Objective To develop a duplex fluorescence RT-PCR assay for detection of scavenger receptor class B, typeⅠ(SRBⅠ) knockout mice. Methods Primers and probes were designed according to knockout region of SRBⅠgene and related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using real-time PCR. SRBⅠgenotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, the sensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygous SRBⅠwild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠknockout genotype was present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typical S amplification curves in both FAM and CY5 channels, respectively. The results showed that the sensitivity reached 4×101 copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The new method is simple, rapid and accurate, which is suitable for genotyping SRBⅠknockout mice.

OBJECTIVETo investigate the effects of hydrogen sulfide preconditioning on myocardial ischemia reperfusion injury in rats.

METHODSSprague-Dawley male rats were divided into 4 groups with 10 in each group: in S group rats received sham operation; in IR group rats were given with NS (1.0 ml/kg iv) 24 h before ischemia; in H group rats were treated with NaHS (0.05 mg/kg iv) 24 h before ischemia; and in D group, NaHS-treated rats received 5-hydroxydecanoate (5-HD) 15 min before ischemia. Rats in IR group,H group and D group were subjected to ischemia by occlusion of coronary artery for 30 min followed by 2 h of reperfusion. At the end of the reperfusion,myocardial infarct size was measured. SAM-s was measured by Western blotting. Plasma SOD activity and MDA were determined at the end of reperfusion.

RESULTSThe infarct size was significantly lesser in H group (25.40 % ± 3.54%) than that in IR group (38.27% ±5.64%,P<0.05). The SAM-s protein expression in myocardium was significantly lower in H group than that in IR group. The plasma MDA content was significantly lower and SOD activity was higher in H group than those in IR group,but there was no difference between IR group and D group.

CONCLUSIONThe hydrogen sulfide preconditioning attenuates myocardial IR injury possibly through down-regulating SAM-s expression,reducing the production of oxygen free radicals and enhancing anti-oxidize effect in rats.

Animals ; Disease Models, Animal ; Hydrogen Sulfide ; pharmacology ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Reperfusion Injury ; metabolism ; pathology ; prevention & control ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

Malignant tumors usually have no obvious clinical symptoms in the early stage. Most patients are already in the advanced stage when they are diagnosed. Some patients have lost the opportunity for operation, resulting in poor prognosis. Therefore, how to find the best therapeutic target for such patients and improve the prognosis of patients has gradually become the focus of scholar′s attention. Recently, Kruppel-like factor (KLF) is a transcriptional regulator that can bind to the target DNA, and its family plays an important role in the occurrence and development of malignant tumors. It has also been confirmed that the KLF family affects the proliferation, differentiation and migration of tumor cells, but the specific mechanism is still not fully elucidate. Consequently, in order to further explored the effect of the KLF family on tumors, this study intends to briefly review the roles and regulatory mechanisms of the KLF family in the cell proliferation, differentiation and migration of malignant tumors, hoping to provide new target for the biological treatment of tumors.

Objective To observe the effect of Sinisan on the learning and memorial ability and the expression of synaptohydin in the hippocampus of exercise-fatigue rats. Methods Rats were made into the fatigue model by Exhaustive Swimming. Learning and memorial ability of the rats were observed by moving-back with current stimulation experiment; expression of synaptohydin protein in hippocampus of exercise-fatigue rats treated with Sinisan for 10 days were detected by immunohistochemistry technique and compared to control group and model group.Results The moving-back time and the error times of the exercise-fatigue rats as follows: model group(73.00±61.96) s/(1.67±1.15), control group(144.25±57.14) s/(0.50±0.80)，Sinisan group (166.17±47.92) s/(0.38±0.49). Comparison of mean optical density of synaptohydin protein in hippocampus of rats in different groups as follows: control group (0.2636±0.10654) and Sinisan group (0.2555±0.163380) were higher than the model group (0.0474±0.1837)(P<0.05). The synaptohydin staining Results showed that synaptohydin-positive granules around the neuron in hippocampus of rats in control group were the darkest and densest, the Sinisan group were darker than the model group rats.Conclusion Sinisan could improve the learning and memorial ability and increase the synaptohydin protein in the hippocampus of the exercise-fatigue rats.

