models of Experimental Renal Calcium Oxalate Stones Formation in Rats

In order to compare the effects of several experimental renal calcium oxalate stones formation models in rats and to find a simple and convenient model with significant effect of calcium oxalate crystals deposition in the kidney, several rat models of renal calcium oxalate stones formation were induced by some crystal-inducing drugs （CID） including ethylene glycol （EG）, ammonium chloride （AC）, vitamin D3 [ 1 α（OH）VitD3, alfacalcidol], calcium gluconate, ammonium oxalate, gentamicin sulfate, L-hydroxyproline. The rats were fed with drugs given singly or unitedly. At the end of experiment, 24-h urines were collected and the serum creatinine （Cr）, blood urea nitrogen （BUN）, the extents of calcium oxalate crystal deposition in the renal tissue, urinary calcium and oxalate excretion were measured. The serum Cr levels in the stone-forming groups were significantly higher than those in the control group except for the group EG＋L-hydroxyproline, group calcium gluconate and group oxalate. Blood BUN concentration was significantly higher in rats fed with CID than that in control group except for group EG＋L-hydroxyproline and group ammonium oxalate plus calcium gluconate. In the group of rats administered with EG plus Vitamin D3, the deposition of calcium oxalate crystal in the renal tissue and urinary calcium excretion were significantly greater than other model groups. The effect of the model induced by EG plus AC was similar to that in the group induced by EG plus Vitamin D3. EG plus Vitamin D3 or EG plus AC could stably and significantly induced the rat model of renal calcium oxalate stones formation .

Effects of C2 hemisection on respiratory and cardiovascular functions in rats

High ce rvical spinal co rd injuries induce permanent neuromotor and autonomic deficits.These injuries impact both central respiratory and cardiovascular functions through modulation of the sympathetic nervous system.So far,cardiovascular studies have focused on models of complete contusion or transection at the lower cervical and thoracic levels and diaphragm activity evaluations using invasive methods.The present study aimed to evaluate the impact of C2 hemisection on different parameters representing vital functions(i.e.,respiratory function,cardiovascular,and renal filtration parameters)at the moment of injury and 7 days post-injury in rats.No ventilatory parameters evaluated by plethys mography were impacted during quiet breathing after 7 days post-injury,whereas permanent diaphragm hemiplegia was observed by ultrasound and confirmed by diaphragmatic electromyography in anesthetized rats.Interestingly,the mean arterial pressure was reduced immediately after C2 hemisection,with complete compensation at 7 days post-injury.Renal filtration was unaffected at 7 days post-injury;however,remnant systolic dysfunction chara cterized by a reduced left ventricular ejection fraction persisted at 7 days post-injury.Taken together,these results demonstrated that following C2 hemisection,diaphragm activity and systolic function are impa cted up to 7 days post-injury,whereas the respiratory and cardiovascular systems display vast ada ptation to maintain ventilatory parameters and blood pressure homeostasis,with the latter likely sustained by the remaining descending sympathetic inputs spared by the initial injury.A better broad characterization of the physiopathology of high cervical spinal cord injuries covering a longer time period post-injury could be beneficial for understanding evaluations of putative therapeutics to further increase cardiorespiratory recovery.

Protective Effects of Different Hypothermal Preservation Solutions on Structure and Function of Isolated Rat Arteries

