Abstract:Objective To study the expression of Growth Hormone (GH) gene in different tissues and different growth stages of Tibet-minipigs. Methods The expression level of GH gene in heart, liver, spleen, lung, kidney, muscle, skin tissue of Tibet mini-pig was analyzed by Real-time PCR in 0 years old (1 days), 0.1 years old (36 days), 0.25 years old (90 days), 0.5 years old (180 days), 1 years old (360 days), 2 years old (720 days), and 3 years old (1080 days). Results The expressions of GH mRNA could be detected in various tissues (heart, liver, spleen, lung, kidney, muscle, skin) in different growth stages (0, 0.1, 0.25, 0.5, 1, 2, 3 years old) in Tibet mini-pigs. The GH gene was most highly expressed in skin in 0, 0.5 years old and most lowly expressed in muscle comparing with other tissues. Moreover, the GH gene was also most highly expressed in lung and liver in 0.25, 1 years old and 0.1, 3 years old, respectively. The expression level of GH gene reached the peak in 0.1 years old among different growth stages. Conclusion The expression of GH gene in Tibet mini-pig showed an obvious temporal and spatial specificity.

Abstract:Objective To analyze the survival curves of presenilin-1/presenilin-2 conditional double knockout mice (dKO mice), and provide a reference for further breeding of this strain of mice and studying the pathogenesis of Alzheimer's disease (AD). Methods dKO mice (50 females and 30 males) and wild-type controls (20 females and 20 males) were included in this study. The survival of all the mice was observed under the same condition of feeding and nutrition for two years. Life span of each mouse was calculated, survival curves were drawn, and their survival was analyzed. Results With the passing of time, the survival probability was decreased in dKO mice. The survival probabilities of female and male dKO mice were significantly worse than that of wild-type controls, respectively (P<0.001, P<0.001; P<0.001, P<0.001). No significant difference was observed between the males and females of dKO mice. Conclusions Double knockout presenilin-1 and presenilin-2 genes may shorten the life span of mice, but there is no significant gender difference between the males and females of dKO mice.

Abstract:Objective To observe the influence of Bcl-2 inhibitor (TW-37) on the alleviation action of astragalus injection (AI) on apoptosis of hippocamal neurons of rats induced by hypoxia/hypoglycemia and reoxygenation (H/R). Methods The hippocampal neurons were isolated from fetal rat and cultured for eight days. The cells were randomly divided into six groups:normal control group, H/R group, AI group, AI solution group (sterile deionized water), TW-37 group, TW-37 with AI group. All groups were tested after 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h since they were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 min except normal control group. The activity of hippocampal neurons were detected with MTT,The expression level of Caspase-3 was measured by western blotting, the expression of mRNA of Caspase-3 was tested by RT-PCR. Results Compared with normal control group, aside from 0 h group, the activity of neurons decreased obviously (P<0.05), moreover, the protein and mRNA expressions of Caspase-3 were enhanced significantly (P<0.05) in H/R groups. Compared with H/R group, the activity of neurons increased obviously in astragalus group (P<0.05), and the expressions of Caspase-3 protein and the expression of Caspase-3 mRNA in the AI groups decreased significantly (P<0.05) besides 0 h group; but there was no significant difference in both AI solution group、TW-37+H/R and TW-37+AI+H/R group (P>0.05). Conclusions Astragalus injection to increase the neurons activity and decrease the expression level of Caspase-3 in hippocampal neurons of rat by activating the effect of anti-apoptosis of Bcl-2. Bcl-2 is one of the targets exerting the inhibitory action of astragalus injection on apoptosis of hippocamal neurons of rats induced by hypoxia/hypoglycemia and reoxygenation.

Abstract:Objective To investigate the effects of chronic restraint stress on the depressive-like behavior of male and female SD rats with different restraint duration. Methods Sprague-Dawley rats (male and female) were subjected to restraint stress 10 h/14 h daily for 28 days. After that, sucrose preference test, open-field test, novel object test, were performed for all the rats to observe the effects of different restraint duration of chronic restraint stress on the depressive-like behaviors in male and female SD rats. Results For the 14 h/28 d CRS group, including male and female rats, the movement time and central activity time were reduced, sugar consumption index were decreased, novelty exploration time and frequency were reduced, latency were increased, numbers of nose pokes were reduced, and these results were significantly different compared with the control group. There were individual indicators that showed significantly difference for all the above-mentioned behavioral tests of the 10h/28 d CRS group. 14h/28 d CRS male group in syrup consumption index decreased significantly, the corresponding female rats did not significantly reduce. 10h/28 d CRS male group in the central motion time less than that of the control group, and latency was prolonged and exploration time was shortened, while the corresponding female rats had no significant difference. Conclusion 14 h/28 d CRS rats in the sucrose preference test, open-field test, novel object test were exhibited depressive-like behavior, but 10 h/28 d CRS male rats showed a trend of depressive-like behavior. Chronic restraint stress was more likely to lead to depressive-like behavior in male rats.

