Specific Pathogen-free Sprague-Dawley Rats Research Articles

Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicle miR-208a-3p Alleviating Spinal Cord Injury via Regulating the Biological Function of Spinal Cord Neurons.

We aim to explore the potential mechanism of bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSCs-Exo) in improving spinal cord injury (SCI). Thirty male 12-week specific pathogen-free (SPF) Sprague-Dawley (SD) rats were used to construct SCI model invivo. Ten male 12-week SPF SD rats were used to extract BMSCs. The Basso, Beattie, Bresnahan (BBB) score was used to evaluate the motor function of rats. Real-time fluorescence quantitative PCR (RT-PCR), western blot (WB), and double luciferase assay were used to explore the regulation between rno-miR-208a-3p and Cdkn1a (p21) in BMSCs. Primary spinal cord neurons were treated with lipopolysaccharide (100 ng/mL) for 30 min to mimic SCI in vitro. Compared with the model group (14 scores), BMSCs-Exo increased BBB score (19 scores) in SCI rats. Compared with the sham group, Cdkn1a was upregulated, whereas rno-miR-208a-3p was downregulated in the model group. However, compared with the model group, Cdkn1a was downregulated, whereas rno-miR-208a-3p was upregulated in the BMSCs-Exo group. In addition, rno-miR-208a-3p inhibited the expression of Cdkn1a via direct binding way. BMSCs-Exo-rno-miR-208a-3p promoted the proliferation of primary spinal neurons via inhibiting apoptosis in vitro. Moreover, BMSCs-Exo-rno-miR-208a-3p promoted cyclin D1, CDK6, and Bcl-2 and inhibited Bax expression in a cell model of SCI. In conclusion, BMSCs-Exo-carried rno-miR-208a-3p significantly protects rats from SCI via regulating the Cdkn1a pathway.

The rapid antidepressant effect of acupuncture on two animal models of depression by increasing the expression of synaptic proteins in the prefrontal cortex: 针刺通过增加前额叶皮层突触蛋白的表达对两种抑郁症动物模型的快速抗抑郁作用

ObjectiveTo investigate the effect of acupuncture onset time and synaptic structure in two different models of depression. MethodsA total of 150 healthy male adult SPF C57BL/6J mice were divided into five time-point groups: 1 h, 3 h, 6 h, 12 h and 24 h. Each time-point group was further divided into three groups: the model, scopolamine and acupuncture groups with 10 rats each. Rats were given forced swimming stimulation (15 min, once) before the intervention. A total of 200 healthy male adult specific pathogen-free (SPF) Sprague-Dawley (SD) rats were divided into 30 normal rats, and 170 modelling ones. A total of 90 rats were successfully modeled, and were randomly divided into three time-point groups: 1 h, 6 h and 24 h. And each time-point group was further divided into the normal, model, scopolamine, and acupuncture group. Chronic unpredictable mild stress (CUMS) combined with individual feeding was used to establish the depressed rat model. The sucrose preference test (SPT) and open field test (OFT) were used to evaluate the success of the model. There were 10 rats in each group. The acupuncture intervention was performed once for 20 min on “Baihui (GV20)”, “Yintang (GV29)”, “Hegu (LI4)”, and “Taichong (LR3)” without any other operations for the corresponding animals. Other groups were given corresponding medications. According to different time points, the forced swimming test (FST) was conducted in mice, and the FST and novelty-suppressed feeding test (NSFT) were measured in rats. Monoamine neurotransmitters (NE, DA, DOPAC, 5-HT, 5-HIAA, HVA) in the prefrontal cortex and hippocampus were detected with high performance liquid chromatography (HPLC). The expression of synapsin I, PSD95, p-mTOR, mTOR, and BDNF in the prefrontal cortex and hippocampus was determined by western blot. ResultsAt the 1 h time point, compared with the model group, the immobility time was significantly decreased in the acupuncture group (P < 0.05). At 3 h, 6 h, 12 h, and 24 h, compared with the model group, the immobility time was significantly reduced in both scopolamine and acupuncture groups (P < 0.05). At the 1 h time point, compared with the normal group, the immobility time of FST and the latency to feed were significantly increased in the model group (P < 0.05). Compared with the model group, the immobility time of FST showed a significant reduction in the acupuncture group (P < 0.05). At the 6 h and 24 h time points, compared with the normal group, the immobility time of FST and the latency to feed were significantly increased in the model group (P < 0.05). Compared with the model group, the immobility time of FST was significantly decreased in the scopolamine and acupuncture groups (P < 0.05). At the 1 h and 24 h time points, compared with the normal group, the expression levels of 5-HT, 5-HIAA, NE, DA, DOPAC, and HVA of the prefrontal cortical and hippocampal tissues were significantly reduced in the model group (P < 0.05). At the 1 h and 24 h time points, compared with the model group, the expression levels of synapsin I, PSD95, p-mTOR, and BDNF of the prefrontal cortex were significantly increased in the acupuncture group (P < 0.05). At the 1 h and 24 h time points, compared with the normal group, the expression levels of synapsin I, PSD95, p-mTOR, and BDNF of the prefrontal cortex were significantly reduced in the model group (P < 0.05). Compared with the model group, the expression levels of synapsin I, PSD95, p-mTOR, and BDNF of the prefrontal cortex were significantly increased in the acupuncture group (P < 0.05). At the 1 h and 24 h time points, compared with the normal group, the expression levels of synapsin I, PSD95, p-mTOR, and BDNF of the hippocampal tissues were significantly reduced in the model group (P < 0.05). Compared with the model group, the expression levels of synapsin I, PSD95, p-mTOR, and BDNF of the hippocampus were not significantly different in the acupuncture group (P > 0.05). ConclusionAcupuncture may play a rapid antidepressant effect by increasing the expression of synaptic plasticity proteins in the prefrontal cortex.

