Effect of abnormal expression of PDK4 on glycolysis and proliferation in prostate cancer cells

AIM To investigate the expression of pyruvate dehydrogenase kinase 4 （PDK4） in prostate cancer tissue and its effect on glycolysis and growth of prostate cancer cells. METHODS Immunohistochemistry was used to compare the expression differences of PDK4 protein in benign prostatic hyperplasia （BPH） and prostate cancer tissues. The expression levels of PDK4 in normal prostatic epithelial cells （RWPE-1） and different prostate cancer cell lines （PC3， LNCaP， DU145 and C4-2） were detected by RT-qPCR and Western blot. Recombinant plasmid carrying PDK4-shRNA was constructed， and the expression of PDK4 in prostate cancer PC3 cells was down-regulated by transfection with PDK4-shRNA. The changes in glycolysis level of PC3 cells before and after transfection were determined by cell glycolysis kit， and the effects of PDK4 on the viability and cell cycle distribution of PC3 cells were detected by CCK-8 assay and flow cytometry. RESULTS In prostate cancer tissues， the expression level of PDK4 protein was significantly higher than that in BPH tissues （P<0.05）， and the analysis of immunohistochemical score showed that prostate cancer tissues with high Gleason score displayed significantly higher PDK4 expression than those with low Gleason score （P<0.05）. Compared with normal prostatic epithelial cells， RT-qPCR and Western blot results indicated that the expression level of PDK4 was also significantly increased in prostate cancer cell lines （P<0.05）. In addition， CCK-8 assay results showed that the viability of prostate cancer PC3 cells was significantly decreased after knockdown of PDK4 expression （P<0.05）. The results of flow cytometry demonstrated that knockdown of PDK4 expression in PC3 cells resulted in a notable increase in G₀/G₁ phase arrest （P<0.05）. CONCLUSION PDK4 is highly expressed in prostate cancer tissues and cell lines， and significantly increases in prostate cancer with high Gleason score. In addition， down-regulation of PDK4 expression significantly inhibits glycolysis and growth of prostate cancer cells， resulting in cell cycle arrest at G₀/G₁ phase.     

STC-1 mediates proliferation and apoptosis of gastric cancer cells through Bcl-2

AIM To investigate the effect of stanniocalcin-1 （STC-1） on the proliferation and apoptosis of gastric cancer AGS cells and the role of Bcl-2 in these processes. METHODS The AGS cells were transfected with the plasmids for STC-1 knockdown or over-expression. The cell proliferation was measured by MTT assay and colony formation assay. The migration ability was detected by scratch assay. Apoptosis was analyzed by Hoechst 33342 staining and flow cytometry with annexin V-FITC/PI double staining. The protein expression of Bcl-2， survivin， caspase-3 and cleared caspase-3 was determined by Western blot. The mRNA expression levels of STC-1 and Bcl-2 in 20 cases of clinical gastric cancer tissues and adjacent tissues were detected by RT-qPCR， and the correlation between them was analyzed by Pearson method. RESULTS After over-expression of STC-1， the proliferation and migration abilities of the AGS cells were increased， the expression of Bcl-2 and survivin was increased， while the expression of caspase-3 and cleared caspase-3 was decreased （P<0.05）. After knockdown of STC-1， the proliferation and migration abilities of the AGS cells were decreased， the expression of Bcl-2 and survivin was decreased， while the expression of caspase-3 and cleared caspase-3 was increased （P<0.05）. The mRNA expression levels of STC-1 and Bcl-2 in the gastric cancer tissues were higher than those in the adjacent tissues. Pearson correlation analysis showed that there was a positive correlation between STC-1 and Bcl-2 mRNA expression in the cancer tissues （r=0.308， P=0.011）. CONCLUSION STC-1 may regulate the biological function of gastric cancer cells by altering the expression level of Bcl-2.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used， and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group， siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12， 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR， and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group， the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）， and the cell cycle was arrested at G₁ phase. The mRNA levels of p53， caspase-3， Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition， the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）， while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Role and potential mechanism of fecal microbiota transplantation in treatment of chronic hepatitis B