Abstract:Objective To observe the effects of Salvianolic acid A (SAA) on heart rate variability (HRV) and myocardial injury in myocardial ischemia reperfusion injured (MI/RI) rats. Methods Sixty SD rats were divided into 6 groups randomly (n=10); the sham group, the MI/RI model group, high, middle and low-dose SAA group (2 mg/kg, 1 mg/kg and 0.5 mg/kg) and positive control group (2 mg/kg Diltiazem). The MI/RI model was made by ligating the left anterior descending branch of coronary artery in rats. Before ligation, the rats were intravenously administered with corresponding drugs. The changes of electrocardiogram of J point and HRV parameters were analyzed. At the end of reperfusion, the myocardial infarct size was measured by using Evans blue and tetrazolium chloride (TTC) double staining. The changes of plasma aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and its isoenzyme (CK-MB), superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (no) content were also detected. Results Compared with MI/RI model group, SAA low, middle and high dose group and Diltiazem group were significantly reduced the total or average value of J point during reperfusion(P<0.05,P<0.01),reduced myocardial infarct size and inhibited the rising of AST, CK-MB and CK activities(P<0.05,P<0.01), and increased the standard deviation of RR intervals (SDNN), the root-mean-square of successive differences (RMSSD), HRV triangle index (tr. Ind) and high frequency (HFnu), reduced the low frequency (LFnu) and the ratio of LF/HF(P<0.05, P<0.01). Moreover, SAA could significantly increase the content of NO and reduce the content of MDA (P<0.05). Conclusion SAA could increase HRV and reduce myocardial infarction area in MI/RI rats, its mechanism of action maybe related with antioxidant effects.

Abstract:Obejective To explore protective effect of miR-29c on cerebral ischemia-reperfusion injury in rats. Methods Forty eight SD rats were randomly divided into sham group, model group, miR-29c NC group and miR-29c inhibitor group. Except the sham operation group, the other three groups were established by using ligation of middle cerebral artery. Twenty four hours after the operation, neurological behavior score, cerebral infarction volume, cell apoptosis, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), cysteine aspartic acid protease 3 (caspase 3), caspase 8, caspase 9, and the expression of miR-29c, Bcl-2, Bax, Bak1, cleaved caspase 3, cleaved caspase 8 and cleaved caspase 9. Results Compared with sham group, neurological behavior score, cerebral infarction volume, cell apoptosis rate was increased, SOD th the expression of miR-29c, activity was decreased, the level of MDA, caspase 3, caspase 8 and caspase 9 activity was increased. The expression of Bcl-2 was down-regulated, the expression of Bax, Bak1, cleaved caspase 3, cleaved caspase 8 and cleaved caspase 9 expression was up-regulated in model group, miR-29c NC group. The differences were also statistically significant (P<0.01). Compared with model group, miR-29c NC group, the expression of miR-29c, neurological behavior score, cerebral infarction volume percentage, cell apoptosis rate was decreased, SOD activity was increased, the level of MDA, caspase 3, caspase 8 and caspase 9 activity was decreased. The expression of Bcl-2 was up-regulated, the expression of Bax, Bak1, cleaved caspase 3, cleaved caspase 8 and cleaved caspase 9 expression was down-regulated in miR-29c inhibitor group. The differences were also statistically significant (P<0.01). Conclusion miR-29c had a protective effect on cerebral ischemia-reperfusion injury in rats, which was related to improve the antioxidant capacity and regulation of apoptosis related protein expression.

