苹果miR396家族鉴定及在不定根发育过程中的表达分析

分析了苹果miR396家族进化特性及其在苹果不定根发育过程中的表达模式。结果表明：苹果miR396家族有4条成熟体和7条前体序列（pre-miRNA）。Mfold预测显示Pre-miR396家族7个成员序列均可形成典型稳定的茎环二级结构,最小折叠自由能介于–62.9 kal ? mol-1（pre-miR396b）~–51.9 kal ? mol-1（pre-miR396g）之间。系统发育进化树分析显示,pre-miR396家族亲缘关系可分为3个亚组（G1、G2、G3）,每个亚组内基因数量不同,分别含有11、9、19个。靶基因预测显示,苹果miR396靶基因包括MdGRF1、MdGRF2和MdGRF5等,降解组测序进一步验证了miR396对其候选靶基因MdGRF1、MdGRF2和MdGRF5的剪切关系。苹果miR396家族成员在侧根和果实中的表达量显著高于其他组织,其候选靶基因表达量则在花芽和腋芽中显著高于其他组织;不定根发育过程中,miR396家族不同成员表达模式存在显著差异,整体上呈上调表达趋势,其候选靶基因呈下调表达趋势;外源IBA处理显著诱导miR396家族成员的表达,尤其是在不定根诱导期和根系生长期更为显著。     

Effects of naringenin on PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways in rats with myocardial ischemia/reperfusion injury

AIM To observe the effect of naringenin on cardiac injury in ischemia/reperfusion （I/R） rats, and to explore whether the role of naringenin is involved in PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways. METHODS SD rats （n=48） were randomly divided into sham operation （sham） group, model （I/R） group, naringenin treatment （NAR） group and naringenin+LY294002 （NL） group. Myocardial I/R injury model was prepared by ligation of left anterior descending coronary artery of rats for 30 min followed by reperfusion for 120 min. After reperfusion, the serum levels of cardiac troponin I （cTnI） was measured by ELISA. HE staining, TTC staining and TUNEL staining were used to detect the myocardial histopathological changes, myocardial infarction area and myocardial cell apoptotic rate. The mRNA levels of endoplasmic reticulum stress-related indicators glucose-regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP） and caspase-12 were detected by RT-qPCR. The protein levels of cleaved caspase-3, GRP78, CHOP, caspase-12, p-PI3K and p-AKT were determined by Western blot. RESULTS Compared with I/R group, the serum content of cTnI, myocardial pathological damage, myocardial infarction area and myocardial cell apoptotic rate were significantly reduced （P<0.05）, the protein levels of cleaved caspase-3, GRP78, CHOP and caspase-12 were decreased （P<0.05）, and the protein levels of p-PI3K and p-AKT were increased in NAR group （P<0.05）. LY294002 attenuated the protective effect of naringenin to some extent. CONCLUSION Naringenin reduces myocardial I/R injury in rats possibly by activating PI3K/AKT signaling pathway and subsequently regulating endoplasmic reticulum stress and its related apoptotic pathways.     

Autophagy induces mitochondrial dysfunction in neurons from neonatal rats with hypoxic ischemic encephalopathy

AIM: To explore the influence of autophagy on the induction of mitochondrial dysfunction in the neurons in a neonatal rat hypoxic ischemic encephalopathy (HIE) model. METHODS: Ten-day-old rat pups (n=30) were randomly divided into sham group and model group. The rats in the latter group were subject to hypoxia-ischemia treatment via unilateral common carotid artery ligation. The rats were sacrificed for brain pathological examination, and the protein levels of cleaved caspase-3 and LC3B-II were detected by immunohistochemical analysis. For the in vitro experiments, the autophagy of primarily cultured rat neurons was observed after hypoxia, and Western blot and mitochondrial function testing were also performed. RESULTS: Compare with sham group, the hypoxia-ischemia treatment caused atrophy and apoptosis of neurons, and ventricular area enlargement of rat brains. Immunohistochemical results demonstrated significantly higher levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase-3 and LC3B-II (P<0.01). In vitro experiments demonstrated that hypoxia induced autophagy and apoptosis in the neurons. Compared with sham group, there were higher levels of reactive oxygen species and mitochondrial superoxide, and lower mitochondrial membrane potential in the model group (P<0.01). CONCLUSION: In neonatal HIE rat model, the hypoxia-induced mitochondrial dysfunction is related to apoptosis and autophagy. It will provide a new idea for administration of autopahgy inducer agents in treatment of HIE.     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