AIM To investigate the effect of fecal microbiota transplantation （FMT） on the treatment of chronic hepatitis B （CHB） and the potential mechanism. METHODS Fifty C57BL/6J mice （6~8 weeks old） were divided into 5 groups： control group， CHB group， entecavir （ETV） group， comprehensive treatment （ETV+FMT， EFMT） group， and blocker （TAK-242+ETV+FMT， EFMT-TAK） group. The mice in each group were given corresponding treatment. The general condition of the mice was observed daily， and fecal specimens were kept every 10 d. The mice were sacrificed after 12 weeks， and the liver tissues and blood samples were collected. HE staining was used for histological scoring. Serum hepatitis B surface antigen （HBsAg） and interleukin-18 （IL-18） levels were measured by ELISA. Toll-like receptor 4 （TLR4） expression was detected by flow cytometry. Intestinal flora diversity was analyzed by high-throughput sequencing. RESULTS （1） Compared with control group， the body weight of the mice in CHB group was significantly reduced （P<0.05）. The body weight loss of the mice in ETV group， EFMT group and EFMT-TAK group was reversed to some extent as compared with CHB group （P<0.05）. （2） The histological score of the mice in CHB group was significantly higher than that in control group （P<0.05）. The score in ETV group was lower than that in CHB group （P<0.05）. The scores in EFMT group and EFMT-TAK group were lower than that in ETV group （P<0.05）， and that in EFMT-TAK group had a further downward trend compared with EFMT group （P<0.05）. （3） Compared with control group， the serum level of HBsAg in the CHB mice was significantly increased （P<0.05） and decreased after ETV treatment （P<0.05）. The HBsAg level in both EFMT group and EFMT-TAK group was significantly lower than that in ETV group （P<0.05）. （4） The IL-18 level in CHB group was significantly higher than that in control group （P<0.05）. After ETV treatment， the IL-18 level was decreased （P<0.05）， and that in both EFMT group and EFMT-TAK group was decreased more than that in ETV group （P<0.05）. （5） TLR4 expression in CHB group was higher than that in control group （P<0.05）， that in ETV group was lower than CHB group （P<0.05）， and that in EFMT group was further decreased （P<0.05）. （6） The heat map analysis at the class level showed that the abundances of Gammaproteobacteria， Deltaproteobacteria and Negativicutes in CHB group were significantly higher than those in control group， and those of Deltaproteobacteria and Negativicutes in EFMT group were close to those in control group. The heat map analysis at the family level indicated that the abundances of Burkholderiaceae， Desulfovibrionaceae and Veillonellaceae in CHB group were significantly higher than those in control group， while those in ETV group and EFMT group gradually approached normal levels. The α diversity index in CHB group was significantly decreased， while the diversity in ETV group was increased， that in EFMT group was further increased， and that in EFMT-TAK group was the highest. CONCLUSION FMT plays an active role in the treatment of CHB. The mechanism may be related to reducing the level of IL-18 and improving the structure and diversity of intestinal flora. The TLR4 signaling pathway is involved.     

Effect of Tripterygium wilfordii multiglucoside on intestinal flora and immune function in IgA nephropathy rats based on C1GALT1/Cosmc pathway