Low-Dose Hexavalent Chromium Exposure Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Rat Liver.

This study investigated the toxic effects of low-dose hexavalent chromium (Cr(VI)) on rat liver. Male specific pathogen-free (SPF) Sprague-Dawley (SD) rats (4-5weeks of age) were randomly divided into groups: saline, 0.05mg/kg Cr(VI), and 0.25mg/kg Cr(VI). The rats were subjected to intratracheal instillation of K2Cr2O7 suspensions or saline once weekly, for a total of five times. The results showed that the accumulation of Cr(VI) in the blood of the 0.25mg/kg K2Cr2O7 group was significantly higher than that in the saline group. Transmission electron microscopy (TEM) showed that exposure to hexavalent chromium caused endoplasmic reticulum (ER) oedema and a disordered arrangement. The levels of endoplasmic reticulum stress (ERS)-related proteins (ATF6, P-PERK, P-IRE1, Grp78, and CHOP) in the 0.25mg/kg K2Cr2O7 group were significantly higher than those in the saline group. The expression of apoptosis-inhibitory protein Bcl-2 was significantly lower in the 0.25mg/kg K2Cr2O7 group than that in the saline group, and the expression of apoptosis protein Bax was significantly higher in the 0.25mg/kg K2Cr2O7 group than that in the saline group, indicating that Cr(VI) increased apoptosis. These findings revealed that Cr(VI) may be involved in rat liver injury by initiating ERS-mediated apoptosis. The expression of ATF6, P-PERK, P-IRE1, and Bax in the 0.05mg/kg K2Cr2O7 group was not significantly different from that in the saline group, and the different effects produced by the two different dose groups provide a possible experimental basis for further study of occupational exposure limits.

Calycosin alleviates cerebral ischemia/reperfusion injury by repressing autophagy via STAT3/FOXO3a signaling pathway

BackgroundAs a common cerebrovascular disease (CVD) of the elderly, ischemic stroke (IS) is characterized by high disability and mortality. Excessive autophagy induced by IS is implicated in neuronal death, therefore, the inhibition of immoderate autophagy is viewed as a potential therapeutic avenue to treat IS. Calysoin (CA) is a bioactive component of Radix Astragali, which has been widely used to treat CVDs. However, the mechanism of the treatment of IS by CA is still problematic. PurposeBased on the result of network pharmacology, whether CA inhibited autophagy by regulating the STAT3/FOXO3a pathway to alleviate cerebral ischemia-reperfusion injury (CIRI) was investigated in vivo and in vitro for the first time. Study designIntegrate computational prediction and experimental validation based on network pharmacology. MethodsIn current study, network pharmacology was applied to predict the mechanism of the treatment of IS by CA, and it was shown that CA alleviated CIRI by inhibiting autophagy via STAT3/FOXO3a signaling pathway. One hundred and twenty adult male specific pathogen-free Sprague-Dawley rats in vivo and PC12 cells in vitro were used to verify the above prediction results. The rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by suture method, and oxygen glucose deprivation/re-oxygenation (OGD/R) model was used to simulate cerebral ischemia in vivo. The content of MDA, TNF-α, ROS and TGF-β1 in rat serum were detected by ELISA kits. The mRNA and protein expressions in brain tissue were detected by RT-PCR and Western Blotting. The expressions of LC3 in brain were detected immunofluorescent staining. ResultsThe experimental results demonstrated that administration of CA dosage-dependently improved rat CIRI as evidenced by the reduction in the cerebral infarct volume, amelioration of the neurological deficits. HE staining and transmission electron microscopy results revealed that CA ameliorated cerebral histopathological damage, abnormal mitochondrial morphology, and damaged mitochondrial cristae structure in MCAO/R rats. CA treatment exerted protective effects in CIRI by inhibiting inflammation response, oxidative stress injury, and cell apoptosis in rat and PC12 cells. CA relieved excessive autophagy induced by MCAO/R or OGD/R through downregulating the LC3Ⅱ/LC3Ⅰ ratio and upregulating the SQSTM1 expression. CA treatment also decreased p-STAT3/STAT3 and p-FOXO3a/FOXO3a ratio in the cytoplasm and modulated the autophagy-related gene expression both in vivo and in vitro. ConclusionTreatment with CA attenuated CIRI by reducing excessive autophagy via STAT3/FOXO3a signal pathway in rat and PC12 cells.