OBJECTIVETo investigate the effect of hydrogen sulfide-induced delayed preconditioning on glutathione S-transferase (GST) expression during myocardial ischemia-reperfusion in rats.

METHODSSprague-Dawley male rats were randomly divided into 4 groups (n= 10 in each): Group S (sham operation group), Group IR (ischemia/reperfusion group), Group H (IR+ NaHS 0.05 mg/kg iv, 24 h before ischemia) and Groups D receiving IR+NaHS 24 h before ischemia and 5-hydroxydecanoate (5-HD)15 min before ischemia. Animals in groups IR, H and D were subjected to ischemia by 30 min of coronary artery occlusion followed by 2 h of reperfusion. At the end of the reperfusion, myocardial infarct size (IS) was examined. Glutathione S-transferase (GST) was measured by Western blotting. The myocardial ultrastructures were observed under the electron microscopy.

RESULTSThe IS was significantly smaller in Group H than that in Group IR [(25.40 ± 3.54)% compared with (38.27 ± 5.64)%, P<0.05]. The GST expression in myocardium was significantly higher in Group H than that in Group IR. Microscopic examination showed less myocardial damage in Group H than in Group IR. The protective effects of delayed preconditioning by hydrogen sulfide was prevented by 5-HD pre-treatment.

CONCLUSIONThe hydrogen sulfide-induced delayed preconditioning attenuates myocardial IR injury possibly through up-regulating glutathione S-transferase expression in rats.

Animals ; Disease Models, Animal ; Glutathione Transferase ; metabolism ; Hydrogen Sulfide ; administration & dosage ; therapeutic use ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Reperfusion Injury ; enzymology ; pathology ; therapy ; Myocardium ; enzymology ; ultrastructure ; Rats ; Rats, Sprague-Dawley

Voltage-dependent potassium channels (Kv) are involved in proliferation and transformation in mammary epithelial cells. In previous studies, several groups have detected various potassium channels in breast cancer cells, and they assumed that potassium channels are related to the development of breast carcinoma, although the precise mechanisms are still unknown. We have previously reported that 4-aminopyridine (4-AP), one kind of potassium channel (K(+) channel) blocker, could affect the proliferation of MCF10A cells. The aim of the present study is to explore the expression and properties of K(+) channels in human mammary epithelial cells (MCF10A) and whether Kv channels are required for the proliferation of MCF10A cell. Electrophysiological, MTT analysis, PCR and Western blot methods were used to identify a K(+) conductance which is involved in tumorigenesis and not yet be described in MCF10A cells. A voltage-dependent, outward rectification and 4-AP-sensitive K(+) current was observed in these cells. The perfusion of 5 mmol/L 4-AP significantly decreased the amplitude of Kv current from (912.5+/-0.6) pA to (275+/-0.8) pA (n=5, P<0.01), when cells were recorded using 800 ms voltage steps from a holding potential of -60 mV to voltage ranging from -60 mV to +60 mV. PCR analysis demonstrated that Kv1.1, Kv1.2, Kv1.3, and Kv1.5 were all expressed in MCF10A and MCF7 cells. Furthermore, the expression of Kv1.5 was much higher in MCF10A than that in MCF7. Inhibitory effect of 4-AP on cell proliferation was dosage-dependent. Incubation with 5 mmol/L 4-AP reduced MCF10A cell proliferation to 25.29% in 48 h. Western blot analysis showed the activation of ERK1/2 which related to cell proliferation was enhanced, while p38 activation was decreased by 4-AP treatment for 10 min. These data provided the first evidence of the Kv channels expression in MCF10A cell and 4-AP could inhibit the proliferation of MCF10A through blocking the potassium channels, and the mechanism may be related to regulating the activity of different members of cell proliferation signaling pathway of MEK/ERK.
4-Aminopyridine ; pharmacology ; Cell Line ; Cell Proliferation ; Epithelial Cells ; physiology ; Humans ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Voltage-Gated ; physiology

