Objective: To explore the mechanism of action of hepatic leukemia factor (HLF) in diabetes mellitus and to construct a conditional animal model of mice with islet β ⁃cell⁃specific HLF gene knockout. Methods: At the cellular level , the effects of HLF inhibition or overexpression on the proliferation of MIN6 cells was verified by the CCK⁃8 assay. The effects of HLF inhibition or overexpression were detected at the mRNA level and protein level by Cre + / - mice (C57BL/6J) to obtain offspring mice. The genotypes of the mice were identified by the PCR method.The differences in the expression levels of the HLF gene at the mRNA and protein levels in islet β ⁃cell knockout mice (HLFflox/flox Cre + / - ) and control mice (HLFflox/flox ) were detected by RT⁃qPCR technology and Western blot technology to verify the knockout effect. At the same time , the islet tissues of the mice in two groups were taken to make paraffin sections and analyzed by hematoxylin⁃eosin (HE) staining. Results: HLF gene inhibition or overex⁃pression had no significant effect on the proliferation of MIN6 cells. When the HLF gene was inhibited in MIN6 cells , the mRNA expression level decreased by 74% compared with the control group , and the protein expression level decreased by 60% compared with the control group. After overexpressing the HLF gene , the mRNA expres⁃sion level was 2. 13 times compared with that of the control group , and the protein expression level was 1. 8 times compared with that of the control group. The mRNA expression level of the HLF gene in the knockout mice de⁃creased by 89% compared with the control group , and the protein expression level decreased by 65% compared with the control group. The results of HE staining showed that there was no significant difference in the cell mor⁃phology in the islet tissues between the knockout mice and the control mice. Inhibiting HLF increased the glycogen content in MIN6 cells by approximately 20% . Conclusion The HLF gene knockout mice are successfully con⁃structed , providing an animal model for studying the role of HLF in the pathogenesis of diabetes mellitus.

Objective To study the role of KRX-725,a specific agonist of Sphingosin-1 phosphate receptor 3,S1PR3),in the function and mechanism of S1PR3 in respect of bacterial clearance.Methods Twenty healthy male C57BL/6 mice were randomly (random number) divided into two groups:KRX-725 group and control group.Septic mice model were established by intraperitoneal injection of E.coli (3 × 106),then KRX-725 (10 mg/kg) or the vehicle was administered intratracheally.Forty-eight-hour survival rate (n =12),bacterial colony numbers in peritoneal cavity and blood (n =5),and lung injury (n =3)were compared between two groups.In vitro,the peritoneal macrophages were stimulated by E.coli (cell∶E.coli=1∶10) with KRX-725 or the vehicle treatment.The reactive oxygen species (ROS) levels were detected by CM-H2DCFDA in macrophages.The bacteria clearance function of KRX-725 was observed by gentamicin protection test.Survival rates were analyzed with the Log-rank test.A 2-tailed student's t test was used to compare difference between two independent groups.Results Compared with the control group,the 48-hour survival rate of KRX-725 group was significantly higher (P ＜ 0.05).Bacterialload in the blood and the peritoneal lavage fluid (PLF) was greatly decreased in the KRX-725 group (blood:t =3.17,P ＜0.05;PLF:t =4.07,P ＜0.01).The lung tissues injury was also obviously reduced in the KRX-725 group of 24 hours after the injection of E.coli (lung injury score:KRX-725 group 1.4 ± 0.25;control group 2.4 ± 0.25) (t =2.89,P ＜ 0.05).In vitro,KRX-725 could up-regulate the ROS levels in macrophage at 20 min and 30 min after E.coli injected intra-peritoneally (20 min fluorescent intensity:KRX-725 group 522.9 ± 38.76,control group 385.9 ± 15.90,P ＜ 0.05;30 min fluorescent intensity:KRX-725 group 519.7 ±25.02,control group 384.5 ± 15.28,P ＜0.01).The bacterial load in the KRX-725 treated macrophage were significantly decreased at 3 h and 6 h after E.coli injected intra-peritoneally (3 h:KRX-725 group 286.5 ±98.35,control group 710.8 ± 107.8,P ＜0.05;6 h:KRX-725 group 72.5 ±6.45,control group 205.8 ±66.76,P ＜0.01).Conclusion In vivo,KRX-725 could improve the survival rate of septic mice,decrease the bacterial lioad in the blood and PLF,and reduce the lung injury.In vitro,KRX-725 could up-regulate the ROS level in macrophages and accelerate the bacterial clearance.