Guishaozichuan granules can attenuate asthma in rats via the MUC5AC/EGFR signaling pathway.

Background: Guishaozichuan (GSZC) granules are a traditional Chinese medicine formulation created by Professor Li (Chinese-Japanese Friendship Hospital, Beijing, China) we studied the effect of GSZC granules in rats suffering from asthma. Methods: Specific pathogen-free Sprague-Dawley rats were divided randomly into seven groups. Ovalbumin (OVA) and Al (OH)3 gel were used to create an asthma model. On day 1, rats were injected with OVA (10mg) and an Al(OH)3 gel suspension (100mg). One week later, rats were sensitized again. On day 15, rats were given aerosolized OVA (1%) for 30min/day for 10days. Gastric administration of OVA was 1h before nebulization. At 24h after the last stimulation, changes in airway resistance (RI) and dynamic compliance (Cdyn) in rat lungs were measured after challenge with methacholine at increasing concentrations. The contents of immunoglobulin (Ig)E, interleukin (IL)-4, IL-5, IL-13, and IL-17 in serum were measured by enzyme-linked immunosorbent assays. The percentage of eosinophils (EOS) and the white blood cell (WBC) count in bronchoalveolar lavage fluid (BALF) were counted under an optical microscope. Pathologic alterations in lung tissue were evaluated by optical microscopy, and lung injury score calculated. Expression of mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) and epidermal growth factor receptor (EGFR) in lung tissue was measured by immunohistochemistry. mRNA expression of MUC5AC and EGFR in lung tissue was measured by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results: GSZC granules reduced RI markedly and improved Cdyn, decreased serum levels of IgE, IL-4, IL-5, IL-13, IL-17, %EOS and the WBC count in BALF. GSZC granules alleviated lung-tissue damage, diminished the Inflammation Score, and reduced mRNA and protein expression of MUC5AC and EGFR in lung tissue. Conclusion: GSZC granules could improve bronchial hyperresponsiveness, bronchial inflammation, and histopathologic damage in the lungs of rats suffering from asthma. This phenomenon may be related to its regulation of cytokine levels and the MUC5AC/EGFR signaling pathway.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways.

To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway. Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot. Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1β and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1β and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (< 0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different ( > 0.05). In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Deer antler extract potentially facilitates xiphoid cartilage growth and regeneration and prevents inflammatory susceptibility by regulating multiple functional genes

BackgroundDeer antler is a zoological exception due to its fantastic characteristics, including amazing growth rate and repeatable regeneration. Deer antler has been used as a key ingredient in traditional Chinese medicine relating to kidney and bone health for centuries. The aim of this study was to dissect the molecular regulation of deer antler extract (DAE) on xiphoid cartilage (XC).MethodsThe DAE used in this experiment was same as the one that was prepared as previously described. The specific pathogen-free (SPF) grade Sprague-Dawley (SD) rats were randomly divided into blank group (n =10) and DAE group (n =10) after 1-week adaptive feeding. The DAE used in this experiment was same as the one that was prepared as previously described. The rats in DAE group were fed with DAE for 3 weeks at a dose of 0.2 g/kg per day according to the body surface area normalization method, and the rats in blank group were fed with drinking water. Total RNA was extracted from XC located in the most distal edge of the sternum. Illumina RNA sequencing (RNA-seq) in combination with quantitative real-time polymerase chain reaction (qRT-PCR) validation assay was carried out to dissect the molecular regulation of DAE on XC.ResultsWe demonstrated that DAE significantly increased the expression levels of DEGs involved in cartilage growth and regeneration, but decreased the expression levels of DEGs involved in inflammation, and mildly increased the expression levels of DEGs involved in chondrogenesis and chondrocyte proliferation.ConclusionsOur findings suggest that DAE might serve as a complementary therapeutic regent for cartilage growth and regeneration to treat cartilage degenerative disease, such as osteoarthritis.