Abstract:Objective To investigate Interleukin 6(IL-6),superoxide dismutase (SOD),malondialdehyde (MDA) in lung tissues of a rat acute pulmonary edema model induced by acute hypoxia. Methods 50 adult Wistar rats were randomly divided into group A (normal),B (acute pulmonary edema,hypoxia for 24 h),C (acute pulmonary edema,hypoxia for 48h),D(acute pulmonary edema,hypoxia for 72 h) and E (dexamethasone treatment,hypoxia for 72h). The model of acute pulmonary edema was established by intraperitoneal injection with 6% ammonium chloride. In group E, dexamethasone (6mg/kg) was injected in tail vein following intraperitoneal injection of 6% ammonium chloride. The rats were killed 24(group B), 48 (C) and 72 hours(D&E) later. Plasma was isolated and lungs were removed. MDA,SOD in lung tissues and IL-6 in plasma were analyzed by ELISA. Results The wet weight of lung tissues were significantly increased in group B, C and D compared to group A(P<0.05). The lung tissues of group A had no obvious congestion and edema,and the morphology of lung tissues was normal(P>0.05). Pulmonary edema,interstitial spaces and alveolar filled fluid can be seen in group B,C,D and transparent membrane formed within alveoli. The lung tissue congestion and edema in group D were the most obvious(P<0.05). The above changes obviously improved in group E. Compared with group A,MDA,IL-6 increased and SOD decreased in lung tissues of group C and D. Compared with group D, MDA was decreased and SOD was increased in lung tissues of group E and IL-6 was also decreased significantly in plasma(P<0.05). Conclusion The occurrence of acute pulmonary edema related to oxidative stress and decreased antioxidant capacity. Increased free radical is an important mechanism of pulmonary edema. SOD,MDA and IL-6 in the lung tissue may be indexes for the prognosis.

Abstract:Objective To explore experimental study of Jiawei Danshen Yin on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into myocardium-like cells in vitro. Methods Rat BMSCs were isolated by adherent method, and their surface antigen were detected by flow cytometry. The maximal non-toxic concentration of Jiawei Danshen Yin was measured by MTT method. The isolated and purified BMSCs were divided into four groups:normal control group, Jiawei Danshen Yin group, 5-azacitidine group, Jiawei Danshen Yin plus 5-azacitidine group. The expression of desmin, α-myosin heavy chain (α-MHC) and cardiac troponin T (cTnT) protein was detected by western blot. The expression of GATA-4, gap junction connexin 43 (CX43) and Nkx2-5 mRNA was detected by RT-PCR. Results The percentage of CD90 positive cells in the rat BMSCs was higher than 95%, and the proportions of CD34 positive and CD45 positive cells was lower than 5%. The maximum toxic concentration of Jiawei Danshen Yin on BMSCs was 0.625 g/L. Compared with the normal control group, the expression of desmin, α-MHC and cardiac cTnT protein was up-regulated, the expression of GATA-4, CX43 and Nkx2-5 mRNA was up-regulated in Jiawei Danshen Yin group, 5-azacitidine group and Jiawei Danshen Yin plus 5-azacitidine group. The difference was statistically significant (P<0.01). Compared with Danshen Yin group or 5-azacitidine group, the expression of desmin, α-MHC and cardiac cTnT protein was up-regulated, the expression of GATA-4, CX43 and Nkx2-5 mRNA was up-regulated in Jiawei Danshen Yin plus 5-azacitidine group. The difference was statistically significant (P<0.01). Conclusion Jiawei Danshen Yin could induce BMSCs differentiation into myocardium-like cells, which had a synergistic effect with 5-azacitidine.

Abstract:Objective To explore effect of siRNA silencing B-cell specific Moloney murine leukemiavirus integration site (Bmi-1) gene expression on cell proliferation of cervical cancer cells. Methods Hela cells were transferred with control and siRNA Bmi-1. The expression of Bmi-1 in Hela cell was examed by Realtime PCR and western blot. The cell viability was detected by MTT. The cell apoptosis was examed by Annexin V-PI flow cytometry and Hoechst staining. Cell cycle was detected by flow cytometry. The expression of p16, p53, CyclinD1 and Rb was detected by western blot. Results Compared with control group, the expression of Bmi-1 protein and mRNA was down-regulated, the cell viability was decreased, cell apoptotic rate was increased, Cell cycle was arrested in the G1 phase, the expression of p16 was up-regulated, the expresson of p53 and CyclinD1 down-regulated, the phosphorylation of Rb was inhibited in siRNA Bmi-1 group. The difference was statistically significant (P<0.01). Conclusion Bmi-1 inhibited Hela cell proliferation via regulation of expression of cell cycle related protein.

Abstract:Objective To explore inhibitory effect of miR-34a on the proliferation of breast cancer cell MCF-7 by targeting Notch1. Methods MCF-7 cells were transferred with miR-34a mimics and miR-34a NC. The expression of miR-34a in MCF-7 cell was examed by RT-PCR. The cell viability was detected by MTT. The cell apoptosis was examed by Hoechst staining. Cell cycle was detected by flow cytometry. Western blot, RT-PCR and dual luciferase reporter gene assay was to detect whether Notch1 was a downstream target gene of miR-34a. Results Compared with miR-34a NC group, the expression of miR-34a was up-regulated, the cell viability was decreased, cell apoptotic rate was increased, cell cycle was arrested in the G1 phase, the expression of Notch1 protein and mRNA was down-regulated (P<0.01). Conclusion miR-34a mimics inhibited MCF-7 cell proliferation and induced cell apoptosis via targeting regulation expression of Notch1.

