Objective To establish a rat model of persistent intestinal mucosal injury.Methods The rat model of persistent intestinal mucosal injury was induced by intraperitoneal injection of methotrexate.The food intake and body weight of all the rats were recorded.Pathological changes were observed using HE staining, the level of D-lactate and diam-ine oxidase in plasma, and myeloperoxidase and malondiadehyde in the intestinal tissue were measured by biochemistry. Results After modeling, the rat body weight and food intake were decreased.On day 4, the scores of mucosal damage, the levels of plasma D-lactate, DAO, MPO and MDA were significantly increased (P<0.05).On day 5, the intestinal damages of rats began to restore, and there was no significant difference among groups on day 6.The symptoms after the secondary injection were similar to those after the first injection, and the rats recovered gradually at day 12.Conclusions Intestinal mucosal injury in rats induced by 20 mg/kg MTX is an acute injury process, the course only lasts for 4-5 days. Intermittent injections twice of 10 mg/kg MTX can cause persistent intestinal mucosal injury in rats.This persistent injury model is more suitable for nutritional therapy evaluation in medium-and long-term studies of nutritional therapy.

Objective To evaluate the effects of peptide-based enteral nutrition (PBEN) on inflammatory response and immune function in rats with intestinal mucositis.Methods 48 Sprague-Dawley male rats were randomly divided into 6 groups (all n =8):basal feed group (BFG),PBEN group (PBENG),intact protein enteral nutrition group (IPENG),methotrexate (MTX) + BFG,MTX + PBENG,and MTX + IPENG.The rats in MTX + BFG,MTX + PENG,and MTX + IPENG were intraperitoneal injected with MTX 10 mg/kg on day 0 and day 6 to induce sustained intestinal injury.From day 1,BFG and MTX + BFG were fed with basal feed,PBENG and MTX + PBENG with PBEN,IPENG and MTX + IPENG with IPEN.The daily energy intake of each rat was 1.80 kJ/g body weight.All the rats were sacrificed on day 11.The pathological changes of intestinal tissue were observed with HE staining,the levels of tissue myeloperoxidase (MPO),nitric oxide (NO),inducible nitric oxide synthase (iNOS),the thymus index and spleen gland index of intestinal tissue were measured using colorimetry,and the serum levels of immunoglobulins IgG,IgA,and IgM were determined with enzyme-linked immunosorbent assay.Results There were no significant differences among BFG,PBENG,and IPENG in each index.Serious injury of intestinal mucosa was observed in MTX groups.Significant differences were noted in all indexes between MTX + BFG and BFG (all P ＜0.05).The mucosal damage score (Chiu score) and the level of MPO and iNOS in MTX + PBENG were significantly lower than those in MTX + BFG [2.3 ± 0.69vs.2.96 ± 0.75,P =0.003 ; (2.30 ± 0.42) U/g tissue vs.(2.98 ± 0.23) U/g tissue,P =0.040 ; (0.37 ±0.06) U/mg prot vs.(0.44 ±0.10) U/rag prot,P =0.030] ; the serum levels of IgG and IgA were significantly higher than those in MTX + BFG (P =0.015,P =0.021) ; however,the levels of NO and IgM were not significantly different between the 2 groups (P =0.597,P =0.160).There were no statistically significant differences between MTX + IPENG and MTX + BFG in terms of the indexes (all P ＞ 0.05).Conclusion PBEN can reduce the inflammation response and improve the immune function in intestinal mucositis rat.