Mechanism of nano-indium-tin oxide inducing pulmonary alveolar proteinosis in Sprague-Dawley rats

Objective: To investigate the pathogenesis of pulmonary alveolar proteinosis in rats induced by nano-indium-tin oxide exposure, and to provide a basis for further determining the limit of occupational exposure to indium and developing related protection measures. Methods: In August 2018, a total of 40 specific pathogen-free Sprague-Dawley rats, with an age of 6-8 weeks and a body weight of (200±10) g, were randomly divided into control group, low-dose group (1.2 mg/kg) , middle-dose group (3 mg/kg) , and high-dose group (6 mg/kg) , with 10 rats in each group. After 1 week of routine feeding, the rats were given non-exposed intratracheal instillation twice every week, with an interval of 3 days, for 12 consecutive weeks. Body weight was measured every week during exposure to observe the change in body weight; The rats were anesthetized and sacrificed by chloral hydrate after the exposure ended, and lung tissue and serum were collected; Hematoxylin-eosin staining and periodic acid-Schiff (PAS) staining were performed for lung tissue to observe pathological results; Inductively coupled plasma mass spectrometry was used to measure the serum level of indium; ELISA was used to measure the levels of surfactant protein A (SP-A) , surfactant protein D (SP-D) , and the type II alveolar cell surface antigen Krebs von den Lungen-6 (KL-6) in lung tissue and the serum level of granulocyte-macrophage colony-stimulating factor (GM-CSF) . Results: The pathological results showed that the rats in the control group had basically complete alveolar structure, and after intratracheal instillation of nano indium-tin oxide, uniform, eosinophilic, and unstructured granular substances were observed in the alveolar space of the low-, middle-, and high-dose exposure groups, with macrophage proliferation and an increase in macrophages, especially in the high-dose group. Negative PAS staining was observed in the control group, while substances with positive PAS staining were observed in lung tissue in each exposure group. The three exposure groups had a significantly higher serum level of indium than the control group (P<0.05) . Compared with the control group, the three exposure groups had significant increases in SP-A, SP-D, and KL-6 in lung tissue and a significant reduction in GM-CSF in serum (P<0.05) . Conclusion: Pulmonary alveolar proteinosis in rats may be associated with the destruction of alveolar macrophages caused by nano-indium-tin oxide and the aggregation of pulmonary surfactants due to disorders in the metabolism and clearance of pulmonary surfactants by macrophages.

Correlations between inflammatory response, oxidative stress, intestinal pathological damage and intestinal flora variation in rats with type 2 diabetes mellitus.

To explore the correlations between inflammatory response, oxidative stress, intestinal pathological damage, and intestinal flora variation in rats with type 2 diabetes mellitus (T2DM). A total of 80 specific pathogen-free (SPF) male Sprague-Dawley (SD) rats purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijiing, China) were randomly divided into two groups, namely T2DM group (n=40) and normal group (n=40). Then, the contents of inflammatory factors [high-sensitivity C-reactive protein (hs-CRP) and tumor necrosis factor-α (TNF-α)] and oxidative stress indicators [malondialdehyde (MDA) and superoxide dismutase (SOD)] were detected. Meanwhile, the distributions of intestinal florae Bifidobacteria, Escherichia coli, Lactobacilli and Enterococcocci and the variation of endotoxin were compared between the two groups. Besides, colon specimens were pathologically examined to observe the occurrence of chronic inflammation variation, intestinal mucosal erosion, intestinal mucosal wall thickening and intestinal mucosal fibrosis. Next, the influences of the glycosylated hemoglobin on levels of hs-CRP, MDA and endotoxin and colonization ability of intestinal florae were analyzed. Additionally, univariate and multivariate analyses were performed for underlying the relations of the pathogenesis of T2DM in rats with their inflammatory response, antioxidant capacity, endotoxin level and intestinal flora colonization capacity. The levels of hs-CRP and TNF-α were significantly higher in T2DM group than those in normal group (p<0.05). T2DM group exhibited an overtly elevated MDA level (p<0.05), and a clearly lowered SOD level (p<0.05) in comparison with normal group. As to intestinal flora-related indicators, the levels of endotoxin, Escherichia coli and Enterococcocci were evidently higher in T2DM group than those in the normal group (p<0.05), while the levels of Bifidobacteria and Lactobacilli in T2DM group were remarkably lowered (p<0.05). Pathological lesions, including chronic inflammation variation, mucosal erosion, mucosal wall thickening and mucosal fibrosis in intestinal mucosal tissues, were worse in T2DM group than those in the normal group (p<0.05). In T2DM rats, the level of glycosylated hemoglobin was positively correlated with changes in the levels of hs-CRP, MDA and endotoxin (p<0.05), and negatively associated with changes in colonization ability of intestinal florae (p<0.05). Aggravated inflammatory response, decreased antioxidant capacity, increased endotoxin level and weakened colonization ability of intestinal florae were independent risk factors for T2DM in rats. Rats with T2DM have significantly aggravated inflammatory response, weakened antioxidant capacity, imbalanced intestinal florae and markedly pathological changes of intestinal mucosa.