Objective To improve the chemotherapy drug delivery to tumor by enhancing the tumor vascular perfusion induced by diagnostic ultrasound combined with microbubbles.Methods Ten healthy male sprague-dawley(SD)rats with total twenty walker-256 tumors implanted in the two back legs were randomized to the two paired groups: controlled group(C,n=10)and treatment group(T,n=10).Tumors in the controlled group were ultrasonic sham operated,while in the treatment group were treated by diagnostic ultrasound combined with microbubbles.The treatment group were taken contrast-enhanced ultrasound(CEUS)before and after treatment and analyzed the quantitative parameters.The microbubbles used in the treatment and CEUS was a kind of self-made lipid microbubbles called Zhifuxian.The 0.02 ml microbubbles were bolus injected at CEUS,while during treatment,0.04 ml microbubbles diluted into 1 ml saline solution were injected slowly at constant speed.Flushed by saline solution after treatment,the rats' tumors were harvested into three parts: one for chemotherapy drug concentration detected by high performance liquid chromatography(HPLC),one for HE detection,and one for Dox fluorescence intensity detected by confocal laser scanning microscopy(CLSM).The peak intensity(PI)values,the area under curve(AUC)values and the Dox concentration of each group were analyzed by pared-samples t test.Results(1)The contrast enhanced ultrasound quantitative analysis of the T group: PI value of the tumors before and after treatment were 66.22±16.25 and 75.74±17.67.The AUC values were 2937.52±677.51 and 3354.91±796.15.There was significant statistical difference between them(t=-5.212,-5.259,all P ＜ 0.05).(2)The Dox concentration of the T and C groups were(1.15±0.25)ug/g and(0.96±0.21)ug/g.There was significant statistical difference between them(t=2.403,P＜0.05).The Dox concentration of the treatment group was 1.2 times of the controlled group.(3)The pathology results of T and C groups: the tumor cells were arranged in cords,with big round deep-stained nucleus.No pathological changes were observed in the controlled group,and there was no significant difference between the two groups.But in the treatment group,tumor vascular congestion and inflammatory cell infiltration could be observed.(4)The confocal laser scanning microscopy(CLSM)detection of the T and C groups: the Dox red fluorescence was distributed in the tumor tissue interstitial,and the fluorescence intensity and distribution area of the treatment group were significant higher than the controlled group.Conclusions Diagnostic ultrasound combined with microbubbles treatment could significantly increase the blood perfusion in the walker-256 tumors of SD rats.Taking advantage of this vascular effect,the chemotherapy drug Dox could be delivered much more to the tumor tissue along with circulating bloodstream.With the addition of the sonoporation effect induced by the cavitation of the microbubbles,the chemotherapy drugs could be released much more to the tumor interstitial tissue.

Objective: To study the relationship between nutrition status and physical fitness in 8-10 year-old children in 3 cities, and to provide a scientific reference for improving physical condition of chidren of pre-school age. Methods: To investigate the weight, height and physical fitness (standing long jump, endurance running, rope skipping in one minute and sit-ups in one minute) of 1 384 children in grade four from 24 primary schools in Beijing, Changzhi, Urumchi in 2018. Evaluated overweight and obesity by using the standard of “Screening for Overweight and Obesity among School-age Children”. Then used mixed linear model to compare the physical fitness of students with different nutritional status and to analyze the relationship between BMI and physical fitness. Results: The prevalence of overweight and obesity were 17.41% and 23.48%, respectively. And the prevalence in boys was both more than that in girls(χ2=9.84，47.68，P<0.01). The increase in BMI of children from the same age and the same gender was related with the decrease in physical fitness by correlation analysis(P<0.05). In comparison method, the performance of the students of normal weight was better than obese students, but the male and female students had got the same results in their physical fitness test(P<0.05). Conclusion There is a negative correlation between nutrition status and physical fitness and the physical fitness in normal weight and obese children are better than the overweight children or obese children.