Objective:With the increasing application of vascular reconstruction in surgical procedures,allogeneic vessels are becoming more popular in clinical practice due to their abundant sources,precise diameter matching,improved histocompatibility,and higher long-term patency rate.This study aimed to investigate the protective effect of various preservation solutions on the function and structure of the isolated rat abdominal aorta preserved under hypothermal conditions.Methods:The study utilized a total of 150 Sprague-Dawley(SD)rats,with 144 rats allocated to the experimental groups and 6 rats allocated to the control groups.The abdominal aorta of the rats was chosen as the subject of our research.The aorta in the experimental groups were randomly assigned to 4 groups:University of Wisconsin(UW)solution group,histidine-tryptophan-ketoglutarate(HTK)solution group,normal saline(NS)group,and sodium lactate Ringer's solution(RS)group.Samples were subjected to examination after preservation periods of 1 day,3 days,5 days,7 days,14 days,30 days,and 90 days.Evaluation of vascular physiological function involved detecting and assessing vasoconstriction ability and measuring cell viability through the MTT test.Evaluation of the vascular wall structure involved tension tolerance tests and pathological staining.Results:The pathogen-positive rate in the HTK group and NS group at 1 month was 16.7%.Regarding the vascular skeleton structure,both the UW group and HTK group exhibited intact structures after 2 weeks of preservation,with slightly edematous collagen and elastic fibers,which was significantly better than that of the NS group and RS group.In terms of cell activity and contractile function,all preservation groups showed similar effects within 2 weeks.However,after 2 weeks,the UW group showed the most favorable preservation effect(P<0.05).In terms of vascular tension,different groups exhibited similar effects within 1 week.However,after 2 weeks,the UW group showed the best preservation effect(P<0.05).Conclusion:All 4 types of preservation solution had a preservation effect on the structure and function of isolated blood vessels during short-term hypothermal preservation.However,after 2-week preservation,the UW solution was found to be the most suitable solution for the preservation of blood vessels.

Mechanism of AiTongXiao granule in the treatment of hepatocellular carcinoma based on network pharmacology and rat transplanted liver cancer model

Objective:To investigate the mechanism of action and material basis of AiTongXiao granule in the treatment of hepatocellular carcinoma(HCC)based on network pharmacology and transplanted liver cancer rat model.Methods:TCMSP database was used to screen out effective components and its corresponding potential pharmaceutical targets,and databases including Gene Cards,OMIM,Drugbank and TTD were further used to collect HCC-related drug targets.The intersecting targets were obtained by mapping the drug and disease targets.The component-targets network was constructed and visualized by Cytoscape 3.8.2 software.Protein-protein interaction(PPI)network was built by STRING online platform,and the topological relationship and core targets was analyzed and screened by using CytoNCA software.In addition,Metascape database was used to perform gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of the core targets.At last,rat liver transplanted liver cancer model was established by using Walker-256 cell line and treated by AiTongXiao granule for 15 days.Western blot was used to further compare the expression levels of AKT,pAKT,p53,p-p53,ERK1/2 and ERK1/2 in the tumor between treatment group and the control group.Results:257 active components were obtained from AiTongXiao granule,corresponding to 294 drug targets.Meanwhile,233 of the 7993 HCC disease targets were screened out between AiTongXiao granule drug and HCC disease targets.11 core targets including AKT1,IL6,TP53,MAPK3,TNF,JUN,CASP3,MAPK1,MYC,PTGS2,MMP9 were further obtained by median screening.GO and KEGG analysis results showed that these core targets enriched to HBV,TNF and cancer related pathways.The rat transplanted liver cancer model results indicated significant down regulation for AKT,p-AKT,pERK1/2,and significant up regulation of p-p53 after AiTongXiao granule treatment(P<0.05).Conclusion:AiTongXiao granule could act to multiple cancer related pathways,and AKT,p53 and ERK1/2 were validated to be regulated by ATXF in rat model.The mechanism may be through the regulation of the above signaling pathways to exert anti-liver cancer effect.

Modeling postpartum depression in rats： theoretic and methodological issues

The postpartum period is when a host of changes occur at molecular, cellular, physiological and behavioral levels to prepare female humans for the challenge of maternity. Alteration or prevention of these normal adaptions is thought to contribute to disruptions of emotion regulation, motivation and cognitive abilities that underlie postpartum mental disorders, such as postpartum depression. Despite the high incidence of this disorder, and the detrimental consequences for both mother and child, its etiology and related neurobiological mechanisms remain poorly understood, partially due to the lack of appropriate animal models. In recent decades, there have been a number of attempts to model postpartum depression disorder in rats. In the present review, we first describe clinical symptoms of postpartum depression and discuss known risk factors, including both genetic and environmental factors. Thereafter, we discuss various rat models that have been developed to capture various aspects of this disorder and knowledge gained from such attempts. In doing so, we focus on the theories behind each attempt and the methods used to achieve their goals. Finally, we point out several understudied areas in this field and make suggestions for future directions.