AIM To investigate the effects of Triptergium wilfordii multiglucoside （TWM） on intestinal flora and immune function in IgA nephropathy （IgAN） rats based on core 1 β1，3-galactosyltransferase （C1GALT1） and its chaperone protein Cosmc （C1GALT1/Cosmc pathway）. METHODS The rat model of IgAN was established， and the animals were randomly divided into model group （IgAN group）， dexamethasone （Dex） group and TWM group. Normal rats served as normal control （NC） group. The levels of serum creatinine （SCr） and blood urea nitrogen （BUN）， 24-hour urinary total protein （24 h UTP） and the number of urinary red blood cells were measured by automatic biochemical analyzer. The levels of serum IgA1， and plasma tumor necrosis factor-α （TNF-α）， B-cell activating factor （Baff） and interleukin-17 （IL-17） were detected by ELISA. The level of galactose-deficient IgA1 （Gd-IgA1） was detected by Vicia villosa lectin affinity ELISA. The intestinal colony was cultured in selective bacterial medium. The ratio of CD4⁺ CD25⁺ regulatory T cells （Treg） to CD4⁺ T cells （Treg proportion） in peripheral blood mononuclear cells （PBMC） was detected by flow cytometry.Western blot was used to determine the protein expression of C1GALT1 and Cosmc in intestinal mucosa. RESULTS Compared with NC group， 24 h UTP， the number of urinary red blood cells， SCr， BUN， serum IgA1 and Gd-IgA1， the numbers of Enterobacteriaceae， Enterococcus and Bacteroides， and the levels of TNF-α， Baff and IL-17 in plasma in IgAN group were significantly increased （P<0.05）， while the numbers of Bifidobacteria and Lactobacilli， the Treg proportion in PBMC， and the protein expression levels of C1GALT1 and Cosmc in intestinal mucosa were significantly decreased （P<0.05）. Compared with IgAN group， 24 h UTP， the number of urinary red blood cells， SCr， BUN， serum IgA1 and Gd-IgA1， the numbers of Enterobacteriaceae， Enterococcus and Bacteroides， and the levels of TNF-α， Baff and IL-17 in plasma in Dex group and TWM group were significantly reduced （P<0.05）， and those in TWM group were lower than those in Dex group （P<0.05）. Moreover， the numbers of Bifidobacteria and Lactobacilli， the Treg proportion in PBMC， and the protein expression levels of C1GALT1 and Cosmc in intestinal mucosa were significantly elevated （P<0.05）， and those in TWM group were higher than those in Dex group （P<0.05）. CONCLUSION TWM reduces the abnormal glycosylation level of IgA in IgAN rats by promoting the activation of C1GALT1/Cosmc pathway， and attenuates the intestinal flora disorder and immune dysfunction in IgAN rats， thus exerting the therapeutic effect.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC， pcDNA-FEZF1-AS1， anti-miR-NC， anti-miR-363-3p， miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1， Ki67 and cleaved caspase-3. RESULTS Compared with control group， the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）， and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group， the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）， the apoptotic rate was significantly reduced （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）， and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group， the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）， the apoptotic rate was significantly reduced （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）， and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group， the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）， the apoptotic rate was significantly increased （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）， and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Alleviating effect of exenatide on ectopic lipid accumulation in skeletal muscle of ob/ob mice is independent on reducing body weight

AIM To investigate the alleviating effect of exenatide （Exe）， a glucagon-like peptide-1 （GLP-1） receptor agonist， on the ectopic lipid accumulation in skeletal muscle of ob/ob mice and its mechanism. METHODS Eight-week-old male ob/ob mice and their wild-type （WT） littermates were randomly divided into 3 groups， ob/ob group， ob/ob+Exe group and WT group， and treated with Exe at 24 nmol/kg or the same volume of saline intraperitoneally once daily for 4 weeks. The body weight， fasting blood glucose （FBG） and fat content were measured after the 4-week treatment. The oil red O staining and the quantification of triglyceride （TG） were performed on the skeletal muscle. The serum levels of TG， total cholesterol and free fatty acid （FFA） were also measured by ELISA. The expression levels of AMP-activated protein kinase （AMPK） and lipid metabolism-related proteins were determined by Western blot. Mouse myoblast C2C12 cells were used as an in vitro model to further investigate the effects of Exe. RESULTS As compared with the ob/ob mice treated with saline， 4-week Exe treatment did not reduce body weight， FBG， food intake and fat content in ob/ob mice （P>0.05）. However， serum FFA was decreased （P<0.05）. Oil red O staining and the quantification of TG showed that 4-week Exe treatment significantly attenuated the ectopic lipid accumulation in the skeletal muscle of ob/ob mice （P<0.05）. The results of Western blot showed that the levels of phosphorylated AMPK （p-AMPK） and lipolysis-related proteins were up-regulated， while the lipid synthesis-related proteins were down-regulated by Exe （P<0.05）. Treatment with Exe alleviated the lipid accumulation in the C2C12 cells induced by sodium palmate （P<0.05）， and the effects of Exe on the levels of p-AMPK and lipid metabolism-related proteins in the C2C12 cells were consistent with those in the ob/ob mice （P<0.05）. Treatment with Exe also up-regulated the protein expression of glucose transporter 4 and improved the ability of glucose uptake in the C2C12 cells （P<0.05）. CONCLUSION Short-term Exe treatment attenuates the ectopic lipid accumulation in skeletal muscle of ob/ob mice by up-regulating lipolysis-related proteins and down-regulating lipid synthesis-related proteins， which is independent on body weight loss.     