Objective: To evaluate the effect of a dietary and exercise intervention on cognition and behaviors among primary school students, and to further explore the mediating role of family support, in order to provide scientific evidence for future effective intervention strategies. Methods: A dietary and exercise intervention program for childhood obesity prevention was carried out in 24 primary schools in Beijing, Changzhi and Urumqi from 2018 to 2019, and 1 392 children in grade four as well as their parents were included. Family support and children s cognition and behaviors were collected through questionnaire. To carry out diet and exercise behavior intervention on the three levels of school, family and individual in the intervention group,and children s dietary and exercise knowledge, eating habits, physical activities, as well as sedentary and screen behaviors were evaluated. The linear mixed model was used to analyze the effect of intervention on children s cognition and behaviors, and the mediation model was used to explore the role of family support. Results: The proportion of children with higher score of dietary and exercise knowledge ( OR= 2.34 , 95%CI =1.71-3.21), eating habits ( OR=2.58, 95%CI =1.75-3.82), and sedentary and screen behaviors ( OR=1.91, 95%CI =1.35-2.68) increased in the intervention group after one year intervention ( P <0.01), compared with the control group, respectively. The intervention also increased the proportion of children s family support in the intervention group compared with the control group ( OR=3.45, 95%CI =2.19-5.45), and the support from children s fathers ( OR=2.70, 95%CI =1.68-4.35), mothers ( OR=3.71, 95%CI =2.28-6.04), paternal grandmothers ( OR=1.65, 95%CI =1.00-2.70), and maternal grandmothers ( OR= 2.14, 95%CI =2.12-2.16) increased significantly ( P <0.05). The mediation analysis showed that family support played a mediating role in association between comprehensive intervention and children s eating habits as well as sedentary and screen behaviors. Conclusion The dietary and exercise intervention effectively promoted children s cognition and behaviors, and family support played an important mediating role.

Using the concepts and methods of epigenetics and metabolomics, to investigate the overall action molecular mechanism of Chrysanthemi indici C (CIC), the anti-hepatitis B virus (HBV) active extracts from Flos chrysanthemi indici. The inhibitory effects of CIC on proliferation and hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg) and HBV-DNA of HepG2.2.15 cells were detected by CCK-8 and antigen kit. The DNA methyltransferases (DNMTs)/ten-eleven-translocation-2 (TET2) equilibrium was detected by ELISA. Illumina 850K methylation chip, pyrosequencing and qPCR were used to determine the action pathway and target of CIC by GO and KEGG analysis. Cell metabolites were extracted with 80% methanol, and the changes of differential metabolites, differential metabolic pathways and cell microenvironment were detected by LC-MS and other metabolomics methods. The results showed that CIC could inhibit the proliferation, HBsAg, HBeAg and HBV-DNA of HepG2.2.15 cells obviously, down-regulate DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a) and DNA methyltransferase 3b (DNMT3b), up-regulate TET2, and restore the balance of DNMTs/TET2. The action targets of CIC were phospholipase C gamma 2 (PLCG2), phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), 5-hydroxytryptamine receptor 2B (HTR2B), nerve growth factor (NGF), mainly involved in lipid metabolism, inflammation mediated regulation of transient receptor potential (TRP), phospholipase D signaling and advanced glycation end product-receptor for AGE (AGE-RAGE) signaling in diabetic complications pathways. CIC could significantly affect fatty acid metabolism and had great influence on phenolic acid, alkaloid and lipid metabolites in cell microenvironment. These results suggest that the action mechanism of CIC may be the synergistic action of multiple pathways and multiple targets, including related inflammatory pathways, immune pathways and lipid metabolism, through regulating epigenetic expression balance and restoring the balance of cell microenvironment.