Serum CTRP1 and CTRP7 levels are associated with coronary atherosclerotic heart disease and diabetes mellitus

AIM To investigate the relationship between the serum levels of C1q/TNF-related protein (CTRP) 1 and CTRP7 and coronary atherosclerotic heart disease (CAD) in the patients with or without type 2 diabetes mellitus (T2DM). METHODS Totally 138 cases of participants were selected, and divided into control group (without T2DM or CAD; n=40), T2DM group (with T2DM, but without CAD; n=30), CAD group (with CAD, but without T2DM; n=40), and CAD+T2DM group (with both T2DM and CAD; n=28). The serum levels of CTRP1 and CTRP7 in these participants were measured by ELISA. RESULTS (1) Compared with control group, serum CTRP1 level in CAD group and CAD+T2DM group was significantly increased (P<0.05 or P<0.01), and serum CTRP7 level in CAD, T2DM and CAD+T2DM groups was significantly decreased (P<0.05 or P<0.01). (2) Increased serum CTRP1 level was a risk factor for CAD [odds ratio (OR)=1.136; 95% confidence interval (CI): 1.010~1.278; P=0.034). Sex, hypertension and serum triglyceride level were also correlated with CAD. Decreased serum CTRP7 level was a risk factor for T2DM (OR=0.984; 95% CI: 0.969~0.999; P=0.038). Sex, hypertension and serum high-density lipoprotein cholesterol level were also correlated with T2DM. Increased serum CTRP1 level was a risk factor for CAD+T2DM (OR=1.009; 95% CI: 1.005~1.203; P=0.040). Hypertension was also a risk factor for CAD+T2DM. (3) In CAD group, serum CTRP1 level had certain diagnostic value, and the area under the receiver operating characteristic (ROC) curve (AUC) was 0.670 (95% CI: 0.580~0.760; P=0.001), but serum CTRP7 level had no diagnostic value for CAD. In T2DM group, both serum CTRP1 and CTRP7 levels had diagnostic value, and the AUC values of their ROC curves were 0.607 (95% CI: 0.505~0.710; P=0.032) and 0.665 (95% CI: 0.574~0.756; P=0.001), respectively. In CAD+T2DM group, both serum CTRP1 and CTRP7 levels had diagnostic value, and the AUC values of their ROC curves were 0.666 (95% CI: 0.552~0.781; P=0.007) and 0.632 (95% CI 0.517~0.747; P=0.032), respectively. CONCLUSION Increased serum CTRP1 level and decreased serum CTRP7 level are associated with increased risk of T2DM and CAD. Increased serum CTRP1 level is a risk factor for CAD and CAD+T2DM, while decreased serum CTRP7 level is a risk factor for T2DM. Serum CTRP1 level has certain specificity and sensitivity for the diagnosis of CAD, T2DM and CAD+T2DM, while serum level of CTRP7 has certain specificity and sensitivity for the diagnosis of T2DM and CAD+T2DM.     

Effects of astragaloside on the levels of sex hormone and oxidative stress injury in rats with polycystic ovary syndrome

AIM To investigate the effects of astragaloside on the levels of sex hormone and oxidative stress in rats with polycystic ovary syndrome （PCOS）. METHODS Female SD rats （n=60） were randomly divided into normal control group, model group, Diane-35 （0.339 2 mg/kg） group, low dose astragaloside （12.5 mg/kg） group and high dose astragaloside （50 mg/kg） group, with 12 rats in each group. The PCOS model was induced by letrozole （1 mg/kg）, which was administered by gavage once a day for 3 weeks. After administration, the estrus cycle of the rats was observed by vaginal smear, and the ovarian index was calculated. HE staining was used to observe the histopathological changes of the ovaries. Serum levels of the sex hormones testosterone （T）, estradiol （E₂）, luteinizing hormone （LH） and follicle-stimulating hormone （FSH） were measured by ELISA. The levels of superoxide dismutase （SOD）, malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） in serum and ovarian tissue were detected by colorimetry, and the protein levels of steroidogenetic acute regulatory protein （StAR） and apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 in ovarian tissue were detected by Western blot. RESULT Compared with control group, the oestrous cycle of the rats in model group was disorder, and the ovarian index was increased, ovary was polycystic. The serum levels of T, LH and MDAwere significantly increased （P<0.05）, while the contents of E2, FSH and the activities of GSH-Px and SOD were significantly decreased （P<0.05）. The levels of MDA, StAR, cleaved caspase-3 and Bax proteins in ovarian tissue were significantly up-regulated （P<0.05）. GSH-Px and SOD activities and Bcl-2 protein levels were significantly down-regulated （P<0.05）. CONCLUSION Astragalosideeffectively balances the levels of sex hormone in PCOS rats and relieves the oxidative stress injury, the mechanism may be related to the inhibition of StAR expression.     