Effect of 27nt-miRNA on apoptosis of human umbilical vein endothelial cells induced by oxidized low-density lipoprotein

AIM To investigate the effect of 27nt-miRNA （27nt-miR） on apoptosis of human umbilical vein endothelial cells （HUVECs） induced by oxidized low-density lipoprotein （Ox-LDL） and its underlying mechanism. METHODS HUVECs were cultured in vitro and grouped as below： normal control group， Ox-LDL group， 27nt-miR+Ox-LDL group， anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group. The cells in Ox-LDL group were treated with Ox-LDL at 40 mg/L for 48 h， while those in normal control group were untreated but cultured normally. The cells in 27nt-miR+Ox-LDL group， anti-27nt-miR+Ox-LDL group and negative control+Ox-LDL group were transfected with their corresponding lentiviral vectors under the same procedure， followed by treatment with Ox-LDL at 40 mg/L for 48 h to induce apoptosis. The cell viability was measured by CCK-8 assay. The migration capacity was detected by scratch assay. The caspase-3 activity was measured by caspase-3 activity assay kit. The apoptotic rate was analyzed by Hoechst 33258 and flow cytometry. The mRNA and protein expression levels of Bcl-2， Bax and caspase-3 were determined by RT-qPCR and Western blot. RESUITS： Compared with negative control+Ox-LDL group， the cell viability and migration ability were significantly decreased by over-expression of 27nt-miR in the HUVECs （P<0.05）， while the activity of caspase-3 and apoptosis induced by Ox-LDL were significantly increased （P<0.05）. Furthermore， the mRNA and protein expression levels of Bax and caspase-3 were significantly up-regulated （P<0.05）， and the mRNA and protein expression level of Bcl-2 was down-regulated in 27nt-miR+Ox-LDL group （P<0.05）. Meanwhile， all the above indexes showed an opposite tendency in anti-27nt-miR+Ox-LDL group. CONCLUSION 27nt-miR promotes Ox-LDL-induced apoptosis and inhibits the viability and migration of HUVECs in vitro， possibly through regulating the expression of apoptotic/anti-apoptotic proteins such as Bax，caspase-3 and Bcl-2.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice， and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice， and the obese mice were randomly divided into 3 groups， including obesity group （treated with saline； n=10）， EET group （treated with 11，12-EET； n=10） and EET inhibitor 14，15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin， tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice， homeostasis model assessment of insulin resistance （HOMA-IR） values were increased， the protein level of CYP2J2 was reduced， and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1， IL-1β， IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11， 12-EET， the HOMA-IR values were decreased compared with vehicle-treated obese mice， HIF-1α expression levels were decreased in the adipose tissue， and the serum levels of MCP-1， IL-1β， IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.     

Niflumic acid inhibits the proliferation,migration and invasion of glioma U87 cells

AIM To investigate the effect of niflumic acid （NFA） on human glioma U87 cells and to clarify the potential mechanism. METHODS The U87 cells were cultured in vitro and divided into blank control group， and 50， 100 and 200 μmol/L NFA groups. MTT assay was performed to determine the viability of cells in various groups. Migration and invasion abilities were measured by real-time cell analysis （RTCA）. RESULTS The results of MTT assay showed that compared with blank control group， the viability of U87 cells was increased after treatment with NFA for 12 h （P<0.05 or P<0.01）， while the viability was significantly decreased after treatment with NFA for 24 and 48 h （P<0.05 or P<0.01） in a concentration-dependent manner. The results of RTCA showed that compared with control group， the cell migration and invasion abilities were inhibited in 100 and 200 μmol/L NFA groups （P<0.05 or P<0.01） and the inhibitory effects were more obvious in 200 μmol/L NFA group （P<0.01）. CONCLUSION NFA inhibits the viability， migration and invasion of human glioma U87 cells.     