Objective To quantitatively assess the cardiopulmonary exercise function of spinal cord injury (SCI) patients and observe the effect of aerobic exercise on cardiopulmonary function, motor function and activities of daily living. Methods From December, 2014 to June, 2016, 34 incomplete SCI patients (ASIA C and D) and 23 healthy controls received cardiopulmonary exercise test (CPET). SCI pa-tients were randomly divided into conventional rehabilitation group (n=17) and aerobic exercise group (n=17). The aerobic exercise group received aerobic exercise for four weeks. They were assessed with CPET, motor and sensory function, walking index for spinal cord injury II (WISCI II) and spinal cord independence measure (SCIM) before and four weeks after training. Results Oxygen uptake (VO2)peak, anaerobic threshold (AT), metabolic equivalent of energy (METpeak), VO2/heart rate (HR)peak, respiratory exchange rate (RER)peak, minute ventilation (VE)peak, work rate (WR)peak and systolic blood pressure (SBP)peak were lower in the patients than in the controls (t>2.714, P<0.05). VO2peak、AT、METpeak、VO2/HRpeak、WRpeak increased in the aerobic exercise group after training (t>2.431, P<0.05). METpeak and WRpeak improved in the conventional rehabilitation group after training (t>3.282, P<0.01). The scores of motor in ASIA and SCIM improved in both groups after training (t>2.985, P<0.05). Conclusion The cardiopulmonary function decreased in incomplete SCI patients, which could be improved by moderate intensity aerobic exercise.

Promoter is one of important elements for gene expression and regulation. In the construction of recombinants for metabolic engineering and synthetic biology, it is necessary to have the promoters with varying strengths for fine-tuning metabolic pathway to reach the metabolic balance, decrease the accumulation of intermediate and increase the production of target metabolite. However, the natural promoters available are not completely suitable for fine-tuning metabolic pathway due to discrete strength, lack of versatility and standardization. To deal with this problem, in this study, a new 88 bp synthetic promoter, which contains the typical -35 box, -10 box as well as ribosome bind site, was designed. Then, the promoter library was constructed by introducing some degenerate base pairs in the sequence of 6 bp in the upstream of the initial transcription site and 14 bp in spacer region between -35 and -10 box. 720 promoters with varying strengths were screened out from a library of more than 5 000 clones via the expression of red fluorescent protein mCherry under the control of the synthetic promoter. The sequence analysis based on 35 promoters with varying strengths showed the promoters with varying strengths are base preference. The purine bases in -13 site and pyrimidine bases in the transcriptional initiation sequence are of high frequency; the purine and pyrimidine bases are of the similar frequency in the spacer sequence between -35 and -10 box in strong promoter. In the end, five characterized promoters with varying strengths were selected to tune the synthetic pathway of cis,cis-muconic acid in Escherichia coli. The results showed that the promoters with varying strengths can regulate the production of cis,cis-muconic acid and the accumulation of the intermediate catechol.
Base Sequence ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Metabolic Engineering ; methods ; Molecular Sequence Data ; Promoter Regions, Genetic ; Sorbic Acid ; analogs & derivatives ; metabolism

[ ABSTRACT] AIM:To investigate the effects of Chaihu Shugan powder ( CSP) on lipid metabolism and the pro-teins involved in adenosine 5’-monophosphate-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) pathway in the liver tissues of the rats with non-alcoholic fatty liver disease (NAFLD).METHODS: Sprague-Dawley rats were randomly di-vided into normal control ( NC) group, with HFD ( HFD) group and CSP group.The NAFLD models were established by feeding with HFD for 16 weeks in the rats.The rats in CSP group were intragastrically administered with CSP extracts (9.6 g· kg-1 · d-1 ) , and blood and liver samples were collected 16 weeks later.Serum and liver levels of total cholesterol ( TC) and triglyceride ( TG) , and serum levels of alanine aminotransferase ( ALT) and aspartate aminotransferase ( AST) were measured using an automatic biochemical analyzer.The histological changes of liver tissues were observed with HE staining, while the lipid deposition was observed with Oil Red O staining.The ultrastructural changes of the liver tissues were observed under transmission electron microscope.Moreover, the protein levels of AMPK, phosphorylated AMPK (pAMPK), SIRT1 and uncoupling protein 2 (UCP2) in the liver were detected by Western blot.RESULTS:The results of HE staining, Oil Red O staining and electron microscopy demonstrated that NAFLD rat model was successfully estab-lished.Compared with NC group, the serum and liver levels of TC and TG, and serum level of AST in model group were markedly elevated ( P<0.01) .Moreover, the protein levels of pAMPK and SIRT1 in HFD group were markedly reduced (P<0.01), whereas UCP2 level was elevated (P<0.01).Furthermore, liver levels of TC and TG, and serum level of AST in GSP group were markedly reduced as compared with HFD group ( P<0.05 ) .The protein levels of pAMPK and SIRT1 were elevated ( P<0.05 ) , whereas the UCP2 level was reduced as compared with HFD group ( P<0.01 ) .The protein level of AMPK between the 3 groups had no significant difference.CONCLUSION: CSP attenuates hepatic lipid disorder and hepatic lipid deposition in NAFLD rats induced by feeding with HFD for 16 weeks, which is associated with the activation of AMPK/SIRT1 pathway.