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.

Changes in expressions of miR-22-3p and MMP-9 in rats with thoracic aortic aneurysm and their significance.

The purpose of this study was to explore the changes in the expressions of micro ribonucleic acid (miR)-22-3p and matrix metalloproteinase-9 (MMP-9) in rats with thoracic aortic aneurysm (TAA) and their significance. A total of 16 specific pathogen-free Sprague-Dawley female rats were randomly divided into normal group (n=8) and angiotensin II (Ang II) group (n=8). Ang II was perfused using the micro pump in Ang II group, while the same amount of normal saline was perfused in the normal group. After continuous intervention, the tumor formation rate in the thoracic aorta was observed, and the expression of miR-22-3p was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) in both groups. Other 16 rats were selected and randomly divided into agomiR-22-3p group (n=8) and control group (n=8). In the agomiR-22-3p group, agomiR-22 and Ang II were continuously injected via angular vein. In the control group, agomiR negative control was injected, and Ang II was continuously perfused. After intervention for 4 weeks, the tumor formation rate in the thoracic aorta was observed, and the expression of MMP-9 was determined via immunofluorescence and immunohistochemistry in both groups. After intervention for 4 weeks, the expression of miR-22-3p in Ang II group was significantly lower than that in normal group (p<0.05). After drug administration for 4 weeks, agomiR-22-3p group had a lower tumor formation rate (p<0.05) and a lower expression of MMP-9 than the control group (p<0.05). The expression of miR-22-3p declines in TAA rats, and miR-22-3p can inhibit the expression of MMP-9, thus suppressing the formation of TAA in rats.

Research on mechanism of sevoflurane in alleviating cerebral ischemia-reperfusion injury in rats through JNK signaling pathway.

To explore the specific mechanism of sevoflurane in alleviating cerebral ischemia-reperfusion injury (CIRI) in rats through the c-Jun N-terminal kinase (JNK) signaling pathway. A total of 60 male specific pathogen-free Sprague-Dawley rats were randomly divided into sham group (n=20), model group (n=20), and sevoflurane group (n=20). In the sevoflurane group, sevoflurane (2.5%) was inhaled for 60 min at 24 h before the blockage of cerebral blood supply. The CIRI model was established using the suture method in the model group and sevoflurane group, while the right common carotid artery and external carotid artery were separated and ligated only, without suture placement, in the sham group. At 24 h after reperfusion, the neurological deficit score in each group was calculated, the water content in brain tissues in each group was detected based on dry-wet weight ratio, the infarction volume of brain tissues in each group was detected via 2,3,5-triphenyltetrazolium chloride (TTC) staining, and the apoptosis rate of brain cells in each group was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the protein levels of JNK, p-JNK, B-cell lymphoma-2 (Bcl-2), and the Bcl-2 associated X protein (Bax) in brain tissues were determined using Western blotting, and the gene expressions of Bax and Bcl-2 in brain tissues were determined through fluorescence quantitative Polymerase Chain Reaction (qPCR). It was found that the water content in brain tissues and the cerebral infarction volume were significantly increased in the model group compared with those in the sham group (p<0.01, p<0.01), while they were notably decreased in the sevoflurane group compared with those in the model group (p<0.05, p<0.01). The neurological deficit score was significantly higher in the model group than that in the sham group (p<0.01), while it was remarkably lower in the sevoflurane group than that in the model group (p<0.01). According to the results of the TUNEL assay, the model group had an evidently higher apoptosis rate of brain cells than the sham group (p<0.01), while the sevoflurane group had a lower apoptosis rate of brain cells than the model group (p<0.05). Besides, the results of Western blotting revealed that the model group exhibited remarkably increased protein levels of JNK, p-JNK, and Bax (p<0.05, p<0.01, p<0.01) and a remarkably decreased protein level of Bcl-2 (p<0.01) compared with the sham group. Sevoflurane group had decreased protein levels of JNK, p-JNK, and Bax (p<0.05, p<0.01, p<0.01) and an increased protein level of Bcl-2 (p<0.05) in comparison with the model group. In addition, the gene expression of Bcl-2 significantly declined (p<0.01), and that of Bax remarkably rose (p<0.01) in the model group compared with those in the sham group, while the contrary is the case in the sevoflurane group compared with those in the model group (p<0.05, p<0.01). Sevoflurane can regulate the protein and gene expressions of Bax and Bcl-2 and reduce apoptosis in CIRI by regulating the JNK signaling pathway, thereby exerting a protective effect on brain tissues and improving the symptoms of neurological deficit.