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.     

Cyanidin attenuates pressure overload-induced cardiac remodeling in mice via mitochondrial fusion enhancement

AIM To investigate the effect of cyanidin （Cyn） on pressure overload-induced cardiac remodeling and the underlying mechanism. METHODS Six-week-old male C57BL/6 mice （n=120） were divided into 4 groups： sham group （n=20）, sham+Cyn group （n=20）, transverse aortic constriction （TAC） group （n=40） and TAC+Cyn group （n=40）. The model of cardiac chronic pressure overload was induced by TAC, and the first day of TAC was defined as day 0. The animals in sham+Cyn group and TAC+Cyn group were treated with Cyn dissolved in DMSO and normal saline （5 mg·kg^-1·d^-1） for 5 d before TAC, while the animals in sham group and TAC group were treated with the same amount of DMSO and normal saline. Four weeks after TAC, the survival rate of the animals in each group was analyzed, the heart function of the mice was measured by ultrasound echocardiography, and the heart weight/body weight and lung weight/body weight were calculated. The cross-sectional area of the cardiomyocytes was measured by wheat germ agglutinin staining and hematoxylin-eosin staining. The degree of cardiac oxidative stress was evaluated by dihydroethidium staining and measurement of superoxide dismutase （SOD） and malondialdehyde （MDA） levels. The cardiomyocyte apoptosis was detected by TUNEL method. The mRNA expression levels of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected by RT-qPCR, and the protein expression levels of Bax, Bcl-2, optic atrophy protein 1 （OPA1） and dynamin-related protein 1 （Drp1） were determined by Western blot. The mitochondrial morphological changes were observed by transmission electron microscopy. RESULTS Compared with TAC group, the survival rate of the mice in TAC+Cyn group was significantly increased （P<0.05）, the myocardial apoptosis, the cross-sectional area of myocardial cells, the heart weight/body weight, the lung weight/body weight, the level of reactive oxygen species and the MDA content were decreased （P<0.05）, and the SOD was activated （P<0.05）. M-mode ultrasound tests showed that Cyn treatment significantly increased left ventricular ejection fraction and left ventricular fractional shortening in the mice after TAC （P<0.05）, while left ventricular end-diastolic diameter and left ventricular posterior wall thickness in diastole were reduced （P<0.05）. Transmission electron microscopic observation showed that the number of myocardial mitochondria was increased and the mitochondrial area was decreased after TAC （P<0.05）, while treatment with Cyn increased the area of myocardial mitochondria and decreased the mitochondrial number （P<0.05）. Compared with sham group, the protein level of OPA1 in TAC group was significantly reduced （P<0.05）, while treatment with Cyn significantly increased the protein level of OPA1. CONCLUSION Cyanidin significantly increases the survival rate, improves the cardiac function and attenuates the cardiac remodeling of the mice after TAC. The mechanism may be related to the inhibition of myocardial mitochondrial OPA1 cleavage and the promotion of mitochondrial fusion.     

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.     

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.     