Effect of continuous renal replacement therapy on mRNA expression of autophagy-related molecules and prognosis in patients with acute kidney injury

AIM To explore the effect of continuous renal replacement therapy （CRRT） on mRNA expression of autophagy-related molecules and the prognosis in the patients with acute kidney injury （AKI）. METHODS A total of 174 patients from our hospital who were diagnosed to have AKI and underwent CRRT between February 2015 and March 2018 were involved in this study. The plasma levels of interleukin-1β （IL-1β） and interleukin-6 （IL-6）， the serum creatinine （SCr） level， and the mRNA expression levels of autophagy-related molecules， including microtubule-associated protein 1 light chain 3-II （LC3-II）， autophagy-related protein 5 （Atg5） and beclin-1， in the monocytes from peripheral blood were compared before and after CRRT. According to the survival of AKI patients after 4 weeks of CRRT， the enrolled patients were divided into death group （n=43） and survival group （n=131）， and the mRNA expression levels of the above molecules were compared between the 2 groups. RESULTS After CRRT treatment， the plasma levels of IL-1β and IL-6， the level of SCr， and the mRNA expression levels of LC3-II， Atg5 and beclin-1 in the monocytes were significantly lower than those before CRRT treatment （P<0.05）. The mRNA expression levels of LC3-II， Atg5 and beclin-1 in death group were significantly higher than those in survival group （P<0.05）. The positive correlation between SCr and IL-1β， IL-6， LC3-II or beclin-1 was observed （P<0.05）， and no correlation between SCr and Atg5 was found （P>0.05）. CONCLUSION CRRT decreases the mRNA expression levels of autophagy-related molecules in the patients with AKI and reduces the autophagy activity， which is protective for the patients. These autophagy-related molecules may be applied as a potential markers to predict the prognosis of CRRT.     

Scutellarin affects LPS-induced oxidative stress and apoptosis of glomerular epithelial cells by up-regulating miR-7-5p

AIM To investigate the effect of scutellarin （SCU） on oxidative stress and apoptosis induced by lipopolysaccharide （LPS） in human glomerular epithelial cells and its mechanism. METHODS Human glomerular epithelial cells were cultured in vitro， and were treated with LPS （1.0 mg/L） to establish a cell injury model. The cells were divided into normal control （NC） group， LPS group， NC+SCU group， LPS+SCU group， LPS+miR-NC group， LPS+microRNA-7-5p （miR-7-5p） group， LPS+SCU+anti-miR-NC group and LPS+SCU+anti-miR-7-5p group. Cell viability was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The intracellular malondialdehyde （MDA） content and superoxide dismutase （SOD） activity， and lactate dehydrogenase （LDH） activity in the cell culture supernatant were determined by kit. RT-qPCR was used to detect the expression level of miR-7-5p. RESULTS Compared with NC group， the cell viability， miR-7-5p expression and SOD activity in LPS group were significantly reduced， and the apoptotic rate， MDA content and LDH activity were significantly increased （P<0.05）. Compared with LPS group， the cell viability， miR-7-5p expression and SOD activity in LPS+SCU group were significantly increased， and the apoptotic rate， MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+miR-NC group， the cell viability and SOD activity in LPS+miR-7-5p group were significantly increased， and the apoptotic rate， MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+SCU+anti-miR-NC group， the cell viability and SOD activity in LPS+SCU+anti-miR-7-5p group were significantly reduced， and the apoptotic rate， MDA content and LDH activity were significantly increased （P<0.05）. CONCLUSION Scutellarin inhibits LPS-induced oxidative stress damage and apoptosis in glomerular epithelial cells via up-regulating miR-7-5p expression.     

