Abstract:Objective To explore the biological indicators of diagnosis and treatment of irritable bowel syndrome (IBS), and to explore the mechanism of action of a Chinese medicine Wuji Pill (WJW) on irritable bowel syndrome (IBS). Methods (1) Postinflammatory irritable bowel syndrome (PI-IBS) rat model was established by acetic acid plus restraint stress method . (2) The colonic motor ability of rats was evaluated by colon motility index (MI), the number of fecal particles discharged within 2 h, and the time of glass pellet discharge. (3) The formation of PI-IBS model rats and the therapeutic effect of WJW were observed. (4) The levels of calcitonin gene-related peptide (CGRP), motilin (MTL), neuropeptide Y (NPY), substance P (SP), somatostatin (SS), vasoactive intestinal peptide (VIP), and cholecystokinin (CCK) in the brain and colon tissues of PI-IBS rats were measured by ELISA. Results (1) The rat PI-IBS model was successfully established. Compared with the normal group, the body weight of the model rats was decreased, the food intake decreased, the amount of feces increased, loose stools and amorphous soft stools appeared, voluntary movements decreased, colon motility index (MI) significantly increased ( P < 0.05), the number of fecal particles discharged significantly increased ( P < 0.05), and the glass pellet discharge time was significantly shortened ( P < 0.05). (2) WJW treatment for 7 days significantly improved a variety of symptoms. Compared with the normal control, the levels of CGRP, SS and VIP in the brain tissue of PI-IBS rats were significantly increased ( P < 0.05), and the NPY concentration was significantly decreased ( P < 0.05). However, the treatment with WJW significantly reduced CGRP, SS and VIP levels ( P < 0.05), and significantly increased the NPY concentration level ( P < 0.05). (3) Compared with the normal control group, the levels of CCK, NPY, MTL, SS and VIP in colonic tissues of PI-IBS rats were significantly decreased ( P < 0.05), while WJW significantly increased the CCK and VIP levels. Conclusions WJW can be used to treat IBS by regulating the levels of various brain-gut peptides in the brain and colon tissues of IBS rats. These anomalous and adjustable brain-gut peptides may become a potential biomarker for the diagnosis and treatment of IBS.

Abstract:Objective Previous studies suggested that overerpression of Slit2 results in abnormal Alzheimer’s disease-like behavior and cognition impairment in mice. The aim of this study is to investigate the relationship between overerpression of Slit2 and accumulation and clearance of amyloid-β in aging mice by comparing the differential expression of genes for accumulation and clearance of amyloid-β in aging Tg-Slit2 and Tg-2576 mice. Methods 14-month old male C57BL/6, Tg-Slit2 and Tg-2576 mice were used to detect the expression of Aβ1- 40 and Aβ1- 42 in brain by immunohistochemistry. Further, the total RNA in the brain of these mice were extracted, identified and inversely transcripted to cDNA, then the cDNA was detected by PCR array. The expression of genes in the brain of Tg-Slit2 and Tg- 2576 mice were analyzed. Results Comparing with the Tg-2576 mice in the same age, accumulation of Aβ was not found in the brain of Tg-Slit2 and C57BL/6 mice. The result from PCR array analysis showed that comparing with the same aged C57BL/6 mice, there were 16 up-regulated genes and 8 down-regulated genes in the brain of Tg-Slit2 mice and 14 up- regulated genes and 17 down-regulated genes in the brain of Tg-Slit2 mice. The expression of amyloid beta precursor protein (APP) in the brain of the three group mice was not changed. The expression of presenilin 2 (Psen2) related with Aβ production was significantly up-regulated in the Tg-2576 mice. In addition, the expression of low density lipoprotein receptor-related protein (LRP) 6 and 9 were markedly decreased in the Tg-2576 mice. Notably, these genes were not changed in the brain of the aging Tg-Slit2 mice. Conclusions The accumulation of Aβ in the brain are not found in 14-month Tg-Slit2 mice, In addition, different from Tg-2576 mice, the significant changes of expression of Aβ-related genes is not found in the brain of Tg-Slit2 mice.