miR-323 regulates hypoxia-induced apoptosis of H9C2 cardiomyocytes through FGF9

AIM: To investigate the effect of microRNA-323 (miR-323) on the apoptosis of hypoxia-induced rat H9C2 cardiomyocytes and its mechanism. METHODS: The hypoxic injury model was established in the H9C2 cells. Anti-miR-323, pcDNA-FGF9 and si-FGF9 were transfected into the H9C2 cells and cultured under hypoxic condition for 48 h. The expression of miR-323 was detected by qPCR. The protein levels of fibroblast growth factor 9 (FGF9), cleaved caspase-3, c-Jun N-terminal kinase (JNK) and p-JNK were determined by Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The method of bioinformatics was applied to predict the target gene of miR-323, and dual-luciferase reporter assay was used for further validation. RESULTS: Hypoxia greatly reduced the viability of H9C2 cells at 12 h, 24 h and 48 h (P<0.05), and remarkably increased apoptotic rate and the protein level of cleaved caspase-3 in a time-dependent manner (P<0.05). The expression of miR-323 and the protein level of p-JNK were up-regulated and the expression of FGF9 was down-regulated in the H9C2 cells exposed to hypoxia (P<0.05). Down-regulation of miR-323 and over-expression of FGF9 obviously increased the viability of the H9C2 cells exposed to hypoxia, and decreased the apoptotic rate and the protein level of cleaved caspase-3 (P<0.05). FGF9 was the target gene of miR-323. Down-regulation of FGF9 reversed the attenuating effect of down-regulation of miR-323 on hypoxia-induced H9C2 cell injury. miR-323 regulated FGF9 and affected p-JNK level. CONCLUSION: miR-323 affects the viability and apoptosis of H9C2 cardiomyocytes under hypoxia by targeting FGF9 and regulating JNK signaling pathway.     

Aberrant SYK expression in cholangiocarcinoma and its effect on M2 macrophage polarization in tumor microenvironment

AIM To investigate the expression of spleen tyrosine kinase (SYK) in 2 murine cholangiocarcinoma (CCA) progressive models and human CCA tissues, and to explore the effects of SYK expression on the polarization of M2 macrophages. METHODS SD rats were given drinking water containing thioacetamide (TAA) daily. BALB/c mice were treated with left median bile duct ligation combined with diethylnitrosamine (DEN) administration. The expression of SYK and M2 macrophage infiltration (CD163) in the animals and human CCA tissues were detected by immunohistochem?istry, and their correlation was analyzed. Ten SD rats, which were given drinking water containing TAA daily for 24 weeks, were randomly divided into control group (n=5) and SYK inhibitor group (n=5). After the rats received SYK inhibitor through gavage for 4 weeks, the effects of SYK inhibitor on tumor growth and macrophage polarization were analyzed. RESULTS The hepatic bile duct hyperplasia, dysplasia (or cholangioma), and CCA occurred at different time points in the rats and mice. During the development of CCA, SYK expression was gradually increased (P<0.01), accompanied by enhanced infiltration of M2 macrophages, while the M1 macrophages were decreased in the hepatic bile duct tissues (P<0.01). SYK and CD163 expression levels were significantly up-regulated (P<0.01), and a positive correlation (r=0.57, P<0.01) in human CCA tissues was observed. In the rat CCA model, the number of tumor nodules and infiltration of M2 macrophages in SYK inhibitor group were decreased compared with its control group (P<0.01). CONCLUSION In the murine CCA models, SYK expression was progressively increased during the evolution of CCA, which may promote tumor progression via the polarization of M2 macrophages, and SYK inhibitor effectively inhibits the tumor growth and M2 macrophage polarization in the rat CCA model.     

Dasatinib inhibits the viability and migration of renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation in vitro

AIM To explore the effect of dasatinib on the viability, apoptosis and migration of human renal carcinoma cell lines 786-O and 769-P, as well as the molecular mechanism in vitro. METHODS 786-O cells and 769-P cells were treated with different concentrations （0~2 μmol/L） of dasatinib, and 0 μmol/L dasatinib was used as blank control group. MTT method was used to detect cell viability. Wound healing assay was used to detect the effect of dasatinib on migration. Hoechst 33258 staining was used to observe the effect of dasatinib on apoptosis. Flow cytometry was used to detect the effect of dasatinib on cell cycle. Western blot method was used to detected cell cycle- and apoptosis-related protein levels. RESULTS Dasatinib inhibited viability and migration of 786-O and 769-P cells, and the inhibitory effect of dasatinib increased with the concentration of dasatinib （P<0.05）. The IC₅₀ values of dasatinib against 786-O and 769-P cell lines were （0.958 7±0.028 8） μmol/L and （0.784 3±0.066 0） μmol/L, respectively. After treatment with dasatinib for 24 h, the expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 increased significantly （P<0.01）, while the expression of cyclin D1 decreased （P<0.05）. The cycle-related pathway proteins p53 and p21 increased （P<0.05）, while the level of p-AKT was decreased （P<0.05）. CONCLUSION Dasatinib impaired the viability and migration ability of human renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation.     