Effects of tripterine on viability and apoptosis of keloid fibroblasts by regulating GINS2

AIM To explore the effects of tripterine on the viability and apoptosis of keloid fibroblasts （KFB） and its molecular mechanism. METHODS The KFB were treated with tripterine at low， medium and high doses. The cell viability and apoptosis were measured by CCK-8 assay and flow cytometry， respectively. RT-qPCR and Western blot were applied to determine the expression of GINS complex subunit 2 （GINS2）. The KFB were divided into si-NC group， si-GINS2 group， Exp+pcDNA group and Exp+pcDNA-GINS2 group， and the changes of cell viability and apoptosis were measured using the above methods. RESULTS After treatment with tripterine， the viability of KFB was decreased， the apoptotic rate was increased， and GINS2 expression was decreased in a dose-dependent manner （P<0.05）. After knockdown of GINS2， the viability of KFB was decreased， and the apoptotic rate was increased （P<0.05）. Over-expression of GINS2 partially reversed the effect of tripterine on the viability and apoptosis of KFB （P<0.05）. CONCLUSION Tripterine inhibits KFB viability and promotes the apoptosis by down-regulating GINS2.     

Differential expression of nesfatin-1 in intestinal tissues of premature infants with necrotizing enterocolitis and and its mechanism

AIM To analyze the expression of nesfatin-1 in intestinal tissues of premature infants with necrotizing enterocolitis （NEC）， and to explore the effect of nesfatin-1 on lipopolysacharide （LPS）-induced enterocytes and its mechanism. METHODS The intestinal tissues were obtained from infants who underwent intestinal surgery for NEC in our hospital from 2017 to 2019. The mRNA expression of nesfatin-1 in the tissue samples of NEC were evaluated by RT-qPCR. Human fetal normal colon epithelial HCoEpiC cells and human colon cancer Caco-2 cells were used as research objects. The effect of nesfatin-1 on the secretion of cytokines was measured by ELISA. Western blot was used to analyze the protein expression of nesfatin-1 and Toll-like receptor 4 （TLR4）， NLRP3， AIM2， caspase-1 and ASC， and co-immunoprecipitation assay were conducted to explore the relation between nesfatin-1 and TLR4. RESULTS The expression of nesfatin-1 in NEC preterm infants was significantly lower than that in the healthy group （P<0.01）. Compared with control group， the expression of nesfatin-1 in HCo Epic cells and HT-29 cells induced by LPS was decreased （P<0.01）， while the transfection of nesfatin-1 reversed the stimulation of LPS， and the over-expression of nesfatin-1 decreased the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）， and increased the level of IL-10 （P<0.05）. In addition， nesfatin-1 over-expression inhibited the expression of NLRP3， AIM2， caspase-1 and ASC. The expression of TLR4 in NEC tissue samples was significantly higher than that in healthy infants （P<0.05）. Pearson correlation analysis showed that there was a significant negative correlation between nesfatin-1 and TLR4 （r=-0.816， P<0.01）. TLR4 was found to co-precipitate with nesfatin-1. CONCLUSION Nesfatin-1 protects intestinal cells from LPS induced inflammation by targeting TLR4， which may be a potential target of anti-NEC therapy.     

Effect of recombinant plasmids encoding IL-1RII and IL-1RAcP on rat experimental autoimmune myocarditis and possible mechanism