Inflammation and apoptosis accelerate progression to irreversible atrophy in denervated intrinsic muscles of the hand compared with biceps: proteomic analysis of a rat model of obstetric brachial plexus palsy

In treating patients with obstetric brachial plexus palsy,we noticed that denervated intrinsic muscles of the hand become irreversibly atrophic at a faster than denervated biceps.In a rat model of obstetric brachial plexus palsy,denervated intrinsic musculature of the forepaw entered the irreversible atrophy far earlier than denervated biceps.In this study,isobaric tags for relative and absolute quantitation were examined in the intrinsic musculature of forepaw and biceps on denervated and normal sides at 3 and 5 weeks to identify dysregulated proteins.Enrichment of pathways mapped by those proteins was analyzed by Kyoto Encyclopedia of Genes and Genomes analysis.At 3 weeks,119 dysregulated proteins in denervated intrinsic musculature of the forepaw were mapped to nine pathways for muscle regulation,while 67 dysregulated proteins were mapped to three such pathways at 5 weeks.At 3 weeks,27 upregulated proteins were mapped to five pathways involving inflammation and apoptosis,while two upregulated proteins were mapped to one such pathway at 5 weeks.At 3 and 5 weeks,53 proteins from pathways involving regrowth and differentiation were downregulated.At 3 weeks,64 dysregulated proteins in denervated biceps were mapped to five pathways involving muscle regulation,while,five dysregulated proteins were mapped to three such pathways at 5 weeks.One protein mapped to inflammation and apoptotic pathways was upregulated from one pathway at 3 weeks,while three proteins were downregulated from two other pathways at 5 weeks.Four proteins mapped to regrowth and differentiation pathways were upregulated from three pathways at 3 weeks,while two proteins were downregulated in another pathway at 5 weeks.These results implicated inflammation and apoptosis as critical factors aggravating atrophy of denervated intrinsic muscles of the hand during obstetric brachial plexus palsy.All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Fudan University,China(approval No.DF-325)in January 2015.

Impact of social isolation and resident intruder stress on aggressive behavior in the male rat

Stress studies frequently utilize physical stressors to establish animal models of stress. In the majority of cases, these models are not consistent with human circumstances. The present study simulated a social isolation plus resident intruder stress model in the rat. The rats were subjected to daily social isolation and resident intruder stress for 2 weeks. Behaviors were then tested. Rats subjected to social stress exhibited different aggressive behavior styles; some rats had greater scores on composite aggression behaviors, as well as locomotor and exploratory activity, but lower scores on latency than others. The high-aggressive group exhibited predominantly anger-out, while the low-aggressive group exhibited anger-in. Results suggest that social isolation plus resident intruder may serve as an appropriate model for anger-in and anger-out emotion modeling in the rat.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats

AIM:To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCI4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang. At wk 6,10,14 and 20 (baseline for CCl4., or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for α-SMA, type I collagen and In situ hybridization of tissue inhibitor of metalloproteinase-1 (TTMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCI4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in μm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and. 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, α-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCI4- and pig serum-induced rat models.

Key factors in developing the trinitrobenzene sulfonic acid-induced post-inflammatory irritable bowel syndrome model in rats