Effects of irisin on osteoblast apoptosis and osteoporosis in postmenopausal osteoporosis rats through upregulating Nrf2 and inhibiting NLRP3 inflammasome.

The nuclear factor E2-related factor 2 (Nrf2)/NLR family, pyrin domain containing protein 3 (NLRP3) plays an important role in osteoporosis (OP), so the effects of irisin on postmenopausal OP rats and osteoblast apoptosis through Nrf2/NLRP3 were explored in the present study. A total of 45 specific pathogen-free Sprague-Dawley rats were selected and divided into OP model group (OP group, n=15), 1 mmol/l irisin treatment group (irisin group, n=15) and normal control group (control group, n=15). After the trial period, the content of serum ALP was detected, the levels of tumor necrosis factor-α (TNF-α) in the serum and bone tissues were observed via ELISA, and the bone microstructure was observed via CT. Osteoblast apoptosis was determined through TUNEL assay, the content of apoptosis genes caspase-3 and Bcl-2, and key genes in Runt-related transcription factor 2 (Runx2), osteocalcin (OC), Nrf2 and NLRP3 was detected via RT-PCR. The protein expression of Bcl-2, Nrf2 and NLRP3 was determined via western blotting. The serum ALP level was increased in OP group compared with that in control group (P<0.05), while it declined in the irisin group. The content of TNF-α and interleukin-6 (IL-6) was significantly higher in OP group, while the content in the irisin group was close to that in the control group. The trabecular thickness, number and bone mineral density in the irisin group were all obviously larger and higher, respectively, than those in the OP group. The mRNA expression of Runx2, OC, Bcl-2 and Nrf2 in the irisin group were obviously higher (P<0.05), while that of caspase-3 and NLRP3 showed the opposite trends. The protein expression of Bcl-2 and Nrf2 in the irisin group was remarkably higher than those in the OP group, while that of NLRP3 was the opposite. irisin can upregulate Nrf2, inhibit NLRP3 inflammasome and lower the content of inflammatory factors, thereby suppressing osteoblast apoptosis in postmenopausal OP rats and reducing the incidence of postmenopausal OP.

Inhibition of the TGF-β/Smad3 signaling pathway by SB431542: A study of the intervention effect of SB431542 on silicotic fibrosis in rats

Objective: To investigate the intervention effect of SB431542, which inhibits the TGF-β/Smad3 signaling pathway, on silicotic fibrosis in rats. Methods: A total of 40 specific pathogen-free Sprague-Dawley rats were divided into normal saline control group, model group, SB431542 inhibitor group, and SB431542 inhibitor control group using a random number table, with 10 rats in each group. All rats except those in the normal saline control group were given non-exposed single intratracheal instillation of free silicon dioxide dust suspension 1 mL (50 mg/mL) ; the rats in the SB431542 inhibitor group were given intraperitoneal injection of SB431542 (5 mg/kg) on days 7 and 30 after dust exposure, those in the SB431542 inhibitor control group were given intraperitoneal injection of SB431542 cosolvent (5 mg/kg) on days 7 and 30 after dust exposure, and those in the normal saline control group were given intratracheal instillation of an equal volume of normal saline (5 mg/kg). On day 60 after dust exposure, the paraffin-embedded section of the right upper lobe of lung was collected for HE staining; the left upper lobe of lung was collected to measure the mRNA levels of fibronectin (FN) , collagen type I (COL-I) , and collagen type III (COL-III) by quantitative real-time PCR; the right inferior lobe of lung was collected to measure the protein levels of FN, COL-I, COL-III, phosphorylated Smad3 (p-Smad3) , and Smad3. Results: Compared with the normal saline control group, the model group had nodules with various sizes in lung tissue, with rupture of some alveolar septa, emphysema changes, and pulmonary interstitial fibrosis, as well as significant increases in the mRNA expression of FN, COL-I, and COL-III and the protein expression of FN, COL-I, COL-III, p-Smad3, and Smad3 in lung tissue (P<0.05) . Compared with the SB431542 inhibitor control group, the SB431542 inhibitor group had a relatively complete structure of lung tissue without marked nodules and with a small amount of exudate in alveolar space and the lumen of bronchioles, as well as significant reductions in the mRNA expression of FN, COL-I, and COL-III and the protein expression of FN, COL-I, COL-III, p-Smad3, and Smad3 in lung tissue (P<0.05) . There were no significant differences in the mRNA expression of FN, COL-I, and COL-III and the protein expression of FN, COL-I, COL-III, p-Smad3, and Smad3 between the model group and the SB431542 inhibitor control group (P>0.05) . Conclusion: SB431542 exerts an intervention effect on silicotic fibrosis by blocking the TGF-β/Smad3 signaling pathway and reducing the expression of the downstream fibrosis factors FN, COL-I, and COL-III.