Abstract:Objective To explore effect of miR-34a on the biological behavior of breast cancer cell MCF-7 by Notch signal pathway. Methods The MCF-7 cells were divided into miR-34a and Control groups. The miR-34a mimics and miR-34a NC were transfected by liposome. The expression of miR-34a in MCF-7 cell was examed by RT-PCR. The cell viability was detected by MTT. The cell apoptosis was examed by Hoechst staining. The cell invasion was detected by transwell method. The expression of Nothch1, Nothc2, Dll1 and Jagged-1 was detected by western blot. Results Compared with Control group, the expression of miR-34a was up-regulated (P<0.01), Cell shrinkage, rounded brighten,the cell viability was decreased (P<0.01), cell apoptotic rate was increased (P<0.01), cell invasion number was reduced (P<0.01), the expression of Notch1, Nothc2, Dll1 and Jagged-1 was down-regulated (P<0.01). Conclusion miR-34a mimics induced MCF-7 cell apoptosis and inhibited cell invasion via inhibition of Notch signal pathway.

Abstract:Objective To explore the effect of vasoactive intestinal peptide (VIP) on the regulation of CD4⁺CD25⁺Treg/Th17 balance in the prevention and treatment of experimental autoimmune cerebrospinal meningitis (EAE). Methods 60 healthy female Wistar rats were randomly divided into normal control group, EAE control group, VIP low-dose group and VIP high-dose group. Used of myelin basic protein (MBP)+completely adjuvant (CFA) to establish EAE model. Since building on, the low and high-dose VIP groups were with intraperitoneal injection of every other day respectively VIP 4 nmol/kg (0.2 mL), 16 nmol/kg (0.8 mL), normal control group and EAE group with 0.8 mL saline, 10 days in a row. Record the incubation period, progression and the peak of neurological dysfunction score (NDS) changes of rats; the pathological changes in brain at the morbidity peak of rats; the proportion of CD4⁺CD25⁺Treg/Th17 cells in spleen tissues with Flow cytometry; the cytokine levels of TGF-β1、IL-17A in brain tissue. Results the incubation period were extended, the progression and the peak nerve dysfunction score were shortened in VIP each dose group; In normal control group, there was no inflammatory cell infiltration in the brain tissue. The degree of infiltration of inflammatory cells in the VIP control groups were significantly lower than that in the EAE group;Compared with the EAE control group, the proportion of CD4⁺CD25⁺Treg cells in the spleen tissue of VIP each dose group increased, the proportion of Th17 cells decreased, and being a dose-response relationship;The cytokine levels of TGF-β1 in brain tissue were increased and the levels of IL-17A were reduced in VIP each dose group, being a dose-response relationship; Conclusion through up-regulating of CD4⁺CD25⁺Treg cells, promoting the secretion of TGF-β1 factor and cut ting the proportion of Th17 cells, inhibiting the expression of cytokines IL-17A, VIP reduced brain tissue inflammatory cell infiltration, and played a role in prevention and treatment of EAE.

Abstract:Objective To observe the changes of microRNA-97a and transforming growth factor β1 (TGF-β1) protein in the myocardium of spontaneously hypertensive rats (SHR). Methods 8 male spontaneously hypertensive(SHR)rats as SHR group, 8 male Wistar rats for control group, Arterial blood pressure was detected by a noninvasive blood pressure measurement and analysis system. 8 weeks after femoral artery,HE staining was used to observe the changes of morphology of the rat heart, the expression of miRNA-97a was checked by Real-time PCR method for detection of rat heart. Western blot was used to detect the TGF-β1, angiotensin II (Ang II) protein and type I collagen (Col I) and type III collagen (Col III) protein expression level. Results In SHR group, systolic blood pressure and diastolic blood pressure increased significantly, TGF-β1 protein and Col I and Col III protein expression levels increased significantly, miRNA-97a expression levels were significantly lower than that of control group (P<0.01). Real-time PCR results showed that the level of miRNA-97a expression in SHR group was (24.6+4.7)% in control group, the expression level of miRNA-97a in SHR group was negatively correlated with the expression of TGF-β1 (r=0.785, P<0.01) in group. Conclusion The level of miRNA-97a is down-regulated along with the up-regulation of TGF-β1 protein expression and collagen synthesis in the myocardial tissues of SHR. miRNA-97a and TGF-β1 may be involved in myocardial fibrosis in SHR.

