Specific Pathogen-free Rats Research Articles

Expression of angiopoietin-like protein 2 in ovarian tissue of rat polycystic ovarian syndrome model and its correlation study

BackgroundThis study investigated the expression of angiopoietin-like protein 2 (ANGPTL2) in the tissues of rat models of polycystic ovary syndrome (PCOS) and its correlation with PCOS.MethodsSix-weeks-old female specific pathogen-free rats (n = 60) were divided into blank control, PCOS model, and metformin groups (n = 20/group). After 21 days of metformin intervention, the serum sex hormones, fasting blood glucose, fasting insulin, and insulin resistance (IR) of rats in each group were measured. The mRNA levels of ANGPTL2, Foxol, and Akt in the ovarian tissues were monitored by real-time fluorescence quantitative PCR.ResultsCompared with the control group, the levels of serum sex hormones, fasting blood glucose, fasting insulin, and IR in the model group showed significant increases, and the levels of ANGPTL2, Foxol, and Akt in the ovarian tissue also showed significant increases. Compared with the PCOS group, the serum sex hormones, fasting blood glucose, fasting insulin, and IR of rats in the metformin group were significantly decreased, and the levels of ANGPTL2, Foxol, and Akt in ovarian tissues also showed significant decreases.ConclusionsThese findings suggest that ANGPTL2 might participate in the development of PCOS through the PI3K/Akt signaling pathway. Metformin improves IR by reducing the expression of ANGPTL2, thus improving the endocrine environment of PCOS and might change the disease outcome.

Ascorbic acid deficiency induces hepatic and intestinal expression of inflammation-related genes irrespective of the presence or absence of gut microbiota in ODS rats

We have previously demonstrated that ascorbic acid (AsA) deficiency causes inflammatory changes in the liver and intestine in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. We have suggested that AsA deficiency increased intestinal interleukine (IL)-6 production, stimulating hepatic acute phase proteins (APPs) expression via the portal vein. In this study, we determined whether these hepatic and intestinal inflammatory changes by AsA deficiency are induced in germ-free (GF) ODS rats. For 18 days, male specific pathogen-free (SPF) ODS rats were fed the basal diet containing 600 mg AsA/kg (control group) or the AsA-free diet (AsA-deficient group) in SPF conditions, while male GF ODS rats were fed the basal diet (control group) or the AsA-free diet (AsA-deficient group) in GF conditions. Firstly, AsA deficiency significantly elevated the hepatic expression of APPs in both SPF and GF rats. In hepatic mRNA levels of some APPs, significant interaction between GF and AsA-deficiency effects was observed. Secondly, AsA deficiency elevated intestinal IL-6 and IL-1β mRNA levels in both SPF and GF rats, and significant interaction between GF and AsA-deficiency effects was observed in these mRNA levels of jejunum and cecum. In SPF and GF rats, AsA deficiency elevated portal IL-6 concentration. These results show that AsA deficiency caused hepatic and intestinal inflammatory changes in both the GF and SPF ODS rats and indicate that AsA deficiency could directly induce intestinal inflammatory changes without the involvement of gut microbiota.

Characterization and anti-uterine tumor effect of extract from Prunella vulgaris L.

BackgroundThe flowers and dried fruit spikes of Prunella vulgaris L. (P. vulgaris L.) have been widely used in traditional Chinese medicine and food. P. vulgaris L. is regarded as a good option for treating uterine myoma (UM). However, scientific evidence of anti-UM activity of the extract of P. vulgaris L. (PVE) is lacking. The present study aimed to characterize the chemical composition of PVE and evaluate the pharmacodynamics and mechanism of PVE against UM.MethodsThe chemical composition of PVE was analyzed by GC-MS. MTT was used to screen and evaluate cell proliferation and toxicity. Double fluorescence flow cytometry method were used to determine the apoptosis and cell cycle progression of UM cells under PVE treatment. The anti-UM activity of PVE was investigated by using a specific-pathogen-free (SPF) rat model of UM. TUNEL staining was used to detect the apoptosis of UM cells. The concentrations of estrogen and progesterone in the serum of SPF rats were detected by ELISA. The expression levels of PCNA, estrogen receptor alpha, estrogen receptor beta, progesterone receptor, survivin, caspase-3, Bax and Bcl-2 in the uterus of SPF rats was detected by immunohistochemistry (IHC).ResultsThe extraction rate of PVE was 8.1%. The main components were squalene (28.3%), linoleic acid (9.96%), linolenic acid (9.95%), stearic acid (6.26%) and oleic acid (5.51%). In vitro, PVE had significant anti-human UM cell activity, exhibited no drug toxicity, promoted the apoptosis of human UM cells, and inhibited the transition of UM cells from the G0/G1 stage into the G2 stage, in which DNA replication occurs. In vivo, PVE had significant anti-UM activity. PVE decreased the concentrations of estrogen and progesterone and downregulated the expression levels of the estrogen and progesterone receptors through the estrogen signaling pathway. PVE also promoted the apoptosis of UM cells by downregulating the expression levels of the survivin and Bcl-2 proteins and upregulating the expression levels of caspase-3 and Bax through the mitochondria-mediated apoptotic pathway.ConclusionPVE has marked anti-UM activity. PVE can be used as an ideal candidate drug to treat UM.