Early Caries in an In Vivo Model: Structural and Nanomechanical Characterization

The utilization of rat models in cariology research has made substantial contributions to decipher mechanisms of caries formation and to develop preventive treatments. The existing rat models still have potential for improvement toward establishing a more accurate standard caries protocol to utilize in testing and/or developing new dental technologies. The current caries-scoring methods rely on optical microscopy–based techniques, which necessitates formation of highly advanced lesions. Moreover, models that facilitate the implementation of cariogenic bacteria by shifting the balance of oral flora through desalivation and/or antibiotic treatment create a nonnatural environment. Furthermore, there is a paucity of detailed structural and mechanical characterization on the resulting carious lesions. The purpose of this study was to develop a rat model that induces formation of mild carious lesions and to provide comprehensive structural and mechanical characterization. With this aim in mind, an in vivo model promoting progression of mild lesions was established with specific pathogen-free Sprague-Dawley rats. Cariogenic bacteria, Streptococcus mutans, was implemented into the oral flora without the use of antibiotics or desalivation surgery. During caries formation, progression of the infection was monitored by quantifying the relative abundance of S. mutans in oral flora with quantitative real-time polymerase chain reaction. A significant increase in colonization efficacy of S. mutans was detected during cariogenic challenge (P < 0.01). The resulting carious lesions were analyzed by conventional light optical and scanning electron microscopy. A detailed structural and morphological characterization on fissure caries with different degrees of severity was provided. The changes in the morphology and demineralization state of the sound and carious tissues were quantified by energy-dispersive X-ray spectroscopy, and local mechanical properties were acquired with nanoindentation. The principles laid out in this work can be utilized in cariology research and developed into a standard protocol for future studies.

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.

Asiaticoside improves spatial learning and memory abilities of Alzheimer's disease rat model by promoting the expression of PPAγ and inhibiting inflammatory factors

Aims: The present study is to investigate the effect of Asiaticoside (AS) on the expression of hippocampal Peroxisome Proliferator Activated Receptor Gamma (PPARγ) and inflammatory factors such as Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α in rats after intracerebral injection of Aβ1-42. Methods: A total of 40 specific pathogen-free Sprague-Dawley rats were evenly divided into shamoperation (control) group, model group, model+AS (15 mg/kg) group and model+AS (45 mg/kg) group. Condensed β-amyloid protein (Aβ)-1-42 was slowly injected into the CA1 region in hippocampus of rats to simulate Alzheimer’s disease. Morris water maze test was used to examine the ability of spatial learning and memory of rats. Immunohistochemistry was performed to detect the deposition of Aβ1-42 in hippocampal tissues. Enzyme-linked immunosorbent assay was carried out to determine the contents of IL-6 and TNF-α in hippocampal tissues. Western blotting was employed to measure the protein expression of PPARγ. Results: Treatment with AS enhanced the spatial learning and memory, reduced the expression of Aβ1-42 in hippocampal region, decreased the contents of IL-6 and TNF-α in hippocampal tissues, and elevated the protein expression of PPARγ in hippocampal tissues of AD model rats. Conclusions: The present study demonstrates that AS improves spatial learning and memory abilities of AD model rats by promoting the expression of PPARγ and inhibiting the contents of inflammatory factors.

Synchronous dynamic research on respiratory and intestinal microflora of chronic bronchitis rat model.

To investigate the mechanism of the Chinese medicine theory that Fei (Lung) and Dachang (Large Intestine) are exteriorly and interiorly related via synchronous observation on the dynamic changes of the respiratory and intestinal microflora. Forty specific pathogen free Sprague-Dawley rats were selected and randomly divided into blank (10 rats) and chronic bronchitis model groups (30 rats). The blank group rats were put into the smoke-free environment and the model group rats were put into the smoke environment in order to establish pulmonary disease (chronic bronchitis) model. Then the corresponding changes of the respiratory and intestinal microflflora of the model on 20th, 50th and 70th days were synchronously observed. The respiratory tract microflflora showed an increase in the total aerobic and Staphylococcus aureus and reduced anaerobic amount signifificantly on 20th day in the respiratory tract microflflora (P<0.05 or 0.01). On 50th day, total aerobic, total anaerobic amount and bififidobacterium signifificantly increased (P<0.05). On 70th day, Staphylococcus aureus reduced and lactobacillus increased signifificantly (P<0.01). The intestinal microflflora showed an increase in the total aerobic, Clostridium perfringens, enterobacter and enterococcus significantly increased on 20th day (P<0.05 or 0.01). Staphylococcus aureus on 50th day increased significantly (P<0.05). Total aerobic and enterococcus increased, total anaerobic and Clostridium perfringens reduced signifificantly on 70th day (P<0.05 or 0.01). The microecosystem of respiratory tract and intestine of rat model during the pathological process showed a dynamic disorder, indicating an interaction between the lung and large intestine which may be one of the connotations as they exteriorly and interiorly related.