Immunohistochemical localization of CD14, IL-4 and TNF-α in periapical tissue of healthy individuals and patients with chronic periapical diseases

AIM: To observe the expression of cluser of differentiation 14 (CD14), tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in periapical tissues of patients with chronic periapical diseases, and to analyze the role of immunopathogenesis of CD14, TNF-α and IL-4 expression in human chronic periapical diseases. METHODS: A total of 88 samples were divided into 3 groups: healthy control group (n=45), chronic periapical abscess group (n=23), and periapical cyst group (n=20). All samples were fixed in 10% buffered formalin and stained with double immunofluorescence for identification of TNF-α-CD14 and IL-4-CD14 double-positive cells in periapical tissues. RESULTS: Compared with healthy control group, the densities of TNF-α-CD14 and IL-4-CD14 double-positive cells in the 2 groups of chronic periapical diseases were increased significantly (P<0.05). Compared with periapical cyst group, the density of TNF-α-CD14 double-positive cells in chronic periapical abscess was increased significantly (P<0.05). The density of IL-4-CD14 double-positive cells in periapical cyst group was significantly higher than that in chronic periapical abscess group (P<0.05). CONCLUSION: There is high expression of TNF-α-CD14 and IL-4-CD14 double-positive cells in the periapical tissues of the patients with chronic periapical abscess and perapical cyst, suggesting that the Th cytokines participate in the immune regulation of periapical diseases, which may be one of the immune mechanisms of the interaction between Th1 and Th2 cytokines.     

矮化中间砧‘宫藤富士’苹果栽植密度对树体生长、冠层光照和果实产量的影响

2011 年春季定植的矮化中间砧苹果成品苗（3 年根 1 年干的‘宫藤富士’/SH6/平邑甜茶）为试材，设置 7 种不同的栽植密度（株行距分别为 1 m × 3 m、1.5 m × 3 m、2 m × 3 m、0.75 m × 4 m、1 m × 4 m、1.25 m × 4 m 和 1.5 m × 4 m），细纺锤形整枝修剪，自栽植第 2 年，连续 7 年调查 7 种栽植密度对树体生长、冠层光照分布、果实产量和品质的影响。随着树龄的增长，不同栽植密度下树干粗度和总枝量逐年增加，不同处理间树干粗度无显著差异，第 7 年 1 m × 3 m 和 0.75 m × 4 m 两个栽植密度下树体总枝量超过 140 万条 · hm-2，第 8 年均超过 140 万条 · hm-2。栽植前期（第 2 ~ 4 年）各栽植密度树体短枝比例不断增加，长枝比例不断减少，第 5 年各栽植密度枝类组成趋于稳定；综合稳产 3 年（第 6 ~ 8 年）树体的枝类组成数据，4 m 行距的短枝比例明显高于 3 m 行距，长枝比例略低。树体冠层平均相对光照强度由高到低的株行距处理依次为 1.5 m × 4 m（63.87%）、1.25 m × 4 m（61.44%）、2 m × 3 m（61.27%）、1 m × 4 m（59.19%）、0.75 m × 4 m（55.79%）、1.5 m × 3 m（53.67%）和 1 m × 3 m（49.37%）；相同栽植株数下，4 m 行距处理低光效（相对光照强度小于 40%）的区域比例显著小于 3 m 行距。比较前 5 年的累计产量，以行距 4 m 和 1 m × 3 m 的最高。综合稳产 3 年的结果情况，大果率（单果质量 > 200 g 的果实产量占总产量的比例）以 4 m 行距和 2 m × 3 m 的最高。各栽植密度下的果实的可溶性固形物含量、固酸比、果形指数和果实硬度均无显著差异。综上，采用 4 m 行距，1 ~ 1.25 m 株距，树体成形快，稳产后树体结构合理，冠层光照充足，低效光区比例少，前期产量高。     

Metformin attenuates nerve injury in stroke rats by regulating SIRT1/PGC-1α signaling pathway via miR-29c