MiR-808 inhibits cardiomyocyte apoptosis and expressions of caspase-3 and caspase-9 in rats with myocardial infarction by regulating TGF-β1 signaling pathway.

To investigate the effects of micro ribonucleic acid (miR)-808 on cardiomyocyte apoptosis and expressions of caspase-3 and caspase-9 in rats with myocardial infarction (MI) by regulating the transforming growth factor-β1 (TGF-β1) signaling pathway. A total of 24 specific pathogen-free female Sprague-Dawley rats were enrolled and randomly divided into normal group, model group, and miR-808 group, 8 rats in each group. In the model group and miR-808 group, MI model was prepared by ligation of the left anterior descending coronary artery in the rats. The miR-808 group was transfected with miR-808 lentivirus after the model was established. After one week of intervention, the expression of TGF-β1 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The cardiac function of rats was determined by echocardiography. The myocardium of rats was observed by Masson staining. The cardiomyocyte apoptosis of rats was examined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. The expression levels of caspase-3 and caspase-9 were detected by Western blotting. The expression of TGF-β1 mRNA was higher in the model group than that in the normal group (p<0.05), but compared with that in the model group, it was lower in the miR-808 group. The myocardial function and cardiomyocyte survival rate in the miR-808 group was better and higher than those in the model group (p<0.05). The expression levels of caspase-3 and caspase-9 in the miR-808 group were lower than those in the model group (p<0.05). MiR-808 can inhibit cardiomyocyte apoptosis in rats with MI by down-regulating TGF-β1 expression and inhibiting the expressions of caspase-3 and caspase-9.

Effect of alprostadil in early wound healing of rats with deep partial-thickness scald

Objective: To explore the effect of alprostadil in early wound healing of rats with deep partial-thickness scald. Methods: Ninety specific pathogen free healthy Sprague-Dawley rats (half males and half females) were divided into sham scald group, simple scald group, and alprostadil group using the random number table with 30 rats in each group. Rats in sham scald group were sham injured, and rats in the other 2 groups were inflicted with deep partial-thickness scald of 30% total body surface area on the back. Immediately after scald, rats in the 3 groups received anti-shock treatment. Two hours post scald, rats in sham scald group and simple scald group were injected intraperitoneally with 1 mL normal saline, and rats in alprostadil group were injected intraperitoneally with 1 mL alprostadil injection, once a day and continued for 14 days. On post scald day (PSD) 3, 7, and 14, 10 rats in each group were collected for observing the general wound conditions and the wound healing rates of rats in 2 scald groups were calculated, abdominal aortic blood of 2 mL from each rat were collected to detect serum thromboxane B2 by enzyme-linked immunosorbent assay, and wound tissue on the back was collected to examine pathomorphological change by hematoxylin-eosin staining and to detect wound microvessel density (MVD) by immunohistochemical staining. Data were statistically analyzed with analysis of variance of factorial design, t test, Kruskal-Wallis H test, Mann-Whitney U test, and Bonferroni correction. Results: (1) There was no scald wound in rats in sham scald group. On PSD 3, wounds of rats in simple scald group and alprostadil group formed dry eschar. On PSD 7 and 14, wound areas of rats in alprostadil group were significantly smaller than those of rats in simple scald group, and with less exudation. (2) On PSD 3, the wound healing rates of rats in simple scald group and alprostadil group were similar (t=1.167, P>0.05). On PSD 7 and 14, the wound healing rates of rats in alprostadil group were significantly higher than those in simple scald group (t=8.657, 33.050, P<0.01). (3) On PSD 3, 7, and 14, the levels of serum thromboxane B2 of rats in simple scald group and alprostadil group were (541±22), (607±47), (688±21), (326±25), (271±21), (135±27) pg/mL, significantly higher than (17±6), (16±4), (16±4) pg/mL of rats in sham scald group (t=72.977, 39.685, 102.076, 37.033, 37.253, 13.845, P<0.01). On PSD 3, 7, and 14, the levels of serum thromboxane B2 of rats in alprostadil group were significantly lower than those in simple scald group (t=20.637, 20.651, 51.680, P<0.01). (4) Normal epidermis and dermis were seen in rats in sham scald group. On PSD 3, a large number of necrotic tissue and inflammatory cells infiltration were seen in wounds of rats in simple scald group, while a little new epithelium formation and some inflammatory cells infiltration were seen in wounds of rats in alprostadil group. On PSD 7 and 14, the new epithelium of rats in alprostadil group was significantly thicker than that in simple scald group, and epidermis formed gradually in alprostadil group. (5) On PSD 3, 7, and 14, the wound MVD of rats in simple scald group and alprostadil group were significantly higher than those in sham scald group (Z=-3.780, -3.781, -3.780, -3.780, -3.781, -3.780, P<0.01). On PSD 3, the wound MVD of rats in simple scald group and alprostadil group were similar (Z=-1.965, P>0.05). On PSD 7 and 14, the wound MVD of rats in alprostadil group were significantly higher than those in simple scald group (Z=-3.780, -3.780, P<0.01). Conclusions: The early intervention with alprostadil can significantly improve microcirculation of deep partial-thickness scald wound, reduce inflammatory cell infiltration, promote the formation of new blood vessels, thus promoting wound healing.