Preparation and analysis of active rat model of rheumatoid arthritis with features of TCM toxic heat-stasis painful obstruction

ObjectiveTo establish a collagen type II-induced rat model of rheumatoid arthritis (RA) presenting characteristics of the human form of the traditional Chinese syndrome pattern of toxic heat-stasis painful obstruction (bi zheng; arthromyodynia) as well as pathologic features of active RA. The Chinese herbal medicine Tengmei decoction was used to validate the animal model. MethodsNinety specific pathogen free Sprague-Dawley rats were randomly divided into a normal group of 6 rats and a model group of 84 rats. To establish the rat model of collagen-induced arthritis (CIA), bovine type II collagen in complete Freund's adjuvant was injected into the model group rats as a priming dose (Day 0) and boosting dose (Day 9). Changes in arthritic index (AI) scores, including limb swelling, were monitored. Thereafter, 24 successfully-established CIA rats were randomly assigned to 4 groups with 6 animals each: model, positive control drug, high-dose traditional Chinese herbal medicine, and traditional Chinese herbal medicine. A blank control group of 6 rats was included. After 12 weeks of intervention with Tengmei decoction, articular synovial tissue and serum specimens were collected to detect interleukin-2 (IL-2) and IL-17 transcription and protein expression levels. ResultsDays 0 through 9, the anterior–posterior and lateral diameters of the limbs in model group rats did not differ significantly when compared to the normal control group (P > .05). On day 16, anterior–posterior, lateral diameters, and footpad thicknesses of both hind limbs in the model group were higher than those of the normal control group (P < .01, P < .05). Total AI, AI of both hind limbs between Days 11 and 16 were significantly higher than those of the normal control group (P < .01). Real-time reverse transcription polymerase chain reaction analyses of rat joint tissues indicated that compared to the normal control group, levels of mRNA transcription of IL-2 and IL-17 were significantly upregulated (P < .01) in the model group. Compared with the model group, levels of mRNA transcription of the aforementioned inflammatory cytokines were significantly downregulated in the positive control drug and traditional Chinese medicine treatment groups (P < .01). Results of ELISA analyses indicated that compared with the normal control group, levels of protein expression of cytokines IL-2, IL-17 were significantly upregulated (P < .01) in the model group. Compared with the model group, levels of protein expression of the aforementioned inflammatory cytokines were significantly downregulated in the positive control drug and traditional Chinese medicine treatment groups (P < .01). ConclusionThe CIA model established in this study presents both active RA pathologic features and characteristics of the symptoms of toxic heat-stasis painful obstruction 12 weeks after successful establishment of an animal model. In addition, this study may be a valuable reference for development of animal studies with combined Eastern and Western medicines in dialectics and identification of diseases.

Seawater immersion aggravates sciatic nerve injury in rats.

The aim of the present study was investigate the impact of seawater immersion on peripheral nerve injury and the underlying mechanisms. A total of 234 specific pathogen-free Sprague-Dawley male rats were randomly divided into a sham group, injury control group and seawater immersion + injury group. The Sciatic Functional Index (SFI) was used to assess nerve function for 6 weeks after injury. Compound muscle action potentials were measured and hematoxylin and eosin (H&E) staining of nerve specimens was carried out at week 6. Levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in nerve tissues were measured by enzyme-linked immunosorbent assay (ELISA), and the expression levels of inducible nitric oxide synthase (iNOS) mRNA and protein were measured by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. The SFI value in the seawater immersion + injury group after 6 weeks was lower than that in the injury control group (P<0.05). The compound muscle action potential in the seawater immersion + injury group had a prolonged latency, and the amplitude and nerve conduction velocity were decreased compared with those in the other groups (P<0.05). H&E staining demonstrated that nerve fiber regeneration was worse in the seawater immersion + injury group. The ROS and MDA levels in the seawater immersion + injury group were higher than those in the other groups (P<0.05). The expression levels of iNOS mRNA and protein gradually increased in the injury and seawater immersion + injury groups and peaked at 48 h after surgery. Immersion in seawater further aggravated sciatic nerve injury and led to worse neuronal recovery. The mechanism may be associated with oxidative stress.