Abstract:Objective To investigate the relationship between different brain developmental stages and changes of cognitive function in rats. Methods 1?month, 2?month and 8?month?old rats were selected to imitate the juvenile, adolescent and adulthood, respectively, and their behavioral functions were compared. The reward operant conditioning and Morris water maze task were used to investigate the differences in exploration interest, executive and recognition ability, spatial learning and memory of the rats at different ages. Results In the reward operant conditioning and Morris water maze task, there was no significant difference in the cognitive function between 1?month and 2?month?old rats. In the reward conditioning phase, the nose pokes numbers of 8?month?old rats were significantly decreased compared with the 1?month?old rats ( P < 0.01). There was no significant difference in nose pokes accuracy. During the operant conditioning phase, the lever press numbers and accuracy of 8?month?old rats were significantly decreased ( P < 0.05 or P < 0.01) and the press latency was longer ( P < 0.05). At the phase of visual identification, the press and reward numbers, and the visual identification index were significantly decreased ( P < 0.05 or P < 0.01). In the Morris water maze test, compared with the 1?month?old rats, the total swimming distance and escape latency of the 8?month?old rats were significantly increased ( P < 0.05), as well as average swimming speed ( P < 0.05 or P < 0.01) in spatial learning phage. In spatial memory phage, the swimming distance and time spent in the target quadrant were obviously decreased ( P < 0.01). Conclusions The cognitive functions of rats at different brain developmental stages are different. The juvenile and adolescent rats have similar cognitive functions, but 8?month?old adult rats appear decline in the exploration interest, executive and recognition ability, and spatial learning and memory function.

Abstract:Objective To investigate the role of vitamin D in the synthesis and degradation of aggrecan in rat articular chondrocytes at cellular level. Methods Rat articular chondrocytes were stimulated by IL-1α, IL-1β and TNF-α, respectively. Normal and inflammatory chondrocytes were treated with different doses of vitamin D, respectively. CCK8, Flow cytometry, real time-PCR and western blot analysis were used to examine the proliferation activity and apoptosis level of chondrocytes, and the expression of aggrecan, ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels. Results IL-1α,IL-1β and TNF-α significantly decreased the proliferation activity and increased the apoptosis level of the chondrocytes. Furthermore, IL-1α, IL-1β and TNF-α significantly decreased the expression of aggrecan, and increased the expressions of ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels in the chondrocytes. 1α,25(OH) ₂ D ₃ supplementation significantly increased the proliferation activity and decreased the apoptosis level of chondrocytes stimulated by IL-1α, IL-1β and TNF-α in a dose-dependent manner, but not affected the normal chondrocytes. Meanwhile, 1α,25(OH) ₂ D ₃ also significantly increased the expression of aggrecan, and decreased the expressions of ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels in the chondrocytes under inflammatory conditions. Conclusions Vitamin D may promote the anabolism of aggrecan and inhibit aggrecanase activity in chondrocytes under inflammatory conditions, which may impact overall protection for articular cartilage.

Abstract:Objective To establish and evaluate a patient?derived orthotopic xenograft ( PDOX) model of pancreatic cancer. Methods Tissues of patient?derived pancreatic tumor were transplanted into nude mouse pancreas by surgery. The PDOX models were evaluated by the small animal near infrared fluorescence (NIRF) optical imaging and PET/ CT. The traceability of PDOX models was detected by STR technology, and the pathological changes were observed by H&E staining, immunohistochemistry, and serum level of CA19?9 was detected by ELISA. Results Apparent NIRF were observed to be accumulated in pancreatic site by optical imaging system. The location and size of the xenografts tumor were revealed by fluorescence intensity. The PET/ CT images with 18 F?FDG molecular probe confirmed the tumor’s location and size. Ex vivo NIRF imaging of isolated organ further showed the tumor formation. The traceability of PDOX models was 99.99% with human origin. H&E staining pathology and immunohistochemistry indicated the pancreatic cancer characteristics. The high serum level of ca19?9 confirmed the mice bearing tumor. Conclusions Pancreatic PDOX modelsare successfully established in this study, and it can be evaluated comprehensively by NIRF optical imaging and PET/ CT, providing an appropriate platform for further research of pancreatic cancer.