AIM To evaluate the effects of recombinant plasmids encoding interleukin-1 type II receptor （IL-1RII） and interleukin-1 receptor accessory protein （IL-1RAcP） on rat experimental autoimmune myocarditis （EAM） and the possible mechanism. METHODS The recombinant plasmids pCAGGS-IL-1RII and pCAGGS-IL-1RAcP were constructed， and pCAGGS-SP （signal peptide） served as the control plasmid. Male Lewis rats （n=29） were divided into 4 groups： control group （rats without immunization or injection， n=5）， EAM+SP group （immunized rats injected with pCAGGS-SP， n=9）， EAM+IL-1RII group （immunized rats injected with pCAGGS-IL-1RII， n=8） and EAM+IL-1RII+IL-1RAcP group （immunized rats injected with pCAGGS-IL-1RII and pCAGGS-IL-1RAcP， n=7）. The rats were immunized to induce EAM on day 0， and injected with recombinant plasmids by hydrodynamics-based delivery on day 6. Echocardiography was performed， and the rats were killed on day 17. The ratio of heart weight to body weight （HW/BW） was evaluated， and the histopathological changes of the myocardial tissues were observed by HE staining. The mRNA expression of atrial natriuretic peptide （ANP）， brain natriuretic peptide （BNP） and inflammatory factors in the myocardial tissues was detected by RT-qPCR. Recombinant plasmids pUC19-IL-1RII-actin and pUC19-IL-1RAcP-tub were transfected into Cos7 cells， and the culture supernatants were collected and added to lipopolysaccharide （LPS）-induced H9c2 cells. The expression of inflammatory genes were detected by RT-qPCR. Recombinant plasmids pEGFP-IL-1RII-actin and pEGFP-IL-1RAcP-tub were transfected into the Cos7 cells to identify the formation of IL-1RII/IL-1RAcP heterodimer by co-immunoprecipitation （Co-IP）. RESULTS Compared with EAM+SP group， injection with plasmids effectively attenuated EAM in EAM+IL-1RII group and EAM+IL-1RII+IL-1RAcP group， as indicated by the decreases in HW/BW， left ventricular end-systolic diameter， and myocardial expression of ANP， BNP， TNF-α， IL-2， IFN-γ and TGF-β， and the increase in expression of IL-4 in the hearts. In LPS-induced H9c2 cells， compared with LPS group， the levels of TGF-β and IL-6 in the culture supernatants were significantly decreased （P<0.01）， and the level of IL-10 was significantly increased （P<0.05） in LPS+IL-1RII group and LPS+IL-1RII+IL-1RAcP group. Compared with LPS+IL-1RII group， the expression of TNF-α and IL-2 was significantly decreased （P<0.05）， and the expression of IL-13 was significantly increased in LPS+IL-1RII+IL-1RAcP group （P<0.01）. The formation of IL-1RII/IL-1RAcP heterodimer was detected by Co-IP. CONCLUSION Plasmids encoding IL-1RII and IL-1RAcP effectively attenuate EAM， and the possible mechanism may be related to the inhibition of inflammatory factor expression and the formation of IL-1RII/IL-1RAcP heterodimer.     

Relationships between lncRNA HOTAIR/H19 SNPs and genetic susceptibility to gastric carcinoma/EBV-related gastric carcinoma

AIM To investigate the potential associations between the single nucleotide polymorphisms （SNPs） of long noncoding RNA （lncRNA） H19/HOTAIR and the susceptibility to gastric carcinoma， especially to Epstein-Barr virus （EBV）-associated gastric carcinoma （EBVaGC）. METHODS Peripheral blood samples from 65 cases of EBV-negative gastric carcinoma （EBVnGC）， 50 cases of EBVaGC and 115 cases of healthy people were collected. A total of 4 TagSNPs， H19 rs3024270 and rs3741219， as well as HOTAIR rs4759314 and rs874945， were selected. The Taq-Man MGB allele typing kit was used to detect the genotype of each SNP locus， and the experimental results were statistically analyzed. RESULTS （1） There were significant differences of both genotypic and allelic frequencies at H19 rs3024270 locus between gastric carcinoma group and control group （P<0.05）. Individuals carrying the G allele at H19 rs3024270 locus had significantly low risk of gastric carcinoma （P<0.01）， indicating that the G allele was protective. （2） People with the GG genotype at HOTAIR rs4759314 locus had significantly high risk of gastric carcinoma （P<0.05）. Carrying the G allele increased the risk of gastric carcinoma， which indicated that the risk gene for gastric carcinoma might be the G allele. （3） No significant difference of the genotypic and allelic frequencies at H19 rs3741219 and HOTAIR rs874945 loci between gastric carcinoma group and control group was observed （P>0.05）.（4） The G allele frequency at HOTAIR rs4759314 locus in EBVaGC group was significantly higher than that in EBVnGC group. However， no difference of the other 3 SNPs was found between EBVaGC group and EBVnGC group （P>0.05）. CONCLUSION The SNPs at H19 rs3024270 and HOTAIR rs4759314 loci are related to the risk of gastric carcinoma， but not significantly related to the risk of EBVaGC.     