AIM:To investigate the key factors in developing the trinitrobenzene sulfonic acid(TNBS)-induced postinflammatory irritable bowel syndrome(PI-IBS)model in rats. METHODS:TNBS was administered to rats at the following conditions:(1)with different doses(20,10,5 mg/0.8 mL per rat);(2)with same dose in different concentrations(20 mg/rat,25,50 mg/mL);(3)in different ethanol percentage(25%,50%);and(4)at depth either 4 cm or 8 cm from anus.At 5 d and 4 wk after TNBS administration,inflammation severity and inflammation resolution were evaluated.At 4 and 8 wk after TNBS application,visceral hyperalgesia and enterochromaffin(EC)cell hyperplasia were assayed by abdominal withdrawal reflex test,silver staining and capillary electrophoresis. RESULTS:Our results showed that:(1)TNBS induced dose-dependent acute inflammation and inflammation resolution.At 5 d post TNBS,the pathological score and myeloperoxidase(MPO)activity in all TNBS treated rats were significantly elevated compared to that of the control(9.48±1.86,8.18±0.67,5.78± 0.77 vs 0,and 3.55±1.11,1.80±0.82,0.97±0.08 unit/mg vs 0.14±0.01 unit/mg,P<0.05).At 4 wk post TNBS,the pathological score in high and median dose TNBS-treated rats were still significantly higher than that of the control(1.52±0.38 and 0.80±0.35 vs 0,P<0.05);(2)Intracolonic TNBS administration position affected the persistence of visceral hyperalgesia.At 4 wk post TNBS,abdominal withdrawal reflex (AWR)threshold pressure in all TNBS-treated groups were decreased compared to that of the control(21.52 ±1.73 and 27.10±1.94 mmHg vs 34.44±1.89 mmHg,P<0.05).At 8 wk post TNBS,AWR threshold pressure in 8 cm administration group was still significantly decreased(23.33±1.33 mmHg vs 36.79±2.29 mmHg,P<0.05);(3)Ethanol percentage affected the TNBS-induced inflammation severity and visceral hyperalgesia.In TNBS-25%ethanol-treated group,the pathological score and MPO activity were significantly lowered compared to that of the TNBS-50%ethanoltreated group,while AWR threshold pressure were significantly elevated(36.33±0.61 mmHg vs 23.33±1.33 mmHg,P<0.05);and(4)TNBS(5 mg/0.8 mL per rat, in 50%ethanol,8 cm from anus)-treated rats recovered completely from the inflammation with acquired visceral hyperalgesia and EC cell hyperplasia at 4 wk after TNBS administration.CONCLUSION:TNBS dosage,concentration,intraco-lonic administration position,and ethanol percentage play important roles in developing visceral hyperalgesia and EC cell hyperplasia of TNBS-induced PI-IBS rats.

Effects of Haobie Yangyin Ruanjian Decoction on hepatic fibrosis induced by carbon tetrachloride in rats

AIM:To explore the anti-fibrotic effect of Haobie Yangyin Ruanjian Decoction(HYRD)on CCl4-induced hepatic fibrosis in rats and its modulation on the transforming growth factor(TGF)β-Smad signaling pathway.METHODS:Fifty-six healthy Wistar rats were randomly divided into five groups:normal control group(n=6),CCl4-induced hepatic fibrosis group(n=14) and three treatment groups(the treated rats received HYRD via oral administration at daily dosages of 8.2,2.5 and 0.82 g/kg,respectively)of HYRD(n=12,respectively).Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride solution(CCl4 dissolved in peanut oil,4:6,V/V)with 0.5 mL/100 g body weight for the first time,and then 0.3 mL/100 g body weight twice a week for 8 wk.In the former 2 wk,rats were raised by feedstuffⅠ(80% corn meal,20%lard,0.5%cholesterol).After 2 wk,they were raised by feedstuffⅡ(corn meal and 0.5% cholesterol).Except for the control group,30%alcohol solution was given orally to each rat every other day from the beginning,1 mL for each rat.Liver function parameters and hepatic hydroxyproline content were detected by chromatometry.Serum levels of hyaluronic acid(HA),typeⅣcollagen(CIV),typeⅢprecollagen(PCⅢ)and laminin(LN)were assayed with radioimmunoassay.Deposition of collagen was observed with hematoxylin-eosin staining and collagen staining.Gene expression of TGFβ1 and Smad3 were detected with real-time reverse transcriptase-polymerase chain reaction and Western blotting,respectively.RESULTS:The serum levels of alanine transaminase and aspartate transaminase were increased in the model group compared with the control group(P<0.01),and they were decreased in the three treatment groups compared with the model group.The serum levels of total protein and albumin were decreased in the model group and increased in the three treatment groups.The hepatic hydroxyproline content and serum levels of PCⅢ,HA,LN and CIV were markedly increased in the model group compared with the control group,and decreased in the treatment groups.The gene expression of TGFβ1 and Smad3 was enhanced in the model group compared with the control group,and HYRD could down regulate their expression.CONCLUSION:HYRD can inhibit hepatic fibrosis induced by CCl4 in rats,which is probably associated with its down-regulation on fibrogenic signal transduction of TGFβ-Smad pathway.