Abstract:Objective The basic biological, echocardiography and gene sequencing parameters of mice overexpressing Slit2 gene (Slit2-Tg mice) were collected and evaluated, and to provide a reference for the application of Slit2-Tg mice in biomedical research. Methods Slit2-Tg and C57BL/6 J mice were inbred. The genotypes of the mice were determined by a PCR assay. The blood samples were collected for blood routine and biochemical tests. The tissues of main organs were collected for protein expression and pathological analysis. Echocardiography and transcriptome sequencing was carried out for analyzing the heart function and gene expression, respectively. Results The litter size was significantly higher in the Slit2-Tg mice than in C57BL/6 J mice. Human Slit2 gene and protein expressions were detected in the main organs of Slit2-Tg mice. Organ coefficient of spleen was significantly increased in Slit2-Tg mice, but the tissue structure appeared normal. There were significant changes in the counts of erythrocytes, platelets, eosinophils, and biochemistry of glucose, globulin, urea nitrogen, triglycerides, HDL, and atherosclerosis index. Echocardiography showed no significant differences in the morphology and function of the Slit2-Tg hearts except in the left ventricular anterior wall thickness at the end-diastolic state. Compared with the C57BL/6 J mice, 535 genes out of 17513 genes in the Slit2-Tg hearts were increased or decreased, mainly involving 15 biological process or signal transduction pathways. Conclusions This study has collected the biological parameters of Slit2-Tg mice and suggests that this model animal is suitable for the studies of cardiovascular diseases.

Abstract:Objective To improve the preparation method of rat model of acute myocardial ischemia reperfusion injury(MI/ RI), to make a comprehensive evaluation and to lay a good model foundation for following MI/ RI research. Methods Thirty?six clean grade Wistar rats were randomly divided into 3 groups: normal group without intervention of the left anterior descending coronary artery (LAD), sham operation group with only wearing thread but no ligation of the coronary artery, and ischemia reperfusion group, with ligation of the LAD for 30 min and reperfusion for 120 min. Rats after anesthesia had continuous ECG recording, and tracheostomy for mechanical auxiliary ventilation. Thoracotomy and LAD intervention were carried out on the rats, and then the rat models were comprehensively evaluated by electrocardiography, detection of myocardial enzyme content, determination of the infarct size, and pathological examination of the myocardium. Results The electrocardiogram of the MI/ RI group showed obvious ST?T dynamic changes. The level of CK?MB in the MI/ RI group was significantly increased at 2 h after surgery. Compared with the sham group, the infarct size of the MI/ RI group was significantly increased, with evident degeneration and necrosis of cardiomyocytes and extensive inflammatory cell infiltration in the myocardium. Conclusions The improved modeling method not only reduced the difficulties of operation, but has also successfully established the rat model of MI/ RI, providing a good foundation for further MI/ RI research.

Abstract:Objective To investigate the inhibitory effect of alantolactone on glioma C6 cells and its possible mechanism. Methods Rat glioma cell line C6 cells were cultured in vitro. The effect of AL on the viability of C6 cells was detected by MTT assay. The effect of AL on migration and invasion of C6 cells was assessed by scratch test and Transwell chamber assay. Flow cytometry was used to detect the effect of AL on the induction of apoptosis in C6 cells, and the effects of AL on mitochondrial membrane potential in C6 cells was detected using JC -1 fluorescent probe. Western blot was used to detect the expression of related proteins in C6 cells after AL treatment. Results AL significantly inhibited the proliferation of C6 cells in a time? and dose?dependent manner. After AL treatment for 24 hours, the migration and invasion of C6 cells were significantly inhibited, N?cadherin protein was significantly decreased and E?cadherin protein was significantly increased, the number of apoptotic cells increased obviously while the mitochondrial membrane potential decreased significantly. The protein expressions of PARP/ cleaved caspase - 3/ cleaved caspase - 9 were significantly up?regulated. Conclusions AL inhibits the proliferation of glioma C6 cells by inhibiting their migration and invasion through regulating the protein expression of cadherin and inducing apoptosis in C6 cells by regulating the cytochrome C/ caspase signaling pathway.