Effects of EGCG on GLUT2/G6PD/GS expression and blood glucose level in type 2 diabetic rats

AIM To investigate whether epigallocatechin gallate （EGCG） improves blood glucose in type 2 diabetic rats through glucose transporter 2 （GLUT2）-glucose-6-phosphate dehydrogenase （G6PD）-glycogen synthase （GS） pathway. METHODS Type 2 diabetes mellitus （T2DM） model was established in male Sprague-Dawley （SD） rats by feeding with high-fat diet and injection of streptozotocin （STZ）. The rats were divided into 5 groups （n=10）： control （Con） group， T2DM model （M） group， metformin （Met； 200 mg/kg， ig） group， T2DM+low-dose （50 mg/kg， ig） EGCG （EL） group， and T2DM+high-dose （100 mg/kg， ig） EGCG （EH） group. Diabetic rats were given drugs for 8 weeks. After 8 weeks of administration， the rats were killed， and the blood and liver tissues were collected. The levels of fasting blood glucose （FBG）， fasting serum insulin （FINS） and serum glycosylated hemoglobin were measured by biochemical tests. Liver glycogen were test by periodic acid-Schiff （PAS） staining. The mRNA expression of G6PD in the liver was detected by real-time PCR. The protein levels of GS and GLUT2 were determined by Western blot and immunohistochemistry. RESULTS T2DM rat model was established successfully. Compared with Con group， the levels of FBG， FINS and serum glycosylated hemoglobin in M group were increased significantly （P<0.05）， while the insulin sensitivity index （ISI）， the liver glycogen， the G6PD mRNA expression， and the protein levels of GS and GLUT2 were decreased significantly （P<0.05）. Compared with M group， the levels of FBG and serum glycosylated hemoglobin in Met group and EH group were decreased significantly （P<0.05）， while the ISI， the liver glycogen， the G6PD mRNA expression， and the protein levels of GS and GLUT2 were increased significantly （P<0.05）. CONCLUSION EGCG reduces the blood glucose level in T2DM rats， which may be related to the regulation of GLUT2-G6PD-GS signaling pathway.     

Expression of Wnt1 and LGR5 in gastric mucosa with stress ulcer after TBI in rats

AIM To investigate the expression of Wnt1 and LGR5 in gastric mucosa with stress ulcer after traumatic brain injury （TBI） in rats, and to study the relationship between Wnt1/LGR5 expression and stress ulcer after TBI. METHODS Healthy 7-week-old male SD rats （n=30） were randomly divided into 3 groups： sham operation （sham） group, mild TBI （mTBI） group, and severe TBI （sTBI） group. The mTBI and sTBI were induced by electronic cortical contusion impactor. Neurological severity score （NSS） was calculated 24 and 48 h after modeling. Mucosal blood flow in gastric fundus, greater curvature, pylorus and cardia of anesthetized rats 48 h after injury was measured by Doppler flowmeter. All rats were sacrificed, and their gastric tissues were harvested after 48 h and then stained with hematoxylin and eosin. The protein expression of Wnt1 and LGR5 was analyzed by Western blot and immunohistochemistry. RESULTS Compared with sham group, the NSS in mTBI group and sTBI group was significantly increased （P<0.01）. Compared with sham group, the gastric mucosal blood flow of the rats after TBI was significantly decreased （P<0.01）, and the decrease in sTBI group was more significant than that in mTBI group （P<0.05）. The protein expression of Wnt1 and LGR5 in mTBI group was significantly higher than that in sham group （P<0.05）, and that in sTBI group was significantly higher than that in mTBI group （P<0.05）. Normal glandular gastric tissue was observed, and no abnormal change was found in sham group, while the infiltration of inflammatory cells in gastric lamina propria, mucosa and submucosa was obvious in mTBI group and sTBI group. CONCLUSION Traumatic brain injury activates Wnt1 and LGR5 expression to induce inflammatory changes in gastric mucosa, and then induces stress ulcer. This results provides experimental basis for the pathogenesis and treatment of stress ulcer after trauma.