Effect of quercetin on bone metabolism and serum osteocalcin in osteoporotic rats

Purpose: To determine the effect of quercetin on bone metabolism and serum osteocalcin in osteoporotic rats.&#x0D; Methods: Sixty specific pathogen-free rats were randomly divided into control group, model group; high, medium and low dose quercetin groups, and diethylstilbestrol group, with 10 rats in each group. The high, middle and low dose quercetin groups were given quercetin suspension at doses of 200, 100, 50 mg/kg/day, respectively; the ethylene estradiol group was given ethylene estradiol (1.0 mg/kg/week), while control rats received ethylene estradiol at doses of 200, 100, 50 mg/kg/day. Rats in the model group were given saline. Samples were taken after 6 weeks of administration. The levels of serum bone-derived alkaline phosphatase (BALP), estradiol (E2) and serum osteocalcin (BGP) in femur tissue were measured using ELISA kits. Bone mineral density (BMD) was determined using BMD tester.&#x0D; Results: Relative to normal rats, BALP and BGP levels in the model rats were markedly increased, while E2 was significantly lower (p &lt; 0.5). Quercetin treatment led to significant increases in BALP and E2 levels in the middle and high dose groups, relative to the model group, while BGP levels in all quercetin treatment groups decreased significantly, when compared to model rats (p &lt; 0.05). There were higher BMD values in quercetin and diethylstilbestrol groups than in model (p &lt; 0.05).&#x0D; Conclusion: Quercetin enhances bone formation and BMD, but decreases osteocalcin levels and maintains bone biomechanics in ovariectomized rats. Thus, it may find therapeutic application in maintaining bone health.&#x0D; Keywords: Quercetin, Osteoporosis, Bone metabolism, Osteocalcin

The expression of β-Defensin-2, IL-22, IL-22R1 and IL-10R2 in rat model of Klebsiella pneumonia and their correlation with histological grades

Backgrounds and Aims: Klebsiella pneumoniae represents the most common opportunistic pathogen contributing to Klebsiella pneumonia in hospital-acquired infections. Klebsiella pneumonia has a rapidly progressive clinical course and multi-drug resistant (MDR). Identification of the effective biochemical markers is crucial for improving early diagnosis and treatment of Klebsiella pneumonia. The aims of our study are to 1) investigate the expression of β-Defensin-2(rβD2), IL-22, IL-22R1 and IL-10R2 in Klebsiella pneumonia-infected rats and 2) their association with the histological grades of Klebsiella pneumonia.Methods and Materials: Fifty specific pathogen free (SPF) male SD rats were randomly divided into two groups: control group (treated with normal saline) and pneumonia group (treated with K. pneumoniae). All animals were sacrificed 1 h, 12 h, 1 d, 3 d, 5 d post infection. The severity and property of pneumonia was evaluated by histopathologic observation and pathogen identification. The mRNA expression of rβD2, IL-22, IL-22R1 and IL-10R2 was measured by RT-qPCR assay. The expression of rβD2 in rat lung tissue was determined by Western blot analysis, and the level of IL-22 in rat serum was determined by ELISA.Results: Histopathologic examination and bacterial counting of lung tissues confirmed the successful establishment of rat pneumonia model. The gene expression of rβD2, IL-22, IL-22R1 and IL-10R2 in pneumonia rats were significantly higher than those in healthy control mice (P < 0.05). The expression of rβD2 was correlated with histological grades of Klebsiella pneumonia and the level of IL-22. RT-qPCR results showed that the peak expression of IL-22R1 appeared earlier than IL-10R2 in rat pneumonia model.Conclusions: The expression of rβD2 and IL-22 was increased significantly at early stage in rat Klebsiella pneumonia model, suggesting that IL-22 and rβD2 might serve as potential biomarkers for the early diagnosis of Klebsiella pneumonia.

