The effect and mechanism of lncRNA XIST targeting miR-150 on LPS-induced apoptosis and secretion of inflammatory factors in mouse lung epithelial MLE-12 cells / 西安交通大学学报(医学版)

【Objective】 To study the effect and mechanism of lncRNA XIST targeting miR-150 on LPS-induced apoptosis and secretion of inflammatory factors in MLE-12 cells. 【Methods】 Mouse lung epithelial MLE-12 cells were divided into control, LPS (LPS treatment), sh-NC+LPS (shRNA control transfection, LPS treatment), and sh-XIST+LPS (XIST shRNA transfection, LPS treatment) groups. We used qRT-PCR method to analyze and detect XIST expression level, Annexin V-FITC/PI double staining method to detect cell apoptosis, Western blotting method to analyze and detect the protein expressions of Bax and Bcl-2 in cells, TBA method to detect MDA content, Xanthine oxidation method to detect SOD activity, DCFH-DA method to detect ROS level, and ELISA method to analyze the levels of IL-1β and TNF-α in the culture supernatant. We used the bioinformatics software Starbase to analyze the possible target genes of XIST (miR-150), and then the luciferase reporter system to identify the target relationship between the two. In MLE-12 cells, XIST shRNA and miR-150 inhibitor were co-transfected, and then treated with LPS. We also measured apoptosis, oxidative damage indicators, and inflammatory factor secretion changes. 【Results】 Compared with control group, XIST level in MLE-12 cells of LPS group increased, apoptosis rate and Bax protein expression level increased, Bcl-2 protein expression level decreased, ROS and MDA level increased, SOD level decreased, and the secretion of IL-1β and TNF-α increased. Compared with the sh-NC+LPS group, the sh-XIST+LPSMLE-12 group had decreased XIST level, decreased apoptosis rate and Bax protein expression level, and increased Bcl-2 protein expression level, decreased ROS and MDA levels, increased SOD levels, and decreased IL-1β and TNF-α secreted by cells. Down-regulating XIST targeting promoted miR-150 expression. miR-150 inhibitor could reverse the effects of XIST shRNA on LPS-induced MLE-12 cell apoptosis, oxidative damage indicators, and inflammatory factor secretion. 【Conclusion】 Down-regulation of lncRNA XIST targeting miR-150 inhibits LPS-induced apoptosis and secretion of inflammatory factors in mouse lung epithelial MLE-12 cells.

Intravenous Injection of miR-34a Inhibitor Alleviates Diabetes Mellitus-Induced Vascular Endothelial Dysfunction by Targeting NOTCH1.

BACKGROUND: miR-34a is a multifunctional post-translational modulator, which is involved in several diabetes-related complications. However, miR-34a remains to be fully elucidated in the diabetic endothelium from rats. In this study, the role of miR-34a/NOTCH1 signaling in the progression of hyperglycemia-vascular endothelial dysfunction was investigated. METHODS: In intravenous injection of miR-34a mimics and inhibitors in streptozotocin (STZ)-induced diabetic rats, the biomarkers of endothelial dysfunction was measured. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. The mRNA and protein levels were assayed by qRT-PCR and western blotting, respectively. Immunohistochemical staining was performed to measure NOTCH1 expression in the diabetic endothelium. RESULTS: miR-34a was significantly up-regulated, and NOTCH1 down-regulated, in the thoracic aorta from STZ-induced diabetic rats compared with control group. As compared to model group, the mRNA of NOTCH1 was significantly decreased or increased by miR-34a mimics or inhibitors ex vivo, respectively. Bioinformatics methods further demonstrated that NOTCH1 was a potential target of miR-34a, which was confirmed by dual-luciferase reporter assay. Moreover, both serum ET and NO were significantly increased in diabetic rats as compared to control group. miR-34a inhibitors ex vivo treatment resulted in significant down-regulation ofserum ET and NO levels in diabetic rats as compared to model group. CONCLUSION: These results provide evidence to support the use of miR-34a inhibitors as a therapeutic approach attenuating hyperglycemia-induced vascular endothelial dysfunction.