Objective:To observe the protective effect of Zhicao Tea Mixture on Müller cells and the expression of inflammatory factors in mice with diabetic retinopathy.Methods:Seventy-five C57BL/6J mice were randomly divided into the normal control group, diabetes mellitus (DM) group, low concentrations group, medium concentrations group and high concentrations group, with 16 mice in each group. The diabetes model of mice in all groups except the normal control group were established by intraperitoneal injection of STZ (60 mg/kg). Four weeks after the successful modeling, the Zhicao Tea Mixture with low (30 ml/kg), medium (60 ml/kg) and high concentrations (120 ml/kg) were respectively administered by gavage. Weight and blood glucose of mice in each group were measured every two weeks. After 8 weeks, Western blot method was used to detect the mice retina Müller cells activation marker gelatinous fibrous acidic protein (GFAP). Immunofluorescence was performed to detect the expression GFAP and glutamine synthetase (GS). Real-time quantitative PCR (RT- qPCR) and ELISA were used to determine the mRNA and protein expression levels of mouse retinal VEGF, TNF-α, IL-1β and IL-6 respectively.Results:The weight of mice in the DM group was lower than that of the normal control group, and the blood glucose was increased. Zhicao Tea Mixture had no effect on the weight of DM mice, but had a significant hypoglycemic effect. The GFAP expression ( t=38.318, P<0.001) in the retina of mice in the DM group was increased and GS expression ( t=29.737, P<0.001) was decreased compared with the control group. The GFAP expression ( t=13.677, 19.387, 16.305; P<0.05) in the retina of mice in the low, medium and high concentrations group were decreased and GS expression ( t=5.170, 19.399, 6.705; P<0.05) were increased compared with the DM group. The expressions of retinal inflammatory factors VEGF, TNF-α, IL-1β and IL-6 in DM group all increased, while the expressions of the above-mentioned inflammatory factors in the retina of mice decreased in the low, medium and high concentrations group. Conclusion:Zhicao Tea Mixture can decrease the blood glucose of DM mice and reduces the diabetic retinal inflammatory response.

Cardiovascular diseases have become a major cause of disability and death among the elderly in China.The continuous improvement of medical care has provided effective treatment for elderly patients with cardiovascular diseases in the acute phase, while how to improve the quality of life and prognosis of patients in the chronic phase has become a medical imperative to be solved.Cardiac rehabilitation, with exercise rehabilitation at its core, is expected to improve cardiopulmonary function, enhance exercise tolerance, alleviate anxiety and depression, and reduce mortality and rehospitalization rates, and has therefore become an effective intervention for cardiovascular diseases in the elderly.In the light of special concerns for the elderly population, such as cognitive impairment, frailty, sarcopenia and polypharmacy, this article systematically introduces the characteristics, obstacles and clinical strategies of cardiac rehabilitation for geriatric cardiovascular diseases, in an effort to provide a clinical reference guide for geriatric cardiac rehabilitation.