Response to mechanical loading in rat Achilles tendon healing is influenced by the microbiome

We have previously shown that changes in the microbiome influence how the healing tendon responds to different treatments. The aim of this study was to investigate if changes in the microbiome influence the response to mechanical loading during tendon healing. 90 Sprague-Dawley rats were used. Specific Opportunist and Pathogen Free (SOPF) rats were co-housed with Specific Pathogen Free (SPF) rats, carrying Staphylococcus aureus and other opportunistic microbes. After 6 weeks of co-housing, the SOPF rats were contaminated which was confirmed by Staphylococcus aureus growth. Clean SOPF rats were used as controls. The rats were randomized to full loading or partial unloading by Botox injections in their calf muscles followed by complete Achilles tendon transection. Eight days later, the healing tendons were tested mechanically. The results were analysed by a 2-way ANOVA with interaction between loading and contamination on peak force as the primary outcome and there was an interaction for both peak force (p = 0.049) and stiffness (p = 0.033). Furthermore, partial unloading had a profound effect on most outcome variables. In conclusion, the response to mechanical loading during tendon healing is influenced by changes in the microbiome. Studies aiming for clinical relevance should therefore consider the microbiome of laboratory animals.

Fibroblast activation induced by common bile duct ligation in rats with obstructive jaundice

Objective To investigate the effect of common bile duct ligation on fibroblast activation in rats with obstructive jaundice. Methods Twenty-four male specific pathogen free (SPF) rats were randomly divided into sham-operated group and experimental group, with 12 rats in each group. All rats underwent common bile duct ligation. Rats The common bile duct in the experimental group was ligated with 3-0 silk thread and cut off. The rats in the two groups were sampled 2 and 7 days after operation. Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of transforming growth factor-β1 (TGF-β1). Immunohistochemistry was used to detect the expression of TGF-β1 protein. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of TGF-β1 gene. Western blotting was used to detect the expression of TGF-β1 protein. Results In sham-operated group, the structure of hepatic lobules was intact, and the hepatic sinuses were normal without congestion. In experimental group, the structure of hepatic lobules was disordered and the hepatic cords were dissociated. The level of serum TGF-β1 in the experimental group at 2nd day [(689.42±58.13) ng/L] and 7th day [(867.53±45.62) ng/L] was significantly higher than that in the sham-operated group [(519.92±32.41) ng/L and (536.50±41.29) ng/L, t=8.822 and 18.635, P<0.05]. The expression of TGF-β1 protein in liver tissues of experimental group at 2nd day [(6.53±1.29)%] and 7th day [(8.97±1.85)%] was significantly higher than that of sham-operated group [(1.17±0.24)% and (1.54±0.39)%, t=8.822 and 18.635, P<0.05]. The expression of TGF-β1 in liver tissues of experimental group at 2nd day [(0.89±0.14)%] and 7th day [(0.95±0.19)%] was significantly higher than that of sham-operated group [(0.52±1.32)% and (0.31±1.76)%, t=14.693 and 18.316, P<0.05]. The gray value of TGF-β1 protein expression in liver tissue of experimental group at 2nd day after operation (0.62±0.13) and 7th day after operation (0.78±0.10) was significantly higher than that of sham-operated group (0.21±0.05 and 0.23±0.06, t=10.197 and 16.337, P<0.05). Conclusion The expression of TGF-β1 in rats with obstructive jaundice induced by common bile duct ligation is significantly up-regulated, and the proliferation of biliary duct epithelial cells induces fibroblast activation. Key words: Common bile duct ligation; Obstructive jaundice; Fibroblast activation

Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis

ObjectivesMethicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy.MethodsAn ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization.ResultsThe results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model.ConclusionIn summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.