Clinical Observation of Addition and Subtraction Therapy of Shenling Baizhu San to Antibiotic-associated Diarrhea with Spleen-stomach Deficiency and Cold Syndrome / 中国实验方剂学杂志

Objective:To discussed the clinical efficacy of addition and subtraction therapy of Shenling Baizhu San to antibiotic-associated diarrhea (AAD) with spleen-stomach deficiency and cold syndrome, and to investigate its effects on immune function and intestinal flora. Method:One hundred and fifteen patients were randomly divided into control group (57 cases) and observation group (58 cases) by random number table. Patients in control group got Shuangqi Ganjun Sanlian Huojun San, 2 bags/time, 2 times/days. Mengtuoshi San, 1 bag/time, 3 times/days, and they also got measures to prevent disturbance of water, electrolyte, acid-base balance and nutritional support. Based on the treatment in control group, patients in observation group also got addition and subtraction therapy of Shenling Baizhu San, 1 dose/day. The course of treatment was 7 days in both groups. Before and after treatment, scores of symptoms, intestinal secretory immunoglobulin (SIgA) levels, peripheral blood immunoglobulin A (IgA), G (IgG), M (IgM) and T lymphocyte subsets (CD3+, CD4+, CD8+and CD4+/CD8+). Detection of bacillus faccalis in feces before and after treatment and the bacteria were cultured to identify and count bifidobacterium, lactobacillus and enterococcus. In addition, diamine oxidase (DAO) and D-lactic acid levels were detected before and after treatment. Result:In rank sum test, clinical efficacy in observation group was better than that in control group (Z=2.268, PPP+, CD4+and CD4+/CD8+were higher than those in control group (P+was lower than that in control group (PPPPD-lactic acid were significantly lower than those in control group (PConclusion:Based on conventional treatment, addition and subtraction therapy of Shenling Baizhu San can alleviate symptoms, improve clinical efficacy, improve immune function, regulate intestinal flora and promote the repair of intestinal mucosal barrier in the treatment of antibiotic-associated diarrhea (AAD) with spleen-stomach deficiency and cold syndrome.

A clinical evaluation of CRRT coupled with ultrasound-guided percutaneous transhepatic gallbladder drainage for the treatment of severe acute biliary pancreatitis / 中华急诊医学杂志

Objective To investigate the value of continuous renal replacement therapy (CRRT) coupled with minimally invasive ultrasound-guided percutaneous transhepatic gallbladder drainage (PTGD) for the treatment of severe acute biliary pancreatitis.Methods Hospitalized patients with severe acute biliary pancreatitis were recruited from the intensive care unit (ICU) of the Mfiliated Hospital of Qingdao University from June 2010 to June 2015,and divided into conventional CRRT alone group (n =30) and CRRT + PTGD group (n =30).Comparisons of postoperatively symptoms (time required for abdominal pain relief,time consumed for,gastrointestinal decompression),laboratory findings (WBC,PLT,PCT,CRP,AMS,TBIL,ALT,ALB,Lac) and acute physiology and chronic health evaluation score (APACHE Ⅱ,Balthazar CT,MODS) were carried out between two groups.The occurrence of complications (ARDS,abdominal infection,bile leakage,abdominal hemorrhage,intestinal injury,catheter translocation,catheter dislocation) was observed.The differences in duration of ventilator support,the length of stay in ICU,and fatality rate were compared between the two groups.Results Compared with the conventional CRRT alone group,the postoperative symptoms were significantly relieved,and time required for abdominal pain relief and time consumed for gastrointestinal decompression were evidently shortened in the CRRT + PTGD group (P ＜ 0.05).There were statistically significant differences in laboratory findings (WBC,PLT,PCT,CRP,AMS,TBIL,ALT) between two groups (P ＜ 0.05).The differences in APACHE Ⅱ,Balthazar CT and MODS score between the two groups also presented statistical significance (P ＜ 0.05).The comparisons of the duration of ventilator support [(6.1 ± 1.3) d vs.(9.5 ± 1.4) d] andthe length of stay [(15.7 ± 1.1) dvs.(21.1 ± 2.5) d] between thetwo groups revealed statistical significance (P ＜ 0.05).Conclusions CRRT coupled with PTGD for the treatment of severe acute biliary pancreatitis can effectively eliminate the inflammatory mediators and toxins from patients.On this basis,the coupled therapy with gallbladder puncture and drainage is capable of decompressing the biliary tract,improving liver function,effectively relieving clinical symptoms,minimizing the changes of laboratory findings an,d APACHE Ⅱ score,and thereby optimizing the prognosis of patients.

Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy.

Fluorofenidone (FD) is a novel pyridone agent with significant antifibrotic effects in vitro. The purpose of this study is to investigate the effects of FD on renal interstitial fibrosis in rats with obstructive nephropathy caused by unilateral ureteral obstruction (UUO). With pirfenidone (PD, 500 mg/kg/day) and enalapril (10 mg/kg/day) as the positive treatment controls, the rats in different experimental groups were administered with FD (500 mg/kg/day) from day 4 to day 14 after UUO. The tubulointerstitial injury, interstitial collagen deposition, and expression of type I and type III collagen, transforming growth factor-ß(1) (TGF-ß(1)), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), α-smooth muscle actin (α-SMA), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were assessed. FD treatment significantly attenuated the prominently increased scores of tubulointerstitial injury, interstitial collagen deposition, and protein expression of type I and type III collagen in ureter-obstructed kidneys, respectively. As compared with untreated rats, FD also significantly reduced the expression of α-SMA, TGF-ß(1), CTGF, PDGF, and inhibitor of TIMP-1 in the obstructed kidneys. Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy through its regulation on fibrogenic growth factors, tubular cell transdifferentiation, and extracellular matrix.

Fluorofenidone attenuates collagen I and transforming growth factor-beta1 expression through a nicotinamide adenine dinucleotide phosphate oxidase-dependent way in NRK-52E cells.

AIM: Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone) is a novel pyridone agent. The aim of the present study is to investigate the effects of fluorofenidone on angiotensin (Ang)II-induced fibrosis and the involved molecular mechanism in rat proximal tubular epithelial cells. METHODS: NRK-52E cells, a rat proximal tubular epithelial cell line, were incubated with medium containing AngII, with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), losartan, fluorofenidone (2, 4 and 8 mmol/L) and pirfenidone (8 mmol/L) for 24 h. Cells in the serum-free medium were controls. The expression of three subunits of NADPH oxidase, including p47phox, Nox-4 and p22phox, were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot. NADPH oxidase activity was measured directly by superoxide dismutase (SOD) inhibitable cytochrome C reduction assay. The generation of reactive oxygen species (ROS) was measured by dichlorofluorescein fluorescence analysis. The mRNA and protein expression of collagen I and transforming growth factor (TGF)-beta1 were determined by real-time RT-PCR and enzyme-linked immunosorbent assay. RESULTS: Fluorofenidone significantly inhibited TGF-beta1 and collagen I expression upregulation induced by AngII or TGF-beta1 respectively. Moreover, fluorofenidone greatly reduced the elevation of expression and activity of NADPH oxidase and inhibited ROS generation induced by AngII in rat proximal tubular epithelial cells. These responses were also attenuated by DPI, losartan, and pirfenidone. CONCLUSION: Fluorofenidone acted as an anti-oxidative and anti-fibrotic agent through the mechanisms of blocking NADPH oxidase-dependent oxidative stress and inhibiting TGF-beta1 expression in rat proximal tubular epithelial cells.