Abstract:Objective To explore the characteristics of immunological changes in tree shrews infected with orthoreovirus, and provide a theoretical basis for the prevention of virus in tree shrews. Methods 40 - 50?day?old tree shrews were divided into three groups: MRV1/ TS/2011 virus?infected and MRV3/ TS/2013 virus?infected groups, and saline?treated control group. On the 1, 8, 14, 21, and 28 days after infection, blood samples were taken from the tail vein and used for RT?PCR, flow cytometry and ELISA detection, to assess the viral load, number of CD4/ CD8/ CD19 cells, and IFN?gamma expression. Results The MRV1/ TS/2011 and MRV3/ TS/2013 viral load in the plasma and the number of CD4⁺ and CD19 ⁺ cells reached a peak at the 14th day after infection. At the first day after MRV1/ TS/2011 infection, the CD4 ⁺ cells had a significantly higher expression compared with the normal group. CD8 ⁺ cells and the IFN?gamma expression reached a peak at the 21st day after infection. The expression of CD4 ⁺ was even higher after MRV1/ TS/2011 infection, and the expression of CD8 ⁺ cells was higher after MRV3/ TS/2013 infection. Conclusions We would conclude that after MRV1/ TS/2011 and MRV3/ TS/2011 virus infection, accompanying the changes of viral load, it shows some regularity of the expression of CD4/ CD8/ CD19 and IFN?gamma in the tree shrews: at the early stage of MRV1/ TS/2011 virus infection, humoral immunity is stimulated, and CD4 ⁺ cells play a major role. MRV3/ TS/2013 virus may mainly affect the cellular immunity, while humoral immunity only plays a role at a high viral expression or the late stage of infection. CD4 ⁺ cells may be more sensitive to type 1 reovirus, and CD19 ⁺ cells may be more sensitive to type 3 reovirus.

Abstract:Objective To evaluate the immunogenicity and safety of inactivated poliovirus vaccine derived from Sabin strain (sIPV) in Banna mini pigs, and to provide experimental evidence for the new animal model. Methods sIPV vaccines which are listed at Institute of Medical Biology at Chinese Academy of Medical Sciences were used in this study. The groups of intramuscular sIPV and the wild strain IPV injections (IPV derived from wild strain, wIPV) were designed, and the saline group was used as a negative control group. The Banna mini pigs in various groups were immunized at 0, 1 and 2 months. Blood samples were collected before immunization and on days 30 after each immunization. Levels of neutralizing antibodies were tested for evaluating immunogenicity. The safety was evaluated by observation of the status and weight of mini pigs. Results After the three dose immunization schedules in the Banna mini pigs, the seroconversion rates of type I, II and III sIPV experimental groups and wIPV group were all up to 100%. The neutralizing antibody levels in all the three types were much higher than the protective titer 1: 8. The weight of mini pigs increased after vaccination. Conclusions The sIPV vaccine has good immunogenicity and safety in Banna mini pigs. Banna mini pigs could be a new animal model for evaluation of sIPV vaccine.

Abstract:Objective To explore the virulence of enterovirus 71 from infected children in neonatal mice. Methods Three strains of EV71 were isolated from the mild, severe and dead patients. Symptoms, weight and death of mice were recorded throughout 14 days. The mice were sacrificed on the first, third, fifth, seventh and ninth days post infection to gain the tissue virus load including the liver, spleen, lung, intestine, brain and muscle tissue which were used to detect the virus tilter by real?time RT?QPCR, and pathological lesions using HE staining. Results < /b> As to the severity of symptoms, no significant difference was found between the severe and mild groups ( P =0.693), which were more serious than that of the fatal group. ( P = 0.000 < 0.05/6, P = 0.000 < 0.05/6). The survival rate of the mice with mild, severe and fatal virus infection was 77.2%, 81.7% and 97.8%, respectively, and there was a significant difference among the three groups ( P =0.0010 < 0.05, P =0.001 < 0.05, P =0.0004 < 0.05). Lung hemorrhage of the mild group was the most serious, and there were no significant differences in pathological lesions of the brain, muscle, spleen and intestine. Virus titer in the liver and muscle was higher than the other tissues and that in mild group of different tissues tended to be higher than the other two groups. Conclusions Neonatal mice infected with the mild strain of enterovirus 71 presents heaviest symptoms, which are not consistent with the outcomes of humans. It is considered to be related to the virus gene, host and other factors.