Extraction optimization and constipation-relieving activity of dietary fiber from Auricularia polytricha

Auricularia polytricha-derived dietary fiber (DF) is a bio-active health-promoting compound, but limited work has been done on optimization of extraction and constipation-relieving activity. In this study, an enzymatic-chemical method to extract DF was optimized, and the results showed that optimal extraction conditions were ratio of water to A. polytricha powder 41 ml/g, 0.4% α-amylase, 1.0% protamex, and enzymolysis at 66 °C, with DF extraction rate of 56 ± 3%. Then, the constipation-relieving activity of DF was evaluated with a constipated rat model. Sprague – Dawley specific pathogen free rats were randomly divided into 6 groups, namely normal, model, Maren boluses treatment, and three DF treatment groups. Constipated rats fed with 5% A. polytricha-derived DF in place of part of the standard diet resulted in increased wet weight of feces and intestinal propulsive rate, which was associated with the functional properties of DF, including high swelling capacity, water binding capacity, fat adsorption capacity, and glucose adsorption capacity. Moreover, A. polytricha-derived DF had a positive effect on gastrointestinal hormones in rats. The results indicated that the enzymatic-chemical extraction method is effective for extracting DF from A. polytricha and the DF has high constipation-relieving activity.

Role of Oxidative Stress and Apoptosis in the Hepatic Toxicity Induced by Cerium Oxide Nanoparticles Following Intratracheal Instillation in Male Sprague-Dawley Rats

Inhaled cerium oxide (CeO₂) nanoparticles have been shown to be capable of translocation to the liver where they can cause dose dependent toxic effects. Herein, we investigate if the deposition of cerium in the liver is linked to increased oxidative stress and cellular apoptosis. Specific pathogen free male Sprague-Dawley rats were instilled with either vehicle (saline) or CeO₂ nanoparticles (7.0 mg/kg) and euthanized 1, 3, 14, 28, 56, or 90 days post exposure.

Conventional and Specific-Pathogen Free Rats Respond Differently to Anesthesia and Surgical Trauma

Specific-pathogen free (SPF) animals were introduced in the 1960s to minimize disease and infection as variables in biomedical research. Our aim was to examine differences in physiological response in rat colonies bred and housed in a conventional versus SPF facility, and implications for research. Sprague-Dawley rats were anesthetized and catheterized for blood and pressure monitoring, and electrocardiogram (ECG) leads implanted. Hematology was assessed, and coagulation profile using rotational thromboelastometry. Health screening was outsourced to Cerberus Sciences. SPF rats had significantly lower pulse pressure (38% decrease), arrhythmias and prolonged QTc (27% increase) compared to conventional rats. No arrhythmias were found in conventional rats. SPF rats had significantly higher white cell, monocyte, neutrophil and lymphocyte counts, and were hyperfibrinolytic, indicated by EXTEM maximum lysis >15%. Independent assessment revealed similar pathogen exclusion between colonies, with the exception of Proteus in SPF animals. Returning to a conventional facility restored normal host physiology. We conclude that SPF animals displayed an abnormal hemodynamic, hematological and hemostatic phenotype in response to anesthesia and surgery, and provide a number of recommendations to help standardize research outcomes and translation.

Cariogenicity of Streptococcus mutans glucan-binding protein deletion mutants.

Streptococcus mutans is a principal etiologic agent in the development of dental caries due to its exceptional aciduric and acidogenic properties, and its ability to adhere and accumulate in large numbers on tooth surfaces in the presence of sucrose. Sucrose-dependent adherence is mediated by glucans, polymers of glucose synthesized from sucrose by glucosyltransferase (Gtf) enzymes. S. mutans makes several proteins that have the property of binding glucans. We hypothesized that three of these glucan-binding proteins (Gbps), Gbps A, C and D, contribute to the cariogenicity of S. mutans. A specific pathogen-free rat model was used to compare the cariogenicity of S. mutans UA130 and a panel of mutants with individual or multiple gbp gene deletions. The mutants were also evaluated in vitro for properties related to cariogenicity, such as acidogenicity, aciduricity, and adhesion to glucan. Only a subset of Gbp mutants were attenuated for cariogenicity, with the combined loss of Gbps A and C most affecting smooth surface caries. The attenuation of Gbp mutant strains was unlikely due to differences in acid-related properties since the mutants were at least as acidogenic and acid-tolerant as the parental strain. Additionally, loss of Gbps did not reduce adhesion to a pre-formed biofilm of S. sanguinis. Analyses of the caries data with in vitro biofilm properties previously determined for the mutant panel found correlations between cariogenicity and biofilm depth and substratum coverage. It is concluded that Gbps contribute to the cariogenicity of S. mutans through a mechanism that may involve alteration of biofilm architecture.