Potential rat model of anxiety-like gastric hypersensitivity induced by sequential stress

AIM To establish a rat model of anxiety-like gastric hyper-sensitivity(GHS) of functional dyspepsia(FD) induced by novel sequential stress.METHODS Animal pups were divided into two groups from postnatal day 2: controls and the sequential-stress-treated. The sequential-stress-treated group received maternal separation and acute gastric irritation early in life and restraint stress in adulthood; controls were reared undisturbed with their mothers. Rats in both groups were followed to adulthood(8 wk) at which point the anxietylike behaviors and visceromotor responses to gastric distention(20-100 mm Hg) and gastric emptying were tested. Meanwhile, alterations in several anxiety-related brain-stomach modulators including 5-hydroxytryptamine(5-HT), γ-aminobutyric acid(GABA), brain-derived neurotrophic factor(BDNF) and nesfatin-1 in the rat hippocampus, plasma and gastric fundus and the 5-HT1 A receptor(5-HT1 AR) in the hippocampal CA1 subfield and the mucosa of the gastric fundus were examined.RESULTS Sequential-stress-treated rats simultaneously demonstrated anxiety-like behaviors and GHS in dose-dependent manner compared with the control group. Although rats in both groups consumed similar amount of solid food, the rate of gastric emptying was lower in the sequentialstress-treated rats than in the control group. Sequential stress significantly decreased the levels of 5-HT(51.91 ± 1.88 vs 104.21 ± 2.88, P < 0.01), GABA(2.38 ± 0.16 vs 5.01 ± 0.13, P < 0.01) and BDNF(304.40 ± 10.16 vs 698.17 ± 27.91, P < 0.01) in the hippocampus but increased the content of nesfatin-1(1961.38 ± 56.89 vs 1007.50 ± 33.05, P < 0.01) in the same site; significantly decreased the levels of 5-HT(47.82 ± 2.29 vs 89.45 ± 2.61, P < 0.01) and BDNF(257.05 ± 12.89 vs 536.71 ± 20.73, P < 0.01) in the plasma but increased the content of nesfatin-1 in it(1391.75 ± 42.77 vs 737.88 ± 33.15, P < 0.01); significantly decreased the levels of 5-HT(41.15 ± 1.81 vs 89.17 ± 2.31, P < 0.01) and BDNF(226.49 ± 12.10 vs 551.36 ± 16.47, P < 0.01) in the gastric fundus but increased the content of nesfatin-1 in the same site(1534.75 ± 38.52 vs 819.63 ± 38.04, P < 0.01). The expressions of 5-HT1 AR in the hippocampal CA1 subfield and the mucosa of the gastric fundus were down-regulated measured by IHC(Optical Density value: Hippocampus 15253.50 ± 760.35 vs 21149.75 ± 834.13; gastric fundus 15865.25 ± 521.24 vs 23865.75 ± 1868.60; P < 0.05, respectively) and WB(0.38 ± 0.01 vs 0.57 ± 0.03, P < 0.01)(n = 8 in each group). CONCLUSION Sequential stress could induce a potential rat model of anxiety-like GHS of FD, which could be used to research the mechanisms of this intractable disease.

Neuroprotective effect of baicalin on focal cerebral ischemia in rats

Baicalin, a flavonoid compound from the root of the herb Scutellaria baicalensis Georgi, has been widely used to treat patients with inflammatory disease. The aim of this study was to assess the efficacy of baicalin in a rat model of focal cerebral ischemia. Adult male Sprague-Dawley rat models of cerebral artery occlusion were established and then randomly and equally divided into three groups： ischemia（cerebral ischemia and reperfusion）, valproic acid（cerebral ischemia and reperfusion ＋ three intraperitoneal injections of valproic acid; positive control）, and baicalin（cerebral ischemia and reperfusion ＋ intraperitoneal injection of baicalin for 21 days）. Neurological deficits were assessed using the postural reflex test and forelimb placing test at 3, 7, 14, and 21 days after ischemia. Rat cerebral infarct volume was measured using 2,3,5-triphenyltetrazolium chloride（TTC） staining method. Pathological change of ischemic brain tissue was assessed using hematoxylin-eosin staining. In the baicalin group, rat neurological function was obviously improved, cerebral infarct volume was obviously reduced, and the pathological impairment of ischemic brain tissue was obviously alleviated compared to the ischemia group. Cerebral infarct volume was similar in the valproic acid and baicalin groups. These findings suggest that baicalin has a neuroprotective effect on cerebral ischemia.