AIM To explore the inhibitory effect of metformin （MET） on nerve injury in rats with stroke and its mechanism. METHODS SD rats were randomly divided into sham group （n=15）, model group （n=30）, MET group （n=30）, MET+agomir-NC group （n=30） and MET+agomir group （n=30）. The modified Puisinelli four-vessel occlusion method was used to prepare the model of global ischemic stroke, while the blood vessels in sham rats were isolated without clamping the common artery. One week before modeling, the rats in MET group, MET+agomir-NC group and MET+agomir group were given intraperitoneal injection of 100 mg·kg^-1·d^-1 MET, 100 mg·kg^-1·d^-1 MET+40 nmol/d agomir-NC, 100 mg·kg^-1·d^-1 MET+40 nmol/d miR-29c agomir, respectively, and the rats in sham group and model group were given intraperitoneal injection of the same amount of normal saline. Each treatment in the above groups was given once a day, 0.2 mL each time, for 7 consecutive days. The neurological deficit scores were measured 24, 48 and 72 h after operation. HE staining was used to observe the morphological changes of the hippocampus, and the living neurons were counted. RT-qPCR was used to detect the expression level of miR-29c, and the mRNA levels of silent information regulator 1 （SIRT1） and peroxisome proliferator-activated receptor γ coactivator 1α （PGC-1α） in hippocampus. The protein expression levels of SIRT1 and PGC-1α were determined by Western blot. RESULTS At the same time point, compared with model group, the neurological deficit score in MET group was significantly decreased, and the survival rate of the neurons was significantly increased （P<0.05）. Compared with MET+agomir-NC group, the neurological deficit score in MET+agomir group was increased, and the survival rate of the neurons was significantly decreased （P<0.05）. With the prolongation of time, except for sham group, the neurological deficit score was increased and the survival rate of the neurons was decreased. At 72 h after operation, compared with sham group, the expression of miR-29c in hippocampus of model group was significantly increased, and the mRNA and protein expression levels of SIRT1 and PGC-1α were significantly decreased （P<0.05）. Compared with model group, the expression of miR-29c in hippocampus of MET group was significantly decreased, and the expression of SIRT1 and PGC-1α at mRNA and protein levels was significantly increased （P< 0.05）. Compared with MET+agomir-NC group, the expression of miR-29c in hippocampus of MET+agomir group was significantly increased, and the mRNA and protein expression of SIRT1 and PGC-1α was significantly decreased （P<0.05）. CONCLUSIONS MET alleviates nerve injury in stroke rats, which may be related to down-regulation of miR-29c and promotion of SIRT1/PGC-1α signaling pathway activation.     

Effect of Herba Taxilli extracts combined with miR-375 on viability and apoptosis of osteoarthritic chondrocytes

AIM To study the effects of extracts of Herba Taxilli (Sangjisheng, SJS) on the viability and apoptosis of osteoarthritic chondrocytes and the underlying mechanism. METHODS Human primary osteoarticular chondrocytes (RPOC) were divided into control group, interleukin-1β (IL-1β) group, IL-1β+low-dose extracts of SJS (SJS-L) group, IL-1β+medium-dose extracts of SJS (SJS-M) group, IL-1β+high-dose extracts of SJS (SJS-H) group, IL-1β+anti-miR-NC group, IL-1β+anti-miR-375 group, IL-1β+SJS-H+miR-NC group, IL-1β+SJS-H+miR-375 group. The cell viability was measured by MTT assay, apoptosis was analyzed by flow cytometry, miR-375 expression was detected by qPCR, and the protein levels of cyclin D1, P21, Bcl-2, Bax and caspase-3 were determined by Western blot. RESULTS Compared with control group, the viability of RPOC at 24 h, 48 h and 72 h and the protein expression levels of cyclin D1 and Bcl-2 were significantly decreased (P<0.05), the protein levels of P21, Bax and caspase-3, the apoptotic rate and the expression level of miR-375 were remarkably increased in IL-1β group(P<0.05). Compared with IL-1β group, the cell viability at 24 h, 48 h and 72 h and the protein expression of cyclin D1 in the RPOC were greatly increased (P<0.05), while the expression of P21 was significantly decreased in IL-1β+SJS-M group and IL-1β+SJS-H group(P<0.05).The apoptotic rate, Bax, caspase-3 protein and miR-375 expression were obviously decreased (P<0.05), and Bcl-2 protein level was significantly increased in IL-1β+SJS-H group compared with IL-1β group(P<0.05). Compared with IL-1β+anti-miR-NC group, the expression of miR-375, the protein levels of P21, Bax, caspase-3 and the apoptotic rate in the RPOC of IL-1β+anti-miR-375 group were markedly decreased (P<0.05), while the cell viability at 24 h, 48 h and 72 h and the protein levels of cyclin D1 and Bcl-2 were significantly increased (P<0.05). Over-expression of miR-375 reversed the effects of extracts of SJS on the viability and apoptosis of RPOC with IL-1β stimulation. CONCLUSION The extracts of Herba Taxilli promotes the viability and inhibits apoptosis of RPOC treated with IL-1β, which is related to the regulation of miR-375 expression.