Stimulation of Tendon Healing With Delayed Dexamethasone Treatment Is Modified by the Microbiome

Background: The immune system reflects the microbiome (microbiota). Modulation of the immune system during early tendon remodeling by dexamethasone treatment can improve rat Achilles tendon healing. The authors tested whether changes in the microbiota could influence the effect of dexamethasone treatment. Hypothesis: A change in microbiome would influence the response to dexamethasone on regenerate remodeling, specifically tendon material properties (peak stress). Study Design: Controlled laboratory study. Methods: Specific opportunist and pathogen-free female rats were housed separately (n = 41) or together with specific pathogen-free rats carrying opportunistic microbes such as Staphylococcus aureus (n = 41). After 6 weeks, all co-housed rats appeared healthy but now carried S aureus. Changes in the gut bacterial flora were tested by API and RapID biochemical tests. All rats (clean and contaminated) underwent Achilles tendon transection under aseptic conditions. Flow cytometry was performed 8 days postoperatively on tendon tissue. Sixty rats received subcutaneous dexamethasone or saline injections on days 5 through 9 after transection. The tendons were tested mechanically on day 12. The predetermined primary outcome was the interaction between contamination and dexamethasone regarding peak stress, tested by 2-way analysis of variance. Results: Dexamethasone increased peak stress in all groups but more in contaminated rats (105%) than in clean rats (53%) (interaction, P = .018). A similar interaction was found for an estimate of elastic modulus (P = .021). Furthermore, dexamethasone treatment reduced transverse area but had small effects on peak force and stiffness. In rats treated with saline only, contamination reduced peak stress by 16% (P = .04) and elastic modulus by 35% (P = .004). Contamination led to changes in the gut bacterial flora and higher levels of T cells (CD3+CD4+) in the healing tendon (P < .05). Conclusion: Changes in the microbiome influence tendon healing and enhance the positive effects of dexamethasone treatment during the early remodeling phase of tendon healing. Clinical Relevance: The positive effect of dexamethasone on early tendon remodeling in rats is strikingly strong. If similar effects could be shown in humans, immune modulation by a few days of systemic corticosteroids, or more specific compounds, could open new approaches to rehabilitation after tendon injury.

Effect of long-term crotonaldehyde exposure on heart damage in male rats

Objective: To observe the effect of long term crotonaldehyde exposure on heart damage in male rats, and to explore the possible mechanism of toxic action. Methods: 24 specific pathogen free healthy male wistar rats were randomly divided into 4 groups with 6 rats in each group. Rats were treated with with 8.5, 4.5, 2.5 and 0.0 mg/kg body weight crotonaldehyde by gavage, once a day for consecutive 150 days. After the last treatment, they were anesthetized and collected blood samples by cardiac puncture. The heart was rapidly separated after cervical dislocation. The cardiac organ coefficient was calculated and the histopathology changes in heart were observed by HE staining. At the same time, the activities of creatine kinase (CK) , lactate dehydrogenase-L (LDH-L) in serum were determined by automatic biochemical analyzer. Moreover, the levels of cardiac troponin (cTnT) , Angiotensin Ⅱ (Ang Ⅱ) , Brain natriuretic peptide (BNP) , Aldosterone (ALD) and interleukin (IL) -6, 8, 1β, interferon (IFN) -γ and tumor necrosis factor (TNF) -α in heart were determined by enzyme linked immunosorbent assay. Results: At the 90d, 120 d, and 150 d exposure, compared with the control group, the body weight gain in 4.5 and 8.5 mg/kg groups were decreased. Moreover, the heart weight in 4.5 and 8.5 mg/kg groups, and heart coefficient in 8.5 mg/kg group were decreased (P<0.05) . With the increasing dosage of crotonaldehyde, the degree of pathological changes in the heart of exposed rats were aggravated. The major pathological changes of heart in 4.5 and 8.5 mg/kg groups could be summarized as lymphocyte infiltration, abnormal cardiac muscle fiber arrangements, necrosis and fibrous connective tissue hyperplasia. Compared with the control group, the serum CK activity in 4.5 mg/kg group, CK and LDH-L activitivies in 8.5 mg/kg group were increased (P<0.05) ; Compared with the control group, the levels of ALD and ANGII in the heart of 4.5 and 8.5 mg/kg groups were increased, BNP level were decreased, and cTNT level in 8.5 mg/kg group were increased (P<0.05) . Compared with the control group, the levels of IL-1β、IL-6、IL-8 in 4.5 mg/kg group and IL-1β、IL-6、IL-8、TNF-α、IFN-γ in 8.5 mg/kg group were increased (P<0.05) . Conclusion: Crotonaldehyde could up-regulate cardiac inflammatory cytokines and alter the balance ofangiotensin-aldosterone-brain natriuretic peptide causing heart damage.