Abstract:Objective To improve fixation methods and tail vein puncture efficiency in conscious rats group. Methods A new four combined holder was designed and made of materials of polystyrene and Polyvinyl chloride.SD rats were randomly divided into A group and B group,twenty rats in each group.Rats in group A fixed by a self-made four combined holder,and rats in group B fixed by common fixture made of organic glass which was commercially available.Then the tail vein injection experiment was conducted respectively by one people. Results It took (9.25±3.70) seconds in group A and(9.95±4.38)seconds in group B to finish the experiment from puncture to see blood return in syringe,and took(47.05±4.65)seconds in group A and(122.45±7.71)seconds in group B to finish the experiment from capture to finish the injection. Futhermore,it was observed(1.4±0.8)times in group A and(2.5±1.2)times in group B with agitation. Conclusions The new four combined rat holder has simple structure, being easy to use, which could significantly shorten the tail vein puncture time and improve the efficiency of the cooperation degree of rats and puncture operation.

Abstract:Objective To observe the effects of different sample process modes on blood biochemical indicator detection in Wistar rats. Methods 40 Wistar rats of specific pathogen free(SPF)level, weighing 180~220 g, were took with half males and half females. After 12 h fasting, rats were anaesthetized by 3% pentobarbital sodium, blood was collected and divided into two samples. One of the samples was for whole blood and another for anticoagulation, with 3mL per serving. Each blood sample was put into three 1.5 mL centrifuge tubes and three heparin anticoagulative tubes with 1mL per, and centrifuged after putting 1 h, 2 h and 4 h in room temperature for testing blood glucose, blood lipid, protein, myocardial enzymes, indexes of liver and renal function. Results The types of blood samples and the standing time after collection and before centrifugation had no significant effect on TC, TG, ALB, ALT, ALP, BUN and CREA in serum or in plasma of Wistar rats(P>0.05).But the value of GLU in serum was significantly higher than that in plasma (P<0.05, P<0.01). With the extension of storage time after collection and before centrifugation, the value of GLU continued to decline. The values of AST, CK, LDH in serum were significantly higher than that in plasma (P<0.05, P<0.01). With the extension of storage time after collection and before centrifugation, the value of AST, CK, LDH continued to increase. However, there was no significant effect on the values of AST, CK, LDH by placing different time after collection and before centrifugation(P>0.05). Conclusion Types of blood samples had no significant effect on blood lipid, protein and indexes of renal function. However, detection value of GLU, AST, CK and LDH would be influenced on a certain degree. The extension of standing time after collection and before centrifugation resulted in plasma GLU decreasing and plasma AST, CK and LDH increasing, but has no obvious effect on the serum AST, CK and LDH.

Abstract:With age, cardiac performance declines progressively and the risk of heart disease, a primary cause of mortality, rises dramatically. As the elderly population continues to increase, it is critical to gain a better understanding of the genetic influences and modulatory factors that impact cardiac aging. As for the human heart, Drosophila heart function also deteriorates with age. Here,In order to better utilize the Drosophila heart model for genetic mechanism studies of cardiac aging, and supply more reliable evidences for molecular-guided diagnostics and therapeutics of age-related heart disease, we sum up the characteristics of cardiac aging of Drosophila in comparative medicine and review genetic factors contributing to cardiac aging, including genes involved in ion exchange, nutrient sensing, and contractile function. These achievements will provide a theoretical basis for molecular guided diagnostics and targeted drug therapied for cardiac aging related diseases, and promote the rapid development of precision medicine of cardiovascular disease.

Abstract:Stress response is the process that our body produces a series of nonspecific physiological and psychological response when dealing with pressureand dangers. The hypothalamus-pituitary-adrenal (HPA) axis, as the central nervous system, plays a critical functional role in regulating stress response.Glucocorticoid receptor (GR) is an important regulatory factor for HPA axis's homeostasis, and its function needs to combine with heat shock protein(Hsp90/70) and co-chaperone proteins (FKBP51or FKBP52). FKBP51,a FK506-binding protein, is a negative regulatory of GR and HPA axis function. In recent years, Fkbp51 has been identified as a significant regulatory in the stress-related psychiatric and metabolic disorders. Here we will review the recent studies of Fkbp51 on stress-related psychiatric and metabolic disorders.