Objective:To investigate the short-term efficacy difference between femoral neck system and cannulated compression screw in the treatment of femoral neck fracture in young and middle-aged patients.Methods:Cochrane Library, PubMed, Web of Science, Embase, CNKI, Wanfang, VIP and CBM databases were searched. All literature on the treatment of femoral neck fractures using the femoral neck system (FNS) or cannulated compression screw (CCS). RevMan 5.4.1 software was used to analyze data.Results:Twelve high-quality cohort studies were included, including 787 patients treated surgically for femoral neck fractures, of whom 369 were treated with FNS and 418 with CCS. Results of meta-analysis showed that FNS had shorter operative time than CCS for femoral neck fracture [ WMD=-8.09, 95% CI (-14.12, -2.06), P=0.009]. FNS had less intraoperative blood loss [ WMD=6.63, 95% CI (1.42, 11.83), P=0.010], Partial weight-bearing time of FNS was earlier postoperative [ WMD=-2.11, 95% CI (-3.00, -1.22), P<0.001], while full weight-bearing time was also earlier postoperative [ WMD=1.01, 95% CI (1.59, 0.43), P<0.001), The postoperative fracture healing time of FNS was shorter [ SMD=0.67, 95% CI (1.04, 0.30), P<0.001]. The Harris score of FNS at the last follow-up was higher [ WMD=4.67, 95% CI (3.26, 6.08), P<0.001]. The number of fluoroscopy during FNS was less [ WMD=-9.05, 95% CI (-10.92, -7.18), P<0.001]. The rate of severe femoral neck shortening after FNS was lower [ RR=0.36, 95% CI (0.19, 0.70), P=0.002]. The incidence of postoperative complications after FNS, including urinary tract infection, venous thromboembolism, bone nonunion, screw loosening, femoral head avascular necrosis, was lower [ RR=0.38, 95% CI (0.27, 0.53), P<0.001]. There was no difference in postoperative hospital stay between FNS and CCS [ WMD=0.01, 95% CI (-0.36, 0.39), P=0.950]. There was no difference in fracture reduction quality (Garden I) [ RR=1.04, 95% CI (0.87, 1.24), P=0.660]. There was no difference in fracture reduction quality (Garden II) [ RR=0.91, 95% CI (0.59, 1.39), P=0.650]. There was no difference in postoperative moderate femoral neck shortening [ RR=0.85, 95% CI (0.58, 1.26), P=0.430]. Conclusion:Compared with cannulated compression screw, femoral neck system had shorter operation time, less intraoperative blood loss, earlier postoperative part load or full load time, shorter fracture healing time higher Harris score at the last follow-up, less number of intraoperative fluoroscopy, and lower rate of severe femoral neck shortening. Besides, FNS had a lower incidence of postoperative complications including urinary tract infection, venous thromboembolism, bone nonunion, screw loosening, and avascular necrosis of femoral head than CCS.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.

Objective To construct Csf1r-CreERT2 R26REYFP reporter gene mice and assess the efficacy of Csf1r-CreERT2-mediated enhancement of CSF1R in CD45+cells labeled with yellow fluorescein protein EYFP.Methods Csf1r-CreERT2 mice were crossbred with R26REYFP homozygous mice,and Csf1r-CreERT2R26REYFP mice were identified through PCR and Western Blot analyses.Flow cytometry was employed to evaluate CSF1R tag-efficiency in CD45+cells across different mouse tissues following tamoxifen induction.Results Csf1r-CreERT2 R26REYFP reporter gene mice were acquired.In addition,it was found that Csf1r-CreERT2-mediated EYFP could effectively mark CSF1R in various tissues of mice and CD45+cells in different locations.Compared to the R26REYF P group,the highest labeling efficiency was observed in the brain tissue(P＜0.001),the lowest in the thymus tissue(P＜0.05),and no sig-nificant difference was observed in the spleen tissue.Conclusion Adult Csf1r-CreERT2 mice and R26REYFP mice are effective ways to obtain Csf1r-CreERT2 R26REYFP induced conditional fluorescence mice.Csf1r-CreERT2 can mediate EYFP to effectively trace CSF1R in CD45+cells in different parts of mice.