Effect of crotonaldehyde long-term exposure induced kidney inflammatory and oxidative injury in male rats

Objective: To observe the effect of crotonaldehyde long-term exposure on kidney injury in male rats, and to explore the specific mechanism of toxic action. Methods: 32 specific pathogen free healthy adult male wistar rats were randomly divided into 4 groups with 8 rats in each group: high-, moderate-, low-dose groups and a control group. Rats were treated with 8.5, 4.5, 2.5 and 0.0 mg/kg body weight crotonaldehyde by gavage, once a day for consecutive 128 days. After the last treatment, they were sacrificed and separated bilateral kidney. Kidney organ coefficients were calculated and the histopathology changes in kidney were observed by HE staining. The activities of malondialdehyde (MDA) , superoxide dismutase (SOD) , glutathion peroxidase (GSH-Px) and the levels of malaondialdehyde uric acid (UA) , urea nitrogen (BUN) , creatinine (CR) in serum were determined in the same time. Moreover, the levels of interleukin (IL) -6, 8, interferon (IFN) -γ and tumor necrosis factor (TNF) -α in kidney were determined by enzyme linked immunosorbent assay. Results: Bilateral kidneys in the 8.5 mg/kg group were reduced in size and dark in color. Under the microscope, the major pathology changes of kidneys could be summarized as summarized as protein cast renal tubule, inflammatory cells and lymphocytes infiltration among kidney cortex. Compared with the control group, the weight gain of rats in 8.5 mg/kg group were smaller, and the weight and organ coefficient of kidney in each groups were significant decreased (P<0.05) . With the increasing dosage of crotonaldehyde, the serum BUN and UA levels in 8.5, 4.5 mg/kg groups, CR level in 8.5 mg/kg group were significant increased (P<0.05) .Compared with the control group, the serum MDA level in 8.5 mg/kg group was significant increased (P<0.05) . However, serum SOD activity in 8.5 mg/kg group and GSH-PX activity in 8.5, 4.5 mg/kg groups was significant decreased (P<0.05) . With the increasing dosage of crotonaldehyde, there are upward trend in the kidney IL-6, IL-8, TNF-α, IFN-γ, β-2 microglobulin levels. Compared with the control group, IL-6, TNF-α, IFN-γ, β-2microglobulin levels in 8.5, 4.5 mg/kg groups and IL-8 level in 8.5 mg/kg group in kidney were significant increased (P<0.05) . Conclusion: Crotonaldehyde could develop the inflammatory factors levels and change the oxidation balance condition in the kidney of male rats, causing the inflammatory and oxidative injures of renal tissues.

Effect of lipopolysaccharide exposure on neuro-immune system of rat offspring with autism spectrum disorder at second trimester

Objective To establish the lipopolysaccharide(LPS)-induced autism spectrum disorder(ASD) models at second trimester in rat offspring, and to investigate the effect of LPS exposure on social interaction behavior and neuro-immune system in rat offspring. Methods Twelve-week-old pregnant specific pathogen free rats were randomly divided into non-exposed group(10 cases) and LPS-exposed group(10 cases). Rats in the exposure group were intraperitoneally injected with LPS 25 mg/kg at 12.5 d, while rats in the non-exposed group were intraperitoneally injected with 9 g/L saline.At the age of 28 days, 10 rats were randomly selected from each group as the experimental group and the control group.The following tests were carried out in two groups of rats.Their offspring were randomly allocated to the control group (10 cases) and the experimental group (10 cases). The autism-like social behaviors were evaluated by the social interaction test.Density of dendritic spine was analyzed by Golgi staining.The expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1(IBA1) were analyzed by immunohistochemisty.The levels of interleukin(IL)-1β, IL-4, IL-6, IL-10, interferon-gamma(IFN-γ), and transforming growth factor-beta(TGF-β) in rat offspring were detected by enzyme-linked immunosorbent assay. Results The score of social interaction test in the experimental group [(-197.81±101.83) scores] was lower than that in the control group [(132.73±114.63) scores], and the difference was statistically significant (t=5.453, P<0.05). Density of dendritic spine in CA1 region of hippocampus was lower in the experimental group[(7.71±1.33) numbers/10 μm] than that in the control group[(9.66±1.76) numbers/10 μm] by Golgi staining, and the difference was statistically significant(t=2.775, P<0.05). Expressions of GFAP and IBA1 in experimental group were higher than those in the control group by immunohistochemisty.Levels of IL-1β, IL-6, IL-10 and IFN-γ in the experimental group were higher than those in the control group, while level of TGF-β was lower than that in the control group, and the differences were statistically significant(all P<0.05). The scores of social interaction test were correlated with the levels of IFN-γ(r=0.756, P<0.05). Conclusion LPS exposure at early pregnancy may affect the neuro-immune system of rat offspring and result in autism-like behavior. Key words: Lipopolysaccharide; Autism spectrum disorder; Cytokines; Neuro-immune system

Influence of periodontitis and scaling and root planing on insulin resistance and hepatic CD36 in obese rats

The aim of the study was to explore the influence of periodontitis and scaling and root planing (SRP) on insulin resistance and hepatic CD36 in obese rats with periodontitis. Thirty-two specific pathogen free Sprague-Dawley rats were randomly divided into four groups of eight animals each as follows: healthy rats (healthy group), obese rats (obesity group), obese rats with periodontitis (non-therapy group), and obese rats with periodontitis who underwent periodontal SRP (therapy group). Rats were fed with a high-fat diet for 16weeks to build an obesity model. Periodontal inflammation was induced by performing periodontal ligation with Porphyromonas gingivalis. The tissue around the maxillary second molars, bilaterally, were collected. The periodontal attachment level (from the cemento-enamel junction to the bottom of the periodontal pocket) of the second molars was measured in all groups. All rats were subjected to fasting blood glucose, insulin, and serum C-reactive protein tests (CRP). Insulin resistance was evaluated by homeostasis model assessment of insulin resistance (HOMA-IR), oral glucose tolerance test (OGTT), and area under the curve (AUC). The liver was excised to detect intrahepatic free fatty acid (FFA) levels and pathologic observation. Real-time quantification PCR, western blot, and immunohistochemistry were applied to detect hepatic CD36 expression. Compared with the obesity group, HOMA-IR, AUC, intrahepatic FFA, and protein expression, and mRNA levels of hepatic CD36 in the non-therapy group were significantly increased (P<0.05). HOMA-IR, AUC, CRP, protein expression, and mRNA levels of hepatic CD36 were all significantly decreased (P<0.05) 2-weeks after SRP. Periodontitis increases insulin resistance while scaling and root planning could improve insulin resistance. Hepatic CD36 regulation may be considered a potential mechanism for this phenomenon.

Effects of S100A12 gene silencing on serum levels of anti-inflammatory/pro-inflammatory cytokines in septic rats through the ERK signaling pathway.

We explored the effect of S100A12 gene on serum levels of anti-inflammatory/pro-inflammatory cytokines in septic rats by activating the extracellular signal-regulated kinase (ERK) signaling pathway. A total of 180 specific pathogen-free (SPF) rats were purchased to establish cecal ligation and puncture (CLP) model. Rats were assigned into the sham, model, empty vector, S100A12 siRNA, epidermal growth factor (EGF), and S100A12 siRNA + EGF groups. The expressions of S100A12, ERK-1, ERK-2, cPLA2, and NF-κB in liver tissues of rats among six groups were detected by RT-qPCR and western blotting. ELISA was used to determine serum levels of IL-1β, IL-10, TNF-α, procalcitonin (PCT), and C-reactive protein (CRP). Pearson correlation analysis was conducted to measure correlations. Cell apoptosis of rats among six groups was detected by Tunel staining. The expressions of S100A12, ERK-1, ERK-2, cPLA2, and NF-κB decreased in the S100A12 siRNA group while increased in the EGF group compared with the model group. S100A12 mRNA expression was positively correlated with mRNA expressions of related genes in the ERK signaling pathway (ERK-1, ERK-2, cPLA2, and NF-κB) in the model and empty vector groups. Expressions of IL-1β, IL-6, TNF-α, PCT, and CRP in the EGF group were higher than those in the model group, but were lower than those in the S100A12 siRNA group. Compared with the model group, cell apoptosis decreased in the S100A12 siRNA group but that increased in the EGF group. We demonstrates that S100A12 gene silencing decreases serum levels of anti-inflammatory/pro-inflammatory cytokines in septic rats by inhibiting the